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Bradyrhizobia are soil bacteria able to establish a symbiotic interaction with a wide range oflegume species. This symbiosis leads to the formation of a new organ, the nodule, in which thebacteria can fix atmospheric dinitrogen for the plant’s benefit. This interaction largely depends onthe plant recognition of bacterial signal molecules, the Nod factors (NFs), that control the infectionand nodule organogenesis processes. Recently, it has been demonstrated that some nonphotosyntheticBradyrhizobium strains, such as B. elkanii USDA61 and Bradyrhizobium sp. ORS3257,are capable to nodulate some legumes (Glycine max, Aeschynomene indica) in the absence of NFsand that this capacity is due to their type III secretion system (T3SS). This discovery suggests thatsome effectors secreted by this secretory machinery, initially believed to solely play a role in thesuppression of plant immunity, can directly activate the nodulation by bypassing the early stages ofthe symbiotic signaling pathway activated by the NFs. The main objective of this thesis was toprogress in the understanding of the mechanisms involved in this novel NF-independent T3SSdependentinteraction using as model the interaction of the strain ORS3257 with the tropical legumeA. indica. It has been shown that this symbiotic process relies on a cocktail of at least 5 effectors thatplay distinct and synergistic roles in the processes of infection, nodule organogenesis andsuppression of the plant immune response. In particular, a novel nuclear effector, that we namedErnA for "Effector required for nodulation-A", is widely distributed in the Bradyrhizobium genus andwas identified as the main inducer of nodule organogenesis. In addition, we have conducted acomparative genomic analysis of 146 Bradyrhizobium genomes in order to better understand thedistribution of T3SS and the main effectors identified to date. This showed that the T3SS iswidespread in the Bradyrhizobium genus and shares a common evolutionary history with the nodgenes. This thesis work constitutes a first step in the understanding of the molecular mechanismsinvolved in this NF-independent T3SS-dependent nodulation and suggests that the T3SS ofbradyrhizobia could play a much larger symbiotic role than originally thought.; Les Bradyrhizobium sont des bactéries du sol ayant la capacité d’établir une interaction symbiotique avec de nombreuses légumineuses. Cette symbiose conduit à la formation d’un nouvel organe, le nodule, au sein duquel la bactérie peut fixer le diazote atmosphérique au bénéfice de la plante. Cette interaction repose largement sur la reconnaissance par la plante de molécules signal,les facteurs Nods (FNs) produits par la bactérie qui contrôlent les processus d’infection et d’organogénèse nodulaire. Récemment, il a été démontré que certaines souches de Bradyrhizobiumnon-photosynthétiques, telles que les souches B. elkanii USDA61 et Bradyrhizobium sp. ORS3257,possèdent la capacité de noduler certaines légumineuses (Glycine max, Aeschynomene indica) enl'absence de facteurs Nods grâce à leur système de sécrétion de type III (T3SS). Cette découverte suggère que certains effecteurs secrétés par cette machinerie de sécrétion, initialement connus pour jouer un rôle dans la répression du système immunitaire de la plante hôte, peuvent directement activer la nodulation en court-circuitant les premières étapes de la voie de signalisation initiée parles FNS. Dans un premier temps, l’objectif principal de ce travail de thèse a consisté à avancer dans la compréhension des mécanismes mis en jeu lors de cette nouvelle interaction FN-indépendante T3SSdépendanteen utilisant la souche ORS3257 en interaction avec la légumineuse tropicale A. indica. Il a été démontré que ce processus symbiotique repose sur un cocktail d'au moins 5 effecteurs quijouent un rôle distinct et complémentaire dans les processus d'infection, d'organogenèse desnodules et de répression de la réponse immune. Plus particulièrement, un nouvel effecteur nucléaire appelé ErnA pour « Effector required for nodulation-A » et largement distribué chez les Bradyrhizobium, a été identifié comme étant le principal inducteur de l’organogenèse nodulaire.Dans un second temps, nous avons mené une analyse génomique comparative sur 146 génomes deBradyrhizobium afin de mieux comprendre la distribution du T3SS et des principaux effecteurs identifiés à ce jours. Il a été mis notamment en évidence que le T3SS est très répandu dans le genreBradyrhizobium et qu’il partage une histoire évolutive commune avec les gènes de nodulation nod. L’ensemble de ce travail de thèse constitue une première étape dans la compréhension des mécanismes moléculaire recrutés pour la mise en place d’un nouveau processus de nodulation indépendante d’une signalisation FNs et suggère, par ailleurs, que le T3SS des Bradyrhizobium pourrait jouer un rôle symbiotique bien plus important qu’estimé jusqu’alors.

Vibrio parahaemolyticus causes severe gastroenteritis following consumption of contaminated seafood. Global warming has allowed this pathogen to spread worldwide, contributing to recent outbreaks. Clinical isolates are known to harbor an array of virulence factors, including T3SS1 and T3SS2; however, the precise role these systems play in intestinal disease remains unclear. There is an urgent need to improve our understanding of how V. parahaemolyticus infects hosts and causes disease. We present a novel mouse model for this facultative intracellular pathogen and observe that the T3SS2 is essential to pathogenicity. Moreover, we show that the T3SS2 effector VopC, previously shown to be a Rac and Cdc42 deamidase that facilitates bacterial uptake by nonphagocytic cells, also plays a key role in the ability of V. parahaemolyticus to invade the intestinal mucosa and cause gastroenteritis. This experimental model thus provides a valuable tool for future elucidation of virulence mechanisms used by this facultative intracellular pathogen during in vivo infection., The Gram-negative marine bacterium Vibrio parahaemolyticus is a common cause of infectious gastroenteritis due to the ingestion of contaminated seafood. Most virulent V. parahaemolyticus strains encode two type III secretion systems (T3SS1 and T3SS2); however, the roles they and their translocated effectors play in causing intestinal disease remain unclear. While studies have identified T3SS1 effectors as responsible for killing epithelial cells in culture, the T3SS2 effectors caused massive epithelial cell disruption in a rabbit ileal loop model. Additional models are thus needed to clarify the pathogen-host interactions that drive V. parahaemolyticus-associated gastroenteritis. Germfree mice were infected with a pathogenic clinical isolate of V. parahaemolyticus, RIMD2210633 (RIMD). The pathogen was found to adhere to as well as invade the cecal mucosa, accompanied by severe inflammation and dramatic mucosal damage, including widespread sloughing of infected epithelial cells. Mice infected with a V. parahaemolyticus strain lacking the T3SS1 (POR2) also developed severe pathology, similar to that seen with RIMD. In contrast, the ΔT3SS2 strain (POR3) appeared unable to invade the intestinal mucosa or cause any mucosal pathology. Confirming a role for TS332 effectors, a strain expressing the T3SS2 but lacking VopC (POR2ΔvopC), a T3SS2 effector implicated in epithelial cell invasion in culture, was strongly attenuated in invading the intestinal mucosa and in causing gastroenteritis, although infection with this mutant resulted in more pathology than the ΔT3SS2 strain. We thus present an experimental system that enables further characterization of T3SS effectors as well as the corresponding host inflammatory response involved in the gastroenteritis caused by invasive V. parahaemolyticus.

