T3SS Genes Research Articles

The type VI secretion system as a potential predictor of subsequent bloodstream infection of carbapenem-resistant Klebsiella pneumoniae strains on intestinal colonization.

The type VI secretion system (T6SS) has been recognized as a novel virulence factor in Klebsiella pneumoniae. This study investigated the occurrence of T6SS genes in carbapenem-resistant Klebsiella pneumoniae (CRKP) strains during intestinal colonization and evaluated their effect on the development of bloodstream infections. The study encompassed 2,385 patients admitted to the intensive care unit (ICU) and subjected to routine screening for intestinal colonization with CRKP. PFGE was employed on CRKP strains isolated from both the patients' intestine and blood cultures, confirming their genetic similarity. PCR was employed to detect the presence of carbapenemase genes, T6SS genes, and virulence genes. Quantitative real-time PCR was conducted to assess the expression levels of the core genes associated with the T6SS. The correlation between T6SS expression and sBSI was further investigated. Approximately 10% (238/2385) of ICU patients tested positive for CRKP colonization. Among patients who tested positive, 10.1% (24/238) developed CRKP-sBSI. Patients carrying T6SS-positive CRKP isolates were more commonly linked to a history of invasive procedures, antibiotic use, and immunosuppression (P < 0.05), and were strongly associated with 28-day mortality (P < 0.001). It indicated that T6SS-positive CRKP strains exhibited a higher prevalence of virulence genes, such as rmpA and iucA, compared to T6SS-negative ones (P < 0.001). Compared to the strains isolated from simple colonization group, there was a significant increase in the mRNA expression of both hcp and vgrG genes (P < 0.05) of strains from the sBSI group, suggesting the key genes of the T6SS may play a significant role in the occurrence and progression of sBSI caused by CRKP. The presence of the T6SS in a CRKP strain from intestinal colonization can serve as a promising predictive marker for sBSI. Conducting screenings for CRKP in patients' intestinal flora and monitoring T6SS carriage can improve the prevention and management of CRKP bloodstream infections.

P-1846. Differences in Type 6 Secretion System Composition among Serogroups of Neonatal Meningitis Escherichia coli

Abstract Background Neonatal meningitis is a public health challenge for pediatricians. Neonatal meningitis Escherichia coli (NMEC) is the most common Gram-negative bacterial cause of sepsis and meningitis in newborns in the United States. NMEC strains can replicate in macrophages, survive in the bloodstream and traverse the blood-brain barrier, resulting in meningitis. Most NMEC cases are associated with serogroups O18, O83, O7, O12, O1, and O45 (Logue et al., 2012). Two clusters of the Type 6 Secretion System (T6SS-I and T6SS-II) were identified in an NMEC O18:K1 strain, and studies have demonstrated that T6SS plays an essential role in the pathogenesis of several bacterial species. However, there is still a lack of knowledge regarding the function of some T6SS genes. Here, we investigated the role of T6SS in different NMEC serogroups to identify specific genes that may be used as a potential target for an efficacious prevention strategy. Characteristics of NMEC strainsFigure 1.Characteristics of NMEC strains used in this study. A) Percentage of serogroups; B) Prevalence of T6SS among the NMEC isolates. Methods A total of 120 NMEC genomes were used in this study (n=78 from the Logue Lab collection; n=42 publicly databases). The T6SS clusters were identified through BLAST analysis using NMEC RS218 and O18 as reference genomes. To establish the presence of a specific cluster, a query cover and percentage identity threshold of 60% and 70% were set. The sequence comparisons were performed using the Artemis Comparison Tool (ACT). Results The serogroup O18 was the most abundant (23%; n=28), followed by O1 (22%; n=26), and O83 (12%; n=14). Analysis examined the prevalence of both T6SS clusters in the NMEC genomes and identified that they were present in 69% (n=83) of the genomes, while 12% (n=14) have only one of the clusters, and 19% (n=23) have no clusters. The presence of both T6SS clusters was observed in the serogroups O18, O14, and O2/O50. In contrast, no gene of any cluster was identified in serogroup O7. To understand the role of variation among the serogroups, in vitro analyses were performed. The results indicate differences in survival within THP-1 macrophages-like among distinct serogroups. Conclusion Our results support the hypothesis that T6SS components differ among the NMEC serogroups, potentially impacting their pathogenicity and ability to cause meningitis. These findings have significant implications for development of a prevention strategy for neonatal meningitis. Disclosures All Authors: No reported disclosures

Effector genes of type III secretion system and biofilm formation in virulent Pseudomonas aeruginosa isolates carrying bla KPC-2 and bla PDC-5 genes in hospital environment.

