Type III Secretion Research Articles

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.

Heterogeneity and Genomic Plasticity of Acinetobacter baumannii and Acinetobacter nosocomialis Isolates Recovered from Clinical Samples in India.

Acinetobacter baumannii and Acinetobacter nosocomialis are the imperious pathogens in the intensive care units. We aimed to explore the genomic features of these pathogens to understand the factors influencing their plasticity. Using next-generation sequencing, two carbapenem-resistant A. baumannii (AbaBS-3, AbaETR-4) isolates and a pan-susceptible A. nosocomialis (AbaAS-5) isolate were characterised. All genomes exhibited 94% similarity with a degree of heterogeneity. AbaBS-3 and AbaETR-4 harboured antibiotic resistance gene (ARG) repertoire to most antibiotic classes. Carbapenem resistance was due to blaOXA-23 and blaOXA-66 besides the antibiotic efflux pumps. Diverse mobile genetic elements (MGE), insertion sequences (IS), prophages and virulence determinants with a plethora of stress response genes were identified in all three genomes. Class-1 integron in AbaETR-4, encoded genes that confer resistance to aminoglycosides, phenicol, sulfonamides and disinfectants. Substitutions in LpxACD and PmrCAB of AbaETR-4 confirmed the colistin resistance in vitro. Novel mutations in piuA, responsible for transporting cefiderocol, were found in AbaBS-3 and AbaETR-4. Plasmids carrying toxin-antitoxin systems, ARGs and ISs were present in these genomes. All three genomes harboured diverse protein secretion systems, virulence determinants related to immune evasion, adherence, biofilm formation and iron acquisition systems. AbaAS-5 exclusively harboured serine protease pkf, and CpaA substrate of type-II secretion system but lacked the acinetobactin-iron acquisition system. Our work delivers a holistic genome characterization of A. baumannii, coupled with a trailblazing attempt to study A. nosocomialis from India. The presence of ARGs and potential virulence factors interspersed with MGE is a cause for concern, depicting the dynamic adaptability mediated by genetic recombination.

Distinct mechanisms of type 3 secretion system recognition control LTB4 synthesis in neutrophils and macrophages.

Leukotriene B4 (LTB4) is an inflammatory lipid produced in response to pathogens that is critical for initiating the inflammatory cascade needed to control infection. However, during plague, Yersinia pestis inhibits the timely synthesis of LTB4 and subsequent inflammation. Using bacterial mutants, we previously determined that Y. pestis inhibits LTB4 synthesis via the action of the Yop effector proteins that are directly secreted into host cells through a type 3 secretion system (T3SS). Here, we show that the T3SS is the primary pathogen associated molecular pattern (PAMP) required for production of LTB4 in response to both Yersinia and Salmonella. However, we also unexpectantly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require the activation of two distinctly different host signaling pathways. We identified that phagocytosis and the NLRP3/CASP1 inflammasome significantly impact LTB4 synthesis by macrophages but not neutrophils. Instead, the SKAP2/PLC signaling pathway is required for T3SS-mediated LTB4 production by neutrophils. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered that a second unrelated PAMP-mediated signal activates the MAP kinase pathway needed for synthesis. Together, these data demonstrate significant differences in the host factors and signaling pathways required by macrophages and neutrophils to quickly produce LTB4 in response to bacteria. Moreover, while macrophages and neutrophils might rely on different signaling pathways for T3SS-dependent LTB4 synthesis, Y. pestis has evolved virulence mechanisms to counteract this response by either leukocyte to inhibit LTB4 synthesis and colonize the host.

Transcriptomes of soybean roots and nodules inoculated with Sinorhizobium fredii with NopP and NopI variants.

The major crop, soybean, forms root nodules with symbiotic rhizobia, providing energy and carbon to the bacteria in exchange for bioavailable nitrogen. The relationship is host-specific and highly host-regulated to maximize energy efficiency. Symbiotic nitrogen fixation (SNF) is greener than synthetic fertilizer for replenishing soil fertility, contributing to yield increase. Nodulation Outer Protein P (NopP) and NopI of the type 3 secretion system (T3SS) of the rhizobium determine host specificity. Sinorhizobium fredii CCBAU25509 (R2) and CCBAU45436 (R4) have different NopP and NopI variants, affecting their respective symbiotic compatibilities with the cultivated soybean C08 and the wild soybean W05. Swapping the NopP variants between R2 and R4 has been shown to switch their compatibility with C08 with the rj2/Rfg1 genotype. To understand the effects of Nops on host compatibility, analyses on the transcriptomic data of W05 roots and nodules inoculated with S. fredii strains containing Nop variants uncovered many differentially expressed genes related to nodulation and nodule functions, providing important information on the effects of Nops on hosts and nodules.

