RESUMEN
Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Enterobacteriaceae/genética , Regulación Bacteriana de la Expresión Génica/genética , Genes Reporteros , Bacterias Gramnegativas/genética , Modelos Genéticos , Mutación , Plásmidos , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genética , Factores de Virulencia/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
Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak in the model plant rice, possesses a hypersensitive response and pathogenicity (hrp), hrp-conserved (hrc), hrp-associated (hpa) cluster (hrp-hrc-hpa) that encodes a type III secretion system (T3SS) through which T3SS effectors are injected into host cells to cause disease or trigger plant defenses. Mutations in this cluster usually abolish the bacterial ability to cause hypersensitive response in nonhost tobacco and pathogenicity in host rice. In Xanthomonas spp., these genes are generally assumed to be regulated by the key master regulators HrpG and HrpX. However, we present evidence that, apart from HrpG and HrpX, HrpD6 is also involved in regulating the expression of hrp genes. Interestingly, the expression of hpa2, hpa1, hpaB, hrcC, and hrcT is positively controlled by HrpD6. Transcriptional expression assays demonstrated that the expression of the hrcC, hrpD5, hrpE, and hpa3 genes was not completely abolished by hrpG and hrpX mutations. As observed in analysis of their corresponding mutants, HrpG and HrpX exhibit contrasting gene regulation, particularly for hpa2 and hrcT. Other two-component system regulators (Zur, LrpX, ColR/S, and Trh) did not completely inhibit the expression of hrcC, hrpD5, hrpE, and hpa3. Immunoblotting assays showed that the secretion of HrpF, which is an HpaB-independent translocator, is not affected by the mutation in hrpD6. However, the mutation in hrpD6 affects the secretion of an HpaB-dependent TAL effector, AvrXa27. These novel findings suggest that, apart from HrpG and HrpX, HrpD6 plays important roles not only in the regulation of hrp genes but also in the secretion of TAL effectors.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Enfermedades de las Plantas/microbiología , Xanthomonas/genética , Proteínas Bacterianas/genética , Células Cultivadas , ADN Bacteriano/genética , Genes Bacterianos/genética , Genes Reguladores/genética , Genes Reporteros , Prueba de Complementación Genética , Familia de Multigenes/genética , Operón/genética , Oryza/microbiología , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genética , Plantones/microbiología , Eliminación de Secuencia , Transcripción Genética , Virulencia , Xanthomonas/metabolismo , Xanthomonas/patogenicidadRESUMEN
Xanthomonas oryzae pv. oryzicola, the causative agent of bacterial leaf streak, injects a plethora of effectors through the type III secretion system (T3SS) into rice cells to cause disease. The T3SS, encoded by the hrp genes, is essential for the pathogen to elicit the hypersensitive response (HR) in nonhost tobacco and for pathogenicity in host rice. Whether or not a putative lytic transglycosylase, Hpa2, interacts with a translocon protein, HrpF, to facilitate bacterial pathogenicity remains unknown. Here we demonstrated that both the hpa2 and hrpF genes are required for the pathogenicity of X. oryzae pv. oryzicola strain RS105 in rice but not for HR induction in tobacco. The expression of hpa2 was positively regulated by HrpG and HrpD6 but not by HrpX. In vivo secretion and subcellular localization analyses confirmed that Hpa2 secretion is dependent on HpaB (a T3SS exit protein) and that Hpa2 binds to the host cell membrane. Protein-protein assays demonstrated that Hpa2 interacts with HrpF. In planta translocation of AvrXa10 indicated that the mutation in hpa2 and hrpF inhibits the injection of the HpaB-dependent transcriptional activator-like (TAL) effector into rice. These findings suggest that Hpa2 and HrpF form a complex to translocate T3S effectors into plant cells for pathogenesis in host rice.
