Diversity, Distribution and Structural Prediction of the Pathogenic Bacterial Effectors EspN and EspS.

BACKGROUND: Many Gram-negative enterobacteria translocate virulence proteins (effectors) into intestinal epithelial cells using a type III secretion system (T3SS) to subvert the activity of various cell functions possess. Many T3SS effectors have been extensively characterized, but there are still some effector proteins whose functional information is completely unknown. METHODS: In this study, two predicted effectors of unknown function, EspN and EspS (Escherichia coli secreted protein N and S), were selected for analysis of translocation, distribution and structure prediction. RESULTS: The TEM1 (ß-lactamase) translocation assay was performed, which showed that EspN and EspS are translocated into host cells in a T3SS-dependent manner during bacterial infection. A phylogenetic tree analysis revealed that homologs of EspN and EspS are widely distributed in pathogenic bacteria. Multiple sequence alignment revealed that EspN and its homologs share a conserved C-terminal region (673-1133 a.a.). Furthermore, the structure of EspN (673-1133 a.a.) was also predicted and well-defined, which showed that it has three subdomains connected by a loop region. EspS and its homologs share a sequence-conserved C-terminal (146-291 a.a.). The predicted structure of EspS (146-291 a.a.) is composed of a ß-sheet consisting of four ß-strands and several short helices, which has a TM score of 0.5014 with the structure of the Vibrio cholerae RTX cysteine protease domain (PDBID: 3eeb). CONCLUSIONS: These results suggest that EspN and EspS may represent two important classes of T3SS effectors associated with pathogen virulence, and our findings provide important clues to understanding the potential functions of EspN and EspS.

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.

FlaG competes with FliS-flagellin complexes for access to FlhA in the flagellar T3SS to control Campylobacter jejuni filament length.

Bacteria power rotation of an extracellular flagellar filament for swimming motility. Thousands of flagellin subunits compose the flagellar filament, which extends several microns from the bacterial surface. It is unclear whether bacteria actively control filament length. Many polarly flagellated bacteria produce shorter flagellar filaments than peritrichous bacteria, and FlaG has been reported to limit flagellar filament length in polar flagellates. However, a mechanism for how FlaG may function is unknown. We observed that deletion of flaG in the polarly flagellated pathogens Vibrio cholerae, Pseudomonas aeruginosa, and Campylobacter jejuni caused extension of flagellar filaments to lengths comparable to peritrichous bacteria. Using C. jejuni as a model to understand how FlaG controls flagellar filament length, we found that FlaG and FliS chaperone-flagellin complexes antagonize each other for interactions with FlhA in the flagellar type III secretion system (fT3SS) export gate. FlaG interacted with an understudied region of FlhA, and this interaction appeared to be enhanced in ΔfliS and FlhA FliS-binding mutants. Our data support that FlaG evolved in polarly flagellated bacteria as an antagonist to interfere with the ability of FliS to interact with and deliver flagellins to FlhA in the fT3SS export gate to control flagellar filament length so that these bacteria produce relatively shorter flagella than peritrichous counterparts. This mechanism is similar to how some gatekeepers in injectisome T3SSs prevent chaperones from delivering effector proteins until completion of the T3SS and host contact occurs. Thus, flagellar and injectisome T3SSs have convergently evolved protein antagonists to negatively impact respective T3SSs to secrete their major terminal substrates.

Epigallocatechin gallate protects mice from Salmonella enterica ser. Typhimurium infection by modulating bacterial virulence through quorum sensing inhibition.

Salmonella enterica ser. Typhimurium is a common pathogen that poses a considerable public health threat, contributing to severe gastrointestinal diseases and widespread foodborne illnesses. The virulence of S. Typhimurium is regulated by quorum sensing (QS) and the type III secretion system (T3SS). This study investigated the inhibitory effects and anti-QS activity of epigallocatechin gallate (EGCG), which is a bioactive ingredient found in green tea, on the virulence of S. Typhimurium. In vitro bacterial experiments demonstrated that EGCG inhibited the production of autoinducers, biofilm formation, and flagellar activity by downregulating the expression of AI-1, AI-2, Salmonella pathogenicity islands (SPI)-1, SPI-2, and genes related to flagella, fimbriae, and curli fibers. In a mouse model of S. Typhimurium-induced enteritis, EGCG considerably reduced intestinal colonization by S. Typhimurium and alleviated intestinal damage. In conclusion, EGCG protects the intestines of mice infected with S. Typhimurium by inhibiting QS-induced virulence gene expression, demonstrating its potential as a therapeutic agent for controlling S. Typhimurium infections.

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.

The Type III Effector XopLXcc in Xanthomonas campestris pv. campestris Targets the Proton Pump Interactor 1 and Suppresses Innate Immunity in Arabidopsis.