Role of the type 6 secretion system on apoptosis and macrophage polarization during Burkholderia pseudomallei infection.

Burkholderia pseudomallei (Bpm) is the causative agent of the disease melioidosis. As a facultative intracellular pathogen, Bpm has a complex lifestyle that culminates in cell-to-cell fusion and multinucleated giant cells (MNGCs) formation. The virulence factor responsible for MNGC formation is the type 6 secretion system (T6SS), a contractile nanomachine. MNGC formation is a cell-to-cell spread strategy that allows the bacteria to avoid the extracellular immune system and our previous data highlighted cell death, apoptosis, and inflammation as pathways significantly impacted by T6SS activity. Thusly, we investigated how the T6SS influences these phenotypes within the macrophage and pulmonary models of infection. Here we report that the T6SS is responsible for exacerbating apoptotic cell death during infection in both macrophages and the lungs of infected mice. We also demonstrate that although the T6SS does not influence differential macrophage polarization, the M2 polarization observed is potentially beneficial for Bpm pathogenesis and replication. Finally, we show that the T6SS contributes to the severity of inflammatory nodule formation in the lungs, which might be potentially connected to the amount of apoptosis that is triggered by the bacteria.

Proteomic analysis of HeLa cells after stable transfection with the Chlamydia trachomatis CT143 gene

BackgroundThe CT143 protein of Chlamydia trachomatis (Ct) is a key immunodominant antigen and candidate type-III secretion substrate. Although CT143 expression has not been detected in the cytosol of infected cells, it is known to interfere with the physiological behavior of HeLa cells. This study aims to investigate how the CT143 protein affects the protein expression profile of HeLa cells, providing a basis for further research into Ct’s pathogenic mechanisms. MethodsWe constructed a stably transfected HeLa cell line, pCD513B-1-CT143-HeLa, and a control cell line, pCD513B-1-HeLa. Protein expression profiles of these cell lines were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins were identified, constructed into a database, and verified using parallel reaction monitoring (PRM). Bioinformatics software facilitated the preliminary analysis of the biological functions of these differential proteins. ResultsA total of 221 host proteins were differentially expressed, with 68 upregulated and 153 downregulated. These variations influence the regulation of peptidase activity and are crucial in biological processes such as cell secretion and protease activity. Significant changes were noted in protein processing, alcohol dehydrogenase activity, Aldo-Keto reductase activity, and peptidase regulator activity. Furthermore, alterations were observed in cellular components like the plasma membrane and cell periphery. Pathways involving the hematopoietic system, glycosaminoglycan degradation, retinol metabolism, and cytochrome P450-mediated exogenous drug metabolism were notably affected. Indirect interactions among differentially expressed proteins included three key nodal proteins: C3, IFIT3, and IFIT1. ConclusionThe successful construction of a host differential protein expression profile was achieved through stable transfection of HeLa cells with the CT143 gene. The differential proteins identified are implicated in regulating various biological processes such as intracellular signal transduction, cell secretion, protein processing, hydrolysis, and enzyme activity. These findings suggest that the CT143 protein may influence the host cell’s biological behavior by altering host protein expression, potentially hindering Ct growth and development.

Enhancing Resistance to Cercospora Leaf Spot in Mung Bean (Vigna radiata L.) through Bradyrhizobium sp. DOA9 Priming: Molecular Insights and Bio-Priming Potential.