Asunto(s)
Proteínas Bacterianas/metabolismo , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Transactivadores/metabolismo , Factores de Virulencia/metabolismo , Xanthomonas/patogenicidad , Glicosiltransferasas/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de Proteínas , Nicotiana/microbiología , Xanthomonas/metabolismoRESUMEN
The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we showed that PNPase downregulates the transcription of T3SS structural and effector genes of the phytopathogenic bacterium Dickeya dadantii. This negative regulation of T3SS by PNPase occurs by repressing the expression of hrpL, encoding a master regulator of T3SS in D. dadantii. By reducing rpoN mRNA stability, PNPase downregulates the transcription of hrpL, which leads to a reduction in T3SS gene expression. Moreover, we have found that PNPase downregulates T3SS by decreasing hrpL mRNA stability. RsmB, a regulatory sRNA, enhances hrpL mRNA stability in D. dadantii. Our results suggest that PNPase decreases the amount of functional RsmB transcripts that could result in reduction of hrpL mRNA stability. In addition, bistable gene expression (differential expression of a single gene that creates two distinct subpopulations) of hrpA, hrpN, and dspE was observed in D. dadantii under in vitro conditions. Although PNPase regulates the proportion of cells in the high state and the low state of T3SS gene expression, it appears that PNPase is not the key switch that triggers the bistable expression patterns of T3SS genes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Dickeya chrysanthemi/enzimología , Exorribonucleasas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , ARN Bacteriano/metabolismo , Proteínas Bacterianas/genética , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/metabolismo , Dickeya chrysanthemi/patogenicidad , Exorribonucleasas/genética , ARN Bacteriano/genéticaRESUMEN
The type III secretion system (T3SS) is considered one of the major virulence factors in many bacterial pathogens. This report demonstrates that RssB, ClpXP, and RpoS play a role in T3SS regulation of Dickeya dadantii 3937. ClpP is a serine-type protease which associates with the ClpX chaperone to form a functional Clp proteolytic complex for degradation of proteins. With the assistance of recognition factor RssB, ClpXP degrades the RpoS sigma factor. RpoS positively regulates the expression of the rsmA gene encoding an RNA-binding regulatory protein. By interacting with the hrpL mRNA, RsmA reduces HrpL production and downregulates the T3SS genes in the HrpL regulon. In addition, ClpXP, RssB, and RpoS affect pectinolytic enzyme production in D. dadantii 3937, probably through RsmA. The ClpXP and RssB proteins are essential for bacterial virulence.
Asunto(s)
Dickeya chrysanthemi/enzimología , Endopeptidasa Clp/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Brassica/microbiología , Dickeya chrysanthemi/metabolismo , Dickeya chrysanthemi/patogenicidad , Endopeptidasa Clp/genética , Regulación Bacteriana de la Expresión Génica/fisiología , VirulenciaRESUMEN
Cyclic diguanylate (c-di-GMP) is a second messenger implicated in the regulation of various cellular properties in several bacterial species. However, its function in phytopathogenic bacteria is not yet understood. In this study we investigated a panel of GGDEF/EAL domain proteins which have the potential to regulate c-di-GMP levels in the phytopathogen Dickeya dadantii 3937. Two proteins, EcpB (contains GGDEF and EAL domains) and EcpC (contains an EAL domain) were shown to regulate multiple cellular behaviours and virulence gene expression. Deletion of ecpB and/or ecpC enhanced biofilm formation but repressed swimming/swarming motility. In addition, the ecpB and ecpC mutants displayed a significant reduction in pectate lyase production, a virulence factor of this bacterium. Gene expression analysis showed that deletion of ecpB and ecpC significantly reduced expression of the type III secretion system (T3SS) and its virulence effector proteins. Expression of the T3SS genes is regulated by HrpL and possibly RpoN, two alternative sigma factors. In vitro biochemical assays showed that EcpC has phosphodiesterase activity to hydrolyse c-di-GMP into linear pGpG. Most of the enterobacterial pathogens encode at least one T3SS, a major virulence factor which functions to subvert host defences. The current study broadens our understanding of the interplay between c-di-GMP, RpoN and T3SS and the potential role of c-di-GMP in T3SS regulation among a wide range of bacterial pathogens.