Introduction. In critically ill patients, the occurrence of multidrug-resistant Pseudomonas aeruginosa infection is a significant concern, given its ability to acquire multidrug-resistant, form biofilms and secrete toxic effectors.Hypothesis or Gap Statement. In Brazil, limited data are available regarding the prevalence of dissemination, and the impact of the type III secretion system (T3SS) on toxin production and biofilm formation in clinical isolates of P. aeruginosa.Aim. This study investigates the dissemination of virulent P. aeruginosa harbouring the bla KPC-2 and bla PDC-5 genes, the presence of T3SS genes and their biofilm-forming capability.Methodology. A total of 128 non-duplicate clinical isolates of carbapenem-resistant P. aeruginosa (CRPA) from different sources collected from eight hospitals were examined. Detection was performed by PCR of the T3SS genes (exoU, exoT, exoS and exoY), carbapenemases (bla KPC, bla GIM and bla NDM) and beta-lactamase gene (bla PDC). PFGE and phenotypic biofilm production (initial adhesion assay and biofilm cell concentration) were performed.Results. We found exoT+ (86%) to be the most frequent genotypic variant, followed by exoY+ (61%). Notably, a substantial proportion of isolates exhibited the simultaneous presence of exoU+ and exoS+ genes, along with a high prevalence of bla KPC-2 + (64%) and bla PDC-5 + (64%) among the disseminated clones in the evaluated region. Additionally, 78% of the isolates demonstrated biofilm-forming capability, and two distinct clonal profiles were identified and disseminated both intra- and inter-hospital. Also, it was revealed that the exoU genotype was significantly more frequent among multidrug-resistant strains.Conclusion. These findings underscore the ability of multiple virulent and biofilm-producing clones of CRPA to propagate effectively.

Klebsiella pneumoniae T6SS impact in IBDs

Abstract Background and aims Our hypothesis is that Klebsiella pneumoniae (Kp) Type VI Secretion System (T6SS), normally used for bacteria competition during host colonization have a collateral impact on the host inflammatory pathway (Fig1). Our specific aims are to: (i) Characterize the impact of bacterial type VI Secretion System (T6SS) on host gut inflammation (ii) Develop a screen to identify T6SS inhibitors. Methods In vivo experiments involve infecting mice with Kp wild type (WT) and T6SS mutants to evaluate gut inflammation and immune response. Metagenomic analysis of IBD and CRC patients' microbiota will be conducted to assess T6SS gene enrichment. The molecular mechanism of T6SS activity and LPS release will be explored using various imaging and biochemical techniques. High-throughput screening of small molecule inhibitors targeting T6SS assembly will be performed, followed by validation in cellular models. The role of bacteria in tumorigenesis is increasingly recognized, yet our understanding of the underlying mechanisms remains incomplete, hindering the identification of new therapeutic strategies. It is therefore critical to decipher the bacterial mechanisms and their impact on the host to identify pathways that can be targeted from both sides. Anticipated impact Our preliminary data have uncovered a novel role of Type VI Secretion System (T6SS) role in gut inflammation. Building on our extensive expertise of this specific system, we will employ a multidisciplinary approach to inhibit T6SS. Inhibiting the T6SS could prevent both gut inflammation and Enterobacteria competition advantage in the gut, therefore reducing dysbiosis. Moreover, inhibition of bacterial virulence factors is an attractive strategy to complement antibiotic and anti-inflammatory treatments already in use.

