Reductions in bacterial viability stimulate the production of Extra-intestinal Pathogenic Escherichia coli (ExPEC) cytoplasm-carrying Extracellular Vesicles (EVs).

Extra-intestinal Pathogenic Escherichia coli (ExPEC) is defined as an extra-intestinal foodborne pathogen, and several dominant sequence types (STs) ExPEC isolates are highly virulent, with zoonotic potential. Bacteria extracellular vesicles (EVs) carry specific subsets of molecular cargo, which affect various biological processes in bacteria and host. The mechanisms of EVs formation in ExPEC remains to be elucidated. Here, the purified EVs of ExPEC strains of different STs were isolated with ultracentrifugation processes. A comparative analysis of the strain proteomes showed that cytoplasmic proteins accounted for a relatively high proportion of the proteins among ExPEC EVs. The proportion of cytoplasm-carrying vesicles in ExPEC EVs was calculated with a simple green fluorescent protein (GFP) expression method. The RecA/LexA-dependent SOS response is a critical mediator of generation of cytoplasm-carrying EVs. The SOS response activates the expression of prophage-associated endolysins, Epel1, Epel2.1, and Epel2.2, which triggered cell lysis, increasing the production of ExPEC cytoplasm-carrying EVs. The repressor LexA controlled directly the expression of these endolysins by binding to the SOS boxes in the endolysin promoter regions. Reducing bacterial viability stimulated the production of ExPEC EVs, especially cytoplasm-carrying EVs. The imbalance in cell division caused by exposure to H2O2, the deletion of ftsK genes, or t6A synthesis defects activated the RecA/LexA-dependent SOS response, inducing the expression of endolysins, and thus increasing the proportion of cytoplasm-carrying EVs in the total ExPEC EVs. Antibiotics, which decreased bacterial viability, also increase the production of ExPEC cytoplasm-carrying EVs through the SOS response. Changes in the proportion of cytoplasm-carrying EVs affected the total DNA content of ExPEC EVs. When macrophages are exposed to a higher proportion of cytoplasm-carrying vesicles, ExPEC EVs were more cytotoxic to macrophages, accompanied with more-severe mitochondrial disruption and a higher level of induced intrinsic apoptosis. In summary, we offered comprehensive insight into the proteome analysis of ExPEC EVs. This study demonstrated the novel formation mechanisms of E. coli cytoplasm-carrying EVs.

Extracellular vesicles produced by avian pathogenic Escherichia coli (APEC) activate macrophage proinflammatory response and neutrophil extracellular trap (NET) formation through TLR4 signaling.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) is the major pathogen causing important avian diseases in poultry. As an important subtype of extraintestinal pathogenic E. coli, APEC has zoonotic potential and is considered a foodborne pathogen. APEC extracellular vesicles (EVs) may play vital roles in the interaction of the pathogen with its host cells. However, the precise roles played by APEC EVs are still not completely clear, especially in immune cells. RESULTS: In this study, we investigated the relationships between APEC EVs and immune cells. The production and characteristics of the EVs of APEC isolate CT265 were identified. Toll like receptor 4 (TLR4) triggered the cellular immune responses when it interacted with APEC EVs. APEC EVs induced a significant release of proinflammatory cytokines in THP-1 macrophages. APEC EVs induced the macrophage inflammatory response via the TLR4/MYD88/NF-κB signaling pathway, which participated in the activation of the APEC-EV-induced NLRP3 inflammasome. However, the loss of lipopolysaccharide (LPS) from APEC EVs reduced the activation of the NLRP3 inflammasome mediated by TLR4/MYD88/NF-κB signaling. Because APEC EVs activated the macrophage inflammatory response and cytokines release, we speculated that the interaction between APEC EVs and macrophages activated and promoted neutrophil migration during APEC extraintestinal infection. This study is the first to report that APEC EVs induce the formation of neutrophil extracellular traps (NETs) and chicken heterophil extracellular traps. Treatment with APEC EVs induced SAPK/JNK activation in neutrophils. The inhibition of TLR4 signaling suppressed APEC-EV-induced NET formation. However, although APEC EVs activated the immune response of macrophages and initiated NET formation, they also damaged macrophages, causing their apoptosis. The loss of LPS from APEC EVs did not prevent this process. CONCLUSION: APEC-derived EVs induced inflammatory responses in macrophages and NETs in neutrophils, and that TLR4 was involved in the APEC-EV-activated inflammatory response. These findings provided a basis for the further study of APEC pathogenesis.

ProQ binding to small RNA RyfA promotes virulence and biofilm formation in avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is a notable subpathotype of the nonhuman extraintestinal pathogenic E. coli (ExPEC). Recognized as an extraintestinal foodborne pathogen, the zoonotic potential of APEC/ExPEC allows for cross-host transmission via APEC-contaminated poultry meat and eggs. ProQ, an RNA binding protein, is evolutionarily conserved in E. coli. However, its regulatory roles in the biofilm formation and virulence of APEC/ExPEC have not been explored. In this study, proQ deletion in the APEC strain FY26 significantly compromised its biofilm-forming ability. Furthermore, animal tests and cellular infection experiments showed that ProQ depletion significantly attenuated APEC virulence, thereby diminishing its capacity for bloodstream infection and effective adherence to and persistence within host cells. Transcriptome analysis revealed a decrease in the transcription level of the small RNA (sRNA) RyfA in the mutant FY26ΔproQ, suggesting a direct interaction between the sRNA RyfA and ProQ. This interaction might indicate that sRNA RyfA is a novel ProQ-associated sRNA. Moreover, the direct binding of ProQ to the sRNA RyfA was crucial for APEC biofilm formation, pathogenicity, adhesion, and intracellular survival. In conclusion, our findings provide detailed insight into the interaction between ProQ and sRNA RyfA and deepen our understanding of the regulatory elements that dictate APEC virulence and biofilm development. Such insights are instrumental in developing strategies to counteract APEC colonization within hosts and impede APEC biofilm establishment on food surfaces.

