Outer Membrane Vesicles Derived from Klebsiella pneumoniae Are a Driving Force for Horizontal Gene Transfer.
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ABSTRACT:
SUBMITTER: Dell'Annunziata F
PROVIDER: S-EPMC8395779 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Outer membrane vesicles (OMVs) secreted by Gram-negative bacteria serve as transporters for the delivery of cargo such as virulence and antibiotic resistance factors. OMVs play a key role in the defense against membrane-targeting antibiotics such as the polymyxin B. Herein, we conducted comparative proteomics of OMVs from paired Klebsiella pneumoniae ATCC 700721 polymyxin-susceptible (polymyxin B MIC = 0.5 mg/L) and an extremely resistant (polymyxin B MIC ≥128 mg/L), following exposure to 2 mg/L of polymyxin B. Comparative profiling of the OMV subproteome of each strain revealed proteins from multiple perturbed pathways, particularly in the polymyxin-susceptible strain, including outer membrane assembly (lipopolysaccharide, O-antigen, and peptidoglycan biosynthesis), cationic antimicrobial peptide resistance, β-lactam resistance, and quorum sensing. In the polymyxin-susceptible strain, polymyxin B treatment reduced the expression of OMV proteins in the pathways related to adhesion, virulence, and the cell envelope stress responses, whereas, in the polymyxin-resistant strain, the proteins involved in LPS biosynthesis, RNA degradation, and nucleotide excision repair were significantly overexpressed in response to polymyxin B treatment. Intriguingly, the key polymyxin resistance enzymes 4-amino-4-deoxy-l-arabinose transferase and the PhoPQ two-component protein kinase were significantly downregulated in the OMVs of the polymyxin-susceptible strain. Additionally, a significant reduction in class A β-lactamase proteins was observed following polymyxin B treatment in the OMVs of both strains, particularly the OMVs of the polymyxin-susceptible strain. These findings shed new light on the OMV subproteome of extremely polymyxin resistant K. pneumoniae, which putatively may serve as active decoys to make the outer membrane more impervious to polymyxin attack. IMPORTANCE OMVs can help bacteria to fight antibiotics not only by spreading antibiotic resistance genes but also by acting as protective armor against antibiotics. By employing proteomics, we found that OMVs have a potential role in shielding K. pneumoniae and acting as decoys to polymyxin attack, through declining the export of proteins (e.g., 4-amino-4-deoxy-l-arabinose transferase) involved in polymyxin resistance. Furthermore, polymyxin B treatment of both strains leads to shedding of the OMVs with perturbed proteins involved in outer membrane remodeling (e.g., LPS biosynthesis) as well as pathogenic potential of K. pneumoniae (e.g., quorum sensing). The problematic extended spectrum beta-lactamases SHV and TEM were significantly reduced in both strains, suggesting that polymyxin B may act as a potentiator to sensitize the bacterium to β-lactam antibiotics. This study highlights the importance of OMVs as "molecular mules" for the intercellular transmission and delivery of resistance and cellular repair factors in the bacterial response to polymyxins.
| S-EPMC9942579 | biostudies-literature
Project description:Klebsiella pneumoniae is an opportunistic pathogen that causes nosocomial and community-acquired infections. The spread of resistant strains of K. pneumoniae represents a growing threat to human health, due to the exhaustion of effective treatments. K. pneumoniae releases outer membrane vesicles (OMVs). OMVs are a vehicle for the transport of virulence factors to host cells, causing cell injury. Previous studies have shown changes of gene expression in human bronchial epithelial cells after treatment with K. pneumoniae OMVs. These variations in gene expression could be regulated through microRNAs (miRNAs), which participate in several biological mechanisms. Thereafter, miRNA expression profiles in human bronchial epithelial cells were evaluated during infection with standard and clinical K. pneumoniae strains. Microarray analysis and RT-qPCR identified the dysregulation of miR-223, hsa-miR-21, hsa-miR-25 and hsa-let-7g miRNA sequences. Target gene prediction revealed the essential role of these miRNAs in the regulation of host immune responses involving NF-?B (miR-223), TLR4 (hsa-miR-21), cytokine (hsa-miR-25) and IL-6 (hsa-let-7g miRNA) signalling pathways. The current study provides the first large scale expression profile of miRNAs from lung cells and predicted gene targets, following exposure to K. pneumoniae OMVs. Our results suggest the importance of OMVs in the inflammatory response.
