Mitesh Dongre

NOD-Like Receptor Activation by Outer Membrane Vesicles from Vibrio cholerae Non-O1 Non-O139 Strains Is Modulated by the Quorum-Sensing Regulator HapR

ABSTRACT Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses, and in particular, V. cholerae strains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. Small interfering RNA (siRNA)-mediated gene knockdown revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain-, leucine-rich repeat-containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum-sensing regulator HapR, which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum-sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs under infective conditions. This work thus identifies a new facet of how Vibrio affects host immune responses and defines a role for the quorum-sensing machinery in this process.

Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains.

Background Outer membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied. Methodology/Principal Findings OMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor. Conclusion/Significance Biological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.

A role for quorum sensing in regulating innate immune responses mediated by Vibrio cholerae outer membrane vesicles (OMVs).

Outer membrane vesicles (OMVs) are released from many Gram-negative bacteria. OMVs interact with and are taken up by human cells. We and others have now showed that OMVs contain peptidoglycan, which is sensed mainly by the pattern-recognition receptor NOD1 in the cytoplasm of host cells. Vibrio cholerae is clinically important as one of the causative agents of severe dehydrating diarrhea in humans. We showed that non-O1 non-O139 V. cholerae (NOVC) strains of V. cholera produce OMVs. Of note, we revealed that NOVC can evade NOD1-mediated immune surveillance by the quorum sensing machinery. Here we review these recent findings and discuss the relevance for our understanding of bacterial infections and innate immune responses.

Evidence on How a Conserved Glycine in the Hinge Region of HapR Regulates Its DNA Binding Ability: LESSONS FROM A NATURAL VARIANT.

HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapRV2) harboring a substitution of glycine to aspartate at position 39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapRV2 to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapRV2 (HapRV2G), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapRV2 and HapRV2G proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins, we acquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a “Y” shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.

Staphylococcus aureus Membrane-Derived Vesicles Promote Bacterial Virulence and Confer Protective Immunity in Murine Infection Models

Staphylococcus aureus produces membrane-derived vesicles (MVs), which share functional properties to outer membrane vesicles. Atomic force microscopy revealed that S. aureus-derived MVs are associated with the bacterial surface or released into the surrounding environment depending on bacterial growth conditions. By using a comparative proteomic approach, a total of 131 and 617 proteins were identified in MVs isolated from S. aureus grown in Luria-Bertani and brain-heart infusion broth, respectively. Purified S. aureus MVs derived from the bacteria grown in either media induced comparable levels of cytotoxicity and neutrophil-activation. Administration of exogenous MVs increased the resistance of S. aureus to killing by whole blood or purified human neutrophils ex vivo and increased S. aureus survival in vivo. Finally, immunization of mice with S. aureus-derived MVs induced production of IgM, total IgG, IgG1, IgG2a, and IgG2b resulting in protection against subcutaneous and systemic S. aureus infection. Collectively, our results suggest S. aureus MVs can influence bacterial–host interactions during systemic infections and provide protective immunity in murine models of infection.

Functional independence of a variant LuxOPL91 from a non-O1 non-O139 Vibrio cholerae over the activity of CsrA and Fis

Functional independence of a variant LuxOPL91 from a non-O1 non-O139 Vibrio cholerae over the activity of CsrA and Fis.

Alanine-scanning mutagenesis of selected residues in the N-terminal region alters the functionality of LuxO: lessons from a natural variant LuxOPL91

Alanine-scanning mutagenesis of selected residues in the N-terminal region alters the functionality of LuxO : lessons from a natural variant LuxOPL91.

Modulation of gene transcription and epigenetics of colon carcinoma cells by bacterial membrane vesicles

Interactions between bacteria and colon cancer cells influence the transcription of the host cell. Yet is it undetermined whether the bacteria itself or the communication between the host and bacteria is responsible for the genomic changes in the eukaryotic cell. Now, we have investigated the genomic and epigenetic consequences of co-culturing colorectal carcinoma cells with membrane vesicles from pathogenic bacteria Vibrio cholerae and non-pathogenic commensal bacteria Escherichia coli. Our study reveals that membrane vesicles from pathogenic and commensal bacteria have a global impact on the gene expression of colon-carcinoma cells. The changes in gene expression correlate positively with both epigenetic changes and chromatin accessibility of promoters at transcription start sites of genes induced by both types of membrane vesicles. Moreover, we have demonstrated that membrane vesicles obtained only from V. cholerae induced the expression of genes associated with epithelial cell differentiation. Altogether, our study suggests that the observed genomic changes in host cells might be due to specific components of membrane vesicles and do not require communication by direct contact with the bacteria.

Bacterial nanotubes for intimate sharing

Classical genetics and biochemical studies on bacterial communication have established that bacteria can exchange small circular plasmids and DNA fragments via conjugative pili or through the agency of a transducing phage. Additionally, homogenous and heterogeneous bacterial population can share chemical information by secreting pheromone-like molecules called as QS auto-inducers or by discharging cytoplasmic and periplasmic proteins, antimicrobial factors, and even DNA in packaged outer membrane vesicles (MVs), which can deliver the cargo to distant cells. Delivery of cellular proteins via a direct cellular contact is also not uncommon in bacteria. Many pathogenic Gram negative bacteria can deliver cytoplasmic effector proteins directly into the host cytoplasm using specialized secretory systems. These secretion systems can employ highly orchestrated needle complexes as in the type III secretion system (T3SS) and the type VI secretion system (T6SS) or pilin channels as in the type IV secretion system, for the delivery of toxins and effector molecules directly into host (Tseng et al., 2009). However, these secretory systems are principally unidirectional delivery apparatus ensembling unique constituent proteins in a highly energy expensive process. Nonetheless, there has been no evidence of bacteria sharing large proteins and DNA fragments through direct cytoplasmic contact. The recent report by Dubey and Ben-Yehuda (2011) in Cell incepted and evidenced the presence of intercellular membranous bridges or nanotubes between proximal bacteria of same as well as different species taking Bacillus subtilis, Streptococcus pyogenes, and Escherichia coli as model Gram positive and Gram negative organisms. This discovery has opened an interesting new arena in the field of bacterial communication. In their study the team characterized hereditary transfer of non-conjugative plasmids and non-hereditary transfer of cytoplasmic traits such as green fluorescent protein (GFP) and antimicrobial resistance protein between neighboring bacteria which was dependent on relative distance between the cells and time. Through high resolution scanning electron microscopy they observed the intercellular membranous conduits between the bacterial cells, facilitating direct cytoplasmic sharing between adjacent bacterial cells. Further, by analyzing thin sections EM of the nanotubes and their disruption by SDS, authors enunciated that these conduits may involve direct cytoplasmic fusions of the adjacent bacteria secured by a multilayer structure comprising cell wall and plasma membrane. This contingency could make these nanotubes as unique rendezvous points between two bacterial cells distinct from other secretory mechanisms in that they may not involve any dedicated cellular machinery for the transfer of cytoplasmic material. Moreover, the diameter of these nanotubes was observed at more than 100 nm which is larger than most pili and fimbriae and is sufficiently large to allow transfer of large protein complexes and DNA fragments. It is interesting to note that most of these features of nanotubes are pertinent to MVs which frequently emerge from the cell wall of Gram negative and Gram positive bacteria. For instance, MVs are membranous structure measuring 20–300 nm in diameter, emerging from the cell wall of Gram negative as well as Gram positive bacteria and carry periplasmic and cytoplasmic proteins, RNA, and DNA fragments along with intrinsic membrane proteins (Wai et al., 2003; Mashburn-Warren and Whiteley, 2006; Lee et al., 2009). Biogenesis of MVs in Gram negative bacteria is not random blebbing of the outer membrane but in fact occur at specific cell surface points excluding the abundant Braun lipoprotein which traverse through outer membrane and peptidoglycan layer (Hoekstra et al., 1976). Moreover, a recent study revealed that in Pseudomonas aeruginosa MVs primarily contain highly charged B-band LPS which is relatively rare in comparison with the abundant A-band LPS in OM (Li et al., 1996). It is proposed that localization of these charged moieties at unlinked areas coupled with the accumulation of cargo proteins and some unidentified curvature-inducing molecules in the periplasm build a substantial inducing force beneath the outer membrane causing an outward curvature which culminates in release of MVs (Kulp and Kuehn, 2010). Further, due to the intrinsic bilayered exterior, these MVs can readily adhere to and fuse with cell wall of other bacteria. Incidentally, Dubey and Ben-Yehuda (2011) also observed similar membrane bulging during initiation of the nanotubes formation. Given the degree of similarity between MVs and nanotubes it is plausible that initiation of these conduits might require similar framework of preferential biogenesis sites on the bacterial cell wall excluding specific membrane components or including preferential cargo which would act as inducing force. Moreover, since nanotube formation is governed by relative distance between two adjacent bacteria, it is possible that these conduits are formed by fusion of two budding MVs from respective bacteria which otherwise would shed these vesicles if not in close proximity of each other. Another fact which stipulate resemblance between nanotubes and MVs is that both allows concurrent delivery of multiple secreted molecules to the host at higher concentration which could be a prerequisite for certain bioprocesses. A major relevance of the discovery of nanotubes is that it can help in improving our understanding about the communication between the inhabitants and accretion of hereditary and non-hereditary traits by constituent bacteria in single and multispecies biofilms. Moreover, since MVs are shown to be a common particulate constituent of the matrix of many Gram negative and multispecies biofilms (Schooling and Beveridge, 2006), it is of further interest to study the correlation between these two coherently analogous modes of bacterial secretion.