Mung bean (Vigna radiata L.), a vital legume in Asia with significant nutritional benefits, is highly susceptible to Cercospora leaf spot (CLS) caused by Cercospora canescens, leading to significant yield losses. As an alternative to chemical fungicides, bio-priming with rhizobacteria can enhance plant resistance. This study explores the potential of Bradyrhizobium sp. strain DOA9 to augment resistance in mung bean against CLS via root priming. The results reveal that short (3 days) and double (17 and 3 days) priming with DOA9 before fungal infection considerably reduces lesion size on infected leaves by activating defense-related genes, including Pti1, Pti6, EDS1, NDR1, PR-1, PR-2, Prx, and CHS, or by suppressing the inhibition of PR-5 and enhancing peroxidase (POD) activity in leaves. Interestingly, the Type 3 secretion system (T3SS) of DOA9 may play a role in establishing resistance in V. radiata CN72. These findings suggest that DOA9 primes V. radiata CN72's defense mechanisms, offering an effective bio-priming strategy to alleviate CLS. Hence, our insights propose the potential use of DOA9 as a bio-priming agent to manage CLS in V. radiata CN72, providing a sustainable alternative to chemical fungicide applications.

Virulent Effector Hasp155 of Puccinia striiformis f. sp. tritici Suppresses Plant Immunity and Promotes Fungus Infection.

As a kind of obligate biotrophic fungus, Puccinia striiformis f. sp. tritici (Pst) secretes vast effectors via haustoria to host cells during the infection to inhibit host defense responses and promote fungal invasion. In this study, based on the completion of genome sequencing and haustorial transcriptome sequencing of Pst, we identified a Pst effector (Hasp155) that is significantly induced in the early stage of Pst infection to wheat. The 18 N-terminal amino acids of Hasp155 encoded a signal peptide with a secretory function. Transient expression of Hasp155 in Nicotiana benthamiana inhibited Bax-induced cell death as well as chitin-triggered callose deposition and defense-related gene expression. Moreover, delivery of the Hasp155 protein into wheat cells via type three secretion systems (TTSS) led to reduced plant immunity to nonpathogenic bacteria and to the avirulent Pst race with decreased H2O2 accumulation and promoted Pst development. Furthermore, transgenic overexpression of Hasp155 significantly renders wheat resistance susceptible, resulting in a decreased defense response and increased Pst pathogenicity. Overall, these results indicate that Hasp155 is an important effector of Pst pathogenicity by suppressing plant immunity.

Yersinia pseudotuberculosis growth arrest during type-III secretion system expression is associated with altered ribosomal protein expression and decreased gentamicin susceptibility.

Slow-growing bacterial cells have reduced antibiotic susceptibility, rendering them very difficult to eliminate during antibiotic treatment. However, for many key virulence factors (bacterial factors required to promote infection), it remains unclear whether expression is sufficient to slow bacterial growth and impact antibiotic susceptibility. Using Yersinia pseudotuberculosis , we found ribosomal protein expression fluctuated based on growth rate, and we generated a fluorescent reporter construct to detect altered ribosomal protein expression within individual bacterial cells. We then asked if expression of a key virulence factor in Yersinia , the type-III secretion system (T3SS), is sufficient to lower ribosomal protein expression, since it has been well established that T3SS induction results in growth arrest. We found high levels of T3SS expression promotes slowed growth and antibiotic tolerance, and bacterial cells that survive treatment with a ribosome-targeting antibiotic, gentamicin, have heightened levels of T3SS and lower levels of S10 expression.

Breaking barriers: pCF10 type 4 secretion system relies on a self-regulating muramidase to modulate the cell wall.

Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional in vitro and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs.IMPORTANCEAntibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from Enterococcus faecalis. This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in E. faecalis as well as other medically relevant Gram-positive bacteria.

Commensal Yeast Promotes Salmonella Typhimurium Virulence.

Enteric pathogens engage in complex interactions with the host and the resident microbiota to establish gut colonization. Although mechanistic interactions between enteric pathogens and bacterial commensals have been extensively studied, whether and how commensal fungi affect pathogenesis of enteric infections remains largely unknown. Here we show that colonization with the common human gut commensal fungus Candida albicans worsened infections with the enteric pathogen Salmonella enterica serovar Typhimurium. Presence of C. albicans in the mouse gut increased Salmonella cecum colonization and systemic dissemination. We investigated the underlying mechanism and found that Salmonella binds to C. albicans via Type 1 fimbriae and uses its Type 3 Secretion System (T3SS) to deliver effector proteins into C. albicans . A specific effector, SopB, was sufficient to manipulate C. albicans metabolism, triggering increased arginine biosynthesis in C. albicans and the release of millimolar amounts of arginine into the extracellular environment. The released arginine, in turn, induced T3SS expression in Salmonella , increasing its invasion of epithelial cells. C. albicans deficient in arginine production was unable to increase Salmonella virulence in vitro or in vivo . In addition to modulating pathogen invasion, arginine also directly influenced the host response to infection. Arginine-producing C. albicans dampened the inflammatory response during Salmonella infection, whereas C. albicans deficient in arginine production did not. Arginine supplementation in the absence of C. albicans increased the systemic spread of Salmonella and decreased the inflammatory response, phenocopying the presence of C. albicans . In summary, we identified C. albicans colonization as a susceptibility factor for disseminated Salmonella infection, and arginine as a central metabolite in the cross-kingdom interaction between fungi, bacteria, and host.

