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In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5′ untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5′-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3′-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements.
Bornyl caffeate (1) was previously isolated by us from Valeriana (V.) wallichii rhizomes and identified as an anti-leishmanial substance. Here, we screened a small compound library of synthesized derivatives 1–30 for activity against schistosomula of Schistosoma (S.) mansoni. Compound 1 did not show any anti-schistosomal activity. However, strong phenotypic changes, including the formation of vacuoles, degeneration and death were observed after in vitro treatment with compounds 23 (thymyl cinnamate) and 27 (eugenyl cinnamate). Electron microscopy analysis of the induced vacuoles in the dying parasites suggests that 23 and 27 interfere with autophagy.
Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii (V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester (1) as the active component against Leishmania major (L. major) promastigotes (IC50 = 48.8 µM). To investigate the structure-activity relationship (SAR), a library of compounds based on 1 was synthesized and tested in vitro against L. major and L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some compounds showed antileishmanial activity in the concentration range of pentamidine and miltefosine which are the standard drugs in use. In the L. major amastigote assay compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM. The Michael system seems not to be essential for antileishmanial activity. Based on the results compound 27 can be regarded as new lead structure for further structure optimization
Coagulase-negative staphylococci (CoNS) are common opportunistic pathogens, but also ubiquitous human and animal commensals. Infection-associated CoNS from healthcare environments are typically characterized by pronounced antimicrobial resistance (AMR) including both methicillin- and multidrug-resistant isolates. Less is known about AMR patterns of CoNS colonizing the general population. Here we report on AMR in commensal CoNS recovered from 117 non-hospitalized volunteers in a region of Germany with a high livestock density. Among the 69 individuals colonized with CoNS, 29 had reported contacts to either companion or farm animals. CoNS were selectively cultivated from nasal swabs, followed by species definition by 16S rDNA sequencing and routine antibiotic susceptibility testing. Isolates displaying phenotypic AMR were further tested by PCR for presence of selected AMR genes. A total of 127 CoNS were isolated and Staphylococcus epidermidis (75%) was the most common CoNS species identified. Nine isolates (7%) were methicillin-resistant (MR) and carried the mecA gene, with seven individuals (10%) being colonized with at least one MR-CoNS isolate. While resistance against gentamicin, phenicols and spectinomycin was rare, high resistance rates were found against tetracycline (39%), erythromycin (33%) and fusidic acid (24%). In the majority of isolates, phenotypic resistance could be associated with corresponding AMR gene detection. Multidrug-resistance (MDR) was observed in 23% (29/127) of the isolates, with 33% (23/69) of the individuals being colonized with MDR-CoNS. The combined data suggest that MR- and MDR-CoNS are present in the community, with previous animal contact not significantly influencing the risk of becoming colonized with such isolates.
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.
Aspergillus is an important fungal genus containing economically important species, as well as pathogenic species of animals and plants. Using eighteen fungal species of the genus Aspergillus, we conducted a comprehensive investigation of conserved genes and their evolution. This also allows us to investigate the selection pressure driving the adaptive evolution in the pathogenic species A. fumigatus. Among single-copy orthologs (SCOs) for A. fumigatus and the closely related species A. fischeri, we identified 122 versus 50 positively selected genes (PSGs), respectively. Moreover, twenty conserved genes of unknown function were established to be positively selected and thus important for adaption. A. fumigatus PSGs interacting with human host proteins show over-representation of adaptive, symbiosis-related, immunomodulatory and virulence-related pathways, such as the TGF-β pathway, insulin receptor signaling, IL1 pathway and interfering with phagosomal GTPase signaling. Additionally, among the virulence factor coding genes, secretory and membrane protein-coding genes in multi-copy gene families, 212 genes underwent positive selection and also suggest increased adaptation, such as fungal immune evasion mechanisms (aspf2), siderophore biosynthesis (sidD), fumarylalanine production (sidE), stress tolerance (atfA) and thermotolerance (sodA). These genes presumably contribute to host adaptation strategies. Genes for the biosynthesis of gliotoxin are shared among all the close relatives of A. fumigatus as an ancient defense mechanism. Positive selection plays a crucial role in the adaptive evolution of A. fumigatus. The genome-wide profile of PSGs provides valuable targets for further research on the mechanisms of immune evasion, antimycotic targeting and understanding fundamental virulence processes.
