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Reactive oxygen species (ROS) play a crucial role in regulating numerous functions in organisms. Among the key regulators of ROS production are NADPH oxidases, primarily referred to as respiratory burst oxidase homologues (RBOHs). However, our understanding of whether and how pathogens directly target RBOHs has been limited. In this study, we revealed that the effector protein RipBJ, originating from the phytopathogenic bacterium Ralstonia solanacearum, was present in low‐ to medium‐virulence strains but absent in high‐virulence strains. Functional genetic assays demonstrated that the expression of ripBJ led to a reduction in bacterial infection. In the plant, RipBJ expression triggered plant cell death and the accumulation of H2O2, while also enhancing host defence against R. solanacearum by modulating multiple defence signalling pathways. Through protein interaction and functional studies, we demonstrated that RipBJ was associated with the plant's plasma membrane and interacted with the tomato RBOH known as SlWfi1, which contributed positively to RipBJ's effects on plants. Importantly, SlWfi1 expression was induced during the early stages following R. solanacearum infection and played a key role in defence against this bacterium. This research uncovers the plant RBOH as an interacting target of a pathogen's effector, providing valuable insights into the mechanisms of plant defence. Summary Statement: This study demonstrates that a tomato plasma membrane‐bound NADPH oxidase is a direct interactor of a bacterial effector protein to mediate plant defence, providing important new information on the interplay between this devastating pathogen and its host plant. [ABSTRACT FROM AUTHOR]

Simple Summary: Type III secretion system (T3SS) is an essential virulence determinant for Vibrio alginolyticus. In this study, we highlighted the significance of T3SS effector protein VopS in the V. alginolyticus infection. The vopS gene, which can promote the virulence of V. alginolyticus to Epinephelus coioides, is required for extracellular protease secretion. Also, it was demonstrated that VopS was involved not only in V. alginolyticus-mediated apoptosis but also in ferroptosis. Collectively, we identified that the VopS effector is a metabolic disruptor, promoting 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. [ABSTRACT FROM AUTHOR]

Background: Plesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors. Results: In this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Luminescence screening assay. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, PstR also positively regulates T3SS expression, which aids in P. shigelloides' capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides. Conclusions: Overall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria. [ABSTRACT FROM AUTHOR]

Bacteria employ two‐component systems (TCSs) to rapidly sense and respond to their surroundings often and during plant infection. Poplar canker caused by Lonsdalea populi is an emerging woody bacterial disease that leads to high mortality and poplar plantation losses in China. Nonetheless, the information about the underlying mechanism of pathogenesis remains scarce. Therefore, in this study, we reported the role of a TCS pair CpxA/CpxR in regulating virulence and stress responses in L. populi. The CpxA/R system is essential during infection, flagellum formation, and oxidative stress response. Specifically, the Cpx system affected flagellum formation by controlling the expression of flagellum‐related genes. CpxR, which was activated by phosphorylation in the presence of CpxA, participated in the transcriptional regulation of a chaperone sctU and the type III secretion system (T3SS)‐related genes, thereby influencing T3SS functions during L. populi infection. Phosphorylated CpxR directly manipulated the transcription of a membrane protein‐coding gene yccA and the deletion of yccA resulted in reduced virulence and increased sensitivity to H2O2. Furthermore, we mutated the conserved phosphorylation site of CpxR and found that CpxRD51A could no longer bind to the yccA promoter but could still bind to the sctU promoter. Together, our findings elucidate the roles of the Cpx system in regulating virulence and reactive oxygen species resistance and provide further evidence that the TCS is crucial during infection and stress response. [ABSTRACT FROM AUTHOR]

Abstract Background Plesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors. Results In this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Luminescence screening assay. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, PstR also positively regulates T3SS expression, which aids in P. shigelloides’ capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides. Conclusions Overall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria.

Abstract The symbiotic relationship between legumes and rhizobia is known to be influenced by specific rhizobial type III effectors (T3Es) in certain cases. In this study, we present evidence that the symbiosis between Vigna radiata and Bradyrhizobium elkanii USDA61 is controlled by a T3E called NopP2, and this interaction is highly dependent on the genetic makeup of the host plant. NopP2 plays a crucial role in promoting nodulation in various V. radiata varieties. Additionally, NopP2 is essential for early infection and the formation of nodules in compatible plants. Through evolutionary analysis, we discovered that bradyrhizobial NopPs can be categorized into two distinct clusters: NopP1 and NopP2. Furthermore, both types of bradyrhizobial NopPs were conserved within their respective groups. Our findings suggest that NopP2 serves as a mechanism for optimizing the symbiotic relationship between V. radiata and B. elkanii USDA61 by interacting with the pathogenesis related-10 (PR10) protein and reducing effector-triggered immunity (ETI) responses.

Aeromonas veronii is an important pathogen in the co-occurrence of humans and fish, causing significant mortality in various aquatic organisms, posing a major threat to aquaculture and public health care. The type III secretion system (T3SS) is one of the most critical virulence mechanisms in determining the pathogenicity of Aeromonas species. The transcription factor ExsA of AraC family serves as the main regulatory factor and activates T3SS assembly and effector protein secretion in response to changes in environmental conditions. In order to explore the effect of exsA on the pathogenicity of the A. veronii, this study constructed an exsA gene knockout strain using homologous recombination strategy and tested its biofilm formation, adhesion capabilities, as well as the cytotoxicity of the spent cultural supernatant. The results showed that the deletion of the exsA gene in A. veronii led to a significant decrease of its biofilm formation capability, its adhesion to ex vivo host tissues, and the cytotoxicity of the spent culture supernatants. Compared with the wild type, the ability of the exsA knockout strain to form biofilm was reduced by 1.5 folds. The adhesion to epithelial cells was not significantly changed in exsA knockout strain as compared with the wild type, while the adhesion to ex vivo mouse intestinal tissues decreased by 3.4 folds and the cytotoxicity of bacterial culture supernatants decreases by 1.2 folds. These results proved that exsA plays an important regulatory role in the virulence mechanism of bacterial pathogen, laying a solid foundation for further research on the function of T3SS in the pathogenic mechanism of A. veronii and provided clues for establishing strategies for the control of the aquatic infection. [ABSTRACT FROM AUTHOR]

Background: Shigellosis, also known as bacillary dysentery, is an acute infection of the intestine. The symptoms can vary from mild watery diarrhoea to severe inflammatory bacillary dysentery, which is characterized by fever, intense abdominal cramps, and the presence of blood and mucus in the stools. While the disease typically resolves on its own, it can become life-threatening in immunocompromised individuals or in the absence of adequate medical care. Shigella is the primary cause of bacillary dysentery worldwide. It is comprised of four distinct species—S. dysenteriae, S. flexneri, S. boydii, and S. sonnei—each with unique genomic characteristics and disease-causing abilities. Shigella spp. have developed resistance to multiple drugs and have also adapted well to the gut environment over time. They have become well-suited to infecting the human gut epithelial cells and causing dysentery. Consequently, numerous studies have investigated the potential application of nanotechnology in the treatment of shigellosis by leveraging its capability for drug delivery and targeted therapy, thereby improving effectiveness while reducing side effects. Short conclusion: It is crucial to maintain ongoing surveillance and develop new strategies to effectively manage this issue. In this review, we shed light on the present comprehension of distinct Shigella spp. and their potential contribution to the pathogenesis of shigellosis, along with their interaction with the gut microbiota. We also provide insight into how nanotechnology may be a major factor in preventing shigellosis in the future. [ABSTRACT FROM AUTHOR]

