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Type III secretion systems (T3SS) are specialized protein secretion systems that allow bacteria to inject toxins into eukaryotic cells. T3SS are important virulence factors, but their expression carries a fitness cost: they slow bacterial growth and make bacteria vulnerable to detection by the innate immune system. Some pathogens, like Pseudomonas aeruginosa, balance the costs and benefits of T3SS expression by restricting T3SS expression to a subset of cells. T3SS-ON cells arise from “primed” bacteria that express the transcriptional activator ExsA and respond immediately to T3SS activating signals. However, the mechanistic basis for priming is unknown. In this study, we tested whether expression of ExsA from a cAMP-dependent promoter could drive cells into the primed state and found this to be true. Whole-cell cryo-electron tomography demonstrated that primed bacteria assembled T3SS injectisomes. This work demonstrates how cAMP inputs into a bistable regulatory switch generate subpopulations of T3SS-primed cells.

Rhizobia are soil bacteria that can establish a symbiotic association with legumes. As a result, plant nodules are formed on the roots of the host plants where rhizobia differentiate to bacteroids capable of fixing atmospheric nitrogen into ammonia. This ammonia is transferred to the plant in exchange of a carbon source and an appropriate environment for bacterial survival. This process is subjected to a tight regulation with several checkpoints to allow the progression of the infection or its restriction. The type 3 secretion system (T3SS) is a secretory system that injects proteins, called effectors (T3E), directly into the cytoplasm of the host cell, altering host pathways or suppressing host defense responses. This secretion system is not present in all rhizobia but its role in symbiosis is crucial for some symbiotic associations, showing two possible faces as Dr. Jekyll and Mr. Hyde: it can be completely necessary for the formation of nodules, or it can block nodulation in different legume species/cultivars. In this review, we compile all the information currently available about the effects of different rhizobial effectors on plant symbiotic phenotypes. These phenotypes are diverse and highlight the importance of the T3SS in certain rhizobium–legume symbioses.

Yersinia pestis, the causative agent of plague, remains a threat to public health worldwide. From the perspective of developing safe and effective vaccines, we present a derived version of our Y. pseudotuberculosis VTnF1 live attenuated vaccine candidate that lacks the pYV virulence plasmid coding for the Type 3 Secretion system (T3SS) and carries no antibiotic resistance cassettes (VTnF1-S). This strain, named VpYV-, fails to cause disease in immunocompromised mice when given orally, and can be considered as avirulent in such conditions. It retains a tropism for Peyer's patches and mesenteric lymph nodes, whilst rarely reaching the spleen and liver. When compared to VTnF1-S, VpYV- elicited equivalent production of IgG directed to the F1 antigen, but less IgG directed to other Yersinia antigens. A single oral dose of VpYV- induced 100 % protection against bubonic and pneumonic forms of plague. Four months after vaccination, the protection induced by VpYV- had decreased more than that induced by VTnF1-S. Furthermore, VpYV- was 30 % less protective against F1-negative Y. pestis, revealing that the T3SS components encoded by pYV are mandatory to obtain a large spectrum protection. Finally, VTnF1-S and VpYV- were compared for their ability to induce immediate immune activity against co-infecting Y. pestis, which could be a potential therapeutic strategy against early-stage infections. Like the historical Y. pestis vaccine EV76, VTnF1-S was able to induce such a protection. The process involved nutritional immunity in serum, indicating a fast activation of innate immune mechanisms. By contrast, VpYV- failed to protect mice, revealing an importance of the T3SS in this mechanism. Overall, VTnF1 and its derivative strain VpYV-, offer a choice between better vaccine performance or greater vaccine safety. They represent useful tools to prevent and treat Y. pestis infection in healthy or immunocompromised individuals.

Sinorhizobium (Ensifer) fredii (S. fredii) is a rhizobial species exhibiting a remarkably broad nodulation host-range. Thus, S. fredii is able to effectively nodulate dozens of different legumes, including plants forming determinate nodules, such as the important crops soybean and cowpea, and plants forming indeterminate nodules, such as Glycyrrhiza uralensis and pigeon-pea. This capacity of adaptation to different symbioses makes the study of the molecular signals produced by S. fredii strains of increasing interest since it allows the analysis of their symbiotic role in different types of nodule. In this review, we analyze in depth different S. fredii molecules that act as signals in symbiosis, including nodulation factors, different surface polysaccharides (exopolysaccharides, lipopolysaccharides, cyclic glucans, and K-antigen capsular polysaccharides), and effectors delivered to the interior of the host cells through a symbiotic type 3 secretion system.

Summary The bacterial stringent response is a response to nutrition deprivation and other stress conditions. In Gram‐negative bacteria, this process is mediated by the small signal molecules guanosine pentaphosphate pppGpp and guanosine tetraphosphate ppGpp (collectively referred to as (p)ppGpp), and the RNA polymerase‐binding transcription factor DksA. The (p)ppGpp synthetase RelA and the bifunctional (p)ppGpp synthase/hydrolase SpoT are responsible for cellular (p)ppGpp levels. Here, we investigated the roles of DksA and (p)ppGpp in the virulence traits of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker. ΔdksA and (p)ppGpp‐deficient ΔspoTΔrelA strains caused reduced virulence and compromised growth in host plants, indicating that DksA and (p)ppGpp are required for full virulence of Xcc. To characterize the effect of stringent response regulators on gene expression, RNA‐seq was conducted using ΔdksA and ΔspoTΔrelA mutant strains grown in hrp‐inducing XVM2 medium. Transcriptome analyses showed that DksA and (p)ppGpp repressed the expression of genes encoding tRNAs, ribosome proteins, iron acquisition and flagellum assembly, and enhanced the expression of genes for histidine metabolism, type 3 secretion system (T3SS), type 2 secretion system (T2SS) and TonB‐dependent transporters. Phenotypically, the ΔdksA and ΔspoTΔrelA strains displayed altered motility, enhanced siderophore production and were unable to cause the hypersensitive response on non‐host plants. In conclusion, stringent response regulators DksA and (p)ppGpp play an important role in virulence, nutrition uptake and host adaptation of Xcc.

