Transcriptome profiling of staphylococcus aureus associated extracellular vesicles reveals presence of small RNA-cargo

Bacterial extracellular vesicles (EVs) have a vital role in bacterial pathogenesis. However, to date, the small RNA-cargo of EVs released by the opportunistic pathogen Staphylococcus aureus has not been characterized. Here, we shed light on the association of small RNAs with EVs secreted by S. aureus MSSA476 cultured in iron-depleted bacteriologic media supplemented with a subinhibitory dosage of vancomycin to mimic infection condition. Confocal microscopy analysis on intact RNase-treated EVs indicated that RNA is associated with EV particles. Transcriptomic followed by bioinformatics analysis of EV-associated RNA revealed the presence of potential gene regulatory small RNAs and high levels of tRNAs. Among the EV-associated enriched small RNAs were SsrA, RsaC and RNAIII. Our finding invites new insights into the potential role of EV-associated RNA as a modulator of host-pathogen interaction. Introductio

Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p>Background: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of <it>Campylobacter jejuni</it>, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment.</p> <p>Results</p> <p>Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs) released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50%) of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form.</p> <p>Conclusion</p> <p>Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of <it>C. jejuni </it>to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.</p

Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

BACKGROUND: Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). METHODOLOGY/PRINCIPAL FINDINGS: We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3), CHA6.8 (FA ratio 1.08+/-0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. CONCLUSION: Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model

Quorum Sensing Regulation of the Two hcp Alleles in Vibrio cholerae O1 Strains

BACKGROUND: The type VI secretion system (T6SS) has emerged as a protein secretion system important to several gram-negative bacterial species. One of the common components of the system is Hcp, initially described as a hemolysin co-regulated protein in a serotype O17 strain of Vibrio cholerae. Homologs to V. cholerae hcp genes have been found in all characterized type VI secretion systems and they are present also in the serotype O1 strains of V. cholerae that are the cause of cholera diseases but seemed to have non-functional T6SS. METHODOLOGY/PRINCIPAL FINDINGS: The serotype O1 V. cholerae strain A1552 was shown to express detectable levels of Hcp as determined by immunoblot analyses using polyclonal anti-Hcp antiserum. We found that the expression of Hcp was growth phase dependent. The levels of Hcp in quorum sensing deficient mutants of V. cholerae were compared with the levels in wild type V. cholerae O1 strain A1552. The expression of Hcp was positively and negatively regulated by the quorum sensing regulators HapR and LuxO, respectively. In addition, we observed that expression of Hcp was dependent on the cAMP-CRP global transcriptional regulatory complex and required the RpoN sigma factor. CONCLUSION/SIGNIFICANCE: Our results show that serotype O1 strains of V. cholerae do express Hcp which is regarded as one of the important T6SS components and is one of the secreted substrates in non-O1 non-O139 V. cholerae isolates. We found that expression of Hcp was strictly regulated by the quorum sensing system in the V. cholerae O1 strain. In addition, the expression of Hcp required the alternative sigma factor RpoN and the cAMP-CRP global regulatory complex. Interestingly, the environmental isolates of V. cholerae O1 strains that showed higher levels of the HapR quorum sensing regulator in comparison with our laboratory standard serotype O1 strain A1552 where also expressing higher levels of Hcp

Кераміка «terra sigillata» з с. Зимне на Волині

Стаття присвячена публікації чотирьох керамічних посудин типу «terra sigillata», знайдених на дні р. Луги у с. Зимне Володимир-Волинського району Волинської області. Попередній аналіз цих знахідок дозволяє віднести їх до Понтійського центру виробництва такого посуду. Вірогідним шляхом потрапляння цієї колекції на Волинь була готська експансія у Північне Причорномор’я

Sub-Optimal Treatment of Bacterial Biofilms

Bacterial biofilm is an emerging clinical problem recognized in the treatment of infectious diseases within the last two decades. The appearance of microbial biofilm in clinical settings is steadily increasing due to several reasons including the increased use of quality of life-improving artificial devices. In contrast to infections caused by planktonic bacteria that respond relatively well to standard antibiotic therapy, biofilm-forming bacteria tend to cause chronic infections whereby infections persist despite seemingly adequate antibiotic therapy. This review briefly describes the responses of biofilm matrix components and biofilm-associated bacteria towards sub-lethal concentrations of antimicrobial agents, which may include the generation of genetic and phenotypic variabilities. Clinical implications of bacterial biofilms in relation to antibiotic treatments are also discussed