Klebsiella pneumoniae employs a type VI secretion system to overcome microbiota-mediated colonization resistance

Microbial species must compete for space and nutrients to persist in the gastrointestinal (GI) tract, and our understanding of the complex pathobiont-microbiota interactions is far from complete. Klebsiella pneumoniae, a problematic, often drug-resistant nosocomial pathogen, can colonize the GI tract asymptomatically, serving as an infection reservoir. To provide insight on how K. pneumoniae interacts with the resident gut microbiome, we conduct a transposon mutagenesis screen using a murine model of GI colonization with an intact microbiota. Among the genes identified were those encoding a type VI secretion system (T6SS), which mediates contact-dependent killing of gram-negative bacteria. From several approaches, we demonstrate that the T6SS is critical for K. pneumoniae gut colonization. Metagenomics and in vitro killing assays reveal that K. pneumoniae reduces Betaproteobacteria species in a T6SS-dependent manner, thus identifying specific species targeted by K. pneumoniae. We further show that T6SS gene expression is controlled by several transcriptional regulators and that expression only occurs in vitro under conditions that mimic the gut environment. By enabling K. pneumoniae to thrive in the gut, the T6SS indirectly contributes to the pathogenic potential of this organism. These observations advance our molecular understanding of how K. pneumoniae successfully colonizes the GI tract.

Genomic analysis of Salmonella isolated from surface water and animal sources in Chile reveals new T6SS effector protein candidates.

Type VI Secretion Systems (T6SS), widely distributed in Gram-negative bacteria, contribute to interbacterial competition and pathogenesis through the translocation of effector proteins to target cells. Salmonella harbor 5 pathogenicity islands encoding T6SS (SPI-6, SPI-19, SPI-20, SPI-21 and SPI-22), in which a limited number of effector proteins have been identified. Previous analyses by our group focused on the identification of candidate T6SS effectors and cognate immunity proteins in Salmonella genomes deposited in public databases. In this study, the analysis was centered on Salmonella isolates obtained from environmental sources in Chile. To this end, bioinformatics and comparative genomics analyses were performed using 695 genomes of Salmonella isolates representing 44 serotypes obtained from surface water and animal sources in Chile to identify new T6SS effector proteins. First, T6SS gene clusters were identified using the SecreT6 server. This analysis revealed that most isolates carry the SPI-6 T6SS gene cluster, whereas the SPI-19 and SPI-21 T6SS gene clusters were detected in isolates from a limited number of serotypes. In contrast, the SPI-20 and SPI-22 T6SS gene clusters were not detected. Subsequently, each ORF in the T6SS gene clusters identified was analyzed using bioinformatics tools for effector prediction, identification of immunity proteins and functional biochemical prediction. This analysis detected 20 of the 37 T6SS effector proteins previously reported in Salmonella. In addition, 4 new effector proteins with potential antibacterial activity were identified in SPI-6: 2 Rhs effectors with potential DNase activity (PAAR-RhsA-NucA_B and PAAR-RhsA-GH-E) and 2 effectors with potential RNase activity (PAAR-RhsA-CdiA and RhsA-CdiA). Interestingly, the repertoire of SPI-6 T6SS effectors varies among isolates of the same serotype. In SPI-19, no new effector protein was detected. Of note, some Rhs effectors of SPI-19 and SPI-6 present C-terminal ends with unknown function. The presence of cognate immunity proteins carrying domains present in bona fide immunity proteins suggests that these effectors have antibacterial activity. Finally, two new effectors were identified in SPI-21: one with potential peptidoglycan hydrolase activity and another with potential membrane pore-forming activity. Altogether, our work broadens the repertoire of Salmonella T6SS effector proteins and provides evidence that SPI-6, SPI-19 and SPI-21 T6SS gene clusters harbor a vast array of antibacterial effectors.