Xanthomonas campestris pathovar campestris (Xcc) is a significant phytopathogen causing black rot disease in crucifers. Xcc injects a variety of type III effectors (T3Es) into the host cell to assist infection or propagation. A number of T3Es inhibit plant immunity, but the biochemical basis for a vast majority of them remains unknown. Previous research has revealed that the evolutionarily conserved XopL-family effector XopLXcc inhibits plant immunity, although the underlying mechanisms remain incompletely elucidated. In this study, we identified proton pump interactor (PPI1) as a specific virulence target of XopLXcc in Arabidopsis. Notably, the C-terminus of PPI1 and the Leucine-rich repeat (LRR) domains of XopLXcc are pivotal for facilitating this interaction. Our findings indicate that PPI1 plays a role in the immune response of Arabidopsis to Xcc. These results propose a model in which XopLXcc binds to PPI1, disrupting the early defense responses activated in Arabidopsis during Xcc infection and providing valuable insights into potential strategies for regulating plasma membrane (PM) H+-ATPase activity during infection. These novel insights enhance our understanding of the pathogenic mechanisms of T3Es and contribute to the development of effective strategies for controlling bacterial diseases.

Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system.

Providencia alcalifaciens is a Gram-negative bacterium found in various water and land environments and organisms, including insects and mammals. Some P. alcalifaciens strains encode gene homologs of virulence factors found in pathogenic Enterobacterales members, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are pathogenic determinants in P. alcalifaciens is not known. In this study, we investigated P. alcalifaciens-host interactions at the cellular level, focusing on the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS1b is widespread in Providencia spp. and encoded on the chromosome. A large plasmid that is present in a subset of P. alcalifaciens strains, primarily isolated from diarrheal patients, encodes for T3SS1a. We show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, lyses its internalization vacuole, and proliferates in the cytosol. This triggers caspase-4-dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS1a in entry, vacuole lysis, and cytosolic proliferation is host cell type-specific, playing a more prominent role in intestinal epithelial cells than in macrophages or insect cells. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa and induces mild epithelial damage with negligible fluid accumulation in a T3SS1a- and T3SS1b-independent manner. However, T3SS1b was required for the rapid killing of Drosophila melanogaster. We propose that the acquisition of two T3SS has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

Differential expression of the type III secretion system genes in Yersinia ruckeri: Preliminary investigations in different environmental conditions.

Type III secretion system (T3SS) is an important virulence system in Gram-negative bacteria. In this investigation, different environmental conditions that regulate the expression of T3SS genes in Yersinia ruckeri were investigated aimed at obtaining a better understanding about its modulation after various environmental challenges. Four isolates of Y. ruckeri CSF007-82, ATCC29473, A7959-11 and YRNC10 were cultivated under the diverse in vitro challenges iron depletion, high salt, low pH and in the presence of fish serum or in the fish cell culture (Chinook Salmon Embryo - CHSE). The transcriptional modulation of the chromosomal genes ysaV, ysaC, ysaJ and prgH of ysa were investigated using quantitative real-time PCR. The expression of prgH, ysaV, ysaC and ysaJ was differentially expressed in all four strains under evaluation. The highest gene expression levels were observed for Y. ruckeri YRNC10 AN after addition of 0.3 M NaCl in Luria Bertani broth. The results obtained from this study provide initial insights into T3SS responses in Y. ruckeri, which pave the way for further studies aimed at expanding our knowledge on the functional roles of the T3SS genes in Y. ruckeri.

Host-Driven Selection, Revealed by Comparative Analysis of Xanthomonas Type III Secretion Effectoromes, Unveils Novel Recognized Effectors.

Xanthomonas species are specialized plant pathogens, often exhibiting a narrow host range. They rely on the translocation of effector proteins through the type III secretion system to colonize their respective hosts. The effector arsenal varies among Xanthomonas spp., typically displaying species-specific compositions. This species-specific effector composition, collectively termed the effectorome, is thought to influence host specialization. We determined the plant host-derived effectoromes of more than 300 deposited genomes of Xanthomonas species associated with either Solanaceae or Brassicaceae hosts. Comparative analyses revealed clear species-specific effectorome signatures. However, Solanaceae or Brassicaceae host-associated effectorome signatures were not detected. Nevertheless, host biases in the presence or absence of specific effector classes were observed. To assess whether host-associated effector absence results from selective pressures, we introduced effectors unique to Solanaceae pathogens to X. campestris pv. campestris and effectors unique to Brassicaceae pathogens to X. euvesicatoria pv. euvesicatoria (Xeue) and evaluated if these introductions hindered virulence on their respective hosts. Introducing the effector XopI into X. campestris pv. campestris reduced virulence on white cabbage leaves without affecting localized or systemic colonization. Introducing the XopAC or XopJ5 effectors into Xeue reduced virulence and colonization on tomato but not on pepper. Additionally, XopAC and XopJ5 induced a hypersensitive response on tomato leaves when delivered by Xeue or through Agrobacterium-mediated transient expression, confirming recognition in tomato. This study demonstrates the role of host-derived selection in establishing species-specific effectoromes, identifying XopAC and XopJ5 as recognized effectors in tomato.