Currently, it is widely accepted that the type III secretion system (T3SS) serves as the transport platform for bacterial virulence factors, while flagella act as propulsion motors. However, there remains a noticeable dearth of comparative studies elucidating the functional disparities between these two mechanisms. Entomopathogenic nematode symbiotic bacteria (ENS), including Xenorhabdus and Photorhabdus, are Gram-negative bacteria transported into insect hosts by Steinernema or Heterorhabdus. Flagella are conserved in ENS, but the T3SS is only encoded in Photorhabdus. There are few reports on the function of flagella and the T3SS in ENS, and it is not known what role they play in the infection of ENS. Here, we clarified the function of the T3SS and flagella in ENS infection based on flagellar inactivation in X. stockiae (flhDC deletion), T3SS inactivation in P. luminescens (sctV deletion), and the heterologous synthesis of the T3SS of P. luminescens in X. stockiae. Consistent with the previous results, the swarming movement of the ENS and the formation of biofilms are dominated by the flagella. Both the T3SS and flagella facilitate ENS invasion and colonization within host cells, with minimal impact on secondary metabolite formation and secretion. Unexpectedly, a proteomic analysis reveals a negative feedback loop between the flagella/T3SS assembly and the type VI secretion system (T6SS). RT-PCR testing demonstrates the T3SS's inhibition of flagellar assembly, while flagellin expression promotes T3SS assembly. Furthermore, T3SS expression stimulates ribosome-associated protein expression. [ABSTRACT FROM AUTHOR]

Despite being considered a normal flora, Providencia alcalifaciens can cause diarrhea. In a previous study, strain 2939/90, obtained from a diarrheal patient, caused invasion and actin condensation in mammalian cells, and diarrhea in a rabbit model. Four TnphoA mutants of 2939/90 produced negligible invasion and actin condensation in mammalian cells. Now, the parent strain and the mutants have been sequenced to locate TnphoA insertion sites and determine the effect on virulence. A TnphoA insertion was detected in the type three secretion system (T3SS) locus on a large plasmid and not in a T3SS locus on the chromosome. In 52 genomes of P. alcalifaciens surveyed, the chromosomal T3SS locus was present in all strains, including both P. alcalifaciens genomic clades, which we classified as group A and group B. Plasmid T3SS was present in 21 of 52 genomes, mostly in group A genomes, which included isolates from an outbreak of hemorrhagic diarrhea in dogs. The TnphoA insertion only in the plasmid T3SS locus affected the invasion phenotype, suggested that this locus is critical for causation of diarrhea. We conclude that a subgroup of P. alcalifaciens that possesses this plasmid-mediated T3SS is an enteric pathogen that can cause diarrheal disease. [ABSTRACT FROM AUTHOR]

ABSTRACT Melioidosis is a serious infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Recently, Rab32-dependent immune vesicles emerge as a critical defense pathway to restrict the intracellular B. pseudomallei. However, B. pseudomallei can evade host immune vesicles and survive in the cytoplasm, although this mechanism is not well understood. In this study, we found Rab32-dependent vesicles could effectively combat B. pseudomallei infection, but not all intracellular B. pseudomallei were encapsulated in Rab32-positive vesicles. To explore how B. pseudomallei counteracted the Rab32-dependent defense pathway, transcriptomic profiling of B. pseudomallei was performed to characterize the response dynamics during infection. We found that the type III secretion system of B. pseudomallei was activated, and a variety of effector proteins were highly upregulated. Among them, BopE, BprD, and BipC were shown to interact with Rab32. Interestingly, BopE directly interacts with host Rab32, potentially suppressing Rab32 function by interfering with nucleotide exchange, which in turn restricts the recruitment of Rab32 to bacterial-containing vesicles. Knocking out of BopE can increase the proportion of Rab32-positive vesicles, suppressing the intracellular replication and virulence of B. pseudomallei. Collectively, our findings have demonstrated that BopE may be an important effector for B. pseudomallei to evade from the Rab32-dependent killing vesicles into the cytosol for survival and replication. Therefore, a deeper understanding of the interaction between BopE and the host Rab32-dependent restriction pathway may provide an effective therapeutic strategy for the elimination of intracellular B. pseudomallei.IMPORTANCEB. pseudomallei is facultative intracellular bacterium that has evolved numerous strategies to evade host immune vesicles and survive in the cytoplasm. Rab32-dependent vesicles are one of these immune vesicles, but the mechanism by which B. pseudomallei escape Rab32-dependent vesicles remains elusive. Here, we find B. pseudomallei infection leading the activation of the type III secretion system (T3SS-3) and increasing the expression of various effectors. Specifically, we identify that BopE, an effector secreted by T3SS-3, triggers vesicle escape to promote B. pseudomallei pathogenicity and survival. Mechanistically, BopE suppresses the activation of Rab32 by interfering with nucleotide exchange, ultimately triggering vesicle escape and intracellular survival. We also find knocking out the bopE gene can increase the proportion of Rab32-positive vesicles that trap B. pseudomallei, dampening the survival of B. pseudomallei both in vitro and in vivo. Taken together, our findings provide insights into the molecular mechanisms of pathogen effector-induced vesicle escape, indicating a potential melioidosis treatment via blocking B. pseudomallei BopE-host Rab32 interaction.

ABSTRACT 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.

Pseudomonas aeruginosa isolates were recovered from surface river water samples in La Rioja region (Spain) to characterise their antibiotic resistance, molecular typing and virulence mechanisms. Fifty-two P. aeruginosa isolates were isolated from 15 different water samples (45.4%) and belonged to 23 different pulsed-field electrophoresis (PFGE) patterns. All isolates were susceptible to all antibiotics tested, except one carbapenem-resistant P. aeruginosa that showed a premature stop codon in OprD porin. Twenty-two sequence types (STs) (six new ones) were detected among 29 selected P. aeruginosa (one strain with a different PFGE pattern per sample), with ST274 (14%) being the most frequent one. O:6 and O:3 were the predominant serotypes (31%). Seven virulotypes were detected, being 59% exoS-exoY-exoT-exoA-lasA-lasB-lasI-lasR-rhlAB - rhlI - rhlR - aprA -positive P. aeruginosa. It is noteworthy that the exlA gene was identified in three strains (10.3%), and the exoU gene in seven (24.1%), exoS in 18 (62.1%), and both exoS and exoU genes in one strain. High motility ranges were found in these strains. Twenty-seven per cent of strains produced more biofilm biomass, 90% more pyorubin, 83% more pyocyanin and 65.5% more than twice the elastase activity compared with the PAO1 strain. These results highlight the importance of rivers as temporary reservoirs and sources of P. aeruginosa transmission, and show the importance of their epidemiological surveillance in the environment. [ABSTRACT FROM AUTHOR]

Bacterial secretion systems, such as the type 3, 4, and 6 are multiprotein nanomachines expressed at the surface of pathogens with Gram‐negative like envelopes. They are known to be crucial for virulence and to translocate bacteria‐encoded effector proteins into host cells to manipulate cellular functions. This facilitates either pathogen attachment or invasion of the targeted cell. Effector proteins also promote evasion of host immune recognition. Imaging by cryo‐electron microscopy in combination with structure determination has become a powerful approach to understand how these nanomachines work. Still, questions on their assembly, the precise secretion mechanisms, and their direct involvement in pathogenicity remain unsolved. Here, we present an overview of the recent developments in in situ cryo‐electron microscopy. We discuss its potential for the investigation of the role of bacterial secretion systems during the host‐bacterial crosstalk at the molecular level. These in situ studies open new perspectives for our understanding of secretion system structure and function. [ABSTRACT FROM AUTHOR]

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.