Type I-E CRISPR-Cas system regulates fimZY and T3SS1 genes expression in Salmonella enterica serovar Pullorum

Clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) provide prokaryotes with adaptive immunity against invasion by plasmids or phages. In Salmonella, the type I-E CRISPR-Cas system is typically considered silent in immunity against foreign genetic elements. To elucidate the role of the CRISPR-Cas system, we chose Salmonella enterica serovar Pullorum S06004 as a model organism due to its four spacers and well-defined biological characteristics observed in previous studies. Western blot analysis revealed expression of Cas3 in S06004 cultured in vitro, but plasmid transformation assays demonstrated that both wild-type (WT) and S06004 strains overexpressing LeuO (a positive regulator of CRISPR-Cas) showed no immunity against the target plasmid. RNA-Seq analysis detected significant downregulation of the fim cluster, encoding type I fimbriae, and T3SS1-related genes in the cas cluster mutant compared to the WT. This downregulation was further confirmed in mutants of CR1 and individual cas genes by qRT-PCR. Consequently, mutants of CR1 and cas clusters exhibited decreased invasion of chicken hepatocellular carcinoma cells. The consistent regulation of T3SS1 genes by the CRISPR-Cas system in S. Pullorum, S. Enteritidis, and S. Typhimurium indicates a common role for the type I-E CRISPR-Cas system in promoting bacterial virulence. However, the specific molecular mechanisms underlying this regulation require further investigation.

Archaeal type six secretion system mediates contact-dependent antagonism.

Microbial communities are shaped by cell-cell interactions. Although archaea are often found in associations with other microorganisms, the mechanisms structuring these communities are poorly understood. Here, we report on the structure and function of haloarchaeal contractile injection systems (CISs). Using a combination of functional assays and time-lapse imaging, we show that Halogeometricum borinquense exhibits antagonism toward Haloferax volcanii by inducing cell lysis and inhibiting proliferation. This antagonism is contact-dependent and requires a functional CIS, which is encoded by a gene cluster that is associated with toxin-immunity pairs. Cryo-focused ion beam milling and imaging by cryo-electron tomography revealed that these CISs are bound to the cytoplasmic membrane, resembling the bacterial type six secretion systems (T6SSs). We show that related T6SS gene clusters are conserved and expressed in other haloarchaeal strains, which exhibit antagonistic behavior. Our data provide a mechanistic framework for understanding how archaea may shape microbial communities and affect the food webs they inhabit.

Vibrio alginolyticus Reprograms CIK Cell Metabolism via T3SS Effector VopS to Promote Host Cell Ferroptosis.

Vibrio alginolyticus is a Gram-negative pathogen of both marine animals and humans, resulting in significant losses for the aquaculture industry. Emerging evidence indicates that V. alginolyticus manipulates cell death for its pathogenicity, but the underlying molecular mechanisms remain unclear. Here, a gene designated vopS in V. alginolyticus HY9901 was identified, which was predicted to encode the T3SS effector protein. To determine whether VopS contributes to the pathogenesis of V. alginolyticus, the ΔvopS mutant strain was constructed and phenotypically characterized. The deletion of VopS not only reduced the ability to secrete extracellular proteases and virulence but also affected the expression of the T3SS genes. Furthermore, VopS was cytotoxic and induced apoptosis, as confirmed by elevated LDH and the activation of caspase-3. Metabolomic analysis revealed considerable metabolomic disruptions upon V. alginolyticus infection. The VopS effector induced host cell ferroptosis by promoting the synthesis of adrenic acid, depleting cellular glutathione, and subsequently increasing the accumulation of ferrous (Fe2+). Taken together, our findings provide that the VopS effector is an essential virulence factor of V. alginolyticus, which can lead to ferroptosis.

Quorum sensing orchestrates parallel cell death pathways in Vibrio cholerae via Type 6 secretion-dependent and -independent mechanisms

Quorum sensing (QS) is a cell-to-cell communication process that enables bacteria to coordinate group behaviors. In Vibrio cholerae colonies, a program of spatial-temporal cell death is among the QS-controlled traits. Cell death occurs in two phases, first along the colony rim, and subsequently, at the colony center. Both cell death phases are driven by the type 6 secretion system (T6SS). Here, we show that HapR, the master QS regulator, does not control t6ss gene expression nor T6SS-mediated killing activity. Nonetheless, a ΔhapR strain displays no cell death at the colony rim. RNA-Sequencing (RNA-Seq) analyses reveal that HapR activates expression of an operon containing four genes of unknown function, vca0646-0649. Epistasis and overexpression studies show that two of the genes, vca0646 and vca0647, are required to drive cell death in both a ΔhapR and a ΔhapR Δt6ss strain. Thus, vca0646-0649 are regulated by HapR but act independently of the T6SS machinery to cause cell death, suggesting that a second, parallel pathway to cell death exists in V. cholerae.

