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FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs.
IMPORTANCE
The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria.
The mechanisms behind carbon dioxide (CO2) dependency in non-autotrophic bacterial isolates are unclear. Here we show that the Staphylococcus aureus mpsAB operon, known to play a role in membrane potential generation, is crucial for growth at atmospheric CO2 levels. The genes mpsAB can complement an Escherichia coli carbonic anhydrase (CA) mutant, and CA from E. coli can complement the S. aureus delta-mpsABC mutant. In comparison with the wild type, S. aureus mps mutants produce less hemolytic toxin and are less virulent in animal models of infection. Homologs of mpsA and mpsB are widespread among bacteria and are often found adjacent to each other on the genome. We propose that MpsAB represents a dissolved inorganic carbon transporter, or bicarbonate concentrating system, possibly acting as a sodium bicarbonate cotransporter.
Defeat of the antibiotic resistance of pathogenic bacteria is one great challenge today and for the future. In the last century many classes of effective antibacterials have been developed, so that upcoming resistances could be met with novel drugs of various compound classes. Meanwhile, there is a certain lack of research of the pharmaceutical companies, and thus there are missing developments of novel antibiotics. Gram-positive bacteria are the most important cause of clinical infections. The number of novel antibacterials in clinical trials is strongly restricted. There is an urgent need to find novel antibacterials. We used synthetic chemistry to build completely novel hybrid molecules of substituted indoles and benzothiophene. In a simple one-pot reaction, two novel types of thienocarbazoles were yielded. Both indole substituted compound classes have been evaluated as completely novel antibacterials against the Staphylococcus and Enterococcus species. The evaluated partly promising activities depend on the indole substituent type. First lead compounds have been evaluated within in vivo studies. They confirmed the in vitro results for the new classes of small-molecule antibacterials.
Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.
In most organisms, ribosomal RNA (rRNA) contributes to >85% of total RNA. Thus, to obtain useful information from RNA-sequencing (RNA-seq) analyses at reasonable sequencing depth, typically, mature polyadenylated transcripts are enriched or rRNA molecules are depleted. Targeted depletion of rRNA is particularly useful when studying transcripts lacking a poly(A) tail, such as some non-coding RNAs (ncRNAs), most bacterial RNAs and partially degraded or immature transcripts. While several commercially available kits allow effective rRNA depletion, their efficiency relies on a high degree of sequence homology between oligonucleotide probes and the target RNA. This restricts the use of such kits to a limited number of organisms with conserved rRNA sequences. In this study we describe the use of biotinylated oligos and streptavidin-coated paramagnetic beads for the efficient and specific depletion of trypanosomal rRNA. Our approach reduces the levels of the most abundant rRNA transcripts to less than 5% with minimal off-target effects. By adjusting the sequence of the oligonucleotide probes, our approach can be used to deplete rRNAs or other abundant transcripts independent of species. Thus, our protocol provides a useful alternative for rRNA removal where enrichment of polyadenylated transcripts is not an option and commercial kits for rRNA are not available.
CRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.
Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa. Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (RomR) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene (farR), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA. All these genes were consequently upregulated in the RomR clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the RomR clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE. FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the RomR clone compared to its parental strain HG001. If farE is deleted in the RomR clone, or, if native farR is expressed in the RomR strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the RomR clone, that FarR is an important regulator, and that the point mutation in farR (RomR clone) makes the clone hyper-virulent.
Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, have been recognised as an important cause of health care-associated infections due to catheterisation, and livestock-associated infections. The colonisation of indwelling medical devices is achieved by the formation of biofilms, which are large cell-clusters surrounded by an extracellular matrix. This extracellular matrix consists mainly of PIA (polysaccharide intercellular adhesin), which is encoded by the icaADBC-operon. The importance of icaADBC in clinical strains provoking severe infections initiated numerous investigations of this operon and its regulation within the last two decades. The discovery of a long transcript being located next to icaADBC, downstream of the regulator gene icaR, led to the hypothesis of a possible involvement of this transcript in the regulation of biofilm formation (Eckart, 2006). Goal of this work was to characterise this transcript, named ncRNA IcaZ, in molecular detail and to uncover its functional role in S. epidermidis.
The ~400 nt long IcaZ is specific for ica-positive S. epidermidis and is transcribed in early- and mid-exponential growth phase as primary transcript. The promotor sequence and the first nucleotides of icaZ overlap with the 3' UTR of the preceding icaR gene, whereas the terminator sequence is shared by tRNAThr-4, being located convergently to icaZ. Deletion of icaZ resulted in a macroscopic biofilm-negative phenotype with highly diminished PIA-biofilm. Biofilm composition was analysed in vitro by classical crystal violet assays and in vivo by confocal laser scanning microscopy under flow conditions to display biofilm formation in real-time. The mutant showed clear defects in initial adherence and decreased cell-cell adherence, and was therefore not able to form a proper biofilm under flow in contrast to the wildtype. Restoration of PIA upon providing icaZ complementation from plasmids revealed inconsistent results in the various mutant backgrounds.
To uncover the functional role of IcaZ, transcriptomic and proteomic analysis was carried out, providing some hints on candidate targets, but the varying biofilm phenotypes of wildtype and icaZ mutants made it difficult to identify direct IcaZ mRNA targets. Pulse expression of icaZ was then used as direct fishing method and computational target predictions were executed with candidate mRNAs from aforesaid approaches. The combined data of these analyses suggested an involvement of icaR in IcaZ-mediated biofilm control. Therefore, RNA binding assays were established for IcaZ and icaR mRNA. A positive gel shift was maintained with icaR 3' UTR and with 5'/3' icaR mRNA fusion product, whereas no gel shift was obtained with icaA mRNA. From these assays, it was assumed that IcaZ regulates icaR mRNA expression in S. epidermidis. S. aureus instead lacks ncRNA IcaZ and its icaR mRNA was shown to undergo autoregulation under so far unknown circumstances by intra- or intermolecular binding of 5' UTR and 3' UTR (Ruiz de los Mozos et al., 2013). Here, the Shine-Dalgarno sequence is blocked through 5'/3' UTR base pairing and RNase III, an endoribonuclease, degrades icaR mRNA, leading to translational blockade. In this work, icaR mRNA autoregulation was therefore analysed experimentally in S. epidermidis and results showed that this specific autoregulation does not take place in this organism. An involvement of RNase III in the degradation process could not be verified here. GFP-reporter plasmids were generated to visualise the interaction, but have to be improved for further investigations.
In conclusion, IcaZ was found to interact with icaR mRNA, thereby conceivably interfering with translation initiation of repressor IcaR, and thus to promote PIA synthesis and biofilm formation. In addition, the environmental factor ethanol was found to induce icaZ expression, while only weak or no effects were obtained with NaCl and glucose. Ethanol, actually is an ingredient of disinfectants in hospital settings and known as efficient effector for biofilm induction. As biofilm formation on medical devices is a critical factor hampering treatment of S. epidermidis infections in clinical care, the results of this thesis do not only contribute to better understanding of the complex network of biofilm regulation in staphylococci, but may also have practical relevance in the future.
Staphylococcus aureus ist ein grampositives Bakterium, welches häufig als kommensaler Besiedler auf der Nasen- und Rachenschleimhaut von Säugetieren vorkommt. Darüber hinaus besitzt dieser fakultativ pathogene Mikroorganismus die Fähigkeit schwer zu behandelnde Krankenhausinfektionen auszulösen. Aufgrund der weiten Verbreitung von Antibiotikaresistenzen und dem Mangel an effektiven Therapien, verursachen S. aureus Infektionen jährlich enorme Kosten für das Gesundheitssystem. S. aureus wird meist von der Nase zum primären Infektionsort übertragen, wodurch zunächst sehr häufig Wund- und Weichteilinfektionen hervor gerufen werden. Von diesem primären Infektionsort ausgehend, kann der Erreger tiefer liegende Gewebsschichten infizieren oder sich über den Blutstrom im gesamten Organismus ausbreiten. Das Spektrum an Krankheitsbildern reicht von leichten Abszessen der Haut bis zu schweren, lebensbedrohlichen Erkrankungen wie Pneumonien und akuter Sepsis.
Für die erfolgreiche Kolonisierung und Infektion des Wirtes exprimiert S. aureus eine Vielzahl unterschiedlicher Virulenzfaktoren. Die wohl größte Gruppe an Virulenzfaktoren umfasst die Proteine, die an der Immunevasion und der Umgehung von verschiedenen Abwehrstrategien des Immunsystems beteiligt sind. Das bisherige Wissen über die Interaktion von S. aureus mit dem Immunsystem des Wirtes und die zugrunde liegenden Pathogenitätsmechanismen ist bisher limitiert.
Um neue Erkenntnisse über die Interaktion von Wirt und Pathogen zu erlangen, wurden im Rahmen dieser Arbeit bislang unbekannte sekretierte und Oberflächen-assoziierte Proteine von S. aureus funktionell charakterisiert. Die Funktion der ausgewählten Proteine wurde in vitro hinsichtlich Einfluss auf Komponenten des Immunsystems, Adhäsion an Wirtsfaktoren und Invasion in eukaryotische Zellen untersucht.
Mit Hilfe der vorangegangenen in-vitro-Charakterisierung der putativen Virulenzfaktoren, konnte für die cytoplasmatische Adenylosuccinat-Synthase PurA eine neuartige Funktion identifiziert werden. PurA ist bekannt als essentielles Enzym der de novo Purin-Synthese. In dieser Arbeit wurde nun gezeigt, dass PurA zudem an der Immunevasion beteiligt ist. Durch die Bindung des humanen Faktor H des Komplementsystems schützt PurA S. aureus vor der lytischen Aktivität des Komplementsystems und verhindert die Opsonisierung des Pathogens. Basierend auf diesen Ergebnissen wurde PurA detailliert charakterisiert. In Bindungsstudien mit rekombinantem Faktor H und PurA wurde eine direkte Interaktion beider Proteine nachgewiesen, wobei Faktor H mit dem N-terminalen Bereich von PurA interagiert. Weiterhin konnte PurA durch Immunfluoreszenz und FACS-Analysen auf der Zelloberfläche nachgewiesen werden, wo es wahrscheinlich mit der Zellwand assoziiert vorliegt. Dort rekrutiert es Faktor H an die bakterielle Oberfläche und verhindert das Fortschreiten der Komplement-Kaskade und damit die Lyse des Pathogens. Aufgrund der Multifunktionalität zählt PurA somit zur Gruppe der Moonlighting Proteine.
Des Weiteren wurde die Rolle von PurA im Infektionsgeschehen in zwei unabhängigen Tiermodellen untersucht. In beiden Modellen wurde ein signifikant reduziertes Virulenzpotential der ΔpurA-Mutante beobachtet. Zukünftig soll geklärt werden, ob die verminderte Virulenz in der fehlenden Komplementevasion oder im Defekt in der Purin-Synthese begründet ist. Aufgrund der sehr starken Attenuation in allen untersuchten Infektionsmodellen sollte PurA als potentielles Target für eine Therapie von S. aureus Infektionen weiter charakterisiert werden. Im Ergebnis dieser Arbeit wurde demnach mit PurA ein neues Moonlighting Protein identifiziert, das als Inhibitor des Komplementsystems wesentlich zur Immunevasion von S. aureus beiträgt.
Für das bessere Verständnis der humoralen S. aureus-spezifischen Immunantwort, Unterschieden in der Antikörperantwort und der gebildeten Antikörperspezifitäten wurde weiterhin das während der Kolonisierung und Infektion gebildete S. aureus-spezifische Antikörperprofil untersucht. Dazu wurden Plasmen von humanen nasalen Trägern und Nicht-Trägern sowie murine Seren von infizierten Tieren untersucht. Insbesondere wurde das Pathogen-spezifische Antikörperprofil in unterschiedlichen Infektionsmodellen mit Hilfe eines Proteinarrays analysiert, der im Rahmen dieser Arbeit in einer Kooperation mit der Firma Alere Technologies (Jena, Deutschland) und universitären Forschergruppen der Universitäten Greifswald, Münster und Jena mitentwickelt wurde. Die Antikörperprofile von intramuskulär und intravenös infizierten Tieren resultierten in jeweils spezifischen Antikörperprofilen. Diese Ergebnisse deuten auf einen Zusammenhang zwischen der Art der Infektion und der gebildeten Antikörperspezifitäten hin. Wahrscheinlich beruht dies auf einer gewebespezifischen Genexpression als Anpassung an die individuellen Bedürfnisse im Wirtsorganismus. Das ausgebildete Antikörperprofil gibt somit einen Einblick in das Expressionsmuster von Virulenzfaktoren von S. aureus unter in vivo Bedingungen und trägt damit zum Verständnis der komplexen Interaktion von Pathogen und Wirt bei. Diese Untersuchungen ergänzen zudem die bisherigen Kenntnisse über die Anpassung der humoralen Immunantwort an eine asymptomatische Kolonisierung im Gegensatz zu einer akuten Infektion durch S. aureus. Darüber hinaus können die gewonnenen Ergebnisse für diagnostische Zwecke und zur Identifikation von neuen Zielstrukturen für eine Vakzin-Entwicklung genutzt werden.
Human herpesvirus 6A (HHV-6A) replicates in peripheral blood mononuclear cells (PBMCs) and various T-cell lines in vitro. Intriguingly, the virus can also establish latency in these cells, but it remains unknown what influences the decision between lytic replication and the latency of the virus. Incoming virus genomes are confronted with the nuclear domain 10 (ND10) complex as part of an intrinsic antiviral response. Most herpesviruses can efficiently subvert ND10, but its role in HHV-6A infection remains poorly understood. In this study, we investigated if the ND10 complex affects HHV-6A replication and contributes to the silencing of the virus genome during latency. We could demonstrate that ND10 complex was not dissociated upon infection, while the number of ND10 bodies was reduced in lytically infected cells. Virus replication was significantly enhanced upon knock down of the ND10 complex using shRNAs against its major constituents promyelocytic leukemia protein (PML), hDaxx, and Sp100. In addition, we could demonstrate that viral genes are more efficiently silenced in the presence of a functional ND10 complex. Our data thereby provides the first evidence that the cellular ND10 complex plays an important role in suppressing HHV-6A lytic replication and the silencing of the virus genome in latently infected cells.