Non-coding RNAs constitute a major class of regulators involved in bacterial gene expression. A group of riboregulators of heterogeneous size and shape referred to as small regulatory RNAs (sRNAs) control trans- or cis-encoded genes through direct base-pairing with their mRNAs. Although mostly inhibiting their target mRNAs, several sRNAs also induce gene expression. An important co-factor for sRNA activity is the RNA chaperone, Hfq, which is able to rearrange intramolecular secondary structures and to promote annealing of complementary RNA sequences. In addition, Hfq protects unpaired RNA from degradation by ribonucleases and thus increases sRNA stability. Co-immunoprecipitation of RNA with the Hfq protein, and further experimental as well as bioinformatical studies performed over the last decade suggested the presence of more than 150 different sRNAs in various Enterobacteria including Escherichia coli and Salmonellae. So-called core sRNAs are considered to fulfill central cellular activities as deduced from their high degree of conservation among different species. Approximately 25 core sRNAs have been implicated in gene regulation under a variety of environmental responses. However, for the majority of sRNAs, both the riboregulators’ individual biological roles as well as modes of action remain to be elucidated. The current study aimed to define the cellular functions of the two highly conserved, Hfq-dependent sRNAs, SdsR and RydC, in the model pathogen Salmonella Typhimurium. SdsR had been known as one of the most abundant sRNAs during stationary growth phase in E. coli. Examination of the conservation patterns in the sdsR promoter region in combination with classic genetic analyses revealed SdsR as the first sRNA under direct transcriptional control of the alternative σ factor σS. In Salmonella, over-expression of SdsR down-regulates the synthesis of the major porin OmpD, and the interaction site in the ompD mRNA coding sequence was mapped by a 3'RACE-based approach. At the post-transcriptional level, expression of ompD is controlled by three additional sRNAs, but SdsR plays a specific role in porin regulation during the stringent response. Similarly, RydC, the second sRNA adressed in this study, was initially discovered in E. coli but appeared to be conserved in many related γ-proteobacteria. An interesting aspect of this Hfq-dependent sRNAs is its secondary structure involving a pseudo-knot configuration, while the 5’ end remains single stranded. A transcriptomic approach combining RydC pulse-expression and scoring of global mRNA changes on microarrays was employed to identify the targets of this sRNA. RydC specifically activated expression of the longer of two versions of the cfa mRNA encoding for the phospholipid-modifying enzyme cyclopropane fatty acid synthase. Employing its conserved single-stranded 5' end, RydC acts as a positive regulator and masks a recognition site of the endoribonuclease, RNase E, in the cfa leader.
Staphylococcus aureus is one of the most frequent causes of nosocomial and community‐acquired infections, with drug‐resistant strains being responsible for tens of thousands of deaths per year. S. aureus sortase A inhibitors are designed to interfere with virulence determinants. We have identified disulfanylbenzamides as a new class of potent inhibitors against sortase A that act by covalent modification of the active‐site cysteine. A broad series of derivatives were synthesized to derive structure‐activity relationships (SAR). In vitro and in silico methods allowed the experimentally observed binding affinities and selectivities to be rationalized. The most active compounds were found to have single‐digit micromolar Ki values and caused up to a 66 % reduction of S. aureus fibrinogen attachment at an effective inhibitor concentration of 10 μM. This new molecule class exhibited minimal cytotoxicity, low bacterial growth inhibition and impaired sortase‐mediated adherence of S. aureus cells.
Background
Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed.
Methods
BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining.
Results
The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages.
Conclusions
Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients.
Azobenzene derivatives with activity against drug‐resistant Candida albicans and Candida auris
(2023)
Increasing resistance against antimycotic drugs challenges anti‐infective therapies today and contributes to the mortality of infections by drug‐resistant Candida species and strains. Therefore, novel antifungal agents are needed. A promising approach in developing new drugs is using naturally occurring molecules as lead structures. In this work, 4,4'‐dihydroxyazobenzene, a compound structurally related to antifungal stilbene derivatives and present in Agaricus xanthodermus (yellow stainer), served as a starting point for the synthesis of five azobenzene derivatives. These compounds prevented the growth of both fluconazole‐susceptible and fluconazole‐resistant Candida albicans and Candida auris strains. Further in vivo studies are required to confirm the potential therapeutic value of these compounds.