Phenotypic diversity of type III secretion system activity in enteropathogenic Escherichia coli clinical isolates.

Introduction. Enteropathogenic Escherichia coli (EPEC) strains pose a significant threat as a leading cause of severe childhood diarrhoea in developing nations. EPEC pathogenicity relies on the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE), facilitating the secretion and translocation of bacterial effector proteins.Gap Statement. While the regulatory roles of PerC (plasmid-encoded regulator) and GrlA (global regulator of LEE-activator) in ler expression and LEE gene activation are well-documented in the EPEC prototype strain E2348/69, understanding the variability in LEE gene expression control mechanisms among clinical EPEC isolates remains an area requiring further investigation.Aim. This study aims to explore the diversity in LEE gene expression control mechanisms among clinical EPEC isolates through a comparative analysis of secretion profiles under defined growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression.Methodology. We compared T3SS-dependent secretion patterns and promoter expression in both typical EPEC (tEPEC) and atypical EPEC (aEPEC) clinical isolates under growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Additionally, we conducted promoter reporter activity assays, quantitative real-time PCR and Western blot experiments to assess gene expression activity.Results. Significant differences in T3SS-dependent secretion were observed among tEPEC and aEPEC strains, independent of LEE sequence variations or T3SS gene functionality. Notably, a clinical tEPEC isolate exhibited increased secretion levels under repressive growth conditions and in the absence of both PerC and GrlA, implicating an alternative mechanism in the activation of Ler (LEE-encoded regulator) expression.Conclusion. Our findings indicate that uncharacterized LEE regulatory mechanisms contribute to phenotypic diversity among clinical EPEC isolates, though their impact on clinical outcomes remains unknown. This challenges the conventional understanding based on reference strains and highlights the need to investigate beyond established models to comprehensively elucidate EPEC pathogenesis.

T6SS in plant pathogens: unique mechanisms in complex hosts.

Type VI secretion systems (T6SSs) are complex molecular machines that allow bacteria to deliver toxic effector proteins to neighboring bacterial and eukaryotic cells. Although initial work focused on the T6SS as a virulence mechanism of human pathogens, the field shifted to examine the use of T6SSs for interbacterial competition in various environments, including in the plant rhizosphere. Genes encoding the T6SS are estimated to be found in a quarter of all Gram-negative bacteria and are especially highly represented in Proteobacteria, a group which includes the most important bacterial phytopathogens. Many of these pathogens encode multiple distinct T6SS gene clusters which can include the core components of the apparatus as well as effector proteins. The T6SS is deployed by pathogens at multiple points as they colonize their hosts and establish an infection. In this review, we describe what is known about the use of T6SS by phytopathogens against plant hosts and non-plant organisms, keeping in mind that the structure of plants requires unique mechanisms of attack that are distinct from the mechanisms used for interbacterial interactions and against animal hosts. While the interactions of specific effectors (such as phospholipases, endonucleases, peptidases, and amidases) with targets have been well described in the context of interbacterial competition and in some eukaryotic interactions, this review highlights the need for future studies to assess the activity of phytobacterial T6SS effectors against plant cells.

A plasmid-mediated type III secretion system associated with invasiveness and diarrheagenicity of Providencia rustigianii.