CRISPR-based subtyping to track the evolutionary history of a global clone of Acinetobacter baumannii

Acinetobacter baumannii global clone 1 (GC1) is the second most common clone in the global population of A. baumannii isolates and a key cause of hospital-acquired infections. In this study, comparative analysis of the clustered regularly interspaced short palindromic repeats (CRISPR)-based sequence types (CST) was performed to determine the genetic relatedness and track patterns of descent among 187 GC1 isolates, as a complement to the evolutionary inferences from their multilocus sequence types and genome-wide single nucleotide polymorphism (SNP)-based phylogeny. The CST2 cluster, CST2 and all the CSTs descending from CST2, corresponded to GC1 lineage 1. This cluster included 143 of the 187 isolates showing a prevalent geographical distribution worldwide. A well-demarcated group of 13 CSTs, accounting for 33 of the 187 isolates, corresponded to GC1 lineage 2. All the CSTs of this group were characterized by the absence of spacer Ab-18. Many of the GC1 lineage 2 isolates had an epidemiological link to the Middle East and/or were obtained in military healthcare facilities. GC1 lineage 3 was a novel lineage that has so far been limited to Afghanistan, Pakistan and India. Diversification of A. baumannii GC1 into lineages and clades has probably been related to a dynamic expansion after passing a migration bottleneck to enter the hospital environment. We conclude that CRISPR-based subtyping is a convenient method to trace the evolutionary history of particular bacterial clones, such as A. baumannii GC1

The VrrA sRNA controls stationary phase survival factor Vrp of Vibrio cholerae

Small non-coding RNAs (sRNAs) are emerging regulatory elements in bacteria. The Vibrio cholerae sRNA VrrA has previously been shown to down-regulate outer membrane proteins (OmpA and OmpT) and biofilmmatrix protein (RbmC) by base-pairing with the 50 region of the corresponding mRNAs. In this study, we present an additional target of VrrA in V. cholerae, the mRNA coding for the ribosome binding protein Vrp. Vrp is homologous to ribosome-associated inhibitor A (RaiA) of Escherichia coli which facilitates stationary phase survival through ribosome hibernation. We show that VrrA downregulates Vrp protein synthesis by base-pairing to the 50 region of vrp mRNA and that the regulation requires the RNA chaperone protein, Hfq. We further demonstrate that Vrp is highly expressed during stationary phase growth and associates with the ribosome of V. cholerae. The effect of the Vrp protein in starvation survival is synergistic with that of the VC2530 protein, a homolog of the E. coli hibernation promoting factor HPF, suggesting a combined role for these proteins in ribosome hibernation in V. cholerae. Vrp and VC2530 are important for V. cholerae starvation survival under nutrient deficient conditions. While VC2530 is down-regulated in cells lacking vrrA, mutation of vrp results in VC2530 activation. This is the first report indicating a regulatory role for an sRNA, modulating stationary factors involved in bacterial ribosome hibernation

Eco-evolutionary feedbacks mediated by bacterial membrane vesicles

Bacterial membrane vesicles (BMVs) are spherical extracellular organelles whose cargo is enclosed by a biological membrane. The cargo can be delivered to distant parts of a given habitat in a protected and concentrated manner. This review presents current knowledge about BMVs in the context of bacterial eco-evolutionary dynamics among different environments and hosts. BMVs may play an important role in establishing and stabilizing bacterial communities in such environments; for example, bacterial populations may benefit from BMVs to delay the negative effect of certain evolutionary trade-offs that can result in deleterious phenotypes. BMVs can also perform ecosystem engineering by serving as detergents, mediators in biochemical cycles, components of different biofilms, substrates for cross-feeding, defense systems against different dangers and enzyme-delivery mechanisms that can change substrate availability. BMVs further contribute to bacteria as mediators in different interactions, with either other bacterial species or their hosts. In short, BMVs extend and deliver phenotypic traits that can have ecological and evolutionary value to both their producers and the ecosystem as a whole