Salmonella Typhimurium exploits host polyamines for assembly of the type 3 secretion machinery.

Bacterial pathogens utilize the factors of their hosts to infect them, but which factors they exploit remain poorly defined. Here, we show that a pathogenic Salmonella enterica serovar Typhimurium (STm) exploits host polyamines for the functional expression of virulence factors. An STm mutant strain lacking principal genes required for polyamine synthesis and transport exhibited impaired infectivity in mice. A polyamine uptake-impaired strain of STm was unable to inject effectors of the type 3 secretion system into host cells due to a failure of needle assembly. STm infection stimulated host polyamine production by increasing arginase expression. The decline in polyamine levels caused by difluoromethylornithine, which inhibits host polyamine production, attenuated STm colonization, whereas polyamine supplementation augmented STm pathogenesis. Our work reveals that host polyamines are a key factor promoting STm infection, and therefore a promising therapeutic target for bacterial infection.

Metabolic specialization drives reduced pathogenicity in Pseudomonas aeruginosa isolates from cystic fibrosis patients.

Metabolism provides the foundation for all cellular functions. During persistent infections, in adapted pathogenic bacteria metabolism functions radically differently compared with more naïve strains. Whether this is simply a necessary accommodation to the persistence phenotype or if metabolism plays a direct role in achieving persistence in the host is still unclear. Here, we characterize a convergent shift in metabolic function(s) linked with the persistence phenotype during Pseudomonas aeruginosa colonization in the airways of people with cystic fibrosis. We show that clinically relevant mutations in the key metabolic enzyme, pyruvate dehydrogenase, lead to a host-specialized metabolism together with a lower virulence and immune response recruitment. These changes in infection phenotype are mediated by impaired type III secretion system activity and by secretion of the antioxidant metabolite, pyruvate, respectively. Our results show how metabolic adaptations directly impinge on persistence and pathogenicity in this organism.

Comparative transcriptomics reveals a highly polymorphic Xanthomonas HrpG virulence regulon.

BACKGROUND: Bacteria of the genus Xanthomonas cause economically significant diseases in various crops. Their virulence is dependent on the translocation of type III effectors (T3Es) into plant cells by the type III secretion system (T3SS), a process regulated by the master response regulator HrpG. Although HrpG has been studied for over two decades, its regulon across diverse Xanthomonas species, particularly beyond type III secretion, remains understudied. RESULTS: In this study, we conducted transcriptome sequencing to explore the HrpG regulons of 17 Xanthomonas strains, encompassing six species and nine pathovars, each exhibiting distinct host and tissue specificities. We employed constitutive expression of plasmid-borne hrpG*, which encodes a constitutively active form of HrpG, to induce the regulon. Our findings reveal substantial inter- and intra-specific diversity in the HrpG* regulons across the strains. Besides 21 genes directly involved in the biosynthesis of the T3SS, the core HrpG* regulon is limited to only five additional genes encoding the transcriptional activator HrpX, the two T3E proteins XopR and XopL, a major facility superfamily (MFS) transporter, and the phosphatase PhoC. Interestingly, genes involved in chemotaxis and genes encoding enzymes with carbohydrate-active and proteolytic activities are variably regulated by HrpG*. CONCLUSIONS: The diversity in the HrpG* regulon suggests that HrpG-dependent virulence in Xanthomonas might be achieved through several distinct strain-specific strategies, potentially reflecting adaptation to diverse ecological niches. These findings enhance our understanding of the complex role of HrpG in regulating various virulence and adaptive pathways, extending beyond T3Es and the T3SS.

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.

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.