Bacteria Regulate Intestinal Epithelial Cell Differentiation Factors Both In Vitro and In Vivo
(2013)
Background: The human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell.
Methods: Expression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/- flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted.
Results: Expression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice.
Conclusions: Intestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch.
We investigated the role of bacterial adherence and hemolysin production from Escherichia coli parent and genetically cloned strains as to their eft'ects on bistaJidne release from rat mast cells and leukotriene generation from human polymorphonuclear granulocytes. These mediators were involved in the induction of inftammatory disease processes and led, for example, to enhancement of vascular permeability, chemotaxis (leukotriene 84 [LTB4]), chemoaggregation, lysosomal enzyme release, and smooth muscle contraction, (LTC4, LTD4 , and LTE4). Washed bacteria (E. coli K-12 Ms+ my=; E. coli 536 Ms+ MR= my=) as weil as their culture supematants were analyzed. Washed E. coli K-12 (Hiy+), unlike Hly- strains, induced high amounts of histamine release from rat mast cells and chemotactic activity from human polymorphonuclear granulocytes. Significant leukotriene releasewas obtained with washed E. coli K-12 my+ strains and their bacterial culture supematants. Leukotriene induction was dependent on the amount of hemolysin activity present in the supematant. However, additional soluble factors should also be considered. The presence of hemolysin appeared to aceeierate and enhance the rate of phagocytosis of bacteria by neutrophUs. When E. coli 536 (MS+ MR= Hly=) strains were analyzed, the simultaneous presence of MR+ pili and hemolysin production led to an increase in histamine release as compared with MR- my+ strains. The genetically cloned MR+ my+ E. coli 536 strain induced higher amounts of IeukotrieDes as compared with the wUd-type strain. Our data soggest a potent role for adhesins and hemolysin as virulence factors in inducing the release of inftammatory mediators.
Asymptomatische Bakteriurie (ABU) stellt eine bakterielle Infektion der Harnblase über einen langen Zeitraum dar, die häufig von Escherichia coli hervorgerufen wird, ohne dass typische Symptome einer Harnwegsinfektion auftreten. Um die Charakteristika von ABU E. coli Isolaten genauer zu untersuchen, wurden die Geno- und Phänotypen von 11 ABU-Isolaten verglichen. Außerdem wurden in mehreren aufeinanderfolgenden in vivo-Reisolaten des Modell-ABU Stammes 83972 die Veränderungen im Transkriptom, Proteom und Genom während einer langfristigen Persistenz in der menschlichen Blase charakterisiert. Schließlich wurde der Effekt des menschlichen Wirtes auf die bakterielle Adaptation durch einen Vergleich von in vitro- mit in vivo-kultivierten Stämmen abgeschätzt. ABU-Isolate stellt eine heterogene Gruppe von Organismen dar. Diese können den vier phylogenetischen Hauptgruppen von E. coli sowie unterschiedlichen klonalen Gruppen zugeordnet werden. Dementsprechend unterscheiden sie sich erheblich bezüglich der Zusammensetzung des Genomes, der Genomgröße und auch der Ausstattung mit UPEC-typischen Virulenz-assoziierten Genen. Multi-Lokus-Sequenz-Typisierung legt nahe, dass bestimmte ABU Stämme sich durch Genomreduktion aus UPEC Stämmen entwickelt haben, die eine Harnwegsinfektion mit charakteristischen Symptomen auslösen konnten. Folglich erlaubt die hohe Genomplastizität von E. coli keine generalisierte Betrachtung einzelner Isolate eines Klons. Genomreduktion über Punktmutationen, Genom-Reorganisation und Deletionen resultierte in der Inaktivierung einiger Gene, die für einige UPEC Virulenz-Faktoren kodieren. Dies stützt die Vorstellung, dass eine verminderte bakterielle Aktivierung der Entzündung der Wirtsschleimhaut den Lebensstil von ABU (bei diesen E. coli-)Isolaten fördert. Genregulation und genetische Diversität sind Strategien, die es Bakterien ermöglichen unter sich fortlaufend ändernden Bedingungen zu leben bzw. zu überleben. Um die anpassungsbedingten Veränderungen bei einem langfristigen Wachstum in der Blase zu