RESUMEN
Mycobacterium tuberculosis (Mtb) possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. Mtb strains deleted for the genes encoding substrates of the ESX-3 T7SS, esxG or esxH, require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of esxG or esxH deletions were isolated, which enabled growth to high titers or restored virulence. Suppression was conferred by mechanisms that cause overexpression of an ESX-3 paralogous region that lacks genes for the secretion apparatus but encodes EsxR and EsxS, apparent ESX-3 orphan substrates that functionally compensate for the lack of EsxG or EsxH. The mechanisms include the disruption of a transcriptional repressor and a massive 38- to 60-fold gene amplification. These data identify an iron acquisition regulon, provide insight into T7SS, and reveal a mechanism of Mtb chromosome evolution involving "accordion-type" amplification.
Asunto(s)
Mycobacterium tuberculosis/genética , Sistemas de Secreción Tipo VII/genética , Animales , Sistemas de Secreción Bacterianos/genética , Evolución Biológica , Evolución Molecular , Amplificación de Genes/genética , Ratones , Mycobacterium tuberculosis/metabolismo , Sistemas de Secreción Tipo VII/fisiología , Virulencia , Factores de Virulencia/genéticaAsunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Farmacorresistencia Bacteriana , Bacterias/genética , Bacterias/metabolismo , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/terapia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/genética , Sistemas de Secreción Bacterianos/metabolismo , Terapia Biológica , Descubrimiento de Drogas , HumanosRESUMEN
Type VI secretion systems (T6SSs) are a class of macromolecular machines that are recognized as an important virulence mechanism in several gram-negative bacteria. The genome of Pantoea ananatis LMG 2665(T), a pathogen of pineapple fruit and onion plants, carries two gene clusters whose predicted products have homology with T6SS-associated gene products from other bacteria. Nothing is known regarding the role of these T6SS-1 and T6SS-3 gene clusters in the biology of P. ananatis. Here, we present evidence that T6SS-1 plays an important role in the pathogenicity of P. ananatis LMG 2665(T) in onion plants, while a strain lacking T6SS-3 remains as pathogenic as the wild-type strain. We also investigated the role of the T6SS-1 system in bacterial competition, the results of which indicated that several bacteria compete less efficiently against wild-type LMG 2665(T) than a strain lacking T6SS-1. Additionally, we demonstrated that these phenotypes of strain LMG 2665(T) were reliant on the core T6SS products TssA and TssD (Hcp), thus indicating that the T6SS-1 gene cluster encodes a functioning T6SS. Collectively, our data provide the first evidence demonstrating that the T6SS-1 system is a virulence determinant of P. ananatis LMG 2665(T) and plays a role in bacterial competition.
Asunto(s)
Sistemas de Secreción Bacterianos/genética , Interacciones Huésped-Patógeno/genética , Pantoea/genética , Pantoea/patogenicidad , Enfermedades de las Plantas/microbiología , Virulencia/genética , Sistemas de Secreción Bacterianos/fisiología , Técnicas de Inactivación de Genes , Genes Bacterianos , Interacciones Huésped-Patógeno/fisiología , Familia de Multigenes , Mutación , Cebollas/microbiología , Pantoea/fisiología , Virulencia/fisiologíaRESUMEN
Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin, several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel diagnostic tool, tilivalline may serve as a biomarker for K oxytoca-induced cytotoxicity in humans and animals through detection in various samples from food to diseased samples using LC-MS/MS. Induction of K. oxytoca cytotoxin by consumption of soy may be in part involved in the pathogenesis of gastrointestinal disease.
