RESUMEN
Cyclic diguanosine monophosphate (c-di-GMP) is widely used by bacteria to control biological functions in response to diverse signals or cues. A previous study showed that potential c-di-GMP metabolic enzymes play a role in the regulation of biofilm formation and motility in Acinetobacter baumannii. However, it was unclear whether and how A. baumannii cells use c-di-GMP signaling to modulate biological functions. Here, we report that c-di-GMP is an important intracellular signal in the modulation of biofilm formation, motility, and virulence in A. baumannii. The intracellular level of c-di-GMP is principally controlled by the diguanylate cyclases (DGCs) A1S_1695, A1S_2506, and A1S_3296 and the phosphodiesterase (PDE) A1S_1254. Intriguingly, we revealed that A1S_2419 (an elongation factor P [EF-P]), is a novel c-di-GMP effector in A. baumannii. Response to a c-di-GMP signal boosted A1S_2419 activity to rescue ribosomes from stalling during synthesis of proteins containing consecutive prolines and thus regulate A. baumannii physiology and pathogenesis. Our study presents a unique and widely conserved effector that controls bacterial physiology and virulence by sensing the second messenger c-di-GMP.
Asunto(s)
Acinetobacter baumannii , Proteínas de Escherichia coli , Acinetobacter baumannii/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Guanosina Monofosfato , Factores de Elongación de Péptidos , Hidrolasas Diéster Fosfóricas/metabolismo , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , VirulenciaRESUMEN
Pseudomonas aeruginosa, a major inhabitant of numerous environmental reservoirs, is a momentous opportunistic human pathogen associated with severe infections even death in the patients suffering from immune deficiencies or metabolic diseases. Type III secretion system (T3SS) employed by P. aeruginosa to inject effector proteins into host cells is one of the pivotal virulence factors pertaining to acute infections caused by this pathogen. Previous studies showed that P. aeruginosa T3SS is regulated by various environmental cues such as calcium concentration and the host signal spermidine. However, how T3SS is regulated and expressed particularly under the ever-changing environmental conditions remains largely elusive. In this study, we reported that a tRNA modification enzyme PA3980, designated as MiaB, positively regulated T3SS gene expression in P. aeruginosa and was essential for the induced cytotoxicity of human lung epithelial cells. Further genetic assays revealed that MiaB promoted T3SS gene expression by repressing the LadS-Gac/Rsm signaling pathway and through the T3SS master regulator ExsA. Interestingly, ladS, gacA, rsmY and rsmZ in the LadS-Gac/Rsm signaling pathway seemed potential targets under the independent regulation of MiaB. Moreover, expression of MiaB was found to be induced by the cAMP-dependent global regulator Vfr as well as the spermidine transporter-dependent signaling pathway and thereafter functioned to mediate their regulation on the T3SS gene expression. Together, these results revealed a novel regulatory mechanism for MiaB, with which it integrates different environmental cues to modulate T3SS gene expression in this important bacterial pathogen.
Asunto(s)
Pseudomonas aeruginosa , Sistemas de Secreción Tipo III , Humanos , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , Pseudomonas aeruginosa/metabolismo , Regulación Bacteriana de la Expresión Génica , Señales (Psicología) , Espermidina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Quorum sensing (QS) is widely employed by bacterial cells to control gene expression in a cell density-dependent manner. A previous study revealed that anthranilic acid from Ralstonia solanacearum plays a vital role in regulating the physiology and pathogenicity of R. solanacearum. We reported here that anthranilic acid controls the important biological functions and virulence of R. solanacearum through the receptor protein RaaR, which contains helix-turn-helix (HTH) and LysR substrate binding (LysR_substrate) domains. RaaR regulates the same processes as anthranilic acid, and both are present in various bacterial species. In addition, anthranilic acid-deficient mutant phenotypes were rescued by in trans expression of RaaR. Intriguingly, we found that anthranilic acid binds to the LysR_substrate domain of RaaR with high affinity, induces allosteric conformational changes, and then enhances the binding of RaaR to the promoter DNA regions of target genes. These findings indicate that the components of the anthranilic acid signaling system are distinguished from those of the typical QS systems. Together, our work presents a unique and widely conserved signaling system that might be an important new type of cell-to-cell communication system in bacteria.