untersuchen, wurden aufeinanderfolgende Reisolate, denen eine langfristige in vivo-Kolonisierung im menschlichen Wirt beziehungsweise eine in vitro-Kultivierung vorausgegangen ist, im Hinblick auf Veränderungen Genexpression und Genomorganisation analysiert. In diesem Zusammenhang konnte gezeigt werden, dass E. coli in der Lage ist, seine metabolischen Netzwerke verschiedenen Wachstumsbedingungen anzupassen und individuelle bakterielle Kolonisierungsstrategien entwickeln kann. Transkriptom- und Proteom-Analysen zeigten verschiedene metabolische Strategien zur Nährstoffbeschaffung und Energieproduktion bei untersuchten in vivo-Reisolaten vom Stamm 83972, die es ihnen ermöglichen, den Wirt zu kolonisieren. Das Zurückgreifen auf D-Serin, Deoxy- und Ribonucleoside sowie die bidirektionale Umwandlung zwischen Pentose und Glucuronat waren hoch-regulierte Stoffwechselwege, die die in vivo-Reisolate mit zusätzlicher Energie für ein effizientes Wachstum in der Blase versorgen. Zudem wurden in dieser Studie die Netzwerke für eine Reaktion auf Abwehrmechanismen des Wirtes erforscht: Erstmals wurde hier die Rolle der Klasse-III-Alkoholdehydrogenase AdhC, bekannt durch ihre Bedeutung bei der Entgiftung von Stickstoffmonoxid, bei der Wirtsantwort während einer asymptomatischen Bakteriurie gezeigt. Aufeinanderfolgende in vivo- und in vitro-Reisolate vom Stamm 83972 wurden ebenfalls bezüglich ihrer Genomstruktur analysiert. Einige Veränderungen in der Genomstruktur der aufeinanderfolgenden Reisolate, die von einer humanen Kolonisierungsstudie stammen, implizieren die Bedeutung einer Interaktion der Bakterien mit dem Wirt bei der Mikroevolution der Bakterien. Dagegen war die Genomstruktur von Reisolaten eines langfristigen in vitro-Kultivierungsexperiments, bei dem sich der Stamm 83972 ohne Wirtskontakt vermehrt hat, nicht von Veränderungen betroffen. Das legt nahe, dass die Immunantwort eine Genomplastizität fördert und somit eine treibende Kraft für den ABU Lebensstil und die Evolution im Harnwegstrakt ist.
Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies
(2011)
Background: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) rena l excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells
Investigations were carried out on the adhesion of cloned S-fimbriated E. coli, labelled with fluoresceinisothiocyanate (FITC) to human buccal epithelial cells. Fluorescence microscopic analysis revealed binding of bacteria to 75-95% of epithelial cells. Inhibition experiments with fetuin, a 1-acid glycoprotein and N-acetyl neuraminic acid confirmed the specificity of bacterial binding to sialoglycoproteins. Further studies using saliva as an inhibitor resulted in a 4-5 times stronger binding inhibition by newborn saliva in comparison to adult saliva coinciding with a 4-5 times higher content of total N-acetyl neuraminic acid in samples of newborn saliva. In Western blot analysis sialoglycoprotein bands with a molecular weight >200 kD reacting with wheat germ agglutinin (WGA), were only identified in samples of newborn saliva. These bands are classified as mucins on account of molecular weight and staining. These data suggest that saliva mucins could represent a major defense mechanism against bacterial infections at a stage of ontogeny where the secretory IgAsystem is not yet developed.
Purified S fimbriae and an Escherichia coli strain carrying the recombinant plasmid pANN801-4 that encodes S fimbriae were tested for adhesion to frozen sections of human kidney. The fimbrlae and the bacteria bound to the same tissue domains, and in both cases the binding was specifically inhibited by the receptor analog of S fimbria, sialyl(a2-3)1actose. S fimbriae bound specifically to the epithelial elements in the kidneys; to the epithelial cells of proximal and distal tubules as weil as of the collecting ducts and to the visceral and parietal glomerular epithelium. In addition, they bound to the vascular endothelium of glomerull and of the renal Interstitium. No blnding to connective tissue elements was observed. The results suggest that the biological functlon of S fimbriae is to mediate the adheslon of E. coli to human epithelial and vascular endothellal ceUs.