We have recently described a clinical isolate of Providencia rustigianii strain JH-1 carrying the genes for cytolethal distending toxin (CDT) in a conjugative plasmid. A cdtB mutant of strain JH-1, which lost CDT activity, was still found to retain invasiveness and diarrheagenicity. The strain was subjected to phenotypic and genetic analyses including whole genome sequencing (WGS) to explore the genetic determinants of the observed invasiveness and diarrheagenic properties. Analysis and annotation of WGS data revealed the presence of two distinct type III secretion systems (T3SS) in strain JH-1, one of which was located on the chromosome designated as cT3SS (3,992,833 bp) and the other on a mega-plasmid designated as pT3SS (168,819 bp). Comparative genomic analysis revealed that cT3SS is generally conserved in Providencia spp. but pT3SS was limited to a subset of Providencia spp., carrying cdt genes. Strain JH-1 was found to invade HeLa cells and induce fluid accumulation with characteristic pathological lesions in rabbit ileal loops. Remarkably, these phenomena were associated with the pT3SS but not cT3SS. The plasmid could be transferred by conjugation from strain JH-1 to other strains of P. rustigianii, Providencia rettgeri, and Escherichia coli with concomitant transfer of these virulence properties. This is the first report of a functional and mobile T3SS in P. rustigianii and its association with invasiveness and diarrheagenicity of this bacterium. These data suggest that P. rustigianii and other CDT-producing Providencia strains might carry T3SS and exert their diarrheagenic effect by exploiting the T3SS nano-machinery.IMPORTANCEThe precise mechanism of virulence of Providencia rustigianii is unclear, although some strains produce cytolethal distending toxin as a putative virulence factor. We have detected the presence of a type III secretion system (T3SS) for the first time on a plasmid in a P. rustigianii strain. Plasmid-mediated T3SS seems to be directly involved in virulence of P. rustigianii and may serve as a means of horizontal transfer of T3SS genes. Our results may have implication in understanding the mechanism of emergence of new pathogenic strains of P. rustigianii.

Gut bacterial type III secretion systems aggravate colitis in mice and serve as biomarkers of Crohn’s disease

Mesenteric adipose tissue (mAT) hyperplasia, known as creeping fat, is a pathologic characteristic of Crohn's disease (CD). In our previously reported cohort, we observed that Achromobacter pulmonis was the most abundant and prevalent bacteria cultivated from creeping fat. A whole genomic sequencing and identification of T3SS orthologs of mAT-derived A. pulmonis were used. A functional type III secretion system (T3SS) mediated the pathogenic potential of A. pulmonis invitro and in mouse colitis model. Furthermore, a T3SS Finder pipeline was introduced to evaluate gut bacterial T3SS orthologs in the feces of CD patients, ulcerative colitis and colorectal cancer patients. Here, we reveal that mAT-derived A. pulmonis possesses a functional T3SS, aggravates colitis in mice via T3SS, and exhibits T3SS-dependent cytotoxicity via a caspase-independent mechanism in macrophages and epithelial cells, which demonstrated the pathogenic potential of the T3SS-harboring A. pulmonis. Metagenomic analyses demonstrate an increased abundance of Achromobacter in the fecal of Crohn's disease patients compared to healthy controls. A comprehensive comparison of total microbial vT3SS abundance in various intestine diseases demonstrated that the specific enrichment of vT3SS genes was shown in fecal samples of CD, neither ulcerative colitis nor colorectal cancer patients, and ten T3SS gene-based biomarkers for CD were discovered and validated in a newly recruited CD cohort. Furthermore, treatment with exclusive enteral nutrition (EEN), an intervention that improves CD patient symptomatology, was found associated with a significant reduction in the prevalence of T3SS genes in fecal samples. These findings highlight the pathogenic significance of T3SSs in the context of CD and identify specific T3SS genes that could potentially function as biomarkers for diagnosing and monitoring the clinical status of CD patients. This work is supported by the National Key Research and Development Program of China (2020YFA0907800), the China Postdoctoral Science Foundation (2023M744089), the National Natural Science Foundation of China (32000096), the Shenzhen Science and Technology Programs (KQTD20200820145822023, RCIC20231211085944057, and ZDSYS20220606100803007), National Key Clinical Discipline, Guangdong Provincial Clinical Research Center for Digestive Diseases (2020B1111170004), Qingfeng Scientific Research Fund of the China Crohn's & Colitis Foundation (CCCF) (CCCF-QF-2022B71-1), and the Sixth Affiliated Hospital, Sun Yat-sen University Clinical Research 1010 Program 1010CG(2023)-08. These funding provided well support for this research work, which involved data collection, analysis, interpretation, patient recruitment and so on.

A pangenomic atlas reveals eco-evolutionary dynamics that shape type VI secretion systems in plant-pathogenic Ralstonia.

