ABSTRACT
Membrane vesicles (MVs) serve as an essential virulence factor in several pathogenic bacteria. The release of MVs by Listeria monocytogenes is only recently recognized; still, the enigmatic role of MVs in pathogenesis is yet to be established. We report the transcriptome response of Caco-2 cells upon exposure to MVs and the L. monocytogenes that leads to observe the up-regulation of autophagy-related genes in the early phase of exposure to MVs. Transcription of inflammatory cytokines is to the peak at the fourth hour of exposure. An array of differentially expressed genes was associated with actin cytoskeleton rearrangement, autophagy, cell cycle arrest, and induction of oxidative stress. At a later time point, transcriptional programs are generated upon interaction with MVs to evade innate immune signals, by modulating the expression of anti-inflammatory genes. KEGG pathway analysis is palpably confirming that MVs appear principally responsible for the induction of immune signaling pathways. Besides, MVs induced the expression of cell cycle regulatory genes, likely responsible for the ability to prolong host cell survival, thus protecting the replicative niche for L. monocytogenes. Notably, we identified several non-coding RNAs (ncRNAs), possibly involved in the regulation of early manipulation of the host gene expression, essential for the persistence of L. monocytogenes.
ABSTRACT
Membrane vesicles (MVs) are naturally secreted by many pathogenic organisms and have various functions that include the release of microbial virulence factors that contributes to pathogenesis. However, very little is known regarding the function of Gram-positive bacteria membrane vesicles. Here, we investigated the functional role of membrane vesicles of Listeria monocytogenes. We found that L. monocytogenes secreted MVs are spherical and diameter size around 192.3 nm. Here, we investigated the role of L. monocytogenes membrane vesicles in interbacterial communication to cope with antibiotic stress. We found that MVs are protecting the bacteria against the antibiotics trimethoprim and streptomycin. These MVs enabled streptomycin-susceptible L. monocytogenes 1143 to survive in the presence of streptomycin. The zeta potential, dynamic light scattering (DLS) and 1-Nphenylnapthylamine (NPN)-uptake assay reveals that MVs protect the bacterium from active antibiotics by different strategies. Exposure to environmental stressors was shown to increase the level of MV production in L. monocytogenes. The biological activity of MV-associated listeriolysin O, internalin B, and phosphatidylinositol-specific phospholipase C (PI-PLC) was investigated using epithelial cell cytotoxicity. The reduced cytotoxicity was observed in Δhly MVs on Caco-2 cells suggesting that MVs are biologically active. It is shown that a potent toxin LLO contributes to the MV mediated pathogenesis of L. monocytogenes.
ABSTRACT
Membrane vesicles (MVs) are produced by various Gram positive and Gram negative pathogenic bacteria and play an important role in virulence. In this study, the membrane vesicles (MVs) of L. monocytogenes were isolated from the culture supernatant. High-resolution electron microscopy and dynamic light scattering analysis revealed that L. monocytogenes MVs are spherical with a diameter of 200 to 300 nm in size. Further, comprehensive proteomic analyses of MVs and whole cells of L. monocytogenes were performed using LC/MS/MS. A total of 1355 and 312 proteins were identified in the L. monocytogenes cells and MVs, respectively. We identified that 296 proteins are found in both whole cells, and MV proteome and 16 proteins were identified only in the MVs. Also, we have identified the virulence factors such as listeriolysin O (LLO), internalin B (InlB), autolysin, p60, NLP/P60 family protein, UPF0356 protein, and PLC-A in MVs. Computational prediction of host-MV interactions revealed a total of 1841 possible interactions with the host involving 99 MV proteins and 1513 host proteins. We elucidated the possible pathway that mediates internalization of L. monocytogenes MV to host cells and the subsequent pathogenesis mechanisms. The in vitro infection assays showed that the purified MVs could induce cytotoxicity in Caco-2 cells. Using endocytosis inhibitors, we demonstrated that MVs are internalized via actin-mediated endocytosis. These results suggest that L. monocytogenes MVs can interact with host cell and contribute to the pathogenesis of L. monocytogenes during infection.