Characterization and engineering of the type 3 secretion system needle monomer from Salmonella through the construction and screening of a comprehensive mutagenesis library.

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) of Salmonella enterica. Despite detailed knowledge of the PrgI needle's assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle's mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS's assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

Inter-species expression of an EF-HAND CALCIUM BINDING PROTEIN in Xanthomonas perforans leads to reduced virulence and decreased immune evasion in tomato plants.

Many phytopathogenic bacteria require a type three secretion system (TTSS) to activate effector triggered immunity (ETI). We identified a calcium binding protein, EfhXXfa, in the citrus pathogen, X. citri subsp. aurantifolii, that does not require a TTSS to activate reactive oxygen species (ROS) and elicit a hypersensitive reaction (HR) in tomato leaves following infection. Purified, recombinant EfhXXfa was shown to bind two moles of calcium per mole of protein, whereas mutation of the first of two EF-hands did not bind calcium . EfhXXfa expression was determined to be inducible in hrp-inducing medium. Additionally, growth of X. perforans transconjugants with and without the efhXXfa gene in hrp-inducing medium differed in intracellular calcium concentration; the transconjugant without efhXXfa yielded higher cell pellet masses and higher increased intracellular calcium concentrations relative to cells expressing EfhXXfa. An EfhXXfa homolog, EfhXXe, present in the pepper pathogen, X. euvesicatoria, when expressed in the tomato pathogen, X. perforans, triggered ROS production and an HR in tomato leaves and is a host-limiting factor. Interestingly, all tested X. perforans and X. euvesicatoria strains pathogenic on tomato contain a stop codon immediately upstream of the first EF-hand domain in the efhXXe gene, whereas most X. euvesicatoria strains pathogenic on pepper do not.

Enterococcus faecalis-derived adenine enhances enterohaemorrhagic Escherichia coli Type 3 Secretion System-dependent virulence.

Interactions between microbiota and enteric pathogens can promote colonization resistance or enhance pathogenesis. The pathobiont Enterococcus faecalis increases enterohaemorrhagic E. coli (EHEC) virulence by upregulating Type 3 Secretion System (T3SS) expression, effector translocation, and attaching and effacing (AE) lesion formation on enterocytes, but the mechanisms underlying this remain unknown. Using co-infection of organoids, metabolomics, supplementation experiments and bacterial genetics, here we show that co-culture of EHEC with E. faecalis increases the xanthine-hypoxanthine pathway activity and adenine biosynthesis. Adenine or E. faecalis promoted T3SS gene expression, while transcriptomics showed upregulation of adeP expression, which encodes an adenine importer. Mechanistically, adenine relieved High hemolysin activity (Hha)-dependent repression of T3SS gene expression in EHEC and promoted AE lesion formation in an AdeP-dependent manner. Microbiota-derived purines, such as adenine, support multiple beneficial host responses; however, our data show that this metabolite also increases EHEC virulence, highlighting the complexity of pathogen-microbiota-host interactions in the gut.

Distinct Mechanisms of Type 3 Secretion System Recognition Control LTB4 Synthesis in Neutrophils versus Macrophages.

Leukotriene B4 (LTB4) is critical for initiating the inflammatory cascade in response to infection. However, Yersinia pestis colonizes the host by inhibiting the timely synthesis of LTB4 and inflammation. Here, we show that the bacterial type 3 secretion system (T3SS) is the primary pathogen associated molecular pattern (PAMP) responsible for LTB4 production by leukocytes in response to Yersinia and Salmonella, but synthesis is inhibited by the Yop effectors during Yersinia interactions. Moreover, we unexpectedly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require two distinct host signaling pathways. We show that the SKAP2/PLC signaling pathway is essential for LTB4 production by neutrophils but not macrophages. Instead, phagocytosis and the NLRP3/CASP1 inflammasome are needed for LTB4 synthesis by macrophages. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered a second unrelated PAMP-mediated signal independently activates the MAP kinase pathway needed for LTB4 synthesis. Together, these data demonstrate significant differences in the signaling pathways required by macrophages and neutrophils to quickly respond to bacterial infections.

