An Investigation into the Effects of Outer Membrane Vesicles and Lipopolysaccharide of Porphyromonas gingivalis on Blood-Brain Barrier Integrity, Permeability, and Disruption of Scaffolding Proteins in a Human in vitro Model

Background: The effects of the key pathogens and virulence factors associated with gum disease such as Porphyromonas gingivalis (P. gingivalis) on the central nervous system is of great interest with respect to development of neuropathologies and hence therapeutics and preventative strategies. Chronic infections and associated inflammation are known to weaken the first line of defense for the brain, the blood-brain barrier (BBB). Objective: The focus of this study is to utilize an established human in vitro BBB model to evaluate the effects of P. gingivalis virulence factors lipopolysaccharide (LPS) and outer membrane vesicles (OMVs) on a primary-derived human model representing the neurovascular unit of the BBB. Methods: Changes to the integrity of the BBB after application of P. gingivalis LPS and OMVs were investigated and correlated with transport of LPS. Additionally, the effect of P. gingivalis LPS and OMVs on human brain microvascular endothelial cells in monolayer was evaluated using immunofluorescence microscopy. Results: The integrity of the BBB model was weakened by application of P. gingivalis LPS and OMVs, as measured by a decrease in electrical resistance and a recovery deficit was seen in comparison to the controls. Application of P. gingivalis OMVs to a monoculture of human brain microvascular endothelial cells showed disruption of the tight junction zona occludens protein (ZO-1) compared to controls. Conclusion: These findings show that the integrity of tight junctions of the human BBB could be weakened by association with P. gingivalis virulence factors LPS and OMVs containing proteolytic enzymes (gingipains).

Outer Membrane Vesicles (OMVs) of Pseudomonas aeruginosa Provide Passive Resistance but Not Sensitization to LPS-Specific Phages

Outer membrane vesicles (OMVs) released from gram-negative bacteria are key elements in bacterial physiology, pathogenesis, and defence. In this study, we investigated the role of Pseudomonas aeruginosa OMVs in the anti-phage defence as well as in the potential sensitization to LPS-specific phages. Using transmission electron microscopy, virion infectivity, and neutralization assays, we have shown that both phages efficiently absorb on free vesicles and are unable to infect P. aeruginosa host. Nevertheless, the accompanying decrease in PFU titre (neutralization) was only observed for myovirus KT28 but not podovirus LUZ7. Next, we verified whether OMVs derived from wild-type PAO1 strain can sensitize the LPS-deficient mutant (Δwbpl PAO1) resistant to tested phages. The flow cytometry experiments proved a quite effective and comparable association of OMVs to Δwbpl PAO1 and wild-type PAO1; however, the growth kinetic curves and one-step growth assay revealed no sensitization event of the OMV-associated phage-resistant P. aeruginosa deletant to LPS-specific phages. Our findings for the first time identify naturally formed OMVs as important players in passive resistance (protection) of P. aeruginosa population to phages, but we disproved the hypothesis of transferring phage receptors to make resistant strains susceptible to LPS-dependent phages.

The Effects of HP0044 and HP1275 Knockout Mutations on the Structure and Function of Lipopolysaccharide in Helicobacter pylori Strain 26695

Helicobacter pylori infection is associated with several gastric diseases, including gastritis, peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma. Due to the prevalence and severeness of H. pylori infection, a thorough understanding of this pathogen is necessary. Lipopolysaccharide, one of the major virulence factors of H. pylori, can exert immunomodulating and immunostimulating functions on the host. In this study, the HP0044 and HP1275 genes were under investigation. These two genes potentially encode GDP-D-mannose dehydratase (GMD) and phosphomannomutase (PMM)/phosphoglucomutase (PGM), respectively, and are involved in the biosynthesis of fucose. HP0044 and HP1275 knockout mutants were generated; both mutants displayed a truncated LPS, suggesting that the encoded enzymes are not only involved in fucose production but are also important for LPS construction. In addition, these two gene knockout mutants exhibited retarded growth, increased surface hydrophobicity and autoaggregation as well as being more sensitive to the detergent SDS and the antibiotic novobiocin. Furthermore, the LPS-defective mutants also had significantly reduced bacterial infection, adhesion and internalization in the in vitro cell line model. Moreover, disruptions of the HP0044 and HP1275 genes in H. pylori altered protein sorting into outer membrane vesicles. The critical roles of HP0044 and HP1275 in LPS biosynthesis, bacterial fitness and pathogenesis make them attractive candidates for drug inventions against H. pylori infection.

Antibiotic action revealed by real-time imaging of the mycobacterial membrane

The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to gaining insight into this time-dependent phenomenon. Herein we describe a fluorogenic chemical probe that reports on mycobacterial cell envelope assembly in real time. We used time-lapse microscopy to reveal distinct spatial and temporal changes in the mycobacterial membrane upon treatment with frontline antibiotics. Differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for monitoring cell envelope construction and remodeling responses simultaneously. Analysis of the imaging data indicates a role for antibiotic-derived outer membrane vesicles in immune modulation.