Asunto(s)
Ralstonia solanacearum , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Ralstonia solanacearum/genética , Virulencia/genética , ortoaminobenzoatosRESUMEN
BACKGROUND: Envelope stress responses (ESRs) are critical for adaptive resistance of Gram-negative bacteria to envelope-targeting antimicrobial agents. However, ESRs are poorly defined in a large number of well-known plant and human pathogens. Dickeya oryzae can withstand a high level of self-produced envelope-targeting antimicrobial agents zeamines through a zeamine-stimulated RND efflux pump DesABC. Here, we unraveled the mechanism of D. oryzae response to zeamines and determined the distribution and function of this novel ESR in a variety of important plant and human pathogens. RESULTS: In this study, we documented that a two-component system regulator DzrR of D. oryzae EC1 mediates ESR in the presence of envelope-targeting antimicrobial agents. DzrR was found modulating bacterial response and resistance to zeamines through inducing the expression of RND efflux pump DesABC, which is likely independent on DzrR phosphorylation. In addition, DzrR could also mediate bacterial responses to structurally divergent envelope-targeting antimicrobial agents, including chlorhexidine and chlorpromazine. Significantly, the DzrR-mediated response was independent on the five canonical ESRs. We further presented evidence that the DzrR-mediated response is conserved in the bacterial species of Dickeya, Ralstonia, and Burkholderia, showing that a distantly located DzrR homolog is the previously undetermined regulator of RND-8 efflux pump for chlorhexidine resistance in B. cenocepacia. CONCLUSIONS: Taken together, the findings from this study depict a new widely distributed Gram-negative ESR mechanism and present a valid target and useful clues to combat antimicrobial resistance.
Asunto(s)
Antiinfecciosos , Clorhexidina , Humanos , Bacterias Gramnegativas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismoRESUMEN
Sporisorium scitamineum and Ustilago maydis are two fungal pathogens causing severe sugarcane and maize diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We showed recently that in the presence of exogenous glucose, the Pseudomonas sp. strain ST4 could block the fungal mating and display a strong disease suppression potency on S. scitamineum. With the aim of conferring strain ST4 the ability to metabolize sucrose in plants for glucose production, we identified a strong native promoter pSsrA in strain ST4 and additional promoter elements to facilitate translation and peptide translocation for the construction of a fusion gene encoding sucrose metabolism. The cscA gene encoding sucrose hydrolase from Pseudomonas protegens Pf-5 was fused to the promoter pSsrA, a translational coupler bicistronic design and a Tat signal peptide, which was then cloned into mini-Tn7 transposon. This synthetic gene cassette was integrated into the chromosome of strain ST4, and the resultant engineered strain ST4E was able to hydrolyze sucrose with high efficiency and displayed elevated inhibitory activity on the mating and virulence of S. scitamineum and U. maydis. The findings from this study provide a valuable device and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens. IMPORTANCE Sporisorium scitamineum and Ustilago maydis are typical dimorphic fungi causing severe sugarcane and maize smut diseases, respectively. Sexual mating of compatible sporidia is essential for these pathogens to form infections dikaryotic mycelia and cause smut diseases. We previously demonstrated that the biocontrol strain Pseudomonas sp. ST4 could block the fungal mating and displays a strong suppression potency on smut diseases, while it was unable to utilize the host-sourced sucrose for glucose production critical for antifungus efficiency. In this study, we constructed a high-expression gene cassette for minitransposon-mediated genome integration and sucrose hydrolysis in the bacterial periplasmic space. The resultant engineered strain ST4E was able to hydrolyze sucrose and inhibit the mating and hyphal growth of S. scitamineum and U. maydis. These findings provide a valuable tool and useful clues for the engineering of sucrose metabolism in non- or weak-sucrose-utilizing bacterial strains and present an improved biocontrol agent against plant smut pathogens.
