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All Staphylococcus aureus genomes contain a genomic island, which is termed vSa\(\alpha\) and characterized by two clusters of tandem repeat sequences, i.e. the exotoxin (set) and 'lipoprotein-like' genes (lpl). Based on their structural similarities the vSa\(\alpha\) islands have been classified as type I to IV. The genomes of highly pathogenic and particularly epidemic S. aureus strains (USA300, N315, Mu50, NCTC8325, Newman, COL, JH1 or JH9) belonging to the clonal complexes CC5 and CC8 bear a type I vSa\(\alpha\) island. Since the contribution of the lpl gene cluster encoded in the vSa\(\alpha\) island to virulence is unclear to date, we deleted the entire lpl gene cluster in S. aureus USA300. The results showed that the mutant was deficient in the stimulation of pro-inflammatory cytokines in human monocytes, macrophages and keratinocytes. Purified lipoprotein Lpl1 was further shown to elicit a TLR2-dependent response. Furthermore, heterologous expression of the USA300 lpl cluster in other S. aureus strains enhanced their immune stimulatory activity. Most importantly, the lpl cluster contributed to invasion of S. aureus into human keratinocytes and mouse skin and the non-invasive S. carnosus expressing the lpl gene cluster became invasive. Additionally, in a murine kidney abscess model the bacterial burden in the kidneys was higher in wild type than in mutant mice. In this infection model the lpl cluster, thus, contributes to virulence. The present report is one of the first studies addressing the role of the vSa\(\alpha\) encoded lpl gene cluster in staphylococcal virulence. The finding that the lpl gene cluster contributes to internalization into non-professional antigen presenting cells such as keratinocytes high-lights the lpl as a new cell surface component that triggers host cell invasion by S. aureus. Increased invasion in murine skin and an increased bacterial burden in a murine kidney abscess model suggest that the lpl gene cluster serves as an important virulence factor.
Two-component systems (TCSs) are the most important sensing mechanisms in bacteria. In Streptomyces, TCSs-mediated responses to environmental stimuli are involved in the regulation of antibiotic production. This study examines the individual role of two histidine kinases (HKs), AbrC1 and AbrC2, which form part of an atypical TCS in Streptomyces coelicolor. gRT-PCR analysis of the expression of both kinases demonstrated that both are expressed at similar levels in NB and NMMP media. Single deletion of abrC1 elicited a significant increase in antibiotic production, while deletion of abrC2 did not have any clear effect. The origin of this phenotype, probably related to the differential phosphorylation ability of the two kinases, was also explored indirectly, analyzing the toxic phenotypes associated with high levels of phosphorylated RR. The higher the AbrC3 regulator phosphorylation rate, the greater the cell toxicity. For the first time, the present work shows in Streptomyces the combined involvement of two different HKs in the response of a regulator to environmental signals. Regarding the possible applications of this research, the fact that an abrC1 deletion mutant overproduces three of the S. coelicolor antibiotics makes this strain an excellent candidate as a host for the heterologous production of secondary metabolites.
Staphylococcus aureus is a commensal of the human nose and skin. Human skin fatty acids, in particular cis-6-hexadecenoic acid (C-6-H), have high antistaphylococcal activity and can inhibit virulence determinant production. Here, we show that sub-MIC levels of C-6-H result in induction of increased resistance. The mechanism(s) of C-6-H activity was investigated by combined transcriptome and proteome analyses. Proteome analysis demonstrated a pleiotropic effect of C-6-H on virulence determinant production. In response to C-6-H, transcriptomics revealed altered expression of over 500 genes, involved in many aspects of virulence and cellular physiology. The expression of toxins (hla, hlb, hlgBC) was reduced, whereas that of host defence evasion components (cap, sspAB, katA) was increased. In particular, members of the SaeRS regulon had highly reduced expression, and the use of specific mutants revealed that the effect on toxin production is likely mediated via SaeRS
Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium.
Two lineages of Salmonella enterica serovar Typhimurium (S. Typhimurium) of multi-locus sequence type ST313 have been linked with the emergence of invasive Salmonella disease across sub-Saharan Africa. The expansion of these lineages has a temporal association with the HIV pandemic and antibiotic usage. We analysed the whole genome sequence of 129 ST313 isolates representative of the two lineages and found evidence of lineage-specific genome degradation, with some similarities to that observed in S. Typhi. Individual ST313 S. Typhimurium isolates exhibit a distinct metabolic signature and modified enteropathogenesis in both a murine and cattle model of colitis, compared to S. Typhimurium outside of the ST313 lineages. These data define phenotypes that distinguish ST313 isolates from other S. Typhimurium and may represent adaptation to a distinct pathogenesis and lifestyle linked to an-immuno-compromised human population.
Background: Despite the limited success after decades of intensive research and development efforts, vaccination still represents the most promising strategy to significantly reduce the disease burden in malaria endemic regions. Besides the ultimate goal of inducing sterile protection in vaccinated individuals, the prevention of transmission by so-called transmission blocking vaccines (TBVs) is being regarded as an important feature of an efficient malaria eradication strategy. Recently, Plasmodium falciparum GAP50 (PfGAP50), a 44.6 kDa transmembrane protein that forms an essential part of the invasion machinery (glideosome) multi-protein complex, has been proposed as novel potential transmission-blocking candidate. Plant-based expression systems combine the advantages of eukaryotic expression with a up-scaling potential and a good product safety profile suitable for vaccine production. In this study we investigated the feasibility to use the transient plant expression to produce PfGAP50 suitable for the induction of parasite specific inhibitory antibodies.