The cell wall synthesis pathway producing peptidoglycan is a highly coordinated and tightly regulated process. Although the major components of bacterial cell walls have been known for decades, the complex regulatory network controlling peptidoglycan synthesis and many details of the cell division machinery are not well understood. The eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp play an important role in cell wall biosynthesis and drug resistance in S. aureus. We show that stp deletion has a pronounced impact on cell wall synthesis. Deletion of stp leads to a thicker cell wall and decreases susceptibility to lysostaphin. Stationary phase Δstp cells accumulate peptidoglycan precursors and incorporate higher amounts of incomplete muropeptides with non-glycine, monoglycine and monoalanine interpeptide bridges into the cell wall. In line with this cell wall phenotype, we demonstrate that the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. Mass spectrometric analyses identify Thr32, Thr36 and Ser415 as phosphoacceptors. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Moreover, Stk interacts with FemA and FemB, but is unable to phosphorylate them. Our data indicate that Stk and Stp modulate cell wall synthesis and cell division at several levels.
The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.
Analysis of the mechanism and the regulation of histatin 5 resistance in \(Candida\) \(albicans\)
(2018)
Antimycotics such as fluconazole are frequently used to treat C. albicans infections of the oral mucosa. Prolonged treatment of the fungal infection with fluconazole pose a risk to resistance development. C. albicans can adapt to these stressful environmental changes by regulation of gene expression or by producing genetically altered variants that arise in the population. Adapted variants frequently carry activating mutations in zinc cluster transcription factors, which cause the upregulation of their target genes, including genes encoding efflux pumps that confer drug resistance. MDR1, regulated by the zinc cluster transcription factor Mrr1, as well as CDR1 and CDR2, regulated by the zinc cluster transcription factor Tac1, are well-known examples of genes encoding efflux pumps that extrude the antimycotic fluconazole from the fungal cell and thus contribute to the survival of the fungus.
In this study, it was investigated if C. albicans can develop resistance to the antimicrobial peptide histatin 5, which serves as the first line of defence in the oral cavity of the human host. Recently, it was shown that C. albicans transports histatin 5 outside of the Candia cell via the efflux pump Flu1. As efflux pumps are often regulated by zinc cluster transcription factors, the Flu1 efflux pump could also be regulated by a zinc cluster transcription factor which could in a hyperactive form upregulate the expression of the efflux pump, resulting in increased export of histatin 5 and consequently in histatin 5 resistance.
In order to find a zinc cluster transcription factor that upregulates FLU1 expression, a comprehensive library of C. albicans strains containing artificially activated forms of zinc cluster transcription factors was screened for suitable candidates. The screening was conducted on medium containing mycophenolic acid because mycophenolic acid is also a substrate of Flu1 and a strain expressing a hyperactive zinc cluster transcription factor that upregulates FLU1 expression should exhibit an easily recognisable mycophenolic acid-resistant phenotype. Further, FACS analysis, quantitative real-time RT-PCR analysis, broth microdilution assays as well as histatin 5 assays were conducted to analyse the mechanism and the regulation of histatin 5 resistance.
Several zinc cluster transcription factors caused mycophenolic acid resistance and upregulated FLU1 expression. Of those, only hyperactive Mrr1 was able to confer increased histatin 5 resistance. Finding Mrr1 to confer histatin 5 resistance was highly interesting as fluconazole-resistant strains with naturally occurring Mrr1 gain of function mutations exist, which were isolated from HIV-infected patients with oral candidiasis. These Mrr1 gain of function mutations as well as artificially activated Mrr1 cause fluconazole resistance by upregulation of the efflux pump MDR1 and other target genes. In the course of the study, it was found that expression of different naturally occurring MRR1 gain-of-function mutations in the SC5314 wild type background caused increased FLU1 expression and increased histatin 5 resistance. The same was true for fluconazole-resistant clinical isolates with Mrr1 gain of function mutations, which also caused the overexpression of FLU1. Those cells were less efficiently killed by histatin 5 dependent on Mrr1. Surprisingly, FLU1 contributed only little to histatin 5 resistance, rather, overexpression of MDR1 mainly contributed to the Mrr1-mediated histatin 5 resistance, but also additional Mrr1-target genes were involved. These target genes are yet to be uncovered. Moreover, if a link between the yet unknown Mrr1-target genes contributing to fluconazole resistance and increased histatin 5 resistance can be drawn remains to be discovered upon finding of the responsible target genes.
Collectively, this study contributes to the understanding of the impact of prolonged antifungal exposure on the interaction between host and fungus. Drug therapy can give rise to resistance evolution resulting in strains that have not only developed resistance to fluconazole but also to an innate host mechanism, which allows adaption to the host niche even in the absence of the drug.
Bacteria adapt to changing environmental conditions by rapid changes in their transcriptome. This is achieved not only by adjusting rates of transcription but also by processing and degradation of RNAs. We applied TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) for the transcriptome-wide identification of RNase E cleavage sites and of 5′ RNA ends, which are enriched when RNase E activity is reduced in Rhodobacter sphaeroides. These results reveal the importance of RNase E for the maturation and turnover of mRNAs, rRNAs, and sRNAs in this guanine-cytosine-rich α-proteobacterium, some of the latter have well-described functions in the oxidative stress response. In agreement with this, a role of RNase E in the oxidative stress response is demonstrated. A remarkably strong phenotype of a mutant with reduced RNase E activity was observed regarding the formation of photosynthetic complexes and phototrophic growth, whereas there was no effect on chemotrophic growth.
To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/.
Staphylococcus epidermidis, the common inhabitant of human skin and mucosal surfaces has emerged as an important pathogen in patients carrying surgical implants and medical devices. Entering the body via surgical sites and colonizing the medical devices through formation of multi-layered biofilms leads to refractory and persistent device-related infections (DRIs). Staphylococci organized in biofilms are more tolerant to antibiotics and immune responses, and thus are difficult-to-treat. The consequent morbidity and mortality, and economic losses in health care systems has strongly necessitated the need for development of new anti-bacterial and anti-biofilm-based therapeutics. In this study, we describe the biological activity of a marine sponge-derived Streptomyces sp. SBT348 extract in restraining staphylococcal growth and biofilm formation on polystyrene, glass, medically relevant titan metal, and silicone surfaces. A bioassay-guided fractionation was performed to isolate the active compound (SKC3) from the crude SBT348 extract. Our results demonstrated that SKC3 effectively inhibits the growth (MIC: 31.25 \(\mu\)g/ml) and biofilm formation (sub-MIC range: 1.95-<31.25 \(\mu\)g/ml) of S. epidermidis RP62A in vitro. Chemical characterization of SKC3 by heat and enzyme treatments, and mass spectrometry (HRMS) revealed its heat-stable and non-proteinaceous nature, and high molecular weight (1258.3 Da). Cytotoxicity profiling of SKC3 in vitro on mouse fibroblast (NIH/3T3) and macrophage (J774.1) cell lines, and in vivo on the greater wax moth larvae Galleria mellonella revealed its non-toxic nature at the effective dose. Transcriptome analysis of SKC3 treated S. epidermidis RP62A has further unmasked its negative effect on central metabolism such as carbon flux as well as, amino acid, lipid, and energy metabolism. Taken together, these findings suggest a potential of SKC3 as a putative drug to prevent staphylococcal DRIs.