Soilborne Ralstonia solanacearum species complex (RSSC) pathogens disrupt microbial communities as they invade roots and fatally wilt plants. RSSC pathogens secrete antimicrobial toxins using a type VI secretion system (T6SS). To investigate how evolution and ecology have shaped the T6SS of these bacterial pathogens, we analyzed the T6SS gene content and architecture across the RSSC and their evolutionary relatives. Our analysis reveals that two ecologically similar Burkholderiaceae taxa, xylem-pathogenic RSSC and Paracidovorax, have convergently evolved to wield large arsenals of T6SS toxins. To understand the mechanisms underlying genomic enrichment of T6SS toxins, we compiled an atlas of 1,066 auxiliary T6SS toxin clusters ("aux" clusters) across 99 high-quality RSSC genomes. We classified 25 types of aux clusters with toxins that predominantly target lipids, nucleic acids, or unknown cellular substrates. The aux clusters were located in diverse genetic neighborhoods and had complex phylogenetic distributions, suggesting frequent horizontal gene flow. Phages and other mobile genetic elements account for most of the aux cluster acquisition on the chromosome but very little on the megaplasmid. Nevertheless, RSSC genomes were more enriched in aux clusters on the megaplasmid. Although the single, ancestral T6SS was broadly conserved in the RSSC, the T6SS has been convergently lost in atypical, non-soilborne lineages. Overall, our data suggest dynamic interplay between the lifestyle of RSSC lineages and the evolution of T6SSes with robust arsenals of toxins. This pangenomic atlas poises the RSSC as an emerging, tractable model to understand the role of the T6SS in shaping pathogen populations.IMPORTANCEWe explored the eco-evolutionary dynamics that shape the inter-microbial warfare mechanisms of a globally significant plant pathogen, the Ralstonia solanacearum species complex. We discovered that most Ralstonia wilt pathogens have evolved extensive and diverse repertoires of type VI secretion system-associated antimicrobial toxins. These expansive toxin arsenals potentially enhance the ability of Ralstonia pathogens to invade plant microbiomes, enabling them to rapidly colonize and kill their host plants. We devised a classification system to categorize the Ralstonia toxins. Interestingly, many of the toxin gene clusters are encoded on mobile genetic elements, including prophages, which may be mutualistic symbionts that enhance the inter-microbial competitiveness of Ralstonia wilt pathogens. Moreover, our findings suggest that the convergent loss of this multi-gene trait contributes to genome reduction in two vector-transmitted lineages of Ralstonia pathogens. Our findings demonstrate that the interplay between microbial ecology and pathogen lifestyle shapes the evolution of a genetically complex antimicrobial weapon.

RNase-mediated reprogramming of Yersinia virulence.

RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we tested the influence of 11 potential RNases in the intestinal pathogen Yersinia pseudotuberculosis on the expression of its type III secretion system (T3SS) and associated effectors (Yops) that are encoded on the Yersinia virulence plasmid. We found that exoribonuclease PNPase and endoribonuclease RNase III inhibit T3SS and yop gene transcription by repressing the synthesis of LcrF, the master activator of Yop-T3SS. Loss of both RNases led to an increase in lcrF mRNA levels. Our work indicates that PNPase exerts its influence via YopD, which accelerates lcrF mRNA degradation. Loss of RNase III, on the other hand, results in the downregulation of the CsrB and CsrC RNAs, thereby increasing the availability of active CsrA, which has been shown previously to enhance lcrF mRNA translation and stability. This CsrA-promoted increase of lcrF mRNA translation could be supported by other factors promoting the protein translation efficiency (e.g. IF-3, RimM, RsmG) that were also found to be repressed by RNase III. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal to virulence plasmid-encoded genes. A similar reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. Overall, our work revealed a complex control system where RNases orchestrate the expression of the T3SS/Yop machinery on multiple levels to antagonize phagocytic uptake and elimination by innate immune cells.

Quorum sensing employs a dual regulatory mechanism to repress T3SS gene expression.