Asunto(s)
Toxinas Bacterianas/toxicidad , Benzodiazepinonas/toxicidad , Infecciones por Klebsiella/veterinaria , Klebsiella oxytoca/patogenicidad , Animales , Sistemas de Secreción Bacterianos/genética , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/química , Toxinas Bacterianas/aislamiento & purificación , Benzodiazepinonas/química , Benzodiazepinonas/aislamiento & purificación , Benzodiazepinonas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Haplorrinos , Células HeLa , Humanos , Infecciones por Klebsiella/microbiología , Klebsiella oxytoca/efectos de los fármacos , Klebsiella oxytoca/aislamiento & purificación , Klebsiella oxytoca/metabolismo , Ratones , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Ratas , Glycine max/química , PorcinosRESUMEN
As a species complex, Pseudomonas syringae exists in both agriculture and natural aquatic habitats. P.viridiflava, a member of this complex, has been reported to be phenotypically largely homogenous. We characterized strains from different habitats, selected based on their genetic similarity to previously described P.viridiflava strains. We revealed two distinct phylogroups and two different kinds of variability in phenotypic traits and genomic content. The strains exhibited phase variation in phenotypes including pathogenicity and soft rot on potato. We showed that the presence of two configurations of the Type III Secretion System [single (S-PAI) and tripartite (T-PAI) pathogenicity islands] are not correlated with pathogenicity or with the capacity to induce soft rot in contrast to previous reports. The presence/absence of the avrE effector gene was the only trait we found to be correlated with pathogenicity of P.viridiflava. Other Type III secretion effector genes were not correlated with pathogenicity. A genomic region resembling an exchangeable effector locus (EEL) was found in S-PAI strains, and a probable recombination between the two PAIs is described. The ensemble of the variability observed in these phylogroups of P.syringae likely contributes to their adaptability to alternating opportunities for pathogenicity or saprophytic survival.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Variación Genética , Genoma Bacteriano , Pseudomonas syringae/patogenicidad , Pseudomonas/patogenicidad , Solanum tuberosum/microbiología , Adaptación Biológica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/genética , Sitios Genéticos , Islas Genómicas , Genotipo , Fenotipo , Filogenia , Enfermedades de las Plantas/microbiología , Pseudomonas/clasificación , Pseudomonas/genética , Pseudomonas/metabolismo , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , VirulenciaRESUMEN
XopN is a type III effector protein from Xanthomonas campestris pathovar vesicatoria that suppresses PAMP-triggered immunity (PTI) in tomato. Previous work reported that XopN interacts with the tomato 14-3-3 isoform TFT1; however, TFT1's role in PTI and/or XopN virulence was not determined. Here we show that TFT1 functions in PTI and is a XopN virulence target. Virus-induced gene silencing of TFT1 mRNA in tomato leaves resulted in increased growth of Xcv ΔxopN and Xcv ΔhrpF demonstrating that TFT1 is required to inhibit Xcv multiplication. TFT1 expression was required for Xcv-induced accumulation of PTI5, GRAS4, WRKY28, and LRR22 mRNAs, four PTI marker genes in tomato. Deletion analysis revealed that the XopN C-terminal domain (amino acids 344-733) is sufficient to bind TFT1. Removal of amino acids 605-733 disrupts XopN binding to TFT1 in plant extracts and inhibits XopN-dependent virulence in tomato, demonstrating that these residues are necessary for the XopN/TFT1 interaction. Phos-tag gel analysis and mass spectrometry showed that XopN is phosphorylated in plant extracts at serine 688 in a putative 14-3-3 recognition motif. Mutation of S688 reduced XopN's phosphorylation state but was not sufficient to inhibit binding to TFT1 or reduce XopN virulence. Mutation of S688 and two leucines (L64,L65) in XopN, however, eliminated XopN binding to TFT1 in plant extracts and XopN virulence. L64 and L65 are required for XopN to bind TARK1, a tomato atypical receptor kinase required for PTI. This suggested that TFT1 binding to XopN's C-terminal domain might be stabilized via TARK1/XopN interaction. Pull-down and BiFC analyses show that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold. This is the first report showing that a type III effector targets a host 14-3-3 involved in PTI to promote bacterial pathogenesis.