Despite being considered a normal flora, Providencia alcalifaciens can cause diarrhea. In a previous study, strain 2939/90, obtained from a diarrheal patient, caused invasion and actin condensation in mammalian cells, and diarrhea in a rabbit model. Four TnphoA mutants of 2939/90 produced negligible invasion and actin condensation in mammalian cells. Now, the parent strain and the mutants have been sequenced to locate TnphoA insertion sites and determine the effect on virulence. A TnphoA insertion was detected in the type three secretion system (T3SS) locus on a large plasmid and not in a T3SS locus on the chromosome. In 52 genomes of P. alcalifaciens surveyed, the chromosomal T3SS locus was present in all strains, including both P. alcalifaciens genomic clades, which we classified as group A and group B. Plasmid T3SS was present in 21 of 52 genomes, mostly in group A genomes, which included isolates from an outbreak of hemorrhagic diarrhea in dogs. The TnphoA insertion only in the plasmid T3SS locus affected the invasion phenotype, suggested that this locus is critical for causation of diarrhea. We conclude that a subgroup of P. alcalifaciens that possesses this plasmid-mediated T3SS is an enteric pathogen that can cause diarrheal disease.

Abstract Xrv proteins are a group of regulators in Xanthomonas spp., belonging to the histone-like nucleoid-structuring (H-NS) proteins of Gram-negative bacteria. The rice bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) harbors three Xrv proteins, the XrvA, XrvB, and XrvC. Here, we report that in Xoc, the XrvB but not XrvA and XrvC is involved in negative regulation of the type III secretion system (T3SS) encoded by hrp genes. As with other Xanthomonas spp., the T3SS is an essential virulence determinant of Xoc and the expression of the hrp genes in Xoc is controlled by the HrpG/HrpX regulatory cascade. HrpG positively regulates the expression of HrpX, which in turn activates the transcription of the hrp genes. We provide evidences to demonstrate that the XrvB binds to the promoter region of hrpG and represses its transcription. Furthermore, we found that XrvB production was induced in the Xoc cells cultured in a nutrient-rich medium compared to a hrp-inducing minimal medium. We also found that in Xoc, the hrpG expression level is inversely correlated with the content of XrvB, and XrvB occupancy at hrpG promoter region is positively correlated with XrvB levels. Our data suggest that XrvB is a determinative factor controlling the expression levels of HrpG. In addition, mutation analysis revealed that the Xoc XrvB also plays positive roles in regulating bacterial growth, cell motility, and stress tolerance. Our findings provide important insights into the molecular mechanism of T3SS expression regulation in Xoc.

ABSTRACTPseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals. Small regulatory RNAs (sRNAs) regulate multiple bacterial adaptations to environmental changes, especially virulence. Our previous study showed that sRNA PrrH negatively regulates the expression of a number of virulence factors, such as pyocyanin, rhamnolipid, biofilm, and elastase in the P. aeruginosa strain PAO1. However, previous studies have shown that the prrH-deficient mutant attenuates virulence in an acute murine lung infection model. All ΔprrH-infected mice survived the entire 28-day course of the experiment, whereas all mice inoculated with the wild-type or the complemented mutant succumbed to lung infection within 4 days of injection, but the specific mechanism is unclear. Herein, we explored how PrrH mediates severe lung injury by regulating the expression of virulence factors. In vivo mouse and in vitro cellular assays demonstrated that PrrH enhanced the pathogenicity of PAO1, causing severe lung injury. Mechanistically, PrrH binds to the coding sequence region of the mRNA of exsA, which encodes the type III secretion system master regulatory protein. We further demonstrated that PrrH mediates a severe inflammatory response and exacerbates the apoptosis of A549 cells. Overall, our results revealed that PrrH positively regulates ExsA, enhances the pathogenicity of P. aeruginosa, and causes severe lung injury.IMPORTANCEPseudomonas aeruginosa is a Gram-negative bacterium and the leading cause of nosocomial pneumonia. The pathogenicity of P. aeruginosa is due to the secretion of many virulence factors. Small regulatory RNAs (sRNAs) regulate various bacterial adaptations, especially virulence. Therefore, understanding the mechanism by which sRNAs regulate virulence is necessary for understanding the pathogenicity of P. aeruginosa and the treatment of the related disease. In this study, we demonstrated that PrrH enhances the pathogenicity of P. aeruginosa by binding to the coding sequence regions of the ExsA, the master regulatory protein of type III secretion system, causing severe lung injury and exacerbating the inflammatory response and apoptosis. These findings revealed that PrrH is a crucial molecule that positively regulates ExsA. Type III-positive strains are often associated with a high mortality rate in P. aeruginosa infections in clinical practice. Therefore, this discovery may provide a new target for treating P. aeruginosa infections, especially type III-positive strains.

ABSTRACT Pseudomonas aeruginosa is a major opportunistic pathogen that can cause severe systemic infections. Here, we found that the type III secretion system effector ExoU was the main determinant of pathogenicity of a highly virulent clinical isolate strain, BSI_S5, of P. aeruginosa, which caused severe bloodstream infections and belongs to ST463/O4. Deletion of exoU showed significantly attenuated cytotoxicity and virulence in vivo, while deletion of two other unique genes from strain BSI_S5, chr_1696 and chr_4238, which encodes type I secretion system permease/ATPase or polysaccharide biosynthesis protein, respectively, caused no apparent loss of toxicity compared to the parent strain, and complementation of the ΔexoU mutant restored its virulence. The presence of ExoU-induced lung lesions and the mucosal damage of gallbladder in mice, and the replication of the ΔexoU mutant was hindered in vivo compared with the parent strain. We show here for the first time that a clinical isolate with intact exoU harbored a mutation of T→G substitution in the adjacent specific Pseudomonas chaperone for ExoU (spcU), which caused phenylalanine to valine change at amino acid 94 of SpcU, resulting in significant loss of cytotoxicity. This finding suggests that intact exoU is not sufficient for cytotoxicity, but a functional downstream SpcU is required for ExoU secretion and cytotoxicity. Finally, we found that BSI_S5 harbored two types of insertion sequences adjacent to exoU, suggesting the potential of exoU to be transferred to other strains. Our study opens an avenue for further mechanistic study of P. aeruginosa systemic infections regarding its high pathogenicity and clinical spread. IMPORTANCE The identification of decisive virulence-associated genes in highly pathogenic P. aeruginosa isolates in the clinic is essential for diagnosis and the start of appropriate treatment. Over the past decades, P. aeruginosa ST463 has spread rapidly in East China and is highly resistant to β-lactams. Given the poor clinical outcome caused by this phenotype, detailed information regarding its decisive virulence genes and factors affecting virulence expression needs to be deciphered. Here, we demonstrate that the T3SS effector ExoU has toxic effects on mammalian cells and is required for virulence in the murine bloodstream infection model. Moreover, a functional downstream SpcU is required for ExoU secretion and cytotoxicity. This work highlights the potential role of ExoU in the pathogenesis of disease and provides a new perspective for further research on the development of new antimicrobials with antivirulence ability.

ABSTRACT Type VI secretion system (T6SS) is a versatile, contact-dependent contractile nano-weapon in Gram-negative bacteria that fires proteinaceous effector molecules directly into prokaryotic and eukaryotic cells aiding in manipulation of the host and killing of competitors in complex niches. In plant pathogenic xanthomonads, T6SS has been demonstrated to play these diverse roles in individual pathosystems. However, the molecular network underlying the regulation of T6SS is still elusive in Xanthomonas spp. To bridge this knowledge gap, we conducted an in vitro transcriptome screen using plant apoplast mimicking minimal medium, XVM2 medium, to decipher the effect of tssM deletion, a core gene belonging to T6SS-cluster i3*, on the regulation of gene expression in Xanthomonas perforans strain AL65. Transcriptomic data revealed that a total of 277 and 525 genes were upregulated, while 307 and 392 genes were downregulated in the mutant strain after 8 and 16 hours of growth in XVM2 medium. The transcript abundance of several genes associated with flagellum and pilus biogenesis as well as type III secretion system was downregulated in the mutant strain. Deletion of tssM of cluster-i3* resulted in upregulation of several T6SS genes belonging to cluster-i3*** and genes involved in biofilm and cell wall biogenesis. Similarly, transcription regulators like rpoN, Pho regulon, rpoE, and csrA were identified to be upregulated in the mutant strain. Our results suggest that T6SS modulates the expression of global regulators like csrA, rpoN, and pho regulons, triggering a signaling cascade, and co-ordinates the expression of suite of virulence factors, stress response genes, and metabolic genes. IMPORTANCE T6SS has received attention due to its significance in mediating interorganismal competition through contact-dependent release of effector molecules into prokaryotic and eukaryotic cells. Reverse-genetic studies have indicated the role of T6SS in virulence in a variety of plant pathogenic bacteria, including the one studied here, Xanthomonas. However, it is not clear whether such effect on virulence is merely due to a shift in the microbiome-mediated protection or if T6SS is involved in a complex virulence regulatory network. In this study, we conducted in vitro transcriptome profiling in minimal medium to decipher the signaling pathways regulated by tssM-i3* in X. perforans AL65. We show that TssM-i3* regulates the expression of a suite of genes associated with virulence and metabolism either directly or indirectly by altering the transcription of several regulators. These findings further expand our knowledge on the intricate molecular circuits regulated by T6SS in phytopathogenic bacteria.

Many pathogens use Type III and Type IV protein secretion systems to secrete virulence factors from the bacterial cytosol into host cells. These systems operate through a one-step mechanism. The secreted substrates (protein or nucleo-protein complexes in the case of Type IV conjugative systems) are guided to the base of the secretion channel, where they are directly delivered into the host cell in an ATP-dependent unfolded state. Despite the numerous disparities between these secretion systems, here we have focused on the structural and functional similarities between both systems. In particular, on the structural similarity shared by one of the main ATPases (EscN and VirD4 in Type III and Type IV secretion systems, respectively). Interestingly, these ATPases also exhibit a structural resemblance to F1-ATPases, which suggests a common mechanism for substrate secretion. The correlation between structure and function of essential components in both systems can provide significant insights into the molecular mechanisms involved. This approach is of great interest in the pursuit of identifying inhibitors that can effectively target these systems. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a variety of acute and chronic infections. Its type III secretion system (T3SS) plays a critical role in pathogenesis during acute infection. ExsA is a master regulator that activates the expression of all T3SS genes. Transcription of exsA is driven by two distinct promoters, its own promoter PexsA and its operon promoter PexsC. Here, in combination with a DNA pull-down assay and mass spectrometric analysis, we found that a histone-like nucleoid-structuring (H-NS) family protein MvaT can bind to the PexsC promoter. Using EMSA and reporter assays, we further found that MvaT directly binds to the PexsC promoter to repress the expression of T3SS genes. The repression of MvaT on PexsC is independent of ExsA, with MvaT binding to the -429 to -380 bp region relative to the transcription start site of the exsC gene. The presented work further reveals the complex regulatory network of the T3SS in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Summary: A prophage tail‐like protein (Bg_9562) of Burkholderia gladioli strain NGJ1 possesses broad‐spectrum antifungal activity, and it is required for the bacterial ability to forage over fungi. Here, we analyzed whether heterologous overexpression of Bg_9562 or exogenous treatment with purified protein can impart disease tolerance in tomato. The physiological relevance of Bg_9562 during endophytic growth of NGJ1 was also investigated.Bg_9562 overexpressing lines demonstrate fungal and bacterial disease tolerance. They exhibit enhanced expression of defense genes and activation of mitogen‐activated protein kinases. Treatment with Bg_9562 protein induces defense responses and imparts immunity in wild‐type tomato.The defense‐inducing ability lies within 18–51 aa region of Bg_9562 and is due to sequence homology with the bacterial flagellin epitope. Interaction studies suggest that Bg_9562 is perceived by FLAGELLIN‐SENSING 2 homologs in tomato. The silencing of SlSERK3s (BAK1 homologs) prevents Bg_9562‐triggered immunity. Moreover, type III secretion system‐dependent translocation of Bg_9562 into host apoplast is important for elicitation of immune responses during colonization of NGJ1.Our study emphasizes that Bg_9562 is important for the endophytic growth of B. gladioli, while the plant perceives it as an indirect indicator of the presence of bacteria to mount immune responses. The findings have practical implications for controlling plant diseases. [ABSTRACT FROM AUTHOR]

Xanthomonas translucens pv. cerealis causes bacterial leaf streak disease on small grain cereals. Type II and III secretion systems (T2SS and T3SS) play a pivotal role in the pathogenicity of the bacterium, while no data are available on the transcriptomic profile of wheat cultivars infected with either wild type (WT) or mutants of the pathogen. In this study, WT, TAL-effector mutants, and T2SS/T3SS mutants of X. translucens pv. cerealis strain NXtc01 were evaluated for their effect on the transcriptomic profile of two wheat cultivars, 'Chinese Spring' and 'Yangmai-158', using Illumina RNA-sequencing technology. RNA-Seq data showed that the number of differentially expressed genes (DEGs) was higher in Yangmai-158 than in Chinese Spring, suggesting higher susceptibility of Yangmai-158 to the pathogen. In T2SS, most suppressed DEGs were related to transferase, synthase, oxidase, WRKY, and bHLH transcription factors. The gspD mutants showed significantly decreased disease development in wheat, suggesting an active contribution of T2SS in virulence. Moreover, the gspD mutant restored full virulence and its multiplication in planta by addition of gspD in trans. In the T3SS-deficient strain, downregulated DEGs were associated with cytochrome, peroxidases, kinases, phosphatases, WRKY, and ethylene-responsive transcription factors. In contrast, upregulated DEGs were trypsin inhibitors, cell number regulators, and calcium transporter. Transcriptomic analyses coupled with quantitative real-time-PCR indicated that some genes are upregulated in Δtal1/Δtal2 compared with the tal-free strain, but no direct interaction was observed. These results provide novel insight into wheat transcriptomes in response to X. translucens infection and pave the way for understanding host-pathogen interactions. [ABSTRACT FROM AUTHOR]

Polymicrobial infections threaten the health of humans and animals but remain understudied in natural systems. We recently described the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease affecting oyster production worldwide. In the French Atlantic coast, the disease involves coinfection with ostreid herpesvirus 1 (OsHV-1) and virulent Vibrio. However, it is unknown whether consistent Vibrio populations are associated with POMS in different regions, how Vibrio contribute to POMS, and how they interact with OsHV-1 during pathogenesis. By connecting field-based approaches in a Mediterranean ecosystem, laboratory infection assays and functional genomics, we uncovered a web of interdependencies that shape the structure and function of the POMS pathobiota. We show that Vibrio harveyi and Vibrio rotiferianus are predominant in OsHV-1-diseased oysters and that OsHV-1 drives the partition of the Vibrio community observed in the field. However only V. harveyi synergizes with OsHV-1 by promoting mutual growth and accelerating oyster death. V. harveyi shows high-virulence potential and dampens oyster cellular defenses through a type 3 secretion system, making oysters a more favorable niche for microbe colonization. In addition, V. harveyi produces a key siderophore called vibrioferrin. This important resource promotes the growth of V. rotiferianus, which cooccurs with V. harveyi in diseased oysters, and behaves as a cheater by benefiting from V. harveyi metabolite sharing. Our data show that cooperative behaviors contribute to synergy between bacterial and viral coinfecting partners. Additional cheating behaviors further shape the polymicrobial consortium. Controlling cooperative behaviors or countering their effects opens avenues for mitigating polymicrobial diseases. [ABSTRACT FROM AUTHOR]

The pyruvate dehydrogenase complex regulator (PdhR) was originally identified as a repressor of the pdhR-aceEF-lpd operon, which encodes the pyruvate dehydrogenase complex (PDHc) and PdhR itself. According to previous reports, PdhR plays a regulatory role in the physiological and metabolic pathways of bacteria. At present, the function of PdhR in Plesiomonas shigelloides is still poorly understood. In this study, RNA sequencing (RNA-Seq) of the wild-type strain and the ΔpdhR mutant strains was performed for comparison to identify the PdhR-controlled pathways, revealing that PdhR regulates ~7.38% of the P. shigelloides transcriptome. We found that the deletion of pdhR resulted in the downregulation of practically all polar and lateral flagella genes in P. shigelloides; meanwhile, motility assay and transmission electron microscopy (TEM) confirmed that the ΔpdhR mutant was non-motile and lacked flagella. Moreover, the results of RNA-seq and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) showed that PdhR positively regulated the expression of the T3SS cluster, and the ΔpdhR mutant significantly reduced the ability of P. shigelloides to infect Caco-2 cells compared with the WT. Consistent with previous research, pyruvate-sensing PdhR directly binds to its promoter and inhibits pdhR-aceEF-lpd operon expression. In addition, we identified two additional downstream genes, metR and nuoA, that are directly negatively regulated by PdhR. Furthermore, we also demonstrated that ArcA was identified as being located upstream of pdhR and lpdA and directly negatively regulating their expression. Overall, we revealed the function and regulatory pathway of PdhR, which will allow for a more in-depth investigation into P. shigelloides pathogenicity as well as the complex regulatory network. [ABSTRACT FROM AUTHOR]

ABSTRACT Vibrio cholerae O1 has caused cholera pandemics. Non-pandemic V. cholerae O1 strains, which are genetically distinctive from the pandemic clones, have been isolated from both human infections and the environment. We aimed to better understand the non-pandemic O1 strains and their pandemic potential. We sequenced 109 non-pandemic O1 isolates from Zhejiang, China (from 1963 to 1996) and compared them with 62 publicly available non-pandemic O1 genomes. The isolates from Zhejiang can be classified into three lineages (L1–L3). All grouped together with L3 sharing the most recent common ancestor with the pandemic clones. L2 and L3 emerged in the 1960s while L1 emerged in the 1970s. L1 and L2 disappeared after the 1990s, but L3 persisted until recently. All isolates contained the type VI secretion system. The Vibrio pathogenicity island was present in all L3 isolates, whereas the type III secretion system was present in all L1 isolates. L2 did not carry any unique virulence genes. An intact CTXφ was present in only two L3 isolates. An intact Vibrio seventh pandemic island 1 was present in only three L3 isolates. The blaCARB-7 gene was identified in 96.3% of L2 isolates. Each of the non-pandemic O1 lineages has unique properties contributing to their capacity to cause disease. Our findings offer new insight into the evolution of O1 V. cholerae for cholera prevention and control. IMPORTANCE It is well recognized that only Vibrio cholerae O1 causes cholera pandemics. However, not all O1 strains cause pandemic-level disease. In this study, we analyzed non-pandemic O1 V. cholerae isolates from the 1960s to the 1990s from China and found that they fell into three lineages, one of which shared the most recent common ancestor with pandemic O1 strains. Each of these non-pandemic O1 lineages has unique properties that contribute to their capacity to cause cholera. The findings of this study enhanced our understanding of the emergence and evolution of both pandemic and non-pandemic O1 V. cholerae.

Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA.In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.

Many pathogens use Type III and Type IV protein secretion systems to secrete virulence factors from the bacterial cytosol into host cells. These systems operate through a one-step mechanism. The secreted substrates (protein or nucleo-protein complexes in the case of Type IV conjugative systems) are guided to the base of the secretion channel, where they are directly delivered into the host cell in an ATP-dependent unfolded state. Despite the numerous disparities between these secretion systems, here we have focused on the structural and functional similarities between both systems. In particular, on the structural similarity shared by one of the main ATPases (EscN and VirD4 in Type III and Type IV secretion systems, respectively). Interestingly, these ATPases also exhibit a structural resemblance to F1-ATPases, which suggests a common mechanism for substrate secretion. The correlation between structure and function of essential components in both systems can provide significant insights into the molecular mechanisms involved. This approach is of great interest in the pursuit of identifying inhibitors that can effectively target these systems.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a variety of acute and chronic infections. Its type III secretion system (T3SS) plays a critical role in pathogenesis during acute infection. ExsA is a master regulator that activates the expression of all T3SS genes. Transcription of exsA is driven by two distinct promoters, its own promoter PexsA and its operon promoter PexsC. Here, in combination with a DNA pull-down assay and mass spectrometric analysis, we found that a histone-like nucleoid-structuring (H-NS) family protein MvaT can bind to the PexsC promoter. Using EMSA and reporter assays, we further found that MvaT directly binds to the PexsC promoter to repress the expression of T3SS genes. The repression of MvaT on PexsC is independent of ExsA, with MvaT binding to the -429 to -380 bp region relative to the transcription start site of the exsC gene. The presented work further reveals the complex regulatory network of the T3SS in P. aeruginosa.