Gram-negative pathogens pose a significant threat due to their propensity for causing various infections, often coupled with formidable resistance to conventional antibiotic treatments. The development of antivirulence (AV) compounds emerges as a promising alternative strategy by disrupting virulence mechanisms rather than targeting bacterial viability. Aurodox has exhibited promising AV properties in previous studies by blocking the expression and function of the LEE-encoded type 3 secretion system (T3SS) in enterohaemorrhagic Escherichia coli, an injectosome that translocates effector proteins directly into host target cells. However, aurodox’s efficacy against the T3SS of other pathogens remained unanswered. Using quantitative real-time polymerase chain reaction, we show that aurodox exerts inhibitory effects on selected T3SS including those of Salmonella Typhimurium, Yersinia pseudotuberculosis and Vibrio parahaemolyticus. Imaging of RAW 264.7 cells infected with S. Typhimurium showed that aurodox protects against late stages of infection by blocking the expression of the SPI-2 T3SS. To elucidate a conserved mechanism of action, we compared transcriptomic datasets from both E. coli and S. Typhimurium treated with aurodox to identify orthologous genes differentially expressed in response to aurodox treatment across both pathogens. This study sheds light on potential mechanisms driving the action of this promising AV compound.

The overuse of antibiotics has led to severe bacterial drug resistance. Blocking pathogen virulence devices is a highly effective approach to combating bacterial resistance worldwide. Type three secretion systems (T3SSs) are significant virulence factors in Gram-negative pathogens. Inhibition of these systems can effectively weaken infection whilst having no significant effect on bacterial growth. Therefore, T3SS inhibitors may be a powerful weapon against resistance in Gram-negative bacteria, and there has been increasing interest in the research and development of T3SS inhibitors. This review outlines several reported small-molecule inhibitors of the T3SS, covering those of synthetic and natural origin, including their sources, structures, and mechanisms of action.

Summary Cases of emergence of novel plant‐pathogenic strains are regularly reported that reduce the yields of crops and trees. However, the molecular mechanisms underlying such emergence are still poorly understood. The acquisition by environmental non‐pathogenic strains of novel virulence genes by horizontal gene transfer has been suggested as a driver for the emergence of novel pathogenic strains. In this study, we tested such an hypothesis by transferring a plasmid encoding the type 3 secretion system (T3SS) and four associated type 3 secreted proteins (T3SPs) to the non‐pathogenic strains of Xanthomonas CFBP 7698 and CFBP 7700, which lack genes encoding T3SS and any previously known T3SPs. The resulting strains were phenotyped on Nicotiana benthamiana using chlorophyll fluorescence imaging and image analysis. Wild‐type, non‐pathogenic strains induced a hypersensitive response (HR)‐like necrosis, whereas strains complemented with T3SS and T3SPs suppressed this response. Such suppression depends on a functional T3SS. Amongst the T3SPs encoded on the plasmid, Hpa2, Hpa1 and, to a lesser extent, XopF1 collectively participate in suppression. Monitoring of the population sizes in planta showed that the sole acquisition of a functional T3SS by non‐pathogenic strains impairs growth inside leaf tissues. These results provide functional evidence that the acquisition via horizontal gene transfer of a T3SS and four T3SPs by environmental non‐pathogenic strains is not sufficient to make strains pathogenic. In the absence of a canonical effector, the sole acquisition of a T3SS seems to be counter‐selective, and further acquisition of type 3 effectors is probably needed to allow the emergence of novel pathogenic strains.

Enteric pathogens rely on a variety of toxins, adhesins and other virulence factors to cause infections. Some of the best studied pathogens belong to the Enterobacterales order; these include enteropathogenic and enterohemorrhagic Escherichia coli, Shigella spp., and the enteropathogenic Yersiniae. The pathogenesis of these organisms involves two different secretion systems, a type 3 secretion system (T3SS) and type 5 secretion systems (T5SSs). The T3SS forms a syringe-like structure spanning both bacterial membranes and the host cell plasma membrane that translocates toxic effector proteins into the cytoplasm of the host cell. T5SSs are also known as autotransporters, and they export part of their own polypeptide to the bacterial cell surface where it exerts its function, such as adhesion to host cell receptors. During infection with these enteropathogens, the T3SS and T5SS act in concert to bring about rearrangements of the host cell cytoskeleton, either to invade the cell, confer intracellular motility, evade phagocytosis or produce novel structures to shelter the bacteria. Thus, in these bacteria, not only the T3SS effectors but also T5SS proteins could be considered “cytoskeletoxins” that bring about profound alterations in host cell cytoskeletal dynamics and lead to pathogenic outcomes.

Bacteria of the genus Xanthomonas cause a wide variety of economically important diseases in most crops. The virulence of the majority of Xanthomonas spp. is dependent on secretion and translocation of effectors by the type 3 secretion system (T3SS) that is controlled by two master transcriptional regulators HrpG and HrpX. Since their discovery in the 1990s, the two regulators were the focal point of many studies aiming to decipher the regulatory network that controls pathogenicity in Xanthomonas bacteria. HrpG controls the expression of HrpX, which subsequently controls the expression of T3SS apparatus genes and effectors. The HrpG/HrpX regulon is activated in planta and subjected to tight metabolic and genetic regulation. In this review, we cover the advances made in understanding the regulatory networks that control and are controlled by the HrpG/HrpX regulon and their conservation between different Xanthomonas spp.