Asunto(s)
Proteínas 14-3-3/metabolismo , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Solanum lycopersicum/microbiología , Transposasas/metabolismo , Xanthomonas campestris/patogenicidad , Proteínas 14-3-3/genética , Proteínas 14-3-3/inmunología , Sistemas de Secreción Bacterianos/genética , Sistemas de Secreción Bacterianos/inmunología , Silenciador del Gen , Solanum lycopersicum/genética , Solanum lycopersicum/inmunología , Solanum lycopersicum/metabolismo , Mutación , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/inmunología , Transposasas/genética , Transposasas/inmunología , Virulencia/genética , Xanthomonas campestris/enzimología , Xanthomonas campestris/genéticaRESUMEN
Antibiotic therapy is the most commonly used strategy to control pathogenic infections; however, it has contributed to the generation of antibiotic-resistant bacteria. To circumvent this emerging problem, we are searching for compounds that target bacterial virulence factors rather than their viability. Pseudomonas aeruginosa, an opportunistic human pathogen, possesses a type III secretion system (T3SS) as one of the major virulence factors by which it secretes and translocates T3 effector proteins into human host cells. The fact that this human pathogen also is able to infect several plant species led us to screen a library of phenolic compounds involved in plant defense signaling and their derivatives for novel T3 inhibitors. Promoter activity screening of exoS, which encodes a T3-secreted toxin, identified two T3 inhibitors and two T3 inducers of P. aeruginosa PAO1. These compounds alter exoS transcription by affecting the expression levels of the regulatory small RNAs RsmY and RsmZ. These two small RNAs are known to control the activity of carbon storage regulator RsmA, which is responsible for the regulation of the key T3SS regulator ExsA. As RsmY and RsmZ are the only targets directly regulated by GacA, our results suggest that these phenolic compounds affect the expression of exoS through the GacSA-RsmYZ-RsmA-ExsA regulatory pathway.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Fenoles/farmacología , Pseudomonas aeruginosa/metabolismo , Factores de Transcripción/metabolismo , Antibacterianos/química , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Genes Reguladores , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento , Humanos , Fenoles/química , Extractos Vegetales/química , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Transcripción Genética/efectos de los fármacos , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Bacterial wilt (brown rot) disease of potato caused by Ralstonia solanacearum is one of the most important bacterial diseases and a major constraint on potato production worldwide. Through a comparative genomic analysis between R. solanacearum'race 3 biovar 2' (R3bv2) strains, we identified a 77 kb region in strain UW551 which is specifically absent in the hypoaggressive strain IPO1609. We proved that IPO1609 indeed carries a 77 kb genomic deletion and provide genetic evidence that occurrence of this deletion is responsible for almost complete loss of pathogenicity of this strain. We carried out a functional analysis of this 77 kb region in strain UW551 using a combination of gene deletion and functional complementation approaches which identified the methionine biosynthesis genes metER as having a major contribution to IPO1609 pathogenesis. Deletion of the metER genes significantly impacts pathogenicity of R3bv2 strains but does not lead to methionine auxotrophy nor reduced ability to multiply in planta. In addition, this study indicated that three type III secretion system effectors or a type VI secretion system present within the 77 kb region have no or very minor contribution to pathogenicity.
Asunto(s)
Genoma Bacteriano , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/genética , Eliminación de Secuencia , Sistemas de Secreción Bacterianos/genética , Secuencia de Bases , Hibridación Genómica Comparativa , Prueba de Complementación Genética , Genómica , Metionina/biosíntesis , Datos de Secuencia Molecular , Fenotipo , Mapeo Físico de Cromosoma , Plásmidos/genética , Ralstonia solanacearum/patogenicidad , Solanum tuberosum/microbiologíaRESUMEN
The immunostimulatory properties conferred by vaccine adjuvants require caspase-1 for processing of IL-1ß and IL-18. Caspase-1 is activated in response to a breach of the cytosolic compartment by microbes and the process is initiated by intracellular pattern recognition receptors within inflammasomes. Listeria monocytogenes is detected in the cytosol by the NLRC4, NLRP3 and AIM2 inflammasomes. NLRC4 is activated by flagellin, and L. monocytogenes evades NLRC4 by repressing flagellin expression. We generated an L. monocytogenes strain that was forced to express flagellin in the host cell cytosol. This strain hyperactivated caspase-1 and was preferentially cleared via NLRC4 detection in an IL-1ß/IL-18 independent manner. We also created a strain of L. monocytogenes with forced expression of another NLRC4 agonist, PrgJ, from the Type III secretion system of Salmonella typhimurium. Forced expression of flagellin or PrgJ resulted in attenuation, yet both strains conferred protective immunity in mice against lethal challenge with L. monocytogenes. This work is the first demonstration of specific targeting of the caspase-1 activation pathway to generate a safe and potent L. monocytogenes-based vaccine. Moreover, the attenuated strains with embedded flagellin or PrgJ adjuvants represent attractive vectors for vaccines aimed at eliciting T-cell responses.