Shigellosis (bacillary dysentery) is a severe gastrointestinal infection with a global incidence of 90 million cases annually. Despite the severity of this disease, there is currently no licensed vaccine against shigellosis. Shigella's primary virulence factor is its type III secretion system (T3SS), which is a specialized nanomachine used to manipulate host cells. A fusion of T3SS injectisome needle tip protein IpaD and translocator protein IpaB, termed DBF, when admixed with the mucosal adjuvant double-mutant labile toxin (dmLT) from enterotoxigenic E. coli was protective using a murine pulmonary model. To facilitate the production of this platform, a recombinant protein that consisted of LTA-1, the active moiety of dmLT, and DBF were genetically fused, resulting in L-DBF, which showed improved protection against Shigella challenge. To extrapolate this protection from mice to humans, we modified the formulation to provide for a multivalent presentation with the addition of an adjuvant approved for use in human vaccines. Here, we show that L-DBF formulated (admix) with a newly developed TLR4 agonist called BECC438 (a detoxified lipid A analog identified as Bacterial Enzymatic Combinatorial Chemistry candidate #438), formulated as an oil-in-water emulsion, has a very high protective efficacy at low antigen doses against lethal Shigella challenge in our mouse model. Optimal protection was observed when this formulation was introduced at a mucosal site (intranasally). When the formulation was then evaluated for the immune response it elicits, protection appeared to correlate with high IFN-γ and IL-17 secretion from mucosal site lymphocytes. [ABSTRACT FROM AUTHOR]

Two of the most fascinating bacterial nanomachines—the broadly disseminated rotary flagellum at the heart of cellular motility and the eukaryotic cell–puncturing injectisome essential to specific pathogenic species—utilize at their core a conserved export machinery called the type III secretion system (T3SS). The T3SS not only secretes the components that self-assemble into their extracellular appendages but also, in the case of the injectisome, subsequently directly translocates modulating effector proteins from the bacterial cell into the infected host. The injectisome is thought to have evolved from the flagellum as a minimal secretory system lacking motility, with the subsequent acquisition of additional components tailored to its specialized role in manipulating eukaryotic hosts for pathogenic advantage. Both nanomachines have long been the focus of intense interest, but advances in structural and functional understanding have taken a significant step forward since 2015, facilitated by the revolutionary advances in cryo-electron microscopy technologies. With several seminal structures of each nanomachine now captured, we review here the molecular similarities and differences that underlie their diverse functions. [ABSTRACT FROM AUTHOR]

Xrv proteins are a group of regulators in Xanthomonas spp., belonging to the histone-like nucleoid-structuring (H-NS) proteins of Gram-negative bacteria. The rice bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) harbors three Xrv proteins, the XrvA, XrvB, and XrvC. Here, we report that in Xoc, the XrvB but not XrvA and XrvC is involved in negative regulation of the type III secretion system (T3SS) encoded by hrp genes. As with other Xanthomonas spp., the T3SS is an essential virulence determinant of Xoc and the expression of the hrp genes in Xoc is controlled by the HrpG/HrpX regulatory cascade. HrpG positively regulates the expression of HrpX, which in turn activates the transcription of the hrp genes. We provide evidences to demonstrate that the XrvB binds to the promoter region of hrpG and represses its transcription. Furthermore, we found that XrvB production was induced in the Xoc cells cultured in a nutrient-rich medium compared to a hrp-inducing minimal medium. We also found that in Xoc, the hrpG expression level is inversely correlated with the content of XrvB, and XrvB occupancy at hrpG promoter region is positively correlated with XrvB levels. Our data suggest that XrvB is a determinative factor controlling the expression levels of HrpG. In addition, mutation analysis revealed that the Xoc XrvB also plays positive roles in regulating bacterial growth, cell motility, and stress tolerance. Our findings provide important insights into the molecular mechanism of T3SS expression regulation in Xoc. [ABSTRACT FROM AUTHOR]

A series of novel 5‐amido‐2‐carboxypyrazine derivatives were designed, synthesized and evaluated for the inhibitory activities against the T3SS of Salmonella enterica serovar Typhimurium. Preliminary results displayed that the compounds 2f, 2g, 2h and 2i showed potent inhibitory activities against T3SS. Compound 2h was identified as the most potent T3SS inhibitor and the SPI‐1 effector secretion was strongly inhibited by 2h in a dose‐dependent manner. The effects of compound 2h on the SPI‐1 genes transcription might be via impacting the SicA/InvF regulatory pathway. [ABSTRACT FROM AUTHOR]

Bacterial injectisomes are crucial nanomachines responsible for the direct secretion into host cells of a variety of substrates, ranging from proteins to DNAs. Prior to being injected, these substrates must be recruited and sometimes processed by large complexes called substrate recruitment platforms. These platforms are characterized by: • their ability to bind effectors and effector/chaperone complexes; • their ability to target the substrates to the injectisome channel for subsequent secretion; • their diversity and complexity, some made of one component, others forming large complexes; • their diverse modes of action, some requiring an ATPase, others where the core components of the injectisome act as the payload carrier. Bacteria use a wide arsenal of macromolecular substrates (DNA and proteins) to interact with or infect prokaryotic and eukaryotic cells. To do so, they utilize substrate-injecting secretion systems or injectisomes. However, prior to secretion, substrates must be recruited to specialized recruitment platforms and then handed over to the secretion apparatus for secretion. In this review, we provide an update on recent advances in substrate recruitment and delivery by gram-negative bacterial recruitment platforms associated with Type III, IV, and VI secretion systems. [ABSTRACT FROM AUTHOR]

Salmonella enterica, a Gram-negative pathogen, has over 2500 serovars that infect a wide range of hosts. In humans, S. enterica causes typhoid or gastroenteritis and is a major public health concern. In this study, SseB (the tip protein of the Salmonella pathogenicity island 2 type III secretion system) was fused with the LTA1 subunit of labile-toxin from enterotoxigenic E. coli to make the self-adjuvanting antigen L-SseB. Two unique nanoparticle formulations were developed to allow multimeric presentation of L-SseB. Mice were vaccinated with these formulations and protective efficacy determined via challenging the mice with S. enterica serovars. The polysaccharide (chitosan) formulation was found to elicit better protection when compared to the squalene nanoemulsion. When the polysaccharide formulation was used to vaccinate rabbits, protection from S. enterica challenge was elicited. In summary, L-SseB in a particulate polysaccharide formulation appears to be an attractive candidate vaccine capable of broad protection against S. enterica. [ABSTRACT FROM AUTHOR]

Pseudomonas syringae pv. aptata is a member of the sugar beet pathobiome and the causative agent of leaf spot disease. Like many pathogenic bacteria, P. syringae relies on the secretion of toxins, which manipulate host-pathogen interactions, to establish and maintain an infection. This study analyzes the secretome of six pathogenic P. syringae pv. aptata strains with different defined virulence capacities in order to identify common and strain-specific features, and correlate the secretome with disease outcome. All strains show a high type III secretion system (T3SS) and type VI secretion system (T6SS) activity under apoplast-like conditions mimicking the infection. Surprisingly, we found that low pathogenic strains show a higher secretion of most T3SS substrates, whereas a distinct subgroup of four effectors was exclusively secreted in medium and high pathogenic strains. Similarly, we detected two T6SS secretion patterns: while one set of proteins was highly secreted in all strains, another subset consisting of known T6SS substrates and previously uncharacterized proteins was exclusively secreted in medium and high virulence strains. Taken together, our data show that P. syringae pathogenicity is correlated with the repertoire and fine-tuning of effector secretion and indicate distinct strategies for establishing virulence of P. syringae pv. aptata in plants. [ABSTRACT FROM AUTHOR]

Abstract Background Sufficient nutrition contributes to rapid translational elongation and protein synthesis in eukaryotic cells and prokaryotic bacteria. Fast synthesis and accumulation of type III secretion system (T3SS) proteins conduce to the invasion of pathogenic bacteria into the host cells. However, the translational elongation patterns of T3SS proteins in pathogenic bacteria under T3SS-inducing conditions remain unclear. Here, we report a mechanism of translational elongation of T3SS regulators, effectors and structural protein in four model pathogenic bacteria (Pseudomonas syringae, Pseudomonas aeruginosa, Xanthomonas oryzae and Ralstonia solanacearum) and a clinical isolate (Pseudomonas aeruginosa UCBPP-PA14) under nutrient-limiting conditions. We proposed a luminescence reporter system to quantitatively determine the translational elongation rates (ERs) of T3SS regulators, effectors and structural protein under different nutrient-limiting conditions and culture durations. Results The translational ERs of T3SS regulators, effectors and structural protein in these pathogenic bacteria were negatively regulated by the nutrient concentration and culture duration. The translational ERs in 0.5× T3SS-inducing medium were the highest of all tested media. In 1× T3SS-inducing medium, the translational ERs were highest at 0 min and then rapidly decreased. The translational ERs of T3SS regulators, effectors and structural protein were inhibited by tRNA degradation and by reduced levels of elongation factors (EFs). Conclusions Rapid translational ER and synthesis of T3SS protein need adequate tRNAs and EFs in nutrient-limiting conditions. Numeric presentation of T3SS translation visually indicates the invasion of bacteria and provides new insights into T3SS expression that can be applied to other pathogenic bacteria.

Ying Zhang,1,* Lingbo Wang,1,* Liqiong Chen,2 Peiwu Zhu,1 Na Huang,1 Tao Chen,1 Lijiang Chen,1 Zhongyong Wang,1 Wenli Liao,1 Jianming Cao,2 Tieli Zhou1 1Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, Wenzhou, People’s Republic of China; 2Department of Medical Laboratory Science, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, People’s Republic of China*These authors contributed equally to this workCorrespondence: Tieli Zhou, Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, Wenzhou, 325035, People’s Republic of China, Tel +86-577-8668-9885, Email wyztli@163.com Jianming Cao, Department of Medical Laboratory Science, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, 325035, People’s Republic of China, Tel +86-577-88069595, Email wzcjming@163.comIntroduction: Globally, Pseudomonas aeruginosa (PA) is emerging as a predominant nosocomial pathogen that often induces aggressive and even deadly infections. Pseudomonas type III repressor A (PtrA) can be activated specifically by copper ions and interacts with type-III transcriptional activator ExsA. This study aims to provide insight into the PtrA-mediated regulation of the pathogenicity and antibiotics resistance of PA.Methods and Results: The results of transcriptome sequencing analyses and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) showed that PtrA plays a dual regulatory role in the virulence systems of PA: negatively regulates the type-III secretion system (T3SS) and positively regulates the quorum-sensing system (QS). The ptrA mutant attenuated extracellular virulence related to QS like pyocyanin, elastase, rhamnolipids, proteolytic activity, and biofilm production. According to adhesion and invasion experiments, PtrA can not only contribute to the adhesiveness but also the invasive of PA. Moreover, the PtrA-mediated regulation of PA pathogenicity was determined both in vivo and in vitro through cytotoxicity and Galleria mellonella survival experiments. In addition, apart from virulence, PtrA was found to influence the carbapenems resistance of PA. After deleting ptrA, the minimum inhibitory concentration (MIC) of carbapenems antibiotics was decreased by 2-fold, while a 2– 8 fold increase was noted for the complemented strain.Conclusion: Our findings establish that PtrA exerts a regulatory role in both pathogenicity and carbapenems resistance of PA. This work may shed light on a novel target for the clinical treatment of PA.Keywords: Pseudomonas aeruginosa, PtrA, T3SS, Quorum-sensing, pathogenicity, carbapenems resistance

Salmonella enterica, a Gram-negative pathogen, has over 2500 serovars that infect a wide range of hosts. In humans, S. enterica causes typhoid or gastroenteritis and is a major public health concern. In this study, SseB (the tip protein of the Salmonella pathogenicity island 2 type III secretion system) was fused with the LTA1 subunit of labile-toxin from enterotoxigenic E. coli to make the self-adjuvanting antigen L-SseB. Two unique nanoparticle formulations were developed to allow multimeric presentation of L-SseB. Mice were vaccinated with these formulations and protective efficacy determined via challenging the mice with S. enterica serovars. The polysaccharide (chitosan) formulation was found to elicit better protection when compared to the squalene nanoemulsion. When the polysaccharide formulation was used to vaccinate rabbits, protection from S. enterica challenge was elicited. In summary, L-SseB in a particulate polysaccharide formulation appears to be an attractive candidate vaccine capable of broad protection against S. enterica.

Pseudomonas syringae pv. aptata is a member of the sugar beet pathobiome and the causative agent of leaf spot disease. Like many pathogenic bacteria, P. syringae relies on the secretion of toxins, which manipulate host-pathogen interactions, to establish and maintain an infection. This study analyzes the secretome of six pathogenic P. syringae pv. aptata strains with different defined virulence capacities in order to identify common and strain-specific features, and correlate the secretome with disease outcome. All strains show a high type III secretion system (T3SS) and type VI secretion system (T6SS) activity under apoplast-like conditions mimicking the infection. Surprisingly, we found that low pathogenic strains show a higher secretion of most T3SS substrates, whereas a distinct subgroup of four effectors was exclusively secreted in medium and high pathogenic strains. Similarly, we detected two T6SS secretion patterns: while one set of proteins was highly secreted in all strains, another subset consisting of known T6SS substrates and previously uncharacterized proteins was exclusively secreted in medium and high virulence strains. Taken together, our data show that P. syringae pathogenicity is correlated with the repertoire and fine-tuning of effector secretion and indicate distinct strategies for establishing virulence of P. syringae pv. aptata in plants.