Asunto(s)
Basidiomycota , Saccharum , Ustilaginales , Ustilago , Ustilaginales/genética , Virulencia , Enfermedades de las Plantas/prevención & control , Enfermedades de las Plantas/microbiología , Saccharum/genética , Saccharum/metabolismo , Saccharum/microbiología , Ustilago/genéticaRESUMEN
The GacS-GacA type two-component system (TCS) positively regulates pathogenicity-related phenotypes in many plant pathogens. In addition, Dickeya oryzae EC1, the causative agent of soft rot disease, produces antibiotic-like toxins called zeamines as one of the major virulence factors that inhibit the germination of rice seeds. The present study identified a GacS-GacA type TCS, named TzpS-TzpA, that positively controls the virulence of EC1, mainly by regulating production of the toxin zeamines. RNA-seq analysis of strain EC1 and its tzpA mutant showed that the TCS regulated a wide range of virulence genes, especially those encoding zeamines. Protein-protein interaction was detected between TzpS and TzpA through the bacterial two-hybrid system and pull-down assay. In trans expression of tzpA failed to rescue the defective phenotypes in both the ΔtzpS and ΔtzpSΔtzpA mutants. Furthermore, TzpA controls target gene expression by direct binding to DNA promoters that contain a Gac-box motif, including a regulatory RNA rsmB and the vfm quorum-sensing system regulator vfmE. These findings therefore suggested that the EC1 TzpS-TzpA TCS system mediates the pathogenicity of Dickeya oryzae EC1 mainly by regulating the production of zeamines.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Asunto(s)
Proteínas Bacterianas , Dickeya , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Macrólidos , Enfermedades de las Plantas/microbiología , Poliaminas , Virulencia/genéticaRESUMEN
It has been demonstrated that quorum sensing (QS) is widely employed by bacterial cells to coordinately regulate various group behaviors. Diffusible signal factor (DSF)-type signals have emerged as a growing family of conserved cell-cell communication signals. In addition to the DSF signal initially identified in Xanthomonas campestris pv. campestris, Burkholderiadiffusible signal factor (BDSF) (cis-2-dodecenoic acid) has been recognized as a conserved DSF-type signal with specific characteristics in both signal perception and transduction from DSF signals. Here, we review the history and current progress of the research on this type of signal, especially focusing on its biosynthesis, signaling pathways, and biological functions. We also discuss and explore the huge potential of targeting this kind of QS system as a new therapeutic strategy to control bacterial infections and diseases.
Asunto(s)
Burkholderia cenocepacia , Burkholderia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia/metabolismo , Burkholderia cenocepacia/metabolismo , Ácidos Grasos Monoinsaturados , Regulación Bacteriana de la Expresión Génica , Percepción de Quorum , Factores Supresores InmunológicosRESUMEN
The type VI secretion system (T6SS) is an important translocation apparatus that is widely employed by Gram-negative bacteria to deliver toxic effectors into eukaryotic and prokaryotic target cells, causing host damage and providing competitive advantages in polymicrobial environments. The genome of Pseudomonas aeruginosa harbors three T6SS clusters (H1-T6SS, H2-T6SS, H3-T6SS). Activities of these systems are tightly regulated by a complicated signaling network which remains largely elusive. In this study, we focused on a previously characterized two-component system FleS/FleR, and performed comparative transcriptome analysis between the PAO1 wild-type strain and its isogenic ΔfleR mutant, which revealed the important role of FleS/FleR in regulating multiple physiological pathways including T6SS. Gene expression and bacterial killing assays showed that the expression and activity of H1-T6SS are repressed in the wild-type strain owing to the high intracellular c-di-GMP content. Further explorations demonstrated that c-di-GMP relies on the transcription factor FleQ to repress H1-T6SS and its synthesis is controlled by a global regulator AmrZ which is induced by the active FleS/FleR. Interestingly, repression of H1-T6SS by FleS/FleR in PAO1 is independent of RetS which is known to regulate H1-T6SS by controlling the central post-transcriptional factor RsmA. Together, our results identified a novel regulator of H1-T6SS and provided detailed mechanisms of this signaling pathway in PAO1. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen distributed widely in the environment. The genome of this pathogen contains three T6SS clusters which contribute significantly to its virulence. Understanding the complex regulatory network that controls the activity of T6SS is essential for the development of effective therapeutic treatments for P. aeruginosa infections. In this study, transcriptome analysis led to the identification of a novel regulator FleS/FleR which inversely regulates H1-T6SS and H2-T6SS in P. aeruginosa PAO1. We further revealed a detailed FleS/FleR-mediated regulatory pathway of H1-T6SS in PAO1 which involves two additional transcriptional regulators AmrZ and FleQ and the second messenger c-di-GMP, providing important implications to develop novel anti-infective strategies and antimicrobial drugs.