Oral Immunogenicity of Enterotoxigenic Escherichia coli Outer Membrane Vesicles Encapsulated into Zein Nanoparticles Coated with a Gantrez® AN–Mannosamine Polymer Conjugate

Enterotoxigenic Escherichia coli (ETEC) represents a major cause of morbidity and mortality in the human population. In particular, ETEC infections affect children under the age of five from low-middle income countries. However, there is no licensed vaccine against this pathogen. ETEC vaccine development is challenging since this pathotype expresses a wide variety of antigenically diverse virulence factors whose genes can be modified due to ETEC genetic plasticity. To overcome this challenge, we propose the use of outer membrane vesicles (OMVs) isolated from two ETEC clinical strains. In these OMVs, proteomic studies revealed the presence of important immunogens, such as heat-labile toxin, colonization factors, adhesins and mucinases. Furthermore, these vesicles proved to be immunogenic after subcutaneous administration in BALB/c mice. Since ETEC is an enteropathogen, it is necessary to induce both systemic and mucosal immunity. For this purpose, the vesicles, free or encapsulated in zein nanoparticles coated with a Gantrez®–mannosamine conjugate, were administered orally. Biodistribution studies showed that the encapsulation of OMVs delayed the transit through the gut. These results were confirmed by in vivo study, in which OMV encapsulation resulted in higher levels of specific antibodies IgG2a. Further studies are needed to evaluate the protection efficacy of this vaccine approach.

Proteomics and lipidomics integrated analysis of outer membrane vesicles indicates that glycerophospholipid metabolism contributes to the early symbiosis between Sinorhizobium fredii HH103 and soybean

Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OMVs have been focused on mammalian-bacterial interactions. However, research on the OMVs of rhizobia is still limited so far. In this work, we isolated and purified OMVs from Sinorhizobium fredii HH103 under free-living conditions that was set as control (C-OMVs) and symbiosis-mimicking conditions that was induced by genistein (G-OMVs). The soybean roots treated with G-OMVs displayed significant deformation of root hairs. G-OMVs significantly induced the expression of nodulation genes related to early symbiosis, while inhibited that of the defense genes of soybean. Proteomics analysis identified a total of 93 differential proteins between C-OMVs and G-OMVs, which are mainly associated with ribosome synthesis, flagellar assembly, two-component system, ABC transporters, oxidative phosphorylation, nitrogen metabolism, quorum sensing, glycerophospholipid metabolism and peptidoglycan biosynthesis. A total of 45 differential lipids were identified in lipidomics analysis. Correlation analysis of OMV proteome and lipidome data revealed that glycerophospholipid metabolism is the enriched KEGG metabolic pathway, and the expression of phosphatidylserine decarboxylase was significantly up-regulated in G-OMVs. The changes in three lipids related to symbiosis in the glycerophospholipid metabolism pathway were verified by ELISA. Our results indicate that glycerophospholipid metabolism contributes to rhizobia-soybean symbiosis via OMVs.

A COVID-19 vaccine candidate composed of SARS-CoV-2 RBD dimer and Neisseria meningitidis outer membrane vesicles

SARS-CoV-2 infection is mediated by the interaction of the spike glycoprotein trimer via its receptor-binding domain (RBD) with the host’s cellular receptor. Vaccines seek to block this interaction by eliciting...

sRNAs enriched in outer membrane vesicles of pathogenic Flavobacterium psychrophilum interact with immune genes of rainbow trout

Outer membrane vesicles (OMVs) released by gram-negative bacteria during host-pathogen interactions harbor cargos, such as DNA, RNA, toxins, and virulence factors. We hypothesized that sRNAs carried within OMVs of Flavobacterium psychrophilum interact with host immune genes and affect their expression. OMVs were isolated from F. psychrophilum and visualized using transmission electron microscopy (TEM). RNA-Seq datasets generated from whole-cell F. psychrophilum and their OMVs indicated enrichment of specific sRNAs in the OMVs compared to the parent cell. Fluorescent in situ hybridization (FISH) and confocal microscopy confirmed the expression of a randomly chosen sRNA. Integrated RNA-Seq analyses of host transcriptome and bacterial sRNAs on day 5 post-infection of F. psychrophilum -resistant and -susceptible rainbow trout genetic lines revealed 516 protein-coding, 595 lncRNA, and 116 bacterial sRNA differentially expressed (DE) transcripts. Integrated and network analyses of these DE transcripts revealed immune genes targeted by bacterial sRNAs. On the top of these genes, an isoform encoding anaphase-promoting complex subunit 13 (ANAPC13_1) was highly upregulated and exhibited interaction and reciprocal expression with 21 DE sRNAs enriched in OMVs and/or located in pathogenicity islands (PAIs). In vitro treatment of the rainbow trout epithelial cell line RTgill-W1 with OMVs showed signs of cell autolysis accompanied by dynamic changes in expression of host genes when profiled 24h following treatment. The OMV-enriched sRNAs, soFE013584 and soFE002123, showed high interactions with the protection of telomeres 1 gene (POT1); essential for chromosome stability and cellular viability. Modulation of the host gene expression following OMV-treatment, which favors elements from the phagocytic, endocytic, and antigen presentation pathways in addition to HSP70, HSP90, and cochaperone proteins, provided evidence for a potential role of OMVs in boosting the host immune response. In conclusion, our work identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections.