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.

Gastroenteritis caused by non-typhoidal Salmonella still prevails resulting in several recent outbreaks affecting many people worldwide. The presence of invasive non-typhoidal Salmonella is exemplified by several characteristic symptoms and their severity relies on prominent risk factors. The persistence of this pathogen can be attributed to its broad host range, complex pathogenicity and virulence and adeptness in survival under challenging conditions inside the host. Moreover, a peculiar aid of the ever-changing climatic conditions grants this organism with remarkable potential to survive within the environment. Abusive use of antibiotics for the treatment of gastroenteritis has led to the emergence of multiple drug resistance, making the infections difficult to treat. This review emphasizes the importance of early detection of Salmonella, along with strategies for accomplishing it, as well as exploring alternative treatment approaches. The exceptional characteristics exhibited by Salmonella, like strategies of infection, persistence, and survival parallelly with multiple drug resistance, make this pathogen a prominent concern to human health.

Shigella spp. are responsible for bacillary dysentery or shigellosis transmitted via the fecal-oral route, causing significant morbidity and mortality, especially among vulnerable populations. There are currently no licensed Shigella vaccines. Shigella spp. use a type III secretion system (T3SS) to invade host cells. We have shown that L-DBF, a recombinant fusion of the T3SS needle tip (IpaD) and translocator (IpaB) proteins with the LTA1 subunit of enterotoxigenic E. coli labile toxin, is broadly protective against Shigella spp. challenge in a mouse lethal pulmonary model. Here, we assessed the effect of LDBF, formulated with a unique TLR4 agonist called BECC470 in an oil-in-water emulsion (ME), on the murine immune response in a high-risk population (young and elderly) in response to Shigella challenge. Dual RNA Sequencing captured the transcriptome during Shigella infection in vaccinated and unvaccinated mice. Both age groups were protected by the L-DBF formulation, while younger vaccinated mice exhibited more adaptive immune response gene patterns. This preliminary study provides a step toward identifying the gene expression patterns and regulatory pathways responsible for a protective immune response against Shigella. Furthermore, this study provides a measure of the challenges that need to be addressed when immunizing an aging population.

This study presents a comprehensive genomic analysis of Burkholderia plantarii, a rice pathogen that causes blight and grain rot in seedlings. The entire genome of B. plantarii KACC 18964 was sequenced, followed by a comparative genomic analysis with other available genomes to gain insights into its virulence, fitness, and interactions with rice. Multiple secondary metabolite gene clusters were identified. Among these, 12 demonstrated varying similarity levels to known clusters linked to bioactive compounds, whereas eight exhibited no similarity, indicating B. plantarii as a source of potentially novel secondary metabolites. Notably, the genes responsible for tropolone and quorum sensing were conserved across the examined genomes. Additionally, B. plantarii was observed to possess three complete CRISPR systems and a range of secretion systems, exhibiting minor variations among the analyzed genomes. Genomic islands were analyzed across the four genomes, and a detailed study of the B. plantarii KACC 18964 genome revealed 59 unique islands. These islands were thoroughly investigated for their gene contents and potential roles in virulence. Particular attention has been devoted to the Type III secretion system (T3SS), a crucial virulence factor. An in silico analysis of potential T3SS effectors identified a conserved gene, aroA. Further mutational studies, in planta and in vitro analyses validated the association between aroA and virulence in rice. Overall, this study enriches our understanding of the genomic basis of B. plantarii pathogenicity and emphasizes the potential role of aroA in virulence. This understanding may guide the development of effective disease management strategies.

BACKGROUND: Type I interferons (IFN-I)-a group of cytokines with immunomodulatory, antiproliferative, and antiviral properties-are widely used as therapeutics for various cancers and viral diseases. Since IFNs are proteins, they are highly susceptible to degradation by proteases and by hydrolysis in the strong acid environment of the stomach, and they are therefore administered parenterally. In this study, we examined whether the intestinal bacterium, enteropathogenic Escherichia coli (EPEC), can be exploited for oral delivery of IFN-Is. EPEC survives the harsh conditions of the stomach and, upon reaching the small intestine, expresses a type III secretion system (T3SS) that is used to translocate effector proteins across the bacterial envelope into the eukaryotic host cells.
RESULTS: In this study, we developed an attenuated EPEC strain that cannot colonize the host but can secrete functional human IFNα2 variant through the T3SS. We found that this bacteria-secreted IFN exhibited antiproliferative and antiviral activities similar to commercially available IFN.
CONCLUSIONS: These findings present a potential novel approach for the oral delivery of IFN via secreting bacteria.

The evolutionary conserved YopJ family comprises numerous type-III-secretion system (T3SS) effectors of diverse mammalian and plant pathogens that acetylate host proteins to dampen immune responses. Acetylation is mediated by a central acetyltransferase domain that is flanked by conserved regulatory sequences, while a nonconserved N-terminal extension encodes the T3SS-specific translocation signal. Bartonella spp. are facultative-intracellular pathogens causing intraerythrocytic bacteremia in their mammalian reservoirs and diverse disease manifestations in incidentally infected humans. Bartonellae do not encode a T3SS, but most species possess a type-IV-secretion system (T4SS) to translocate Bartonella effector proteins (Beps) into host cells. Here we report that the YopJ homologs present in Bartonellae species represent genuine T4SS effectors. Like YopJ family T3SS effectors of mammalian pathogens, the \"Bartonella YopJ-like effector A\" (ByeA) of Bartonella taylorii also targets MAP kinase signaling to dampen proinflammatory responses, however, translocation depends on a functional T4SS. A split NanoLuc luciferase-based translocation assay identified sequences required for T4SS-dependent translocation in conserved regulatory regions at the C-terminus and proximal to the N-terminus of ByeA. The T3SS effectors YopP from Yersinia enterocolitica and AvrA from Salmonella Typhimurium were also translocated via the Bartonella T4SS, while ByeA was not translocated via the Yersinia T3SS. Our data suggest that YopJ family T3SS effectors may have evolved from an ancestral T4SS effector, such as ByeA of Bartonella. In this evolutionary scenario, the signal for T4SS-dependent translocation encoded by N- and C-terminal sequences remained functional in the derived T3SS effectors due to the essential role these sequences coincidentally play in regulating acetyltransferase activity.

Shigellosis is a severe gastrointestinal disease that annually affects approximately 270 million individuals globally. It has particularly high morbidity and mortality in low-income regions; however, it is not confined to these regions and occurs in high-income nations when conditions allow. The ill effects of shigellosis are at their highest in children ages 2 to 5, with survivors often exhibiting impaired growth due to infection-induced malnutrition. The escalating threat of antibiotic resistance further amplifies shigellosis as a serious public health concern. This review explores Shigella pathology, with a primary focus on the status of Shigella vaccine candidates. These candidates include killed whole-cells, live attenuated organisms, LPS-based, and subunit vaccines. The strengths and weaknesses of each vaccination strategy are considered. The discussion includes potential Shigella immunogens, such as LPS, conserved T3SS proteins, outer membrane proteins, diverse animal models used in Shigella vaccine research, and innovative vaccine development approaches. Additionally, this review addresses ongoing challenges that necessitate action toward advancing effective Shigella prevention and control measures.

Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.

Bacteria express lytic enzymes such as glycosidases, which have potentially self-destructive peptidoglycan (PG)-degrading activity and, therefore, require careful regulation in bacteria. The PG glycosidase EtgA is regulated by localization to the assembling type III secretion system (T3SS), generating a hole in the PG layer for the T3SS to reach the outer membrane. The EtgA localization was found to be mediated via EtgA interacting with the T3SS inner rod protein EscI. To gain structural insights into the EtgA recognition of EscI, we determined the 2.01 Å resolution structure of an EscI (51-87)-linker-EtgA fusion protein designed based on AlphaFold2 predictions. The structure revealed EscI residues 72-87 forming an α-helix interacting with the backside of EtgA, distant from the active site. EscI residues 56-71 also were found to interact with EtgA, with these residues stretching across the EtgA surface. The ability of the EscI to interact with EtgA was also probed using an EscI peptide. The EscI peptide comprising residues 66-87, slightly larger than the observed EscI α-helix, was shown to bind to EtgA using microscale thermophoresis and thermal shift differential scanning fluorimetry. The EscI peptide also had a two-fold activity-enhancing effect on EtgA, whereas the EscI-EtgA fusion protein enhanced activity over four-fold compared to EtgA. Our studies suggest that EtgA regulation by EscI could be trifold involving protein localization, protein activation, and protein stabilization components. Analysis of the sequence conservation of the EscI EtgA interface residues suggested a possible conservation of such regulation for related proteins from different bacteria.

The Vibrio genus includes bacteria widely distributed in aquatic habitats and the infections caused by these bacteria can affect a wide range of hosts. They are able to adhere to numerous surfaces, which can result in biofilm formation that helps maintain them in the environment. The involvement of the biofilm lifestyle in the virulence of Vibrio pathogens of aquatic organisms remains to be investigated. Vibrio harveyi ORM4 is a pathogen responsible for an outbreak in European abalone Haliotis tuberculata populations. In the present study, we used a dynamic biofilm culture technique coupled with laser scanning microscopy to characterize the biofilm formed by V. harveyi ORM4. We furthermore used RNA-seq analysis to examine the global changes in gene expression in biofilm cells compared to planktonic bacteria, and to identify biofilm- and virulence-related genes showing altered expression. A total of 1565 genes were differentially expressed, including genes associated with motility, polysaccharide synthesis, and quorum sensing. The up-regulation of 18 genes associated with the synthesis of the type III secretion system suggests that this virulence factor is induced in V. harveyi ORM4 biofilms, providing indirect evidence of a relationship between biofilm and virulence.