Bacterial motility depends on a critical flagellum length and energy-optimised assembly.

Our study demonstrates how protein secretion of the bacterial flagellum is finely tuned to optimize filament assembly rate and flagellum function while minimizing energy consumption. By measuring flagellar filament lengths and bacterial swimming after initiation of flag-ellum assembly, we were able to establish the minimal filament length necessary for swimming motility, which we rationalized physically as resulting from an elasto-hydrodynamic instability of the swimming cell. Our bio-physical model of flagellum growth further illustrates how the physiological flagellin secretion rate is optimized to maximize filament elongation while conserving energy. These findings illuminate the evolutionary pressures that have shaped the function of the bacterial flagellum and type-III secretion system, driving improvements in bacterial motility and overall fitness.

Genomic insights into antimicrobial resistant Salmonella in internationally traded chicken meat: First baseline findings in the United Arab Emirates

Non-typhoidal Salmonella is among the most prevalent foodborne zoonoses, challenging food safety and One Health worldwide. A dearth of knowledge exists regarding non-typhoidal Salmonella prevalence and genomic features within the United Arab Emirates (UAE), one of the top markets in chicken meat consumption worldwide. In this study, Salmonella was detected in 16 out of 254 (6.30 %, 95 % confidence interval: 3.64 %, 10.03 %) samples of imported frozen chicken carcasses sampled from UAE retails. Among the recovered serotypes, S. Minnesota; ST 548 (10/16) emerged as the most prevalent, trailed by S. Heidelberg; ST 15 (4/16), and then S. Kentucky; ST 198, and ST 152 (2/16). Antimicrobial resistance (AMR) was most prevalent against tetracycline (93.7 %), followed by ampicillin (68.7 %), and extended-spectrum cephalosporins (ceftriaxone and cefoxitin) (56.2 %); with 68.7 % (11/16) of the isolates classified as multidrug resistant (MDR). Whole-genome sequencing (WGS) analysis revealed the existence of 14 AMR genes, among which the blaCMY-2, an AmpC cephalosporinases resistance gene, presented in 10 of the 16 isolates. Among the ten out of the eleven MDR Salmonella isolates, both IncC and Col(pHAD28) plasmid incompatibility types were concurrently featured. The range of virulence genes varied from 149 to 165 genes, with an average of 168 genes per isolate. Except for one isolate, all other isolates possessed type III secretion system (TTSS) related genes known to be encoded by the Salmonella pathogenicity island-1 (SPI-1). This study contributes to our global understanding of Salmonella epidemiology, specifically focusing on the Middle East. The insights gained from this study are significant in shaping import risk analyses aimed at mitigating Salmonella exposure risks through globally traded chicken. The research underscores the value of WGS as a crucial tool for substantiating evidence-based food safety hazard assessment.

Vibrio pathogens and their toxins in aquaculture: A comprehensive review

AbstractAquaculture is an indispensable food source for the growing world population. In the last decades, its intensification has increased the incidence of viral and bacterial infections, emphasizing the need for novel disease management strategies. Vibrionaceae bacteria are widespread in aquatic environments, affecting various host species, including economically important fish, shrimp and bivalves. Also, human consumption of undercooked aquatic food contaminated with Vibrionaceae and/or their toxins poses a threat to human health, leading to conditions such as gastroenteritis, wound infection and sepsis. In addition, small fish and shrimp, in open aquaculture systems, can be eaten by birds that can then carry and spread this pathogen to livestock and humans. Many Vibrionaceae produce toxins, some detrimental or even lethal to the hosts, others affecting surrounding bacteria. This review provides a comprehensive overview of Vibrio toxins affecting aquatic (in)vertebrate, summarizing findings on molecular structures, mechanisms of action and regulation and secretion processes involved. Important toxin classes for microbe–host interactions, such as haemolytic, proteolytic and MARTX toxins, are discussed, along with other toxins like PirAB produced by the AHPND‐causing Vibrio parahaemolyticus. The review also emphasizes the importance of microbe–microbe interactions, particularly the production of bacteriocins by Vibrio species (Vibriocins) and antibacterial effectors produced by the Type 6 Secretion System (T6SS). While whole genome sequencing has identified putative virulence factors that are toxins, many toxins remain partially characterized or uncharacterized. Elucidating their regulation, secretion and molecular characteristics can provide valuable information and lead to novel solutions for managing Vibrio pathogens in aquaculture.

A novel chaperone-effector-immunity system identified in uropathogenic Escherichia coli UMN026.

Urinary tract infections (UTIs) are very common worldwide. According to their symptomatology, these infections are classified as pyelonephritis, cystitis, or asymptomatic bacteriuria (AB). Approximately 75-95% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which is an extraintestinal bacterium that possesses virulence factors for bacterial adherence and invasion in the urinary tract. In addition, UPEC possesses type 6 secretion systems (T6SS) as virulence mechanisms that can participate in bacterial competition and in bacterial pathogenicity. UPEC UMN026 carries three genes, namely, ECUMN_0231, ECUMN_0232, and ECUMN_0233, which encode three uncharacterized proteins related to the T6SS that are conserved in strains from phylogroups B2 and D and have been proposed as biomarkers of UTIs. To analyze the frequency of the ECUMN_0231, ECUMN_0232, ECUMN_0233, and vgrG genes in UTI isolates, as well as their expression in Luria Bertani (LB) medium and urine; to determine whether these genes are related to UTI symptoms or bacterial competence and to identify functional domains on the putative proteins. The frequency of the ECUMN and vgrG genes in 99 clinical isolates from UPEC was determined by endpoint PCR. The relationship between gene presence and UTI symptomatology was determined using the chi2 test, with p < 0.05 considered to indicate statistical significance. The expression of the three ECUMN genes and vgrG was analyzed by RT-PCR. The antibacterial activity of strain UMN026 was determined by bacterial competence assays. The identification of functional domains and the docking were performed using bioinformatic tools. The ECUMN genes are conserved in 33.3% of clinical isolates from patients with symptomatic and asymptomatic UTIs and have no relationship with UTI symptomatology. Of the ECUMN+ isolates, only five (15.15%, 5/33) had the three ECUMN and vgrG genes. These genes were expressed in LB broth and urine in UPEC UMN026 but not in all the clinical isolates. Strain UMN026 had antibacterial activity against UPEC clinical isolate 4014 (ECUMN-) and E. faecalis but not against isolate 4012 (ECUMN+). Bioinformatics analysis suggested that the ECUMN genes encode a chaperone/effector/immunity system. The ECUMN genes are conserved in clinical isolates from symptomatic and asymptomatic patients and are not related to UTI symptoms. However, these genes encode a putative chaperone/effector/immunity system that seems to be involved in the antibacterial activity of strain UMN026.

Virulence phenotypes differ between toxigenic Vibrio parahaemolyticus isolated from western coasts of Europe

Vibrio parahaemolyticus is the leading bacterial cause of gastroenteritis associated with seafood consumption worldwide. Not all members of the species are thought to be pathogenic, thus identification of virulent organisms is essential to protect public health and the seafood industry. Correlations of human disease and known genetic markers (e.g. thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH)) appear complex. Some isolates recovered from patients lack these factors, while their presence has become increasingly noted in isolates recovered from the environment. Here, we used whole-genome sequencing in combination with mammalian and insect models of infection to assess the pathogenic potential of V. parahaemolyticus isolated from European Atlantic shellfish production areas. We found environmental V. parahaemolyticus isolates harboured multiple virulence-associated genes, including TDH and/or TRH. However, carriage of these factors did not necessarily reflect virulence in the mammalian intestine, as an isolate containing TDH and the genes coding for a type 3 secretion system (T3SS) 2α virulence determinant, appeared avirulent. Moreover, environmental V. parahaemolyticus lacking TDH or TRH could be assigned to groups causing low and high levels of mortality in insect larvae, with experiments using defined bacterial mutants showing that a functional T3SS1 contributed to larval death. When taken together, our findings highlight the genetic diversity of V. parahaemolyticus isolates found in the environment, their potential to cause disease and the need for a more systematic evaluation of virulence in diverse V. parahaemolyticus to allow better genetic markers.