Results: We performed the transient expression of recombinant PfGAP50 in Nicotiana benthamiana leaves using endoplasmatic reticulum (ER) and plastid targeting. After IMAC-purification the protein yield and integrity was investigated by SDS-PAGE and Western Blot. Rabbit immune IgG derived by the immunization with the plastidtargeted variant of PfGAP50 was analyzed by immune fluorescence assay (IFA) and zygote inhibition assay (ZIA). PfGAP50 could be produced in both subcellular compartments at different yields IMAC (Immobilized Metal Affinity Chromatography) purification from extract yielded up to 4.1 mu g/g recombinant protein per fresh leaf material for ER-retarded and 16.2 mu g/g recombinant protein per fresh leave material for plasmid targeted PfGAP50, respectively. IgG from rabbit sera generated by immunization with the recombinant protein specifically recognized different parasite stages in immunofluorescence assay. Furthermore up to 55 % inhibition in an in vitro zygote inhibition assay could be achieved using PfGAP50-specific rabbit immune IgG.
Conclusions: The results of this study demonstrate that the plant-produced PfGAP50 is functional regarding the presentation of inhibitory epitopes and could be considered as component of a transmission-blocking malaria vaccine formulation.
Phenotypic heterogeneity at the cellular level in response to various stresses, e.g., antibiotic treatment has been reported for a number of bacteria. In a clonal population, cell-to-cell variation may result in phenotypic heterogeneity that is a mechanism to survive changing environments including antibiotic therapy. Stenotrophomonas rnaltophilia has been frequently isolated from cystic fibrosis patients, can cause numerous infections in other organs and tissues, and is difficult to treat due to antibiotic resistances. S. maltophilia K279a produces the Li and L2 beta-lactamases in response to beta-lactam treatment. Here we report that the patient isolate S. rnaltophilia K279a diverges into cellular subpopulations with distinct but reversible morphotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of elongated chains of bacteria during exponential growth phase and the occurrence of mainly rod-shaped cells in liquid media. RNA-seq analysis of small versus big colonies revealed differential regulation of at least seven genes among the colony morphotypes. Among those, bleu and bla(L2) were transcriptionally the most strongly upregulated genes. Promoter fusions of b/a(L1) and b/a(L2) genes indicated that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additionally, the comE homolog was found to be differentially expressed in homogenously versus heterogeneously bla(L2) expressing cells as identified by RNA(seq) analysis. Overexpression of cornE in S. maltophilia K279a reduced the level of cells that were in a bla(L2)-ON mode to 1% or lower. Taken together, our data provide strong evidence that S. maltophilia K279a populations develop phenotypic heterogeneity in an ampicillin challenged model. This cellular variability is triggered by regulation networks including b/a(L1), b/a(L2), and comE.
Background
Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed.
Methodology/Principal
Findings Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB.
Conclusions
Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.
Background:
Ethiopia, a high tuberculosis (TB) burden country, reports one of the highest incidence rates of extra-pulmonary TB dominated by cervical lymphadenitis (TBLN). Infection with Mycobacterium bovis has previously been excluded as the main reason for the high rate of extra-pulmonary TB in Ethiopia.
Methods:
Here we examined demographic and clinical characteristics of 953 pulmonary (PTB) and 1198 TBLN patients visiting 11 health facilities in distinct geographic areas of Ethiopia. Clinical characteristics were also correlated with genotypes of the causative agent, Mycobacterium tuberculosis.
Results:
No major patient or bacterial strain factor could be identified as being responsible for the high rate of TBLN, and there was no association with HIV infection. However, analysis of the demographic data of involved patients showed that having regular and direct contact with live animals was more associated with TBLN than with PTB, although no M. bovis was isolated from patients with TBLN. Among PTB patients, those infected with Lineage 4 reported "contact with other TB patient" more often than patients infected with Lineage 3 did (OR = 1.6, CI 95% 1.0-2.7; p = 0.064). High fever, in contrast to low and moderate fever, was significantly associated with Lineage 4 (OR = 2.3; p = 0.024). On the other hand, TBLN cases infected with Lineage 4 tended to get milder symptoms overall for the constitutional symptoms than those infected with Lineage 3.
Conclusions:
The study suggests a complex role for multiple interacting factors in the epidemiology of extra-pulmonary TB in Ethiopia, including factors that can only be derived from population-based studies, which may prove to be significant for TB control in Ethiopia.
Biofilm formation by Staphylococcus aureus represents a problem in both the medical field and the food industry, because the biofilm structure provides protection to embedded cells and it strongly attaches to surfaces. This circumstance is leading to many research programs seeking new alternatives to control biofilm formation by this pathogen. In this study we show that a potent inhibition of biofilm mass production can be achieved in community-associated methicillin-resistant S. aureus (CA-MRSA) and methicillin-sensitive strains using plant compounds, such as individual constituents (ICs) of essential oils (carvacrol, citral, and (+)-limonene). The Crystal Violet staining technique was used to evaluate biofilm mass formation during 40 h of incubation. Carvacrol is the most effective IC, abrogating biofilm formation in all strains tested, while CA-MRSA was the most sensitive phenotype to any of the ICs tested. Inhibition of planktonic cells by ICs during initial growth stages could partially explain the inhibition of biofilm formation. Overall, our results show the potential of EOs to prevent biofilm formation, especially in strains that exhibit resistance to other antimicrobials. As these compounds are food additives generally recognized as safe, their anti-biofilm properties may lead to important new applications, such as sanitizers, in the food industry or in clinical settings.