The probiotic escherichia coli strain Nissle 1917 combats lambdoid bacteriophages stx and lambda
(2018)
Shiga toxin (Stx) producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC) are the major cause of foodborne illness in humans. In vitro studies showed the probiotic Escherichia coil strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype 0104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coll strains which got infected by stx2-encoding lambdoid phages turning the E. coil into lysogenic and subsequently Stx producing strains. Since antibiotics induce stx genes and Stx production, EHEC infected persons are not recommended to be treated with antibiotics. Therefore, EcN might be an alternative medication. However, because even commensal E. coli strains might be converted into Stx-producers after becoming host to a stx encoding prophage, we tested EcN for stx-phage genome integration. Our experiments revealed the resistance of EcN toward not only stx-phages but also against lambda-phages. This resistance was not based on the lack of or by mutated phage receptors. Rather it involved the expression of a phage repressor (pr) gene of a defective prophage in EcN which was able to partially protect E. coli K-12 strain MG1655 against stx and lambda phage infection. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx- as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx-phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.
Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans, a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans, candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin. IMPORTANCE Candida albicans, usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer.
Many evolutionarily distant pathogenic organisms have evolved similar survival strategies to evade the immune responses of their hosts. These include antigenic variation, through which an infecting organism prevents clearance by periodically altering the identity of proteins that are visible to the immune system of the host1. Antigenic variation requires large reservoirs of immunologically diverse antigen genes, which are often generated through homologous recombination, as well as mechanisms to ensure the expression of one or very few antigens at any given time. Both homologous recombination and gene expression are affected by three-dimensional genome architecture and local DNA accessibility2,3. Factors that link three-dimensional genome architecture, local chromatin conformation and antigenic variation have, to our knowledge, not yet been identified in any organism. One of the major obstacles to studying the role of genome architecture in antigenic variation has been the highly repetitive nature and heterozygosity of antigen-gene arrays, which has precluded complete genome assembly in many pathogens. Here we report the de novo haplotype-specific assembly and scaffolding of the long antigen-gene arrays of the model protozoan parasite Trypanosoma brucei, using long-read sequencing technology and conserved features of chromosome folding4. Genome-wide chromosome conformation capture (Hi-C) reveals a distinct partitioning of the genome, with antigen-encoding subtelomeric regions that are folded into distinct, highly compact compartments. In addition, we performed a range of analyses—Hi-C, fluorescence in situ hybridization, assays for transposase-accessible chromatin using sequencing and single-cell RNA sequencing—that showed that deletion of the histone variants H3.V and H4.V increases antigen-gene clustering, DNA accessibility across sites of antigen expression and switching of the expressed antigen isoform, via homologous recombination. Our analyses identify histone variants as a molecular link between global genome architecture, local chromatin conformation and antigenic variation.
Metabolic conversion of CI-1040 turns a cellular MEK-inhibitor into an antibacterial compound
(2018)
Influenza virus (IV) infections cause severe respiratory illnesses that can be complicated by bacterial super-infections. Previously, we identified the cellular Raf-MEK-ERK cascade as a promising antiviral target. Inhibitors of MEK, such as CI-1040, showed potent antiviral activity. However, it remained unclear if this inhibitor and its active form, ATR-002, might sensitize host cells to either IV or secondary bacterial infections. To address these questions, we studied the anti-pathogen activity of ATR-002 in comparison to CI-1040, particularly, its impact on Staphylococcus aureus (S. aureus), which is a major cause of IV super-infections. We analysed IV and S. aureus titres in vitro during super-infection in the presence and absence of the drugs and characterized the direct impact of ATR-002 on bacterial growth and phenotypic changes. Importantly, neither CI-1040 nor ATR-002 treatment led to increased bacterial titres during super-infection, indicating that the drug does not sensitize cells for bacterial infection. In contrast, we rather observed reduced bacterial titres in presence of ATR-002. Surprisingly, ATR-002 also led to reduced bacterial growth in suspension cultures, reduced stress- and antibiotic tolerance without resistance induction. Our data identified for the first time that a particular MEK-inhibitor metabolite exhibits direct antibacterial activity, which is likely due to interference with the bacterial PknB kinase/Stp phosphatase signalling system.
Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia
(2017)
MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.
Preclinical development of an immunotherapy against antibiotic-resistant Staphylococcus aureus
(2017)
The Gram-positive bacterium Staphylococcus aureus is the leading cause of nosocomial infections. In particular, diseases caused by methicillin-resistant S. aureus (MRSA) are associated with higher morbidity, mortality and medical costs due to showing resistance to several classes of established antibiotics and their ability to develop resistance mechanisms against new antibiotics rapidly. Therefore, strategies based on immunotherapy approaches have the potential to close the gap for an efficient treatment of MRSA.
In this thesis, a humanized antibody specific for the immunodominant staphylococcal antigen A (IsaA) was generated and thoroughly characterized as potential candidate for an antibody based therapy. A murine monoclonal antibody was selected for humanization based on its binding characteristics and the ability of efficient staphylococcal killing in mouse infection models. The murine antibody was humanized by CDR grafting and mouse and humanized scFv as well as scFv-Fc fragments were constructed for comparative binding studies to analyse the successful humanization. After these studies, the full antibody with the complete Fc region was constructed as isotype IgG1, IgG2 and IgG4, respectively to assess effector functions, including antibody-dependent killing of S. aureus. The biological activity of the humanized antibody designated hUK-66 was analysed in vitro with purified human PMNs and whole blood samples taken from healthy donors and patients at high risk of S. aureus infections, such as those with diabetes, end-stage renal disease, or artery occlusive disease (AOD).
Results of the in vitro studies show, that hUK-66 was effective in antibody-dependent killing of S. aureus in blood from both healthy controls and patients vulnerable to S. aureus infections. Moreover, the biological activity of hUK-66 and hUK-66 combined with a humanized anti-alpha-toxin antibody (hUK-tox) was investigated in vivo using a mouse pneumonia model. The in vivo results revealed the therapeutic efficacy of hUK-66 and the antibody combination of hUK-66 and hUK-tox to prevent staphylococcal induced pneumonia in a prophylactic set up.
Based on the experimental data, hUK-66 represents a promising candidate for an antibody-based therapy against antibiotic resistant MRSA.