The quorum sensing regulator RhlR positively controls the expression of the type III secretion system in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunist bacterium that causes acute and chronic infections. During acute infections, the type III secretion system (T3SS) plays a pivotal role in allowing the bacteria to translocate effectors such as ExoS, ExoT, and ExoY into host cells for colonization. Previous research on the involvement of quorum sensing systems Las and Rhl in controlling the T3SS gene expression produced ambiguous results. In this study, we determined the role of the Las and Rhl systems and the PqsE protein on T3SS expression. Our results show that in the wild-type PAO1 strain, the deletion of lasR or pqsE do not affect the secretion of ExoS. However, rhlI inactivation increases the expression of T3SS genes. In contrast to the rhlI deletion, rhlR inactivation decreases both T3SS genes expression and ExoS secreted protein levels, and this phenotype is restored when this mutant is complemented with the exsA gene, which codes for the master regulator of the T3SS. Additionally, cytotoxicity is affected in the rhlR mutant strain compared with its PAO1 parental strain. Overall, our results indicate that neither the Las system nor PqsE are involved in regulating the T3SS. Moreover, the Rhl system components have opposite effects, RhlI participates in negatively controlling the T3SS expression, while RhlR does it in a positive way, and this regulation is independent of C4 or PqsE. Finally, we show that rhlR, rhlI, or pqsE inactivation abolished pyocyanin production in T3SS-induction conditions. The ability of RhlR to act as a positive T3SS regulator in the absence of its cognate autoinducer and PqsE shows that it is a versatile regulator that controls different virulence traits allowing P. aeruginosa to compete for a niche.

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

BACKGROUND: 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. METHODS: 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 in vitro 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: 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.

Identification of homologs of the Chlamydia trachomatis effector CteG reveals a family of Chlamydiaceae type III secreted proteins that can be delivered into host cells.

Chlamydiae are a large group of obligate endosymbionts of eukaryotes that includes the Chlamydiaceae family, comprising several animal pathogens. Among Chlamydiaceae, Chlamydia trachomatis causes widespread ocular and urogenital infections in humans. Like many bacterial pathogens, all Chlamydiae manipulate host cells by injecting them with type III secretion effector proteins. We previously characterized the C. trachomatis effector CteG, which localizes at the host cell Golgi and plasma membrane during distinct phases of the chlamydial infectious cycle. Here, we show that CteG is a Chlamydiaceae-specific effector with over 60 homologs phylogenetically categorized into two distinct clades (CteG I and CteG II) and exhibiting several inparalogs and outparalogs. Notably, cteG I homologs are syntenic to C. trachomatis cteG, whereas cteG II homologs are syntenic among themselves but not with C. trachomatis cteG. This indicates a complex evolution of cteG homologs, which is unique among C. trachomatis effectors, marked by numerous events of gene duplication and loss. Despite relatively modest sequence conservation, nearly all tested CteG I and CteG II proteins were identified as type III secretion substrates using Yersinia as a heterologous bacterial host. Moreover, most of the type III secreted CteG I and CteG II homologs were delivered by C. trachomatis into host cells, where they localized at the Golgi region and cell periphery. Overall, this provided insights into the evolution of bacterial effectors and revealed a Chlamydiaceae family of type III secreted proteins that underwent substantial divergence during evolution while conserving the capacity to localize at specific host cell compartments.

Structural and functional characterization of the IpaD π-helix reveals critical roles in DOC interaction, T3SS apparatus maturation, and Shigella virulence.

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella's T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein invasion plasmid antigen D (IpaD). Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.

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.

A novel small non-coding RNA 562 mediates the virulence of Pseudomonas plecoglossicida by regulating the expression of fliP, a key component of flagella T3SS.

Pseudomonas plecoglossicida is a vital pathogen that poses a substantial risk to aquaculture. Small RNAs (sRNAs) are non-coding regulatory molecules capable of sensing environmental changes and modulating virulence-associated signaling pathways, such as the assembly of flagella. However, the relevant researches on P. plecoglossicida are an urgent need. Here, we report a novel sRNA, sRNA562, which has potential to regulate the post-transcriptional of fliP, a key component of the lateral flagellar type III secretion system. In this study, the effects of sRNA562 on the virulence of P. plecoglossicida and its role in regulating the pathogenic process were investigated through the use of a constructed sRNA562 deletion strain. The deletion of sRNA562 resulted in an up-regulation of fliP in P. plecoglossicida, and leading to increased swarming motility and enhanced the ability of biofilm formation, adhesion and chemotaxis. Subsequent artificial infection experiment demonstrated that the deletion of sRNA562 increased the virulence of P. plecoglossicida towards hybrid grouper, as evidenced by a reduction in survival rate, elevation of tissue bacterial load, and the exacerbation of histopathological damage. Further studies have found that the deletion of sRNA562 lead to an up-regulation of fliP expression during hybrid grouper infection, thereby enhancing bacterial swarming ability and ultimately heightening pathogenicity, leading to a dysregulated host response to infection, tissue damage and eventually death. Our work revealed a sRNA that exerts negative regulation on the expression of lateral flagella in P. plecoglossicida, thereby impacting its virulence. These findings provide a new perspective on the virulence regulation mechanism of P. plecoglossicida, contributing to a more comprehensive understanding in the field of pathogenicity research.