Binding sites in the rat brain for Escherichia coli S fimbriae associated with neontal meningitis
(1988)
Escherichia coli strains that cause sepsis and meningitis in neonatal infants carry S fimbriae that bind to sialyl galactoside units of cell surface glycoproteins. To investigate the possible role of S fimbriae in determining the tissue tropism of neonatal menlngitis, we have studied the preselice of binding sites for S fimbriae in different tissues of the neonatal rat which is susceptible to meningitis caused by S-fimbriated E. coli. Purified S fimbriae were incubated on cryostat sections of different rat oipns and their bindina was assessed by indirect immunofluorescence. In the bnin of the neonatal rat, S fimbriae specifically bound to the luminal surfaces of the vascular endothelium and of the epithelium lining the choroid plexuses and bnin ventricles. The · bindlog W.s completely inhibited by the trisaccharide NeuAca2-3Ga)ßl-4Gic, a receptor analogue of S fimbriae, and by a preceding neuraminidase treatment of the sections. A recombinant E. coli strain expressina S fimbriae adhered in large numbers to the same tissue sites in the neonatal brain sections as did the purified fimbriae, · whereas the nonfimbriated host strahi and a recombiiuuit strain expresslog P fi.mbriae did not adhere to brain tissues. The results soggest that adhesion of S-fimbriated bacteria to the binding sites observed in the neonatai bnin has a pathogenetic roJe durlog bacterial Invasion from cii'culation into the cerebrospinal fluid.
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/.
Inflammation in the brain and gut is a critical component of several neurological diseases, such as Parkinson’s disease (PD). One trigger of the immune system in PD is aggregation of the pre-synaptic protein, α-synuclein (αSyn). Understanding the mechanism of propagation of αSyn aggregates is essential to developing disease-modifying therapeutics. Using a brain-first mouse model of PD, we demonstrate αSyn trafficking from the brain to the ileum of male mice. Immunohistochemistry revealed that the ileal αSyn aggregations are contained within CD11c+ cells. Using single-cell RNA sequencing, we demonstrate that ileal CD11c\(^+\) cells are microglia-like and the same subtype of cells is activated in the brain and ileum of PD mice. Moreover, by utilizing mice expressing the photo-convertible protein, Dendra2, we show that CD11c\(^+\) cells traffic from the brain to the ileum. Together these data provide a mechanism of αSyn trafficking between the brain and gut.
Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are exploited by human-specific pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella and Haemophilus species. As opsonin-independent phagocytosis of CEACAM-binding Neisseria depends on Src-family protein tyrosine kinase (PTK) phosphorylation of the CEACAM3 cytoplasmic domain, we hypothesized that an SH2-containing protein might be involved in CEACAM3-initiated, phagocytosis-promoting signals. Accordingly, we screened glutathione-S-transferase (GST) fusion proteins containing SH2 domains derived from a panel of signaling and adapter molecules for their ability to associate with CEACAM3. In vitro pull-down assays demonstrated that the SH2 domain of the adapter molecule Nck (GST-Nck SH2), but not other SH2 domains such as the Grb2 SH2 domain, interact with CEACAM3 in a phosphotyrosine-dependent manner. Either deletion of the cytoplasmic tail of CEACAM3, or point-mutation of a critical arginine residue in the SH2 domain of Nck (GST-NckSH2R308K) that disrupts phosphotyrosine binding, both abolished CEACAM3-Nck-SH2 interaction. Upon infection of human cells with CEACAM-binding Neisseria, full-length Nck comprising an SH2 and three SH3 domains co-localized with tyrosine phosphorylated CEACAM3 and associated bacteria as analyzed by immunofluorescence staining and confocal microscopy. In addition, Nck could be detected in CEACAM3 immunoprecipitates confirming the interaction in vivo. Importantly, overexpression of a GFP-fusion protein of the isolated Nck SH2 domain (GFP-Nck-SH2), but not GFP or GFP-Nck SH2 R308K reduced CEACAM3-mediated phagocytosis of CEACAM-binding Neisseria suggesting that the adaptor molecule Nck plays an important role in CEACAM3-initiated signaling leading to internalization and elimination of human-specific pathogens.