| S-EPMC7764071 | biostudies-literature
Project description:Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important multidrug resistance (MDR) pathogen that threatens human health and is the main source of hospital-acquired infection. Outer membrane vesicles (OMVs) are extracellular vesicles derived from Gram-negative bacteria and contain materials involved in bacterial survival and pathogenesis. They also contribute to cellular communication to nearby or distant recipient cells and influence their functions and phenotypes. In this study, we sought to understand the mechanism of bacterial response to meropenem pressure and explore the relationship between pathogenic proteins and the high pathogenicity of bacteria. We performed whole-genome PacBio sequencing on a clinical CRKP strain, and its OMVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and proteomic analysis. Thousands of vesicle proteins have been identified in mass spectrometry-based high-throughput proteomics analyses of K. pneumoniae OMVs. Protein functionality analysis showed that the OMVs were predominantly involved in metabolic, intracellular compartments, nucleic acid binding, survival, defense, and antibiotic resistance, such as Chromosome partition protein MukB, 3-methyl-2-oxobutanoate hydroxymethyltransferase, methionine-tRNA ligase, Heat shock protein 60 family chaperone GroEL, and Gamma-glutamyl phosphate reductase. Additionally, a protein-protein interaction network demonstrated that OMVs from meropenem-treated K. pneumoniae showed the highest connectivity in DNA polymerase I, phenylalanine-tRNA ligase beta subunit, DNA-directed RNA polymerase subunit beta, methionine-tRNA ligase, DNA-directed RNA polymerase subunit beta, and DNA-directed RNA polymerase subunit alpha. The OMVs proteome expression profile indicates increased secretion of stress proteins released from meropenem-treated K. pneumoniae, which provides clues for revealing the biogenesis and pathophysiological functions of Gram-negative bacteria OMVs. The significant differentially expressed proteins identified in this study are of great significance for exploring effective control strategies for CRKP infection.IMPORTANCEMeropenem is one of the main antibiotics used in the clinical treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP). This study demonstrated that some important metabolic changes occurred in meropenem-induced CRKP-outer membrane vesicles (OMVs), The OMVs proteome expression profile indicates increased secretion of stress proteins released from meropenem-induced Klebsiella pneumoniae. Furthermore, this is the first study to discuss the protein-protein interaction network of the OMVs released by CRKP, especially under antibiotic stress.
| S-EPMC10846168 | biostudies-literature
Project description:Hypervirulent Klebsiella pneumoniae (hvKp) is a notorious clinical pathogen that is more likely to cause severe primary and metastatic abscesses. The dissemination of antimicrobial-resistant hvKp isolates has been reported worldwide, posing a great challenge and severe clinical threat. However, the mechanisms of antimicrobial-resistant hvKp isolates prevalent worldwide are not well precise. Outer membrane vesicles (OMVs) secreted from gram-negative bacteria are an important vehicle for delivering effector molecules inter- and intra-species. To explore whether OMVs as the vector of virulence genes horizontal transfer among Klebsiella pneumoniae and to explain the potential mechanism for the development of antimicrobial-resistant hvKp isolates, we isolated OMVs from hvKp and classical Klebsiella pneumoniae (cKp) by sequential differential centrifugation, respectively. Then, the characteristics and contents of hvKp-OMVs and cKp-OMVs were analyzed. These hvKp-OMVs contain virulence genes, which could be transferred from hvKp horizontally to extended-spectrum beta lactamase (ESBL)-producing cKp, leading to the production of antimicrobial-resistant hypervirulent transformants. Further experiments confirmed the transformants exhibited antimicrobial resistance and hypervirulent phenotypes in vitro and in vivo. In short, this work demonstrated that hvKp-OMVs facilitated virulence genes transfer, allowing an increase in the virulence level of ESBL-producing cKp and providing a new mechanism for the emergence of antimicrobial-resistant hvKp isolates.
| S-EPMC9132476 | biostudies-literature
Project description:Gram-negative bacteria produce outer membrane vesicles (OMVs) as delivery vehicles for nefarious bacterial cargo such as virulence factors, which are antibiotic resistance determinants. This study aimed to investigate the impact of polymyxin B treatment on the OMV lipidome from paired polymyxin-susceptible and -resistant Klebsiella pneumoniae isolates. K. pneumoniae ATCC 700721 was employed as a reference strain in addition to two clinical strains, K. pneumoniae FADDI-KP069 and K. pneumoniae BM3. Polymyxin B treatment of the polymyxin-susceptible strains resulted in a marked reduction in the glycerophospholipid, fatty acid, lysoglycerophosphate and sphingolipid content of their OMVs. Conversely, the polymyxin-resistant strains expressed OMVs richer in all of these lipid species, both intrinsically and increasingly under polymyxin treatment. The average diameter of the OMVs derived from the K. pneumoniae ATCC 700721 polymyxin-susceptible isolate, measured by dynamic light scattering measurements, was ~90.6 nm, whereas the average diameter of the OMVs isolated from the paired polymyxin-resistant isolate was ~141 nm. Polymyxin B treatment (2 mg/L) of the K. pneumoniae ATCC 700721 cells resulted in the production of OMVs with a larger average particle size in both the susceptible (average diameter ~124 nm) and resistant (average diameter ~154 nm) strains. In light of the above, we hypothesize that outer membrane remodelling associated with polymyxin resistance in K. pneumoniae may involve fortifying the membrane structure with increased glycerophospholipids, fatty acids, lysoglycerophosphates and sphingolipids. Putatively, these changes serve to make the outer membrane and OMVs more impervious to polymyxin attack.