Dissemination of virulence and resistance genes among Klebsiella pneumoniae via outer membrane vesicle: An important plasmid transfer mechanism to promote the emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae.

Klebsiella pneumoniae is well-known opportunistic enterobacteria involved in complex clinical infections in humans and animals. The domestic animals might be a source of the multidrug-resistant virulent K. pneumoniae to humans. K. pneumoniae infections in domestic animals are considered as an emergent global concern. The horizontal gene transfer plays essential roles in bacterial genome evolution by spread of virulence and resistance determinants. However, the virulence genes can be transferred horizontally via K. pneumoniae-derived outer membrane vesicles (OMVs) remains to be unreported. In this study, we performed complete genome sequencing of two K. pneumoniae HvK2115 and CRK3022 with hypervirulent or carbapenem-resistant traits. OMVs from K. pneumoniae HvK2115 and CRK3022 were purified and observed. The carriage of virulence or resistance genes in K. pneumoniae OMVs was identified. The influence of OMVs on the horizontal transfer of virulence-related or drug-resistant plasmids among K. pneumoniae strains was evaluated thoroughly. The plasmid transfer to recipient bacteria through OMVs was identified by polymerase chain reaction, pulsed field gel electrophoresis and Southern blot. This study revealed that OMVs could mediate the intraspecific and interspecific horizontal transfer of the virulence plasmid phvK2115. OMVs could simultaneously transfer two resistance plasmids into K. pneumoniae and Escherichia coli recipient strains. OMVs-mediated horizontal transfer of virulence plasmid phvK2115 could significantly enhance the pathogenicity of human carbapenem-resistant K. pneumoniae CRK3022. The CRK3022 acquired the virulence plasmid phvK2115 could become a CR-hvKp strain. It was critically important that OMVs-mediated horizontal transfer of phvK2115 lead to the coexistence of virulence and carbapenem-resistance genes in K. pneumoniae, resulting in the emerging of carbapenem-resistant hypervirulent K. pneumoniae.

Complete genomic analysis of ST117 lineage extraintestinal pathogenic Escherichia coli (ExPEC) to reveal multiple genetic determinants to drive its global transmission: ST117 E. coli as an emerging multidrug-resistant foodborne ExPEC with zoonotic potential.

Avian pathogenic Escherichia coli (APEC) is recognized as a primary source of foodborne extraintestinal pathogenic E. coli (ExPEC), which poses a significant risk of extraintestinal infections in humans. The potential of human infection with ST117 lineage APEC/ExPEC from poultry is particularly concerning. However, relatively few whole-genome studies have focused on ST117 as an emerging ExPEC lineage. In this study, the complete genomes of 11 avian ST117 isolates and the draft genomes of 20 ST117 isolates in China were sequenced to reveal the genomic islands and large plasmid composition of ST117 APEC. With reference to the extensive E. coli genomes available in public databases, large-scale comprehensive genomic analysis of the ST117 lineage APEC/ExPEC was performed to reveal the features of the ST117 pan-genome and population. The high variability of the accessory genome emphasized the diversity and dynamic traits of the ST117 pan-genome. ST117 isolates recovered from different hosts and geographic sources were randomly located on a phylogeny tree, suggesting that ST117 E. coli lacked host specificity. A time-scaled phylogeny tree showed that ST117 was a recent E. coli lineage with a relatively short evolutionary period. Further characterization of a wide diversity of ExPEC-related virulence genes, pathogenicity islands (PAIs), and resistance genes of the ST117 pan-genome provided insights into the virulence and resistance of ST117 APEC/ExPEC. The results suggested zoonotic potential of ST117 APEC/ExPEC between birds and humans. Moreover, genomic analysis showed that a pool of diverse plasmids drove the virulence and multidrug resistance of ST117 APEC/ExPEC. Several types of large plasmids were scattered across the ST117 isolates, but there was no strong plasmid-clade adaptation. Combined with the pan-genome analysis, a double polymerase chain reaction (PCR) method was designed for rapid and cost-effective detection of ST117 isolates from various avian and human APEC/ExPEC isolates. Overall, this study addressed a gap in current knowledge about the ST117 APEC/ExPEC genome, with significant implications to understand the success and spread of ST117 APEC/ExPEC.

Genomics Analysis to Identify Multiple Genetic Determinants That Drive the Global Transmission of the Pandemic ST95 Lineage of Extraintestinal Pathogenic Escherichia coli (ExPEC).

Extraintestinal pathogenic Escherichia coli (ExPEC) is a pathogen that causes host extraintestinal diseases. The ST95 E. coli lineage is one of the dominant ExPEC lineages in humans and poultry. In this study, we took advantage of extensive E. coli genomes available through public open-access databases to construct a detailed understanding of the phylogeny and evolution of ST95. We used a high variability of accessory genomes to highlight the diversity and dynamic traits of ST95. Isolates from diverse hosts and geographic sources were randomly located on the phylogenetic tree, which suggested that there is no host specificity for ST95. The time-scaled phylogeny showed that ST95 is an ancient and long-lasting lineage. The virulence genes, resistance genes, and pathogenicity islands (PAIs) were characterized in ST95 pan-genomes to provide novel insights into the pathogenicity and multidrug resistance (MDR) genotypes. We found that a pool of large plasmids drives virulence and MDR. Based on the unique genes in the ST95 pan-genome, we designed a novel multiplex PCR reaction to rapidly detect ST95. Overall, our study addressed a gap in the current understanding of ST95 ExPEC genomes, with significant implications for recognizing the success and spread of ST95.