RNA-binding proteins (RBPs) have been extensively studied in eukaryotes, where they post-transcriptionally regulate many cellular events including RNA transport, translation, and stability. Experimental techniques, such as cross-linking and co-purification followed by either mass spectrometry or RNA sequencing has enabled the identification and characterization of RBPs, their conserved RNA-binding domains (RBDs), and the regulatory roles of these proteins on a genome-wide scale. These developments in quantitative, high-resolution, and high-throughput screening techniques have greatly expanded our understanding of RBPs in human and yeast cells. In contrast, our knowledge of number and potential diversity of RBPs in bacteria is comparatively poor, in part due to the technical challenges associated with existing global screening approaches developed in eukaryotes.
Genome- and proteome-wide screening approaches performed in silico may circumvent these technical issues to obtain a broad picture of the RNA interactome of bacteria and identify strong RBP candidates for more detailed experimental study. Here, I report APRICOT (“Analyzing Protein RNA Interaction by Combined Output Technique”), a computational pipeline for the sequence-based identification and characterization of candidate RNA-binding proteins encoded in the genomes of all domains of life using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences of an input proteome using position-specific scoring matrices and hidden Markov models of all conserved domains available in the databases and then statistically score them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them according to functionally relevant structural properties. APRICOT performed better than other existing tools for the sequence-based prediction on the known RBP data sets. The applications and adaptability of the software was demonstrated on several large bacterial RBP data sets including the complete proteome of Salmonella Typhimurium strain SL1344. APRICOT reported 1068 Salmonella proteins as RBP candidates, which were subsequently categorized using the RBDs that have been reported in both eukaryotic and bacterial proteins. A set of 131 strong RBP candidates was selected for experimental confirmation and characterization of RNA-binding activity using RNA co-immunoprecipitation followed by high-throughput sequencing (RIP-Seq) experiments. Based on the relative abundance of transcripts across the RIP-Seq libraries, a catalogue of enriched genes was established for each candidate, which shows the RNA-binding potential of 90% of these proteins. Furthermore, the direct targets of few of these putative RBPs were validated by means of cross-linking and co-immunoprecipitation (CLIP) experiments.
This thesis presents the computational pipeline APRICOT for the global screening of protein primary sequences for potential RBPs in bacteria using RBD information from all kingdoms of life. Furthermore, it provides the first bio-computational resource of putative RBPs in Salmonella, which could now be further studied for their biological and regulatory roles. The command line tool and its documentation are available at https://malvikasharan.github.io/APRICOT/.
The opportunistic fungal pathogen Candida albicans frequently produces genetically altered variants to adapt to environmental changes and new host niches in the course of its life-long association with the human host. Gain-of-function mutations in zinc cluster transcription factors, which result in the constitutive upregulation of their target genes, are a common cause of acquired resistance to the widely used antifungal drug fluconazole, especially during long-term therapy of oropharyngeal candidiasis. In this study, we investigated if C. albicans also can develop resistance to the antimicrobial peptide histatin 5, which is secreted in the saliva of humans to protect the oral mucosa from pathogenic microbes. As histatin 5 has been shown to be transported out of C. albicans cells by the Flu1 efflux pump, we screened a library of C. albicans strains that contain artificially activated forms of all zinc cluster transcription factors of this fungus for increased FLU1 expression. We found that a hyperactive Mrr1, which confers fluconazole resistance by upregulating the multidrug efflux pump MDR1 and other genes, also causes FLU1 overexpression. Similarly to the artificially activated Mrr1, naturally occurring gain-of-function mutations in this transcription factor also caused FLU1 upregulation and increased histatin 5 resistance. Surprisingly, however, Mrr1-mediated histatin 5 resistance was mainly caused by the upregulation of MDR1 instead of FLU1, revealing a previously unrecognized function of the Mdr1 efflux pump. Fluconazole-resistant clinical C. albicans isolates with different Mrr1 gain-of-function mutations were less efficiently killed by histatin 5, and this phenotype was reverted when MRR1 was deleted. Therefore, antimycotic therapy can promote the evolution of strains that, as a consequence of drug resistance mutations, simultaneously have acquired increased resistance against an innate host defense mechanism and are thereby better adapted to certain host niches.
RNA-binding proteins (RBPs) have been established as core components of several post-transcriptional gene regulation mechanisms. Experimental techniques such as cross-linking and co-immunoprecipitation have enabled the identification of RBPs, RNA-binding domains (RBDs) and their regulatory roles in the eukaryotic species such as human and yeast in large-scale. In contrast, our knowledge of the number and potential diversity of RBPs in bacteria is poorer due to the technical challenges associated with the existing global screening approaches. We introduce APRICOT, a computational pipeline for the sequence-based identification and characterization of proteins using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences using position-specific scoring matrices and Hidden Markov Models of the functional domains and statistically scores them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them by several biological properties. Here we demonstrate the application and adaptability of the pipeline on large-scale protein sets, including the bacterial proteome of Escherichia coli. APRICOT showed better performance on various datasets compared to other existing tools for the sequence-based prediction of RBPs by achieving an average sensitivity and specificity of 0.90 and 0.91 respectively. The command-line tool and its documentation are available at https://pypi.python.org/pypi/bio-apricot.
The primary transcriptome of Neisseria meningitidis and its interaction with the RNA chaperone Hfq
(2017)
Neisseria meningitidis is a human commensal that can also cause life-threatening meningitis and septicemia. Despite growing evidence for RNA-based regulation in meningococci, their transcriptome structure and output of regulatory small RNAs (sRNAs) are incompletely understood. Using dRNA-seq, we have mapped at single-nucleotide resolution the primary transcriptome of N. meningitidis strain 8013. Annotation of 1625 transcriptional start sites defines transcription units for most protein-coding genes but also reveals a paucity of classical σ70-type promoters, suggesting the existence of activators that compensate for the lack of −35 consensus sequences in N. meningitidis. The transcriptome maps also reveal 65 candidate sRNAs, a third of which were validated by northern blot analysis. Immunoprecipitation with the RNA chaperone Hfq drafts an unexpectedly large post-transcriptional regulatory network in this organism, comprising 23 sRNAs and hundreds of potential mRNA targets. Based on this data, using a newly developed gfp reporter system we validate an Hfq-dependent mRNA repression of the putative colonization factor PrpB by the two trans-acting sRNAs RcoF1/2. Our genome-wide RNA compendium will allow for a better understanding of meningococcal transcriptome organization and riboregulation with implications for colonization of the human nasopharynx.
Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis. Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5′ ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases.
Many pathogenic bacteria utilize specialized secretion systems to deliver proteins called effectors into eukaryotic cells for manipulation of host pathways. The vast majority of known effector targets are host proteins, whereas a potential targeting of host nucleic acids remains little explored. There is only one family of effectors known to target DNA directly, and effectors binding host RNA are unknown. Here, we take a two-pronged approach to search for RNA-binding effectors, combining biocomputational prediction of RNA-binding domains (RBDs) in a newly assembled comprehensive dataset of bacterial secreted proteins, and experimental screening for RNA binding in mammalian cells. Only a small subset of effectors were predicted to carry an RBD, indicating that if RNA targeting was common, it would likely involve new types of RBDs. Our experimental evaluation of effectors with predicted RBDs further argues for a general paucity of RNA binding activities amongst bacterial effectors. We obtained evidence that PipB2 and Lpg2844, effector proteins of Salmonella and Legionella species, respectively, may harbor novel biochemical activities. Our study presenting the first systematic evaluation of the RNA-targeting potential of bacterial effectors offers a basis for discussion of whether or not host RNA is a prominent target of secreted bacterial proteins.
Scaffold proteins are ubiquitous chaperones that promote efficient interactions between partners of multi-enzymatic protein complexes; although they are well studied in eukaryotes, their role in prokaryotic systems is poorly understood. Bacterial membranes have functional membrane microdomains (FMM), a structure homologous to eukaryotic lipid rafts. Similar to their eukaryotic counterparts, bacterial FMM harbor a scaffold protein termed flotillin that is thought to promote interactions between proteins spatially confined to the FMM. Here we used biochemical approaches to define the scaffold activity of the flotillin homolog FloA of the human pathogen Staphylococcus aureus, using assembly of interacting protein partners of the type VII secretion system (T7SS) as a case study. Staphylococcus aureus cells that lacked FloA showed reduced T7SS function, and thus reduced secretion of T7SS-related effectors, probably due to the supporting scaffold activity of flotillin. We found that the presence of flotillin mediates intermolecular interactions of T7SS proteins. We tested several small molecules that interfere with flotillin scaffold activity, which perturbed T7SS activity in vitro and in vivo. Our results suggest that flotillin assists in the assembly of S. aureus membrane components that participate in infection and influences the infective potential of this pathogen.
\(Enterococcus\) species cause increasing numbers of infections in hospitals. They contribute to the increasing mortality rates, mostly in patients with comorbidities, who suffer from severe diseases. \(Enterococcus\) resistances against most antibiotics have been described, including novel antibiotics. Therefore, there is an ongoing demand for novel types of antibiotics that may overcome bacterial resistances. We discovered a novel class of antibiotics resulting from a simple one-pot reaction of indole and \(o\)-phthaldialdehyde. Differently substituted indolyl benzocarbazoles were yielded. Both the indole substitution and the positioning at the molecular scaffold influence the antibacterial activity towards the various strains of \(Enterococcus\) species with the highest relevance to nosocomial infections. Structure-activity relationships are discussed, and the first lead compounds were identified as also being effective in the case of a vancomycin resistance.
High-throughput sequencing (HTS) has revolutionized bacterial genomics. Its unparalleled sensitivity has opened the door to analyzing bacterial evolution and population genomics, dispersion of mobile genetic elements (MGEs), and within-host adaptation of pathogens, such as Escherichia coli.
One of the defining characteristics of intestinal pathogenic E. coli (IPEC) pathotypes is a specific repertoire of virulence factors (VFs). Many of these IPEC VFs are used as typing markers in public health laboratories to monitor outbreaks and guide treatment options. Instead, extraintestinal pathogenic E. coli (ExPEC) isolates are genotypically diverse and harbor a varied set of VFs -- the majority of which also function as fitness factors (FFs) for gastrointestinal colonization.
The aim of this thesis was the genomic characterization of pathogenic and commensal E. coli with respect to their virulence- and antibiotic resistance-associated gene content as well as phylogenetic background. In order to conduct the comparative analyses, I created a database of E. coli VFs, ecoli_VF_collection, with a focus on ExPEC virulence-associated proteins (Leimbach, 2016b). Furthermore, I wrote a suite of scripts and pipelines, bac-genomics-scripts, that are useful for bacterial genomics (Leimbach, 2016a). This compilation includes tools for assembly and annotation as well as comparative genomics analyses, like multi-locus sequence typing (MLST), assignment of Clusters of Orthologous Groups (COG) categories, searching for protein homologs, detection of genomic regions of difference (RODs), and calculating pan-genome-wide association statistics.
Using these tools we were able to determine the prevalence of 18 autotransporters (ATs) in a large, phylogenetically heterogeneous strain panel and demonstrate that many AT proteins are not associated with E. coli pathotypes. According to multivariate analyses and statistics the distribution of AT variants is instead significantly dependent on phylogenetic lineages. As a consequence, ATs are not suitable to serve as pathotype markers (Zude et al., 2014).
During the German Shiga toxin-producing E. coli (STEC) outbreak in 2011, the largest to date, we were one of the teams capable of analyzing the genomic features of two isolates. Based on MLST and detection of orthologous proteins to known E. coli reference genomes the close phylogenetic relationship and overall genome similarity to enteroaggregative E. coli (EAEC) 55989 was revealed. In particular, we identified VFs of both STEC and EAEC pathotypes, most importantly the prophage-encoded Shiga toxin (Stx) and the pAA-type plasmid harboring aggregative adherence fimbriae. As a result, we could show that the epidemic was caused by an unusual hybrid pathotype of the O104:H4 serotype. Moreover, we detected the basis of the antibiotic multi-resistant phenotype on an extended-spectrum beta-lactamase (ESBL) plasmid through comparisons to reference plasmids. With this information we proposed an evolutionary horizontal gene transfer (HGT) model for the possible emergence of the pathogen (Brzuszkiewicz et al., 2011).
Similarly to ExPEC, E. coli isolates of bovine mastitis are genotypically and phenotypically highly diverse and many studies struggled to determine a positive association of putative VFs. Instead the general E. coli pathogen-associated molecular pattern (PAMP), lipopolysaccharide (LPS), is implicated as a deciding factor for intramammary inflammation. Nevertheless, a mammary pathogenic E. coli (MPEC) pathotype was proposed presumably encompassing strains more adapted to elicit bovine mastitis with virulence traits differentiating them from commensals.
We sequenced eight E. coli isolates from udder serous exudate and six fecal commensals (Leimbach et al., 2016). Two mastitis isolate genomes were closed to a finished-grade quality (Leimbach et al., 2015). The genomic sequence of mastitis-associated E. coli (MAEC) strain 1303 was used to elucidate the biosynthesis gene cluster of its O70 LPS O-antigen. We analyzed the phylogenetic genealogy of our strain panel plus eleven bovine-associated E. coli reference strains and found that commensal or MAEC could not be unambiguously allocated to specific phylogroups within a core genome tree of reference E. coli. A thorough gene content analysis could not identify functional convergence of either commensal or MAEC, instead both have only very few gene families enriched in either pathotype. Most importantly, gene content and ecoli_VF_collection analyses showed that no virulence determinants are significantly associated with MAEC in comparison to bovine fecal commensals, disproving the MPEC hypothesis. The genetic repertoire of bovine-associated E. coli, again, is dominated by phylogenetic background. This is also mostly the case for large virulence-associated E. coli gene cluster previously associated with mastitis. Correspondingly, MAEC are facultative and opportunistic pathogens recruited from the bovine commensal gastrointestinal microbiota (Leimbach et al., 2017). Thus, E. coli mastitis should be prevented rather than treated, as antibiotics and vaccines have not proven effective.
Although traditional E. coli pathotypes serve a purpose for diagnostics and treatment, it is clear that the current typing system is an oversimplification of E. coli's genomic plasticity. Whole genome sequencing (WGS) revealed many nuances of pathogenic E. coli, including emerging hybrid or heteropathogenic pathotypes. Diagnostic and public health microbiology need to embrace the future by implementing HTS techniques to target patient care and infection control more efficiently.
A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen Staphylococcus aureus, which generates the phenotypic bifurcation of the cells into two genetically identical but different cell types during the course of an infection. Whereas one cell type promotes the formation of biofilms that contribute to chronic infections, the second type is planktonic and produces the toxins that contribute to acute bacteremia. We identified a bimodal switch in the agr quorum sensing system that antagonistically regulates the differentiation of these two physiologically distinct cell types. We found that extracellular signals affect the behavior of the agr bimodal switch and modify the size of the specialized subpopulations in specific colonization niches. For instance, magnesium-enriched colonization niches causes magnesium binding to S. aureusteichoic acids and increases bacterial cell wall rigidity. This signal triggers a genetic program that ultimately downregulates the agr bimodal switch. Colonization niches with different magnesium concentrations influence the bimodal system activity, which defines a distinct ratio between these subpopulations; this in turn leads to distinct infection outcomes in vitro and in an in vivo murine infection model. Cell differentiation generates physiological heterogeneity in clonal bacterial infections and helps to determine the distinct infection types.
Background
The lytic cycle of the protozoan parasite \(Toxoplasma\) \(gondii\), which involves a brief sojourn in the extracellular space, is characterized by defined transcriptional profiles. For an obligate intracellular parasite that is shielded from the cytosolic host immune factors by a parasitophorous vacuole, the brief entry into the extracellular space is likely to exert enormous stress. Due to its role in cellular stress response, we hypothesize that translational control plays an important role in regulating gene expression in \(Toxoplasma\) during the lytic cycle. Unlike transcriptional profiles, insights into genome-wide translational profiles of \(Toxoplasma\) \(gondii\) are lacking.
Methods
We have performed genome-wide ribosome profiling, coupled with high throughput RNA sequencing, in intracellular and extracellular \(Toxoplasma\) \(gondii\) parasites to investigate translational control during the lytic cycle.
Results
Although differences in transcript abundance were mostly mirrored at the translational level, we observed significant differences in the abundance of ribosome footprints between the two parasite stages. Furthermore, our data suggest that mRNA translation in the parasite is potentially regulated by mRNA secondary structure and upstream open reading frames.
Conclusion
We show that most of the \(Toxoplasma\) genes that are dysregulated during the lytic cycle are translationally regulated.
Oligopeptides incorporating \(N3\)-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP), an inhibitor of glucosamine-6-phosphate synthase, exhibited growth inhibitory activity against \(Candida\) \(albicans\), with minimal inhibitory concentration values in the 0.05–50 μg mL\(^{-1}\) range. Uptake by the peptide permeases was found to be the main factor limiting an anticandidal activity of these compounds. Di- and tripeptide containing FMDP (F2 and F3) were transported by Ptr2p/Ptr22p peptide transporters (PTR) and FMDP-containing hexa-, hepta-, and undecapeptide (F6, F7, and F11) were taken up by the oligopeptide transporters (OPT) oligopeptide permeases, preferably by Opt2p/Opt3p. A phenotypic, apparent resistance of \(C. albicans\) to FMDP-oligopeptides transported by OPT permeases was triggered by the environmental factors, whereas resistance to those taken up by the PTR system had a genetic basis. Anticandidal activity of longer FMDP-oligopeptides was strongly diminished in minimal media containing easily assimilated ammonium sulfate or L-glutamine as the nitrogen source, both known to downregulate expression of the OPT genes. All FMDP-oligopeptides tested were more active at lower pH and this effect was slightly more remarkable for peptides F6, F7, and F11, compared to F2 and F3. Formation of isolated colonies was observed inside the growth inhibitory zones induced by F2 and F3 but not inside those induced by F6, F7, and F11. The vast majority (98%) of those colonies did not originate from truly resistant cells. The true resistance of 2% of isolates was due to the impaired transport of di- and to a lower extent, tripeptides. The resistant cells did not exhibit a lower expression of \(PTR2\), \(PTR22\), or \(OPT1–3\) genes, but mutations in the \(PTR2\) gene resulting in T422H, A320S, D119V, and A320S substitutions in the amino acid sequence of Ptr2p were found.
Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation
(2017)
Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to absence of cell toxicity, the extract might represent a good starting material to develop a future remedy to block staphylococcal biofilm formation on contact lenses and thereby to prevent intractable contact lens-mediated ocular infections.
The transcriptome is a powerful proxy for the physiological state of a cell, healthy or diseased. As a result, transcriptome analysis has become a key tool in understanding the molecular changes that accompany bacterial infections of eukaryotic cells. Until recently, such transcriptomic studies have been technically limited to analyzing mRNA expression changes in either the bacterial pathogen or the infected eukaryotic host cell. However, the increasing sensitivity of high-throughput RNA sequencing now enables “dual RNA-seq” studies, simultaneously capturing all classes of coding and noncoding transcripts in both the pathogen and the host. In the five years since the concept of dual RNA-seq was introduced, the technique has been applied to a range of infection models. This has not only led to a better understanding of the physiological changes in pathogen and host during the course of an infection but has also revealed hidden molecular phenotypes of virulence-associated small noncoding RNAs that were not visible in standard infection assays. Here, we use the knowledge gained from these recent studies to suggest experimental and computational guidelines for the design of future dual RNA-seq studies. We conclude this review by discussing prospective applications of the technique.
The yeast form of the fungus Candida albicans promotes persistence in the gut of gnotobiotic mice
(2017)
Many microorganisms that cause systemic, life-threatening infections in humans reside as harmless commensals in our digestive tract. Yet little is known about the biology of these microbes in the gut. Here, we visualize the interface between the human commensal and pathogenic fungus Candida albicans and the intestine of mice, a surrogate host. Because the indigenous mouse microbiota restricts C. albicans settlement, we compared the patterns of colonization in the gut of germ free and antibiotic-treated conventionally raised mice. In contrast to the heterogeneous morphologies found in the latter, we establish that in germ free animals the fungus almost uniformly adopts the yeast cell form, a proxy of its commensal state. By screening a collection of C. albicans transcription regulator deletion mutants in gnotobiotic mice, we identify several genes previously unknown to contribute to in vivo fitness. We investigate three of these regulators—ZCF8, ZFU2 and TRY4—and show that indeed they favor the yeast form over other morphologies. Consistent with this finding, we demonstrate that genetically inducing non-yeast cell morphologies is detrimental to the fitness of C. albicans in the gut. Furthermore, the identified regulators promote adherence of the fungus to a surface covered with mucin and to mucus-producing intestinal epithelial cells. In agreement with this result, histology sections indicate that C. albicans dwells in the murine gut in close proximity to the mucus layer. Thus, our findings reveal a set of regulators that endows C. albicans with the ability to endure in the intestine through multiple mechanisms.
Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.
Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle.
Model enteric bacteria such as Escherichia coli and Salmonella enterica express hundreds of small non-coding RNAs (sRNAs), targets for most of which are yet unknown. Some sRNAs are remarkably well conserved, indicating that they serve cellular functions that go beyond the necessities of a single species. One of these ‘core sRNAs’ of largely unknown function is the abundant ∼100-nucleotide SdsR sRNA which is transcribed by the general stress σ-factor, σ\(^{S}\) and accumulates in stationary phase. In Salmonella, SdsR was known to inhibit the synthesis of the species-specific porin, OmpD. However, sdsR genes are present in almost all enterobacterial genomes, suggesting that additional, conserved targets of this sRNA must exist. Here, we have combined SdsR pulse-expression with whole genome transcriptomics to discover 20 previously unknown candidate targets of SdsR which include mRNAs coding for physiologically important regulators such as the carbon utilization regulator, CRP, the nucleoid-associated chaperone, StpA and the antibiotic resistance transporter, TolC. Processing of SdsR by RNase E results in two cellular SdsR variants with distinct target spectra. While the overall physiological role of this orphan core sRNA remains to be fully understood, the new SdsR targets present valuable leads to determine sRNA functions in resting bacteria.
Motivation:
Next generation sequencing technologies have provided us with a wealth of information on genetic variation, but predi cting the functional significance of this variation is a difficult task. While many comparative genomics studies have focused on gene flux and large scale changes, relatively little attention has been paid to quantifying the effects of single nucleotide polymorphisms and indels on protein function, particularly in bacterial genomics.
Results:
We present a hidden Markov model based approach we call delta-bitscore (DBS) for identifying orthologous proteins that have diverged at the amino acid sequence level in a way that is likely to impact biological function. We benchmark this approach with several widely used datasets and apply it to a proof-of-concept study of orthologous proteomes in an investigation of host adaptation in Salmonella enterica. We highlight the value of the method in identifying functional divergence of genes, and suggest that this tool may be a better approach than the commonly used dN/dS metric for identifying functionally significant genetic changes occurring in recently diverged organisms.
Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions.
Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed.
Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments.
Advances in stem cell research have allowed the development of 3-dimensional (3D) primary cell cultures termed organoid cultures, as they closely mimic the in vivo organization of different cell lineages. Bridging the gap between 2-dimensional (2D) monotypic cancer cell lines and whole organisms, organoids are now widely applied to model development and disease. Organoids hold immense promise for addressing novel questions in host-microbe interactions, infectious diseases and the resulting inflammatory conditions. Researchers have started to use organoids for modeling infection with pathogens, such as Helicobacter pylori or Salmonella enteritica, gut- microbiota interactions and inflammatory bowel disease. Future studies will broaden the spectrum of microbes used and continue to establish organoids as a standard model for human host-microbial interactions. Moreover, they will increasingly exploit the unique advantages of organoids, for example to address patient-specific responses to microbes.
An expanded evaluation of protein function prediction methods shows an improvement in accuracy
(2016)
Background
A major bottleneck in our understanding of the molecular underpinnings of life is the assignment of function to proteins. While molecular experiments provide the most reliable annotation of proteins, their relatively low throughput and restricted purview have led to an increasing role for computational function prediction. However, assessing methods for protein function prediction and tracking progress in the field remain challenging.
Results
We conducted the second critical assessment of functional annotation (CAFA), a timed challenge to assess computational methods that automatically assign protein function. We evaluated 126 methods from 56 research groups for their ability to predict biological functions using Gene Ontology and gene-disease associations using Human Phenotype Ontology on a set of 3681 proteins from 18 species. CAFA2 featured expanded analysis compared with CAFA1, with regards to data set size, variety, and assessment metrics. To review progress in the field, the analysis compared the best methods from CAFA1 to those of CAFA2.
Conclusions
The top-performing methods in CAFA2 outperformed those from CAFA1. This increased accuracy can be attributed to a combination of the growing number of experimental annotations and improved methods for function prediction. The assessment also revealed that the definition of top-performing algorithms is ontology specific, that different performance metrics can be used to probe the nature of accurate predictions, and the relative diversity of predictions in the biological process and human phenotype ontologies. While there was methodological improvement between CAFA1 and CAFA2, the interpretation of results and usefulness of individual methods remain context-dependent.
While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans, the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK. Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein.
Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169) in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments.
Sepsis caused by Gram-positive bacterial pathogens is a major fatal disease but its molecular basis remains elusive. Toll-like receptor 2 (TLR2) has been implicated in the orchestration of inflammation and sepsis but its role appears to vary for different pathogen species and clones. Accordingly, Staphylococcus aureus clinical isolates differ substantially in their capacity to activate TLR2. Here we show that strong TLR2 stimulation depends on high-level production of phenol-soluble modulin (PSM) peptides in response to the global virulence activator Agr. PSMs are required for mobilizing lipoproteins, the TLR2 agonists, from the staphylococcal cytoplasmic membrane. Notably, the course of sepsis caused by PSM-deficient S. aureus is similar in wild-type and TLR2-deficient mice, but TLR2 is required for protection of mice against PSM-producing S. aureus. Thus, a crucial role of TLR2 depends on agonist release by bacterial surfactants. Modulation of this process may lead to new therapeutic strategies against Gram-positive infections.
Background
Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii.
Results
Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs).
Conclusion
This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated.
Background
Differential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. However, using dRNA-seq data to map transcriptional start sites (TSSs) and promoters genome-wide is a bioinformatics challenge. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters.
Results
A specialized machine learning procedure for TSS recognition allowed us to map 15,923 TSSs: 14,360 in free-living bacteria, 4329 in symbiosis with soybean and 2766 in both conditions. Further, we provide proteomic evidence for 4090 proteins, among them 107 proteins corresponding to new genes and 178 proteins with N-termini different from the existing annotation (72 and 109 of them with TSS support, respectively). Guided by proteomics evidence, previously identified TSSs and TSSs experimentally validated here, we assign a score threshold to flag 14 % of the mapped TSSs as a class of lower confidence. However, this class of lower confidence contains valid TSSs of low-abundant transcripts. Moreover, we developed a de novo algorithm to identify promoter motifs upstream of mapped TSSs, which is publicly available, and found motifs mainly used in symbiosis (similar to RpoN-dependent promoters) or under both conditions (similar to RpoD-dependent promoters). Mapped TSSs and putative promoters, proteomic evidence and updated gene annotation were combined into an annotation file.
Conclusions
The genome-wide TSS and promoter maps along with the extended genome annotation of B. japonicum represent a valuable resource for future systems biology studies and for detailed analyses of individual non-coding transcripts and ORFs. Our data will also provide new insights into bacterial gene regulation during the agriculturally important symbiosis between rhizobia and legumes.
The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state.