| S-EPMC6121281 | biostudies-literature
Project description:Outer membrane vesicles (OMV) are critical elements in many host-cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that within 12?h of colonizing crypts deep within the squid's light organ, the symbionts trigger an irreversible programme of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage-like cells called haemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin released by the symbionts, which can induce haemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that haemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host's detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis.
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Project description:Conjugation is the process by which plasmids, including those that carry antibiotic-resistance genes, are mobilized from one bacterium (the donor) to another (the recipient). The conjugation efficiency of IncF-like plasmids relies on the formation of mating-pair stabilization via intimate interactions between outer membrane proteins on the donor (a plasmid-encoded TraN isoform) and recipient bacteria. Conjugation of the R100-1 plasmid into Escherichia coli and Klebsiella pneumoniae (KP) recipients relies on pairing between the plasmid-encoded TraNα in the donor and OmpW in the recipient. Here, the crystal structure of K. pneumoniae OmpW (OmpWKP) is reported at 3.2 Å resolution. OmpWKP forms an eight-stranded β-barrel flanked by extracellular loops. The structures of E. coli OmpW (OmpWEC) and OmpWKP show high conservation despite sequence variability in the extracellular loops.
| S-EPMC10833342 | biostudies-literature
Project description:The outer membrane (OM) of Gram-negative bacteria is an evolving antibiotic barrier composed of a glycerophospholipid (GP) inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The two-component regulatory system CrrAB has only recently been reported to confer high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Mutations in crrB have been shown to lead to the modification of the lipid A moiety of LPS through CrrAB activation. However, functions of CrrAB activation in the regulation of other lipids are unclear. Work here demonstrates that CrrAB activation not only stimulates LPS modification but also regulates synthesis of acyl-glycerophosphoglycerols (acyl-PGs), a lipid species with undefined functions and biosynthesis. Among all possible modulators of acyl-PG identified from proteomic data, we found expression of lipid A palmitoyltransferase (PagP) was significantly upregulated in the crrB mutant. Furthermore, comparative lipidomics showed that most of the increasing acyl-PG activated by CrrAB was decreased after pagP knockout with CRISPR-Cas9. These results suggest that PagP also transfers a palmitate chain from GPs to PGs, generating acyl-PGs. Further investigation revealed that PagP mainly regulates the GP contents within the OM, leading to an increased ratio of acyl-PG to PG species and improving OM hydrophobicity, which may contribute to resistance against certain cationic antimicrobial peptides resistance upon LPS modification. Taken together, this work suggests that CrrAB regulates the palmitoylation of PGs and lipid A within the OM through upregulated PagP, which functions together to form an outer membrane barrier critical for bacterial survival.
| S-EPMC9403492 | biostudies-literature
Project description:Leptospires belong to a genus of parasitic bacterial spirochetes that have adapted to a broad range of mammalian hosts. Mechanisms of leptospiral molecular evolution were explored by sequence analysis of four genes shared by 38 strains belonging to the core group of pathogenic Leptospira species: L. interrogans, L. kirschneri, L. noguchii, L. borgpetersenii, L. santarosai, and L. weilii. The 16S rRNA and lipL32 genes were highly conserved, and the lipL41 and ompL1 genes were significantly more variable. Synonymous substitutions are distributed throughout the ompL1 gene, whereas nonsynonymous substitutions are clustered in four variable regions encoding surface loops. While phylogenetic trees for the 16S, lipL32, and lipL41 genes were relatively stable, 8 of 38 (20%) ompL1 sequences had mosaic compositions consistent with horizontal transfer of DNA between related bacterial species. A novel Bayesian multiple change point model was used to identify the most likely sites of recombination and to determine the phylogenetic relatedness of the segments of the mosaic ompL1 genes. Segments of the mosaic ompL1 genes encoding two of the surface-exposed loops were likely acquired by horizontal transfer from a peregrine allele of unknown ancestry. Identification of the most likely sites of recombination with the Bayesian multiple change point model, an approach which has not previously been applied to prokaryotic gene sequence analysis, serves as a model for future studies of recombination in molecular evolution of genes.
| S-EPMC387810 | biostudies-literature
Project description:In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.
| S-EPMC7017139 | biostudies-literature