Pathogens find diverse niches for survival inside host cells where replication occurs in a relatively protected environment. Vibrio parahaemolyticus, a facultative intracellular pathogen, uses its type 3 secretion system 2 (T3SS2) to invade and replicate inside host cells. However, after extensive analysis, the T3SS2 pathogenicity island appeared to lack a mechanism for egress of this bacterium from the invaded host cell. Using a combination of cell biology, microbial genetics and lipid biochemistry, we found that VPA0226, a constitutively secreted lipase, is required for escape of Vibrio parahaemolyticus from host cells. Remarkably, this lipase must be delivered into the host cytoplasm where it preferentially uses fatty acids associated with innate immune response (i.e. arachidonic acid, 20:4) to esterify cholesterol, weakening the plasma membrane and allowing egress of the bacteria. This study reveals the resourcefulness of microbes and the interplay between virulence systems to evolve an ingenious scheme for survival and escape.Impact StatementConsidering the course of a pathogen’s evolution, there appears to be interplay between secretion systems, providing unique, synergistic mechanisms to support a successful lifestyle for possibly pathogenesis, symbiosis and/or parasitosis.

International audience

International audience; Salmonella is a facultative intracellular pathogen that invades epithelial cells of the intestine using the SPI-1 Type 3 secretion System (T3SS). Insertion of the SPI-1 T3SS translocon is facilitated by acylation of the translocator SipB, which involves a protein-protein interaction with the acyl carrier protein IacP. Using nuclear magnetic resonance and biological tests, we identified the residues of IacP that are involved in the interaction with SipB. Our results suggest that the 4 0-phosphopantetheine group that functionalizes IacP participates in the interaction. Its solvent exposition may rely on two residues highly conserved in acyl carrier proteins associated with T3SS. This study is the first to address the specificity of acyl carrier proteins associated with T3SS.

Background A rice associated bacterium Burkholderia gladioli strain NGJ1 demonstrates mycophagy, a phenomenon wherein bacteria feed on fungi. Previously, we have reported that NGJ1 utilizes type III secretion system (T3SS) to deliver a prophage tail-like protein (Bg_9562) into fungal cells to establish mycophagy. Results In this study, we report that calcium ion concentration influences the mycophagous ability of NGJ1 on Rhizoctonia solani, an important fungal pathogen. The calcium limiting condition promotes mycophagy while high calcium environment prevents it. The expression of various T3SS apparatus encoding genes of NGJ1 was induced and secretion of several potential T3SS effector proteins (including Bg_9562) into extracellular milieu was triggered under calcium limiting condition. Using LC-MS/MS proteome analysis, we identified several calcium regulated T3SS effector proteins of NGJ1. The expression of genes encoding some of these effector proteins was upregulated during mycophagous interaction of NGJ1 with R. solani. Further, mutation of one of these genes (endo-β-1, 3- glucanase) rendered the mutant NGJ1 bacterium defective in mycophagy while complementation with full length copy of the gene restored its mycophagous activity. Conclusion Our study provides evidence that low calcium environment triggers secretion of various T3SS effectors proteins into the extracellular milieu and suggests the importance of cocktail of these proteins in promoting mycophagy.

Xanthomonas translucens pv. cerealis (Xtc) causes bacterial leaf streak (BLS) of important cereal crops, including wheat (Triticum aestivum) and barley (Hordeum vulgare). Transcription activator-like effectors (TALEs) play vital roles in many plant diseases caused by Xanthomonas spp., however, TALEs have not been previously characterized in Xtc. In this study, the whole genome of NXtc01, a virulent strain of Xtc from Xinjiang, China, was sequenced and compared with genomes of other Xanthomonas spp. Xtc NXtc01 consists of a single 4,622,298 bp chromosome that encodes 4,004 genes. Alignment of the NXtc01 sequence with the draft genome of Xtc strain CFBP 2541 (United States) revealed a single giant inversion and differences in the location of two tal genes, which were designated tal1 and tal2. In NXtc01, both tal genes are located on the chromosome, whereas tal2 is plasmid-encoded in CFBP 2541. The repeat variable diresidues (RVDs) at the 12th and 13th sites within Tal2 repeat units were identical in both strains, whereas Tal1 showed differences in the third RVD. Xtc NXtc01 and CFBP 2541 encoded 35 and 33 non-TALE type III effectors (T3Es), respectively. tal1, tal2, and tal-free deletion mutants of Xtc NXtc01 were constructed and evaluated for virulence. The tal1 and tal-free deletion mutants were impaired with respect to symptom development and growth in wheat, suggesting that tal1 is a virulence factor in NXtc01. This was confirmed in gain-of-function experiments that showed the introduction of tal1, but not tal2, restored virulence to the tal-free mutant. Furthermore, we generated a hrcC deletion mutant of NXtc01; the hrcC mutant was non-pathogenic on wheat and unable to elicit a hypersensitive response in the non-host Nicotiana benthamiana. Our data provide a platform for exploring the roles of both TALEs and non-TALEs in promoting BLS on wheat.

Several bacterial pathogens including Shigella (shigellosis), Escherichia coli (urinary tract infections), Pseudomonas (lung infections), Salmonella (food poisoning), and Yersinia (plague) critically rely on a complex type three secretion system (T3SS) for infection. With the rise in multi-antibiotic resistant strains of several of these pathogens, we turn to the T3SS as a promising target for the development of novel therapeutics. The Dickenson lab at Utah State University has been the first to identify and characterize the ATPase Spa47, the energy source of the Shigella infection system. We show that Spa47 is necessary for proper T3SS formation and function, being ultimately responsible for overall Shigella virulence. We find that proper ATPase function and in turn T3SS apparatus formation can be affected by something as simple as a single mutation to the removal of a non-catalytic domain. The insights gained from this work expands our understanding of the powerhouse that fuels these infection systems and brings us a step closer to developing novel therapeutics to combat infection.