Vibrio parahaemolyticus is a moderately halophilic foodborne pathogen that is mainly distributed in marine and freshwater environments. The transition of V. parahaemolyticus between aquatic ecosystems and hosts is essential for infection. Both freshwater and host environments have low salinity. In this study, we sought to further investigate the effects of low salinity (0.5% NaCl) on the fitness and virulence of V. parahaemolyticus. We found that V. parahaemolyticus could survive in Luria–Bertani (LB) and M9 mediums with different NaCl concentrations, except for the M9 medium containing 9% NaCl. Our results further showed that V. parahaemolyticus cultured in M9 medium with 0.5% NaCl had a higher cell density than that cultured at other NaCl concentrations when it entered the stationary phase. Therefore, we compared the transcriptomes of V. parahaemolyticus wild type (WT) cultured in an M9 medium with 0.5% and 3% NaCl at the stationary phase using RNA-seq. A total of 658 genes were significantly differentially expressed in the M9 medium with 0.5% NaCl, including regulators, osmotic adaptive responses (compatible solute synthesis systems, transporters, and outer membrane proteins), and virulence factors (T3SS1 and T6SS1). Furthermore, a low salinity concentration in the M9 medium induced the expression of T3SS1 to mediate the cytotoxicity of V. parahaemolyticus to HeLa cells. Similarly, low salinity could also induce the secretion of the T3SS2 translocon protein VPA1361. These factors may result in the high pathogenicity of V. parahaemolyticus in low-salinity environments. Taken together, these results suggest that low salinity (0.5% NaCl) could affect gene expression to mediate fitness and virulence, which may contribute to the transition of V. parahaemolyticus between aquatic ecosystems and the host. [ABSTRACT FROM AUTHOR]

Erwinia amylovora causes a devastating fire blight disease in apples and pears. One of the main virulence determinants in E. amylovora is the hypersensitive response (HR) and pathogenicity (hrp)-type III secretion system (T3SS), which is activated by the RpoN-HrpL sigma factor cascade. However, the RpoN regulon in E. amylovora has not been investigated. In this study, we determined the RpoN regulon in E. amylovora by combining RNA-seq transcriptomic analysis with in silico binding site analysis. RNA-seq revealed that 262 genes, approximately 7.5% genes in the genome of E. amylovora, were differentially transcribed in the rpoN mutant as compared with the wild type. Specifically, genes associated with virulence, motility, nitrogen assimilation, the PspF system, stress response, and arginine biosynthesis are positively regulated by RpoN, whereas genes associated with biosynthesis of amino acids and sorbitol transport are negatively regulated by RpoN. In silico binding site analysis identified 46 potential target genes with a putative RpoN binding site, and the upstream sequences of six, three, and three genes also contain putative GlnG, PspF, and YfhA binding sites, respectively. Overall, RpoN directly regulates genes associated with virulence, nitrogen assimilation, the PspF system, motility and the YfhA/YfhK two-component regulatory system. [ABSTRACT FROM AUTHOR]

The nanomachine referred to as the type III secretion system (T3SS) is used by many Gram-negative pathogens or symbionts to inject their effector proteins into host cells to promote their infections or symbioses. Among the genera possessing T3SS is Vibrio, which consists of diverse species of Gammaproteobacteria including human pathogenic species and inhabits aquatic environments. We describe the genetic overview of the T3SS gene clusters in Vibrio through a phylogenetic analysis from 48 bacterial strains and a gene order analysis of the two previously known categories in Vibrio (T3SS1 and T3SS2). Through this analysis we identified a new T3SS category (named T3SS3) that shares similar core and related proteins (effectors, translocons, and chaperones) with the Ssa-Esc family of T3SSs in Salmonella, Shewanella, and Sodalis. The high similarity between T3SS3 and the Ssa-Esc family suggests a possibility of genetic exchange among marine bacteria with similar habitats. [ABSTRACT FROM AUTHOR]

Infections caused by Gram-negative pathogens pose a major health burden. Both respiratory and gastrointestinal infections are commonly associated with these pathogens. With the increase in antimicrobial resistance (AMR) over the last decades, bacterial infections may soon become the threat they have been before the discovery of antibiotics. Many Gram-negative pathogens encode virulence-associated Type III and Type IV secretion systems, which they use to inject bacterial effector proteins across bacterial and host cell membranes into the host cell cytosol, where they subvert host cell functions in favor of bacterial replication and survival. These secretion systems are essential for the pathogens to cause disease, and secretion system mutants are commonly avirulent in infection models. Hence, these structures present attractive targets for anti-virulence therapies. Here, we review previously and recently identified inhibitors of virulence-associated bacterial secretions systems and discuss their potential as therapeutics. [ABSTRACT FROM AUTHOR]

Traditional antibiotics target essential cellular components or metabolic pathways conserved in both pathogenic and nonpathogenic bacteria. Unfortunately, long-term antibiotic use often leads to antibiotic resistance and disruption of the overall microbiota. In this work, we identified a phenylamino acetamide compound, named 187R, that strongly inhibited the expression of the type III secretion system (T3SS) encoding genes and the secretion of the T3SS effector proteins in Pseudomonas aeruginosa. T3SS is an important virulence factor, as T3SS-deficient strains of P. aeruginosa are greatly attenuated in virulence. We further showed that 187R had no effect on bacterial growth, implying a reduced selective pressure for the development of resistance. 187R-mediated repression of T3SS was dependent on ExsA, the master regulator of T3SS in P. aeruginosa. The impact of 187R on the host-associated microbial community was also tested using the Arabidopsis thaliana phyllosphere as a model. Both culture-independent (Illumina sequencing) and culture-dependent (Biolog) methods showed that the application of 187R had little impact on the composition and function of microbial community compared to the antibiotic streptomycin. Together, these results suggested that compounds that target virulence factors could serve as an alternative strategy for disease management caused by bacterial pathogens. [ABSTRACT FROM AUTHOR]

Abstract Edwardsiella piscicida is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish. The type III secretion system (T3SS) is one of its two most important virulence islands. T3SS protein EseJ inhibits E. piscicida adhesion to epithelioma papillosum cyprini (EPC) cells by negatively regulating type 1 fimbria. Type 1 fimbria helps E. piscicida to adhere to fish epithelial cells. In this study, we characterized a functional unknown protein (Orf1B) encoded within the T3SS gene cluster of E. piscicida. This protein consists of 122 amino acids, sharing structural similarity with YscO in Vibrio parahaemolyticus. Orf1B controls secretion of T3SS translocon and effectors in E. piscicida. By immunoprecipitation, Orf1B was shown to interact with T3SS ATPase EsaN. This interaction may contribute to the assembly of the ATPase complex, which energizes the secretion of T3SS proteins. Moreover, disruption of Orf1B dramatically decreased E. piscicida adhesion to EPC cells due to the increased steady-state protein level of EseJ within E. piscicida. Taken together, this study partially unraveled the mechanisms through which Orf1B promotes secretion of T3SS proteins and contributes to E. piscicida adhesion. This study helps to improve our understanding on molecular mechanism of E. piscicida pathogenesis.