Asunto(s)
Pseudomonas aeruginosa , Sistemas de Secreción Tipo VI , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , GMP Cíclico/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Pseudomonas aeruginosa/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/metabolismo , Virulencia/genéticaRESUMEN
It is highly intriguing how bacterial pathogens can quickly shut down energy-costly infection machinery once successful infection is established. This study depicts that mutation of repressor SghR increases the expression of hydrolase SghA in Agrobacterium tumefaciens, which releases plant defense signal salicylic acid (SA) from its storage form SA ß-glucoside (SAG). Addition of SA substantially reduces gene expression of bacterial virulence. Bacterial vir genes and sghA are differentially transcribed at early and later infection stages, respectively. Plant metabolite sucrose is a signal ligand that inactivates SghR and consequently induces sghA expression. Disruption of sghA leads to increased vir expression in planta and enhances tumor formation whereas mutation of sghR decreases vir expression and tumor formation. These results depict a remarkable mechanism by which A. tumefaciens taps on the reserved pool of plant signal SA to reprogram its virulence upon establishment of infection.
Asunto(s)
Agrobacterium tumefaciens/patogenicidad , Arabidopsis/microbiología , Interacciones Huésped-Patógeno , Factores de Virulencia/genética , Agrobacterium tumefaciens/genética , Arabidopsis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Hidrolasas/genética , Hidrolasas/metabolismo , Ácido Salicílico/metabolismo , Transducción de Señal , Sacarosa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Agrobacterium tumefaciens infects various plants and causes crown gall diseases involving temporal expression of virulence factors. SghA is a newly identified virulence factor enzymatically releasing salicylic acid from its glucoside conjugate and controlling plant tumor development. Here, we report the structural basis of SghR, a LacI-type transcription factor highly conserved in Rhizobiaceae family, regulating the expression of SghA and involved in tumorigenesis. We identified and characterized the binding site of SghR on the promoter region of sghA and then determined the crystal structures of apo-SghR, SghR complexed with its operator DNA, and ligand sucrose, respectively. These results provide detailed insights into how SghR recognizes its cognate DNA and shed a mechanistic light on how sucrose attenuates the affinity of SghR with DNA to modulate the expression of SghA. Given the important role of SghR in mediating the signaling cross-talk during Agrobacterium infection, our results pave the way for structure-based inducer analog design, which has potential applications for agricultural industry.
Asunto(s)
Agrobacterium tumefaciens/metabolismo , Proteínas Bacterianas/metabolismo , Tumores de Planta/microbiología , Elementos de Respuesta , Transducción de Señal , Agrobacterium tumefaciens/genética , Proteínas Bacterianas/genéticaRESUMEN
Nuclear import of proteins relies on nuclear import receptors called importins/karyopherins (Kaps), whose functions were reported in yeasts, fungi, plants, and animal cells, including cell cycle control, morphogenesis, stress sensing/response, and also fungal pathogenecity. However, limited is known about the physiological function and regulatory mechanism of protein import in the rice-blast fungus Magnaporthe oryzae. Here, we identified an ortholog of ß-importin in M. oryzae encoded by an ortholog of KAP119 gene. Functional characterisation of this gene via reverse genetics revealed that it is required for vegetative growth, conidiation, melanin pigmentation, and pathogenicity of M. oryzae. The mokap119Δ mutant was also defective in formation of appressorium-like structure from hyphal tips. By affinity assay and liquid chromatography-tandem mass spectrometry, we identified potential MoKap119-interacting proteins and further verified that MoKap119 interacts with the cyclin-dependent kinase subunit MoCks1 and mediates its nuclear import. Transcriptional profiling indicated that MoKap119 may regulate transcription of infection-related genes via MoCks1 regulation of MoSom1. Overall, our findings provide a novel insight into the regulatory mechanism of M. oryzae pathogenesis likely by MoKap119-mediated nuclear import of the cyclin-dependent kinase subunit MoCks1.
