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Bordetella bronchiseptica and Bordetella pertussis are two closely related respiratory pathogens that employ their T3SS injectisome to deliver the BteA effector into host cells. In this study, we visualized the needle tip filament of their T3SS injectisome, a structure formed by the Bsp22 protein. We demonstrate that during Bordetella cultivation in Stainer-Scholte medium, Bsp22 filaments are abundant and can dynamically extend up to several micrometers in length through the incorporation of new subunits at their distal ends. In contrast, these filaments become shorter and/or less abundant during infection of host cells. This reduction correlates with decreased bsp22 mRNA expression and lower Bsp22 protein levels, while the levels of bscD mRNA, which encodes the inner membrane ring protein of the injectisome, remain stable. These results highlight the adaptability of the Bordetella T3SS injectisome and show how its tip filament structure changes in response to different environments.

Enteropathogenic and enterohemorrhagic are diarrheagenic bacterial human pathogens that cause severe gastroenteritis. These enteric pathotypes, together with the mouse pathogen , belong to the family of attaching and effacing pathogens that form a distinctive histological lesion in the intestinal epithelium. The virulence of these bacteria depends on a type III secretion system (T3SS), which mediates the translocation of effector proteins from the bacterial cytosol into the infected cells. The core architecture of the T3SS consists of a multi-ring basal body embedded in the bacterial membranes, a periplasmic inner rod, a transmembrane export apparatus in the inner membrane, and cytosolic components including an ATPase complex and the C-ring. In addition, two distinct hollow appendages are assembled on the extracellular face of the basal body creating a channel for protein secretion: an approximately 23 nm needle, and a filament that extends up to 600 nm. This filamentous structure allows these pathogens to get through the host cells mucus barrier. Upon contact with the target cell, a translocation pore is assembled in the host membrane through which the effector proteins are injected. Assembly of the T3SS is strictly regulated to ensure proper timing of substrate secretion. The different type III substrates coexist in the bacterial cytoplasm, and their hierarchical secretion is determined by specialized chaperones in coordination with two molecular switches and the so-called sorting platform. In this review, we present recent advances in the understanding of the T3SS in attaching and effacing pathogens.

A syringe‐like type III secretion system (T3SS) plays essential roles in the pathogenicity of Ralstonia solanacearum, which is a causal agent of bacterial wilt disease on many plant species worldwide. Here, we characterized functional roles of a CysB regulator (RSc2427) in R. solanacearum OE1‐1 that was demonstrated to be responsible for cysteine synthesis, expression of the T3SS genes, and pathogenicity of R. solanacearum. The cysB mutants were cysteine auxotrophs that failed to grow in minimal medium but grew slightly in host plants. Supplementary cysteine substantially restored the impaired growth of cysB mutants both in minimal medium and inside host plants. Genes of cysU and cysI regulons have been annotated to function for R. solanacearum cysteine synthesis; CysB positively regulated expression of these genes. Moreover, CysB positively regulated expression of the T3SS genes both in vitro and in planta through the PrhG to HrpB pathway, whilst impaired expression of the T3SS genes in cysB mutants was independent of growth deficiency under nutrient‐limited conditions. CysB was also demonstrated to be required for exopolysaccharide production and swimming motility, which contribute jointly to the host colonization and infection process of R. solanacearum. Thus, CysB was identified here as a novel regulator on the T3SS expression in R. solanacearum. These results provide novel insights into understanding of various biological functions of CysB regulators and complex regulatory networks on the T3SS in R. solanacearum. Ralstonia solanacearum CysB controls cysteine synthesis and positively regulates the type III secretion system (T3SS) expression through the PrhG to HrpB pathway. The cysB mutant is cysteine auxotroph, while its impact on the T3SS is independent of growth deficiency.

As extrachromosomal elements, plasmids are well known for their role in conferring advantageous attributes to hosts, including antibiotic resistance, heavy metal resistance, the ability to degrade xenobiotics, and osmotolerance. Although several host chromosomally encoded proteins are required for the replication and maintenance of plasmids in their hosts, few proteins from plasmids have been reported to affect the host chromosomal gene expression. In this study, we identified TrfA, a plasmid replication initiation protein, as a novel repressor of the T3SS in P. aeruginosa . We also elucidated the regulatory mechanism of T3SS repression mediated by the TrfA. The significance of this work is in the identification of the replication initiation protein of a naturally occurring plasmid from P. aeruginosa functioning as a regulator of the expression of chromosomal genes in its host P. aeruginosa .

Salmonella enterica is an increasing global public health threat. As part of its virulence arsenal, Salmonella relies on a type III secretion system (T3SS) or injectisome, a molecular injection device that translocates effector proteins into host cells to promote invasion and inflammation. A central component of this machine is the SpaO protein, which is produced in two forms: a full-length form and a shorter variant. Here, by studying the functional and structural relationship between the two SpaO forms in their native cellular environment, we define how and when they assemble within the injectisome. Employing quantitative injection assays in cultured cells, we define the shorter SpaO variant as an accessory structural piece that boosts effector delivery. These findings refine our understanding of injectisome assembly and function and provide mechanistic insight to inform future efforts to target T3SS-dependent pathogens through antivirulence strategies.