Vibrio campbellii utilizes the type III secretion system (T3SS) as a mechanism of pathogenesis, which is a highly studied 'injectisome' complex that delivers exotoxins into host cells during infection. The T3SS pathogenicity island in V. campbellii comprises ∼40 genes that are organized into four structural operons. In this study, we determined that quorum sensing - a method of bacterial communication - regulates T3SS genes both at the transcriptional and post-translational levels to shut down T3SS gene expression at high population densities.

Comparative transcriptome reveals importance of export apparatus subunit (ascR) in type III secretion system and its roles on biological properties, gene expression profiles, virulence and colonization of Aeromonas veronii

Aeromonas veronii, an opportunistic pathogen, is known to cause serious infections across various species. In our previous study, we discovered that A. veronii GL2 exhibited a virulence up to ten times greater than that of FO1. To ascertain the factors contributing to the disparity in virulence between the two strains, we conducted a comparative transcriptome analysis. This analysis reveals a significant upregulation (P < 0.05) of the ascR gene in GL2 compared with FO1. Additionally, six differentially expressed genes (DEGs) were identified within the “Bacterial secretion system” pathway (map03070), with ascR being an essential component of type III secretion system (T3SS). AscR, considered as SctR family export apparatus subunit within the T3SS, has ambiguous roles in the biological properties, gene expression profiles, virulence and colonization of A. veronii. Therefore, we constructed a mutant strain (ΔascR) by homologous recombination. Comparative analysis with the wide-type GL2 reveals no significant differences in terms of colony morphology, growth curve, hemolytic activity and protease activity. However, significant reductions (P < 0.01) were observed in the abilities of biofilm formation and swimming mobility. No remarkable difference was noted in the lengths of flagella. The LD50 value of ΔascR was to be 5.15 times higher than that of GL2. Interestingly, the mRNA expression of ascC, ascD, ascJ and ascI genes in the T3SS, and mshB, mshE, mshK and mshP genes in the MSHA type pili were significantly upregulated (P < 0.05) in ΔascR, potentially due to transcriptional compensation. Further analysis of enzymatic biomarkers revealed that ΔascR might not destruct the recognition of innate immune response in host remarkably, but the colonization levels of A.veronii were significantly suppressed (P < 0.01) in ΔascR group. In conclusion, the ascR gene may be a key determinant in regulating the virulence of A. veronii, and the destruction of the T3SS caused by ascR deficiency results in these notable changes.

Genomic Insights into Edwardsiella ictaluri: Molecular Epidemiology and Antimicrobial Resistance in Striped Catfish (Pangasianodon hypophthalmus) Aquaculture in Vietnam

Edwardsiella ictaluri is responsible for causing bacillary necrosis (BNP) in striped catfish (Pangasianodon hypophthalmus) in Vietnam. This study offers a comprehensive genomic characterization of E. ictaluri to enhance understanding of the molecular epidemiology, virulence, and antimicrobial resistance. E. ictaluri isolates were collected from diseased striped catfish in the Mekong Delta. The species was confirmed through PCR. Antimicrobial susceptibility testing was conducted using minimum inhibitory concentrations for commonly used antimicrobials. Thirty representative isolates were selected for whole genome sequencing to delineate their genomic profiles and phylogeny. All strains belonged to ST-26 and exhibited genetic relatedness, differing by a maximum of 90 single nucleotide polymorphisms. Most isolates carried multiple antimicrobial resistance genes, with the tet(A) gene present in 63% and floR in 77% of the genomes. The ESBL gene, blaCTX-M-15, was identified in 30% of the genomes. Three plasmid replicon types were identified: IncA, p0111, and IncQ1. The genomes clustered into two clades based on their virulence gene profile, one group with the T3SS genes and one without. The genetic similarity among Vietnamese isolates suggests that disease spread occurs within the Mekong region, underscoring the importance of source tracking, reservoir identification, and implementation of necessary biosecurity measures to mitigate spread of BNP.

CopG1, a Novel Transcriptional Regulator Affecting Symbiosis in Bradyrhizobium sp. SUTN9-2.

The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of T4SS1 were examined. We generated deleted mutants and tested them in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be recently restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of the nodulation gene and ttsI but exhibited high expression levels of the T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild-type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and the C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.