Asunto(s)
Transporte Activo de Núcleo Celular , Ascomicetos/patogenicidad , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Fúngicas/metabolismo , Ascomicetos/genética , Núcleo Celular , Quinasas Ciclina-Dependientes/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Hordeum/microbiología , Carioferinas , Filogenia , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Genética Inversa , Virulencia/genéticaRESUMEN
Taro [Colocasia esculenta (L.) Schott.] is an important root crop in the world with great economic value. In recent years, outbreaks of soft rot were observed on taro plants in several plantation areas located in Shaoguan, Guangdong Province, China (25°7'57" N, 113°19'5" E). Root tubers of taro (Paodan variety) infected by soft rot had water-soaked lesions with a dark brown-black margin including a rotten smell, they also had internal rot that was also found in root tubers with no external symptoms. In some areas, the incidence of soft rot can reach up to 30%. To isolate the causal agent, ten pieces of taro root tubers with typical symptoms were surface-sterilized with 75% ethanol and 0.1% HgCl2 solution and then washed thrice with sterile water. The tuber slices were soaked in 50 ml sterile water and shaken at 28°C, 200 rpm for 2 h, and 100 µl was streaked onto the modified Yeast Extract Beef (YEB) agar medium (1% peptone, 0.5% yeast extract, 0.5% sucrose, 0.5% NaCl, 1 Mmol/L MgSO4â¢7H2O, 1.5% agar, pH 7.0) plates (Zhou et al. 2011) and incubated at 28°C for 24 h. Single colonies grown on YEB were selected for preliminary inoculation onto healthy taro (Paodan variety) slices. Two of the Gram-negative bacteria, named as ZXC1 and MPC2, developed symptoms consistent in rotted decay inside the root tubers after incubation for 24h at 30°C. ZXC1 and MPC2 were biochemically profiled using a Biolog Gen III MicroPlate (Microlog 3, 5.2) (Shen et al. 2019) and resulted Dickeya sp. (SIM 0.856 and 0.704). To determine the species of the Dickeya isolates, 16S rRNA sequences were amplified by primers 27F and 1492R (Hauben et al. 1998). Housekeeping genes including gyrB, atpD, rpoB, and infB were also amplified using degenerate primers (Brady et al. 2008). Results from the BLASTn analysis of the 16S rRNA (GenBank accession numbers MN853405, MN853406), gyrB (GenBank accession numbers MN866299, MN866303), atpD (GenBank accession numbers MN866298, MN866302), rpoB (GenBank accession numbers MN866301, MN866305), and infB (GenBank accession numbers MN866300, MN866304) genes in the isolates ZXC1 and MPC2 showed 99% identities to those of the previously reported D. fangzhongdai isolates from Phalaenopsis (Zhang et al. 2018). Multilocus sequence analysis (MLSA) by MEGA 7.0 performed with four housekeeping genes (gyrB, atpD, rpoB, infB) showed that they clustered with D. fangzhongdai isolates. Analyses using scanning and transmission electron microscopy showed that ZXC1 and MPC2 bacteria were rod-shaped, 0.5-1.0 µm × 1.0-3.0 µm, with peritrichous flagella. Pathogenicity tests were performed thrice using surface-sterilized 2-month-old taro seedlings (Paodan variety). Six individual seedlings were inoculated using a sterile syringe with ten microliters of bacterial suspension (108 CFU/ml) in Tris buffer (0.1 mol/L Tris and 0.1 mol/L HCl, pH 7.4). Taro seedlings injected with sterile Tris buffer were used as the negative control. These taro seedlings were grown in the greenhouse (30 ± 2°C, 90 ± 5% relative humidity). At the 25th day post inoculation, soft rot symptoms were observed in inoculated taro, while all control taro plants remained symptom-free. Small and pale yellow with irregular margins colonies consistent with morphological characteristics of those of D. fangzhongdai were re-isolated from symptomatic taro tubers and the housekeeping genes presence was verified by sequencing as described above, fulfilling Koch's postulates. D. fangzhongdai is a newly emerging bacterial pathogen, which causes bleeding cankers in pear trees (Tian et al. 2016), and soft rot of Phalaenopsis (Zhang et al. 2018). This is the first report of D. fangzhongdai causing soft rot disease in taro. Considering the high incidence of soft rot, this pathogen might pose a significant threat to taro and other economically important crops. Therefore, further researches are needed to investigate host range of the pathogen and develop appropriate integrated management to contain this disease spreading.
