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Background: A prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet. Methods: Escherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses. Results: The T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition. Conclusions: Our study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae. General significance: We envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.

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.

Background Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells. We present here an analysis of the Vibrio cholerae OMV-associated protease PrtV. Methodology/Principal Findings In this study, we demonstrated that PrtV was secreted from the wild type V. cholerae strain C6706 via the type II secretion system in association with OMVs. By immunoblotting and electron microscopic analysis using immunogold labeling, the association of PrtV with OMVs was examined. We demonstrated that OMV-associated PrtV was biologically active by showing altered morphology and detachment of cells when the human ileocecum carcinoma (HCT8) cells were treated with OMVs from the wild type V. cholerae strain C6706 whereas cells treated with OMVs from the prtV isogenic mutant showed no morphological changes. Furthermore, OMV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37. Conclusion/Significance Our findings suggest that OMVs released from V. cholerae can deliver a processed, biologically active form of PrtV that contributes to bacterial interactions with target host cells.

VibriotasmaniensisLGP32, a facultative intracellular pathogen of oyster haemocytes, was shown here to release outer membrane vesicles (OMVs) both in the extracellular milieu and inside haemocytes. Intracellular release of OMVs occurred inside phagosomes of intact haemocytes having phagocytosed few vibrios as well as in damaged haemocytes containing large vacuoles heavily loaded with LGP32. The OMV proteome of LGP32 was shown to be rich in hydrolases (25%) including potential virulence factors such as proteases, lipases, phospholipases, haemolysins and nucleases. One major caseinase/gelatinase named Vsp for vesicular serine protease was found to be specifically secreted through OMVs in which it is enclosed. Vsp was shown to participate in the virulence phenotype of LGP32 in oyster experimental infections. Finally, OMVs were highly protective against antimicrobial peptides, increasing the minimal inhibitory concentration of polymyxin B by 16-fold. Protection was conferred by OMV titration of polymyxin B but did not depend on the activity of Vsp or another OMV-associated protease. Altogether, our results show that OMVs contribute to the pathogenesis of LGP32, being able to deliver virulence factors to host immune cells and conferring protection against antimicrobial peptides.

There are at least four different variants of ΔclyA, suggesting that such deletions in clyA have arisen at more than one occasion. On the basis of this occurrence of the truncated clyA genes, we considered that there may be a patho-adaptive selection for deletions in clyA in extraintestinal pathogenic E. coli. In E. coli K-12 the clyA gene has been viewed as “cryptic” since it is tightly silenced by the nucleoid structuring protein H-NS. We constructed a restored clyA⁺ locus in derivatives of the UPEC strain 536 for further investigation of this hypothesis and, in particular, how the gene would be expressed. Our results show that the level of clyA⁺ expression is highly increased in the UPEC derivatives in comparison with the non-pathogenic E. coli K-12. Transcription of the clyA⁺ gene was induced to even higher levels when the SfaX regulatory protein was overproduced. The derivative with a restored clyA⁺ locus displayed a somewhat slower growth than the parental UPEC strain 536 when a sub-inhibitory concentration of the antimicrobial peptide Polymyxin B was added to the growth medium.

The Pacific oyster Crassostrea gigas is an important cultured species whose production has suffered from recurrent summer mortality events worldwide over the past two decades. In France, these mortality outbreaks have become devastating for the production of juvenile stages since 2008, and have resulted in great economic losses that currently threaten this activity. Studies on oyster immunity have been performed to better understand its response to pathogens, in particular to bacteria of the Vibrio genus and Herpes viruses, both found in moribund oysters. The immune response of C. gigas relies entirely on the innate immune system, in which hemocytes, the oyster circulating blood cells, are the main cellular mediators of the defense system. Activated when pathogen-associated molecular patterns are recognized by plasma soluble or cell surface pattern recognition receptors, hemocytes operate in a coordinated manner with soluble factors in the hemolymph to circumvent the infection. Over the past years, research efforts have substantially increased the knowledge of the molecular bases of oyster immunity, from non-self recognition proteins, to cytokines, signaling pathways and defense effectors including protease inhibitors, hydrolytic enzymes and antimicrobial peptides/proteins. This review describes the present state of our knowledge on the cellular and molecular effectors of oyster immunity, and describes the oyster responses to pathogenic Vibrio and Herpes species. 

Vibrios are associated with a broad diversity of hosts that produce antimicrobial peptides (AMPs) as part of their defense against microbial infections. In particular, vibrios colonize epithelia, which function as protective barriers and express AMPs as a first line of chemical defense against pathogens. Recent studies have shown they can also colonize phagocytes, key components of the animal immune system. Phagocytes infiltrate infected tissues and use AMPs to kill the phagocytosed microorganisms intracellularly, or deliver their antimicrobial content extracellularly to circumvent tissue infection. We review here the mechanisms by which vibrios have evolved the capacity to evade or resist the potent antimicrobial defenses of the immune cells or tissues they colonize. Among their strategies to resist killing by AMPs, primarily vibrios use membrane remodeling mechanisms. In particular, some highly resistant strains substitute hexaacylated Lipid A with a diglycine residue to reduce their negative surface charge, thereby lowering their electrostatic interactions with cationic AMPs. As a response to envelope stress, which can be induced by membrane-active agents including AMPs, vibrios also release outer membrane vesicles to create a protective membranous shield that traps extracellular AMPs and prevents interaction of the peptides with their own membranes. Finally, once AMPs have breached the bacterial membrane barriers, vibrios use RND efflux pumps, similar to those of other species, to transport AMPs out of their cytoplasmic space.