Virulence-associated type III secretion systems (T3SS) serve the injection of bacterial effector proteins into eukaryotic host cells. They are able to secrete a great diversity of substrate proteins in order to modulate host cell function, and have evolved to sense host cell contact and to inject their substrates through a translocon pore in the host cell membrane. T3SS substrates contain an N-terminal signal sequence and often a chaperone-binding domain for cognate T3SS chaperones. These signals guide the substrates to the machine where substrates are unfolded and handed over to the secretion channel formed by the transmembrane domains of the export apparatus components and by the needle filament. Secretion itself is driven by the proton motive force across the bacterial inner membrane. The needle filament measures 20-150 nm in length and is crowned by a needle tip that mediates host-cell sensing. Secretion through T3SS is a highly regulated process with early, intermediate and late substrates. A strict secretion hierarchy is required to build an injectisome capable of reaching, sensing and penetrating the host cell membrane, before host cell-acting effector proteins are deployed. Here, we review the recent progress on elucidating the assembly, structure and function of T3SS injectisomes.

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.

Type IV pili (T4P) are virulence factors in various pathogenic bacteria of animals and plants that play important roles in twitching motility, swimming motility, biofilm formation, and adhesion to host cells. Here, we genetically characterized functional roles of a putative T4P assembly protein TapV (Rsc1986 in reference strain GMI1000) and its homologue Rsp0189, which shares 58% amino acid identity with TapV, in Ralstonia solanacearum. Deletion of tapV, but not rsp0189, resulted in significantly impaired twitching motility, swimming motility, and adhesion to tomato roots, which are consistent as phenotypes of the pilA mutant (a known R. solanacearum T4P‐deficient mutant). However, unlike the pilA mutant, the tapV mutant produced more biofilm than the wild‐type strain. Our gene expression studies revealed that TapV, but not Rsp0189, is important for expression of a type III secretion system (T3SS, a pathogenicity determinant of R. solanacearum) both in vitro and in planta, but it is T4P independent. We further revealed that TapV affected the T3SS expression via the PhcA–TapV–PrhG–HrpB pathway, consistent with previous reports that PhcA positively regulates expression of pilA and prhG. Moreover, deletion of tapV, but not rsp0189, significantly impaired the ability to migrate into and colonize xylem vessels of host plants, but there was no alteration in intercellular proliferation of R. solanacearum in tobacco leaves, which is similar to the pilA mutant. The tapV mutant showed significantly impaired virulence in host plants. This is the first report on the impact of T4P components on the T3SS, providing novel insights into our understanding of various biological functions of T4P and the complex regulatory pathway of T3SS in R. solanacearum. A novel type 4 pili (T4P) assembly protein (TapV) was identified to contribute to full virulence of Ralstonia solanacearum, which affects the T3SS expression via the PhcA–TapV–PrhG–HrpB pathway but is T4P independent.

The type III secretion system with its delivered type III effectors (T3Es) is one of the main virulence determinants of Ralstonia solanacearum, a worldwide devastating plant pathogenic bacterium affecting many crop species. The pan‐effectome of the R. solanacearum species complex has been exhaustively identified and is composed of more than 100 different T3Es. Among the reported strains, their content ranges from 45 to 76 T3Es. This considerably large and varied effectome could be considered one of the factors contributing to the wide host range of R. solanacearum. In order to understand how R. solanacearum uses its T3Es to subvert the host cellular processes, many functional studies have been conducted over the last three decades. It has been shown that R. solanacearum effectors, as those from other plant pathogens, can suppress plant defence mechanisms, modulate the host metabolism, or avoid bacterial recognition through a wide variety of molecular mechanisms. R. solanacearum T3Es can also be perceived by the plant and trigger immune responses. To date, the molecular mechanisms employed by R. solanacearum T3Es to modulate these host processes have been described for a growing number of T3Es, although they remain unknown for the majority of them. In this microreview, we summarize and discuss the current knowledge on the characterized R. solanacearum species complex T3Es. This review compiles all the current functional knowledge on the large and diverse repertoire of type III effectors from bacteria belonging to the Ralstonia solanacearum species complex.

is a Gram-negative intracellular pathogen and the causative agent of melioidosis, a severe disease of both humans and animals. Melioidosis is an emerging disease which is predicted to be vastly under-reported. Type III Secretion Systems (T3SSs) are critical virulence factors in Gram negative pathogens of plants and animals. The genome of encodes three T3SSs. T3SS-1 and -2, of which little is known, are homologous to Hrp2 secretion systems of the plant pathogens and . T3SS-3 is better characterized and is homologous to the Inv/Mxi-Spa secretion systems of spp. and , respectively. Upon entry into the host cell, requires T3SS-3 for efficient escape from the endosome. T3SS-3 is also required for full virulence in both hamster and murine models of infection. The regulatory cascade which controls T3SS-3 expression and the secretome of T3SS-3 have been described, as well as the effect of mutations of some of the structural proteins. Yet only a few effector proteins have been functionally characterized to date and very little work has been carried out to understand the hierarchy of assembly, secretion and temporal regulation of T3SS-3. This review aims to frame current knowledge of T3SSs in the context of other well characterized model T3SSs, particularly those of and .