Although predicted on the basis of sequence conservation, the work presented here formally demonstrates that all classes of type III secretion systems, flagellar or virulence, share the same architecture at the level of the core structures. This absolute conservation of the unusual extramembrane structure of the core export gate complex now allows work to move to focusing on both mechanistic studies of type III but also on fundamental studies of how such a complex is assembled., Export of proteins through type III secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported through a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 Å that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assembles into an extramembrane helical assembly that likely seeds correct assembly of the rod. Here we present the structure of an equivalent complex from the Shigella virulence system at 3.5 Å by cryo-electron microscopy. This higher-resolution structure yields a more precise description of the structure and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also suggests how the SctS/FliQ subunits sequentially assemble in the complex.

Bordetella pertussis is a Gram-negative strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Previously, we have shown that RNA chaperone Hfq is required for virulence of B. pertussis. Furthermore, microarray analysis revealed that a large number of genes are affected by the lack of Hfq. This study represents the first attempt to characterize the Hfq regulon in bacterial pathogen using an integrative omics approach. Gene expression profiles were analyzed by RNA-seq and protein amounts in cell-associated and cell-free fractions were determined by LC-MS/MS technique. Comparative analysis of transcriptomic and proteomic data revealed solid correlation (r2 = 0.4) considering the role of Hfq in post-transcriptional control of gene expression. Importantly, our study confirms and further enlightens the role of Hfq in pathogenicity of B. pertussis as it shows that &Delta, hfq strain displays strongly impaired secretion of substrates of Type III secretion system (T3SS) and substantially reduced resistance to serum killing. On the other hand, significantly increased production of proteins implicated in transport of important metabolites and essential nutrients observed in the mutant seems to compensate for the physiological defect introduced by the deletion of the hfq gene.

While considered solely an extracellular pathogen, increasing evidence indicates that Pseudomonas aeruginosa encounters intracellular environment in diverse mammalian cell types, including macrophages. In the present study, we have deciphered the intramacrophage fate of wild-type P. aeruginosa PAO1 strain by live and electron microscopy. P. aeruginosa first resided in phagosomal vacuoles and subsequently could be detected in the cytoplasm, indicating phagosomal escape of the pathogen, a finding also supported by vacuolar rupture assay. The intracellular bacteria could eventually induce cell lysis, both in a macrophage cell line and primary human macrophages. Two bacterial factors, MgtC and OprF, recently identified to be important for survival of P. aeruginosa in macrophages, were found to be involved in bacterial escape from the phagosome as well as in cell lysis caused by intracellular bacteria. Strikingly, type III secretion system (T3SS) genes of P. aeruginosa were down-regulated within macrophages in both mgtC and oprF mutants. Concordantly, cyclic di-GMP (c-di-GMP) level was increased in both mutants, providing a clue for negative regulation of T3SS inside macrophages. Consistent with the phenotypes and gene expression pattern of mgtC and oprF mutants, a T3SS mutant (ΔpscN) exhibited defect in phagosomal escape and macrophage lysis driven by internalized bacteria. Importantly, these effects appeared to be largely dependent on the ExoS effector, in contrast with the known T3SS-dependent, but ExoS independent, cytotoxicity caused by extracellular P. aeruginosa towards macrophages. Moreover, this macrophage damage caused by intracellular P. aeruginosa was found to be dependent on GTPase Activating Protein (GAP) domain of ExoS. Hence, our work highlights T3SS and ExoS, whose expression is modulated by MgtC and OprF, as key players in the intramacrophage life of P. aeruginosa which allow internalized bacteria to lyse macrophages., Author summary The ability of professional phagocytes to ingest and kill microorganisms is central to host defense and Pseudomonas aeruginosa has developed mechanisms to avoid being killed by phagocytes. While considered an extracellular pathogen, P. aeruginosa has been reported to be engulfed by macrophages in animal models. Here, we visualized the fate of P. aeruginosa within cultured macrophages, revealing macrophage lysis driven by intracellular P. aeruginosa. Two bacterial factors, MgtC and OprF, recently discovered to be involved in the intramacrophage survival of P. aeruginosa, appeared to play a role in this cytotoxicity caused by intracellular bacteria. We provided evidence that type III secretion system (T3SS) gene expression is lowered intracellularly in mgtC and oprF mutants. We further showed that intramacrophage P. aeruginosa uses its T3SS, specifically the ExoS effector, to promote phagosomal escape and cell lysis. We thus describe a transient intramacrophage stage of P. aeruginosa that could contribute to bacterial dissemination.

Bordetella pertussis is a Gram-negative strictly human pathogen of the respiratory tract and the etiological agent of whooping cough (pertussis). Previously, we have shown that RNA chaperone Hfq is required for virulence of B. pertussis. Furthermore, microarray analysis revealed that a large number of genes are affected by the lack of Hfq. This study represents the first attempt to characterize the Hfq regulon in bacterial pathogen using an integrative omics approach. Gene expression profiles were analyzed by RNA-seq and protein amounts in cell-associated and cell-free fractions were determined by LC-MS/MS technique. Comparative analysis of transcriptomic and proteomic data revealed solid correlation (r2 = 0.4) considering the role of Hfq in post-transcriptional control of gene expression. Importantly, our study confirms and further enlightens the role of Hfq in pathogenicity of B. pertussis as it shows that Δhfq strain displays strongly impaired secretion of substrates of Type III secretion system (T3SS) and substantially reduced resistance to serum killing. On the other hand, significantly increased production of proteins implicated in transport of important metabolites and essential nutrients observed in the mutant seems to compensate for the physiological defect introduced by the deletion of the hfq gene.

The plant pathogen Pseudomonas savastanoi invades host plants through a type III secretion system, which is strictly regulated by a two-component system called RhpRS. The orthologues of RhpRS are widely distributed in the bacterial kingdom. The master regulator RhpR specifically depends on the phosphorylation state to regulate the majority of the virulence-related genes. Under nutrient-rich conditions, it modulates many important metabolic pathways, which consist of one-fifth of the genome. We propose that RhpRS uses phosphorylation- and nutrition-dependent mechanisms to switch between regulating virulence and metabolism, and this functionality is widely conserved among bacterial species., Pseudomonas savastanoi uses a type III secretion system (T3SS) to invade host plants. Our previous studies have demonstrated that a two-component system (TCS), RhpRS, enables P. savastanoi to coordinate the T3SS gene expression, which depends on the phosphorylation state of RhpR under different environmental conditions. Orthologues of RhpRS are distributed in a wide range of bacterial species, indicating a general regulatory mechanism. How RhpRS uses external signals and the phosphorylation state to exercise its regulatory functions remains unknown. We performed chromatin immunoprecipitation sequencing (ChIP-seq) assays to identify the specific binding sites of RhpR and RhpRD70A in either King’s B medium (KB [a T3SS-inhibiting medium]) or minimal medium (MM [a T3SS-inducing medium]). We identified 125 KB-dependent binding sites and 188 phosphorylation-dependent binding sites of RhpR. In KB, RhpR directly and positively regulated cytochrome c550 production (via ccmA) and alcohol dehydrogenase activity (via adhB) but negatively regulated anthranilate synthase activity (via trpG) and protease activity (via hemB). In addition, phosphorylated RhpR (RhpR-P) directly and negatively regulated the T3SS (via hrpR and hopR1), swimming motility (via flhA), c-di-GMP levels (via PSPPH_2590), and biofilm formation (via algD). It positively regulated twitching motility (via fimA) and lipopolysaccharide production (via PSPPH_2653). Our transcriptome sequencing (RNA-seq) analyses identified 474 and 840 new genes that were regulated by RhpR in KB and MM, respectively. We showed nutrient-rich conditions allowed RhpR to directly regulate multiple metabolic pathways of P. savastanoi and phosphorylation enabled RhpR to specifically control virulence and the cell envelope. The action of RhpRS switched between virulence and regulation of multiple metabolic pathways by tuning its phosphorylation and sensing environmental signals in KB, respectively.

Summary Bacterial canker of kiwifruit, is a severe global disease caused by Pseudomonas syringae pv. actinidiae (Psa). Here, we found that Psa biovar 3 (Psa3) was the only biovar consisting of three widely distributed clades in the largest Chinese kiwifruit cultivated area. Comparative genomics between the three clades revealed 13 polymorphic genes, each of which had multiple intra‐clade variations. For instance, we confirmed that the polymorphic copA gene, which encodes a periplasmic protein CopA that is translocated by the Twin‐arginine targeting (Tat) system, was involved in copper tolerance. We also found extensive variation in pathogenicity amongst strains within each genetically monomorphic clade. Accordingly, the pathogenic determinants of Psa3 were identified via a genomic comparison of phenotypically different strains within each clade. A case study of the high‐ and low‐virulence strains in the clade 2 of Psa3 revealed that an hfq variant involved in in vitro growth and virulence, while a conserved locus 930 bp upstream of the hrpR gene in the Type III secretion system (T3SS) cluster was required for full pathogenicity on kiwifruit and elicitation of the hypersensitivity response on non‐host Nicotiana benthamiana. The ‘‐930’ locus is involved in transcriptional regulation of hrpR/S and modulates T3SS function via the hierarchical ‘HrpR/S‐HrpL‐T3SS/effector’ regulatory cascade in Psa. Our results provide insights into the molecular basis underlying the genetic diversification and evolution of pathogenicity in Psa3 since kiwifruit canker emerged in China in the 1980s.

Submitted by Jéssica Alcimari Ferreira Mello (je_fmello@hotmail.com) on 2019-04-24T15:34:33Z No. of bitstreams: 1 Dissertação_JessicaMello_final.pdf: 3053563 bytes, checksum: c57c9c61af33a7b3c5d1dae4940c9d96 (MD5) Approved for entry into archive by Tatiana Camila Gricio (tatiana.gricio@unesp.br) on 2019-04-24T17:47:09Z (GMT) No. of bitstreams: 1 mello_jaf_me_jabo.pdf: 3053563 bytes, checksum: c57c9c61af33a7b3c5d1dae4940c9d96 (MD5) Made available in DSpace on 2019-04-24T17:47:09Z (GMT). No. of bitstreams: 1 mello_jaf_me_jabo.pdf: 3053563 bytes, checksum: c57c9c61af33a7b3c5d1dae4940c9d96 (MD5) Previous issue date: 2019-03-12 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Xanthomonas citri subsp. citri (Xac) é um fitopatógeno responsável por causar o Cancro Cítrico, relacionado a grandes perdas econômicas para a citricultura. Esta bactéria utiliza diversos mecanismos moleculares para infectar o hospedeiro, dentre eles, o Sistema de Secreção Tipo III (T3SS), considerado o principal fator de virulência expresso in planta pela Xac. Sabe-se que a ativação do T3SS é controlada por dois reguladores: HrpG e HrpX. Porém, os mecanismos que controlam estes reguladores e posteriormente a expressão do T3SS in planta ainda são pouco compreendidos. Estudos realizados por nosso grupo de pesquisa sugerem que a concentração de fosfato inorgânico (Pi) presente em meio de cultura ou em espaço apoplástico, onde a Xac causa a infecção em plantas, pode estar relacionado com a ativação do T3SS, sugerindo uma possível relação entre o Pi com os reguladores HrpG e HrpX. Em Xac, a detecção e transportorte do Pi é realizada pelo sistema de dois componentes (TCS) PhoR/PhoB, codificado pelo operon pho. Afim de averiguar a possível relação entre PhoR/PhoB com os reguladores do T3SS HrpG e HrpX, ensaios de modelagem molecular foram realizados, indicando que a proteína PhoR poderia interagir fisicamente com HrpG, fosforilando-a, e sugerindo um possível mecanismo de interação entre o TCS PhoR/PhoB e os genes reguladores do T3SS. Para testar a hipótese de interação física entre HrpG e PhoR, as proteínas recombinantes PhoR, PhoB e HrpG de Xac foram expressas em células de Escherichia coli BL21 com a finalidade de realização de ensaios de pull-down. Através da purificação por cromatografia de afinidade em coluna de Ni2+-Sepharose, as proteínas HrpG e PhoB recombinantes foram obtidas com alto grau de pureza e na forma solúvel. Entretanto a proteína PhoR se mostrou insolúvel em todos os testes de purificação e de renaturação realizados, impossibilitando a realização dos ensaios de interação física até o momento. De qualquer modo, este trabalho representa importantes avanços sobre a regulação do T3SS em Xac, tendo em vista que até o atual momento configura-se como o primeiro estudo relacionando o TCS PhoR/PhoB com HrpG. Xanthomonas citri subsp. citri (Xac) is a plant pathogen that causes citrus canker, responsible for significant economic losses in citriculture. This bacteria uses several molecular mechanisms to infect the host, such as the Type III Secretion System (T3SS), which is considered the main factor of virulence expressed in plant by Xac. Activation of T3SS is controlled by two regulators, HrpG and HrpX, however, the mechanisms that control these regulators and subsequently the expression of T3SS in planta are poorly understood. Previosuly, our research group identified that the concentration of inorganic phosphate (Pi) present in culture media or in the apoplastic space, where Xac causes infection in plants, may be related to the activation of T3SS, suggesting a possible relationship between Pi and the HrpG and HrpX regulators. In Xac, the detection and transport of Pi is performed by the two-component system (TCS) PhoR/PhoB, encoded by the operon pho. In order to investigate the possible relationship between PhoR/PhoB and the regulators of T3SS HrpG and HrpX, molecular modeling analyses were performed and indicated that the PhoR protein could physically interact with HrpG and phosphorylate it, suggesting a possible mechanism of interaction between TCS PhoR / PhoB and the T3SS regulatory genes with subsequent activation of T3SS. To test the hypothesis, recombinant forms of PhoR, PhoB and HrpG were expressed in Escherichia coli BL21 cells for the purpose of performing pull-down assays. By using Ni2+-Sepharose affinity chromatography, recombinant HrpG and PhoB proteins were obtained with high purity and in their soluble forms. However, the PhoR protein was insoluble in all the purification and renaturation tests performed, making the subsequent physical tests impossible. Nevertheless, this work represents important towards the understanding of T3SS regulation in Xac, since this is the first study correlating the TSC PhoR /PhoB proteins and HrpG. 1703888

A pathogenic lifestyle is inextricably linked with the constant necessity of facing various challenges exerted by the external environment (both within and outside the host). To successfully colonize the host and establish infection, pathogens have evolved sophisticated systems to combat the host defense mechanisms and also to be able to withstand adverse environmental conditions. Proteases, as crucial components of these systems, are involved in a variety of processes associated with infection. In phytopathogenic bacteria, they play important regulatory roles and modulate the expression and functioning of various virulence factors. Secretory proteases directly help avoid recognition by the plant immune systems, and contribute to the deactivation of the defense response pathways. Finally, proteases are important components of protein quality control systems, and thus enable maintaining homeostasis in stressed bacterial cells. In this review, we discuss the known protease functions and protease-regulated signaling processes associated with virulence of plant pathogenic bacteria.

Background Among the diverse roles of the Type III secretion-system (T3SS), one of the notable functions is that it serves as unique nano machineries in gram-negative bacteria that facilitate the translocation of effector proteins from bacteria into their host. These effector proteins serve as potential targets to control the pathogenicity conferred to the bacteria. Despite being ideal choices to disrupt bacterial systems, it has been quite an ordeal in the recent times to experimentally reveal and establish a concrete sequence-structure-function relationship for these effector proteins. This work focuses on the disease-causing spectrum of an effector protein, HopS2 secreted by the phytopathogen Pseudomonas syringae pv. tomato DC3000. Results The study addresses the structural attributes of HopS2 via a bioinformatics approach to by-pass some of the experimental shortcomings resulting in mining some critical regions in the effector protein. We have elucidated the functionally important regions of HopS2 with the assistance of sequence and structural analyses. The sequence based data supports the presence of important regions in HopS2 that are present in the other functional parts of Hop family proteins. Furthermore, these regions have been validated by an ab-initio structure prediction of the protein followed by 100 ns long molecular dynamics (MD) simulation. The assessment of these secondary structural regions has revealed the stability and importance of these regions in the protein structure. Conclusions The analysis has provided insights on important functional regions that may be vital to the effector functioning. In dearth of ample experimental evidence, such a bioinformatics approach has helped in the revelation of a few structural regions which will aid in future experiments to attain and evaluate the structural and functional aspects of this protein family. Electronic supplementary material The online version of this article (10.1186/s12859-018-2551-1) contains supplementary material, which is available to authorized users.

Type III secretion systems (T3SSs) are tightly regulated key virulence mechanisms shared by many Gram-negative pathogens. YopN, one of the substrates, is also crucial in regulation of expression, secretion and activation of the T3SS of pathogenic Yersinia species. Interestingly, YopN itself is also targeted into host cells but so far no activity or direct role for YopN inside host cells has been described. Recently, we were able show that the central region of YopN is required for efficient translocation of YopH and YopE into host cells. This was also shown to impact the ability of Yersinia to block phagocytosis. One difficulty in studying YopN is to generate mutants that are not impaired in regulation of the T3SS. In this study we extended our previous work and were able to generate specific mutants within the central region of YopN. These mutants were predicted to be crucial for formation of a putative coiled-coil domain (CCD). Similar to the previously described deletion mutant of the central region, these mutants were all impaired in translocation of YopE and YopH. Interestingly, these YopN variants were not translocated into host cells. Importantly, when these mutants were introduced in cis on the virulence plasmid, they retained full regulatory function of T3SS expression and secretion. This allowed us to evaluate one of the mutants, yopNGAGA, in the systemic mouse infection model. Using in vivo imaging technology we could verify that the mutant was also attenuated in vivo and highly impaired to establish systemic infection.

The present work comprehensively analyzed the transcriptome and phenotypes that were regulated by c-di-GMP in P. syringae. Given that the majority of diguanylate cyclases and phosphodiesterases have not been characterized in P. syringae, this work provided a very useful database for the future study on regulatory mechanism (especially its relationship with T3SS) of c-di-GMP in P. syringae. In particular, we identified three promoters that were sensitive to elevated c-di-GMP levels and inserted them into luciferase-based reporters that effectively respond to intracellular levels of c-di-GMP in P. syringae, which could be used as an economic and efficient way to measure relative c-di-GMP levels in vivo in the future., Although the ubiquitous bacterial secondary messenger cyclic diguanylate (c-di-GMP) has important cellular functions in a wide range of bacteria, its function in the model plant pathogen Pseudomonas syringae remains largely elusive. To this end, we overexpressed Escherichia coli diguanylate cyclase (YedQ) and phosphodiesterase (YhjH) in P. syringae, resulting in high and low in vivo levels of c-di-GMP, respectively. Via genome-wide RNA sequencing of these two strains, we found that c-di-GMP regulates (i) fliN, fliE, and flhA, which are associated with flagellar assembly; (ii) alg8 and alg44, which are related to the exopolysaccharide biosynthesis pathway; (iii) pvdE, pvdP, and pvsA, which are associated with the siderophore biosynthesis pathway; and (iv) sodA, which encodes a superoxide dismutase. In particular, we identified three promoters that are sensitive to elevated levels of c-di-GMP and inserted them into luciferase-based reporters that respond effectively to the c-di-GMP levels in P. syringae; these promoters could be useful in the measurement of in vivo levels of c-di-GMP in real time. Further phenotypic assays validated the RNA sequencing (RNA-seq) results and confirmed the effect on c-di-GMP-associated pathways, such as repressing the type III secretion system (T3SS) and motility while inducing biofilm production, siderophore production, and oxidative stress resistance. Taken together, these results demonstrate that c-di-GMP regulates the virulence and stress response in P. syringae, which suggests that tuning its level could be a new strategy to protect plants from attacks by this pathogen. IMPORTANCE The present work comprehensively analyzed the transcriptome and phenotypes that were regulated by c-di-GMP in P. syringae. Given that the majority of diguanylate cyclases and phosphodiesterases have not been characterized in P. syringae, this work provided a very useful database for the future study on regulatory mechanism (especially its relationship with T3SS) of c-di-GMP in P. syringae. In particular, we identified three promoters that were sensitive to elevated c-di-GMP levels and inserted them into luciferase-based reporters that effectively respond to intracellular levels of c-di-GMP in P. syringae, which could be used as an economic and efficient way to measure relative c-di-GMP levels in vivo in the future.

The Type III Secretion System (T3SS) is a protein superstructure, consisting of hundreds of subunits, which self-assemble into a molecular needle and syringe that allows bacteria to inject effector proteins directly into eukaryotic host cells. These effector proteins promote virulence in many pathogenic bacteria. The extracellular needle filament is capped by a pentameric tip complex that is involved in environmental sensing and regulation of a pore-forming complex that is secreted upon host-cell contact. Tip complexes do not self-assemble from in vitro solutions of their subunits alone suggesting that in vivo, other environmental factors are required. During in vivo assembly, each tip complex subunit unfolds within the bacteria and secrets through the needle filament whereupon they refold and localize to the filaments end. We attempt to recapitulate these conditions in vitro. The influence of unfolding and refolding on tip complex assembly was investigated using pH and temperature denaturation combined with small angle X-ray scattering (SAXS). This data confirmed that in vitro refolding can promote oligomer formation in some species but is an inefficient process. To test the effects of needle localisation we developed a novel DNA-templating approach. Nanoscale DNA templates with the same dimensions of the needle filament (~8 nm) were designed, synthesised, and measured with electron microscopy, molecular dynamics simulations, and SAXS. Tip complex subunits were then chemically localized to these DNA templates drastically increasing their relative concentration and interaction frequency. Electron micrographs of these DNA-templated assemblies revealed some particles with compact structures similar to those observed in vivo. Chemical cross-linking and mass-spectroscopy provided distance restraints between residues of subunits in DNA-templated assemblies. These restraints allowed the generation of rigid body models of DNA-templated assemblies that resemble current models of in vitro tip complexes. These results present a promising future for the ability to rationally control protein assembly of difficult to access structures using the DNA-templating approach developed here.

Presentación oral La especie Pseudomonas savastanoi pertenece al complejo Pseudomonas syringae, el cual está constituido por un conjunto de bacterias fitopatógenas Gram (-) de alto interés agrícola y económico. Dentro de esta especie se han descrito hasta la fecha cuatro patovares capaces de infectar huéspedes leñosos: pv. savastanoi (aislados de olivo), pv. nerii (aislados de adelfa), pv. fraxini (aislados de fresno) y pv. retacarpa (aislados de retama). La cepa NCPPB 3335 del patovar savastanoi (Psv), establecida como modelo para el estudio de la interacción de Pseudomonas patógenas con plantas leñosas, es el agente causal de la tuberculosis del olivo, enfermedad caracterizada por la aparición de tumores en las partes aéreas de la planta. Uno de los factores de patogenicidad de Psv más relevante es el sistema de secreción tipo III (T3SS) y su repertorio de efectores (T3E). La disponibilidad de la secuencia de Psv NCPPB 3335 nos ha permitido la predicción bioinformática de los genes que codifican su T3SS y su repertorio de T3E en base a la homología con otros efectores previamente descritos. Esta cepa presenta un repertorio de efectores constituido por 28 T3E. Con el objetivo de analizar la expresión del repertorio de T3E en Psv, se ha llevado a cabo un análisis RNAseq comparativo entre la cepa silvestre Psv NCPPB 3335 y un mutante de la misma del gen hrpL, el cual codifica un activador transcripcional de los genes del T3SS y de la mayoría de sus T3E. Las secuencias procedentes de este RNAseq se están analizando actualmente y los resultados que se obtengan del mismo nos permitirá no sólo analizar la expresión de los T3E que hemos identificado en esta cepa, sino también caracterizar el regulón HrpL completo en la misma y quizás identificar nuevos posibles T3E. Los resultados obtenidos, se compararán con una predicción de promotores regulados por HrpL (hrp-box), que se llevará a cabo utilizando una herramienta bioinformática generada por nuestro equipo. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

Significance Legumes have a tremendous ecological and agronomic importance due to their ability to interact symbiotically with nitrogen-fixing rhizobia. In most of the rhizobial–legume symbioses, the establishment of the interaction requires the plant perception of the bacterial lipochitooligosaccharide Nod factor signal. However, some bradyrhizobia can activate the symbiosis differently, thanks to their type III secretion system, which delivers effector proteins into the host cell. Here, we demonstrate that this symbiotic process relies on a small set of effectors playing distinct and complementary roles. Most remarkably, a nuclear-targeted effector named ErnA conferred the ability to form nodules. The understanding of this alternative pathway toward nitrogen-fixing symbiosis could pave the way for designing new strategies to transfer nodulation into cereals., Several Bradyrhizobium species nodulate the leguminous plant Aeschynomene indica in a type III secretion system-dependent manner, independently of Nod factors. To date, the underlying molecular determinants involved in this symbiotic process remain unknown. To identify the rhizobial effectors involved in nodulation, we mutated 23 out of the 27 effector genes predicted in Bradyrhizobium strain ORS3257. The mutation of nopAO increased nodulation and nitrogenase activity, whereas mutation of 5 other effector genes led to various symbiotic defects. The nopM1 and nopP1 mutants induced a reduced number of nodules, some of which displayed large necrotic zones. The nopT and nopAB mutants induced uninfected nodules, and a mutant in a yet-undescribed effector gene lost the capacity for nodule formation. This effector gene, widely conserved among bradyrhizobia, was named ernA for “effector required for nodulation-A.” Remarkably, expressing ernA in a strain unable to nodulate A. indica conferred nodulation ability. Upon its delivery by Pseudomonas fluorescens into plant cells, ErnA was specifically targeted to the nucleus, and a fluorescence resonance energy transfer–fluorescence lifetime imaging microscopy approach supports the possibility that ErnA binds nucleic acids in the plant nuclei. Ectopic expression of ernA in A. indica roots activated organogenesis of root- and nodule-like structures. Collectively, this study unravels the symbiotic functions of rhizobial type III effectors playing distinct and complementary roles in suppression of host immune functions, infection, and nodule organogenesis, and suggests that ErnA triggers organ development in plants by a mechanism that remains to be elucidated.

The species Pseudomonas savastanoi, a member of the Pseudomonas syringae complex, includes four pathovars causing knots or excrescences in woody hosts: P. savastanoi pv. savastanoi (Psv), pv. fraxini (Psf), pv. nerii (Psn) and pv. retacarpa (Psr), comprising isolates from olive, ash, oleander and broom plants, respectively. One of the better characterized pathogenicity factor in this bacterial complex is the type III secretion system (T3SS) and its effector (T3E) repertoire. Transcription of the T3SS structural genes and most of its T3E genes is positively regulated HrpL. We have constructed ∆hrpL mutants in a model strain belonging to each of this four pathovars and analyzed the role of this gene in the pathogenicity of P. savastanoi. As previously reported for Psv and Psn, the hrpL gene is also necessary in Psf and Psr for both symptoms production during a compatible interaction and the induction of the hypersensitive response in a resistant host. To characterize the HrpL regulon, we are currently following two strategies (i) the bioinformatics identification of HrpL-dependent promoters in the genomes of several P. savastanoi pathovars and, (ii) a comparative RNAseq analysis of a wild-type Psv strain and its ∆hrpL mutant. The results obtained will be compared with those published for other P. syringae strains isolated from herbaceous hosts, in order to identify specific HrpL-dependent proteins involved in the interaction with woody hosts. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.

The type III secretion system (T3SS) is a principal virulence determinant of the model bacterial plant pathogen Pseudomonas syringae. T3SS effector proteins inhibit plant defense signaling pathways in susceptible hosts and elicit evolved immunity in resistant plants. The extracytoplasmic function sigma factor HrpL coordinates the expression of most T3SS genes. Transcription of hrpL is dependent on sigma-54 and the codependent enhancer binding proteins HrpR and HrpS for hrpL promoter activation. hrpL is oriented adjacently to and divergently from the HrpL-dependent gene hrpJ, sharing an intergenic upstream regulatory region. We show that association of the RNA polymerase (RNAP)-HrpL complex with the hrpJ promoter element imposes negative autogenous control on hrpL transcription in P. syringae pv. tomato DC3000. The hrpL promoter was upregulated in a ΔhrpL mutant and was repressed by plasmid-borne hrpL. In a minimal Escherichia coli background, the activity of HrpL was sufficient to achieve repression of reconstituted hrpL transcription. This repression was relieved if both the HrpL DNA-binding function and the hrp-box sequence of the hrpJ promoter were compromised, implying dependence upon the hrpJ promoter. DNA-bound RNAP-HrpL entirely occluded the HrpRS and partially occluded the integration host factor (IHF) recognition elements of the hrpL promoter in vitro, implicating inhibition of DNA binding by these factors as a cause of negative autogenous control. A modest increase in the HrpL concentration caused hypersecretion of the HrpA1 pilus protein but intracellular accumulation of later T3SS substrates. We argue that negative feedback on HrpL activity fine-tunes expression of the T3SS regulon to minimize the elicitation of plant defenses., IMPORTANCE The United Nations Food and Agriculture Organization has warned that agriculture will need to satisfy a 50% to 70% increase in global food demand if the human population reaches 9 billion by 2050 as predicted. However, diseases caused by microbial pathogens represent a major threat to food security, accounting for over 10% of estimated yield losses in staple wheat, rice, and maize crops. Understanding the decision-making strategies employed by pathogens to coordinate virulence and to evade plant defenses is vital for informing crop resistance traits and management strategies. Many plant-pathogenic bacteria utilize the needle-like T3SS to inject virulence factors into host plant cells to suppress defense signaling. Pseudomonas syringae is an economically and environmentally devastating plant pathogen. We propose that the master regulator of its entire T3SS gene set, HrpL, downregulates its own expression to minimize elicitation of plant defenses. Revealing such conserved regulatory strategies will inform future antivirulence strategies targeting plant pathogens.

Bradyrhizobium elkanii USDA61 possesses a functional type III secretion system (T3SS) that controls host-specific symbioses with legumes. Here, we demonstrated that B. elkanii T3SS is essential for the nodulation of several southern Asiatic Vigna mungo cultivars. Strikingly, inactivation of either Nod factor synthesis or T3SS in B. elkanii abolished nodulation of the V. mungo plants. Among the effectors, NopL was identified as a key determinant for T3SS-dependent symbiosis. Mutations of other effector genes, such as innB, nopP2, and bel2-5, also impacted symbiotic effectiveness, depending on host genotypes. The nopL deletion mutant formed no nodules on V. mungo, but infection thread formation was still maintained, thereby suggesting its pivotal role in nodule organogenesis. Phylogenetic analyses revealed that NopL was exclusively conserved among Bradyrhizobium and Sinorhizobium (Ensifer) species and showed a different phylogenetic lineage from T3SS. These findings suggest that V. mungo evolved a unique symbiotic signaling cascade that requires both NFs and T3Es (NopL).

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic Escherichia coli (EPEC) are attaching and effacing (A/E) pathogens, which translocate effector proteins to intestinal enterocytes through a type III secretion system (T3SS). T3SS and most of its effector proteins are encoded in a pathogenicity island called LEE. Recently, new effectors have been located outside the LEE. This study aimed to characterize EspY3, a novel non-LEE encoded T3SS effector of EHEC. EspY3 shares homology with SopD and PipB2 effector proteins of Salmonella&rsquo, s T3SS-1 and T3SS-2, respectively. The presence of recombinant EspY3 in the supernatant samples demonstrated that EspY3 was secreted by the T3SS of EHEC and EPEC. Through infection assays, we demonstrated the translocation of EspY3 into Caco-2 cells by T3SS of EPEC. The subcellular localization of EspY3 was determined in the pedestal region, where its presence generates a significant increase in the size of the pedestals area. The EspY3 effector induced the elongation of polymerized actin pedestals in infected Caco-2 by EPEC. This study confirmed that EspY3 is part of the repertoire of T3SS effectors of EHEC O157:H7, and that it participates in modeling cellular actin during the infection.

Many Gram-negative bacterial symbionts and pathogens employ a type III secretion system (T3SS) to live in contact with eukaryotic cells. Because T3SSs inject bacterial proteins (effectors) directly into host cells, the switching of secretory substrates between translocators and effectors in response to host cell attachment is a crucial step for the effective delivery of effectors. Here, we show that the protein secretion switch of Vibrio parahaemolyticus T3SS2, which is a main contributor to the enteropathogenicity of a food poisoning bacterium, is regulated by two gatekeeper proteins, VgpA and VgpB. In the absence of these gatekeepers, effector secretion was activated, but translocator secretion was abolished, causing the loss of virulence. We found that the K+ concentration, which is high inside the host cell but low outside, is a key factor for VgpA- and VgpB-mediated secretion switching. Exposure of wild-type bacteria to K+ ions provoked both gatekeeper and effector secretions but reduced the level of secretion of translocators. The secretion protein profile of wild-type bacteria cultured with 0.1 M KCl was similar to that of gatekeeper mutants. Furthermore, depletion of K+ ions in host cells diminished the efficiency of T3SS2 effector translocation. Thus, T3SS2 senses the high intracellular concentration of K+ of the host cell so that T3SS2 effectors can be effectively injected., IMPORTANCE The pathogenesis of many Gram-negative bacterial pathogens arises from a type III secretion system (T3SS), whereby bacterial proteins (effectors) are directly injected into host cells. The injected effectors then modify host cell functions. For effective delivery of effector proteins, bacteria need to both recognize host cell attachment and switch the type of secreted proteins. Here, we identified gatekeeper proteins that play important roles in a T3SS2 secretion switch of Vibrio parahaemolyticus, a causative agent of food-borne gastroenteritis. We also found that K+, which is present in high concentrations inside the host cell but in low concentrations outside, is a key factor for the secretion switch. Thus, V. parahaemolyticus senses the high intracellular K+ concentration, triggering the effective injection of effectors.

Gram-negative bacteria utilize the type III secretion system (T3SS) to inject effector proteins into the host cell cytoplasm, where they subvert cellular functions and assist pathogen invasion. The conserved type III-associated ATPase is critical for the separation of chaperones from effector proteins, the unfolding of effector proteins and translocating them through the narrow channel of the secretion apparatus. However, how ATP hydrolysis is coupled to the mechanical work of the enzyme remains elusive. Herein, we present a complete description of nucleoside triphosphate binding by surface presentation antigens 47 (Spa47) from Shigella flexneri, based on crystal structures containing ATPγS, a catalytic magnesium ion and an ordered water molecule. Combining the crystal structures of Spa47-ATPγS and unliganded Spa47, we propose conformational changes in Spa47 associated with ATP binding, the binding of ATP induces a conformational change of a highly conserved luminal loop, facilitating ATP hydrolysis by the Spa47 ATPase. Additionally, we identified a specific hydrogen bond critical for ATP recognition and demonstrated that, while ATPγS is an ideal analog for probing ATP binding, AMPPNP is a poor ATP mimic. Our findings provide structural insight pertinent for inhibitor design.

Many strains of Escherichia coli carry a 29,250-bp ETT2 pathogenicity island (PAI), which includes genes predicted to encode type III secretion system (T3SS) components. Because it is similar to the Salmonella pathogenicity island 1 (SPI-1) system, encoding a T3SS in Salmonella enterica, it was assumed that ETT2 also encodes a secretion system injecting effectors into host cells. This assumption was checked in E. coli serotype O2—associated with urinary tract infections and septicemia—which has an intact ETT2 gene cluster, in contrast to most strains in which this cluster carries deletions and mutations. A proteomic search did not reveal any putative secreted effector. Instead, the majority of the secreted proteins were identified as flagellar proteins. A deletion of the ETT2 gene cluster significantly reduced the secretion of flagellar proteins, resulting in reduced motility. There was also a significant reduction in the transcriptional level of flagellar genes, indicating that ETT2 affects the synthesis, rather than secretion, of flagellar proteins. The ETT2 deletion also resulted in additional major changes in secretion of fimbrial proteins and cell surface proteins, resulting in relative resistance to detergents and hydrophobic antibiotics (novobiocin), secretion of large amounts of outer membrane vesicles (OMVs), and altered multicellular behavior. Most important, the ETT2 deletion mutants were sensitive to serum. These major changes indicate that the ETT2 gene cluster has a global effect on cell surface and physiology, which is especially important for pathogenicity, as it contributes to the ability of the bacteria to survive serum and cause sepsis., IMPORTANCE Drug-resistant extraintestinal pathogenic E. coli (ExPEC) strains are major pathogens, especially in hospital- and community-acquired infections. They are the major cause of urinary tract infections and are often involved in septicemia with high mortality. ExPEC strains are characterized by broad-spectrum antibiotic resistance, and development of a vaccine is not trivial because the ExPEC strains include a large number of serotypes. It is therefore important to understand the virulence factors that are involved in pathogenicity of ExPEC and identify new targets for development of antibacterial drugs or vaccines. Such a target could be ETT2, a unique type III secretion system present (complete or in parts) in many ExPEC strains. Here, we show that this system has a major effect on the bacterial surface—it affects sensitivity to drugs, motility, and secretion of extracellular proteins and outer membrane vesicles. Most importantly, this system is important for serum resistance, a prerequisite for septicemia.

Dickeya dadantii is a bacterial plant pathogen that causes soft rot disease on a wide range of host plants. The type III secretion system (T3SS) is an important virulence factor in D. dadantii. Expression of the T3SS is induced in the plant apoplast or in hrp-inducing minimal medium (hrp-MM), and is repressed in nutrient-rich media. Despite the understanding of induction conditions, how individual cells in a clonal bacterial population respond to these conditions and modulate T3SS expression is not well understood. In our previous study, we reported that in a clonal population, only a small proportion of bacteria highly expressed T3SS genes while the majority of the population did not express T3SS genes under hrp-MM condition. In this study, we developed a method that enabled in situ observation and quantification of gene expression in single bacterial cells in planta. Using this technique, we observed that the expression of the T3SS genes hrpA and hrpN is restricted to a small proportion of D. dadantii cells during the infection of potato. We also report that the expression of T3SS genes is higher at early stages of infection compared to later stages. This expression modulation is achieved through adjusting the ratio of T3SS ON and T3SS OFF cells and the expression intensity of T3SS ON cells. Our findings not only shed light into how bacteria use a bi-stable gene expression manner to modulate an important virulence factor, but also provide a useful tool to study gene expression in individual bacterial cells in planta.

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors—a type III secretion system (T3SS) and Shiga toxins (Stxs)—that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection., IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors—a type III secretion system (T3SS) and Shiga toxins (Stxs)—that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3) and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogen’s key virulence factors.