RESUMEN
Flavonoids are polyphenolic secondary metabolites that function as signaling molecules, allopathic compounds, phytoalexins, detoxifying agents and antimicrobial defensive compounds in plants. Blast caused by the fungus Magnaporthe oryzae is a serious disease affecting rice cultivation. In this study, we revealed that a natural flavonoid, tangeretin, substantially delays the formation of M. oryzae appressoria and blocks the development of blast lesions on rice plants. Our data suggest that tangeretin has antioxidant activity that interferes with conidial cell death/ferroptosis, which is critical for M. oryzae pathogenicity. Tangeretin showed a ferroptosis inhibition efficacy comparable to the well-established liproxstatin-1. Furthermore, overexpression of the NADPH oxidases NOX1 or NOX2 significantly decreased sensitivity toward tangeretin treatment, suggesting Nox-mediated lipid peroxidation as a possible target for tangeretin in regulating redox signaling and ferroptosis in M. oryzae. Our nursery and field tests showed that application of tangeretin can effectively mitigate overall disease symptoms and prevent leaf blast. Our study reveals the plant-derived fungal ferroptosis inhibitor tangeretin as a potential and novel antifungal agrochemical for the sustainable prevention of the devastating blast disease in important cereal crops.
Asunto(s)
Ferroptosis , Magnaporthe , Oryza , Flavonas , Proteínas Fúngicas/metabolismo , Oryza/metabolismo , Enfermedades de las Plantas/microbiologíaRESUMEN
Bacterial pathogen Dickeya zeae strain EC1 produces antibiotics-like phytotoxins called zeamines, which are major virulence determinants encoded by the zms gene cluster. In this study, we identified a zeamine-deficient mutant with a Tn5 insertion in a gene designated as vfmI encoding a two-component system (TCS) sensor histidine kinase (HK), which is accompanied by vfmH encoding a response regulator (RR) at the same genetic locus. Domain analysis shows this TCS is analogous to the VfmIH of D. dadantii, with typical characteristics of sensor HK and RR, respectively, and sharing the same operon. Deletion of either vfmI or vfmH resulted in decreased production of zeamines and cell wall degrading enzymes (CWDEs), and alleviated virulence on rice seeds and potato tubers. In D. dadantii 3937, VfmH was shown to bind to the promoters of vfmA and vfmE, while in D. zeae EC1, VfmH could bind to the promoters of vfmA, vfmE and vfmF. RNA-seq analysis of strain EC1 and its vfmH mutant also showed that the TCS positively regulated a range of virulence genes, including zms, T1SS, T2SS, T3SS, T6SS, flagellar and CWDE genes.
Asunto(s)
Proteínas Bacterianas/genética , Gammaproteobacteria/genética , Gammaproteobacteria/patogenicidad , Factores de Virulencia/genética , Proteínas Bacterianas/metabolismo , Dickeya , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Histidina Quinasa/genética , Macrólidos/metabolismo , Familia de Multigenes , Operón , Fenotipo , Enfermedades de las Plantas/microbiología , Poliaminas/metabolismo , Regiones Promotoras Genéticas , Percepción de Quorum , Análisis de Secuencia de ARN , Virulencia/genéticaRESUMEN
Quorum quenching, which disrupts quorum sensing (QS) by either degradation of QS signals or interference of signal generation or perception, is a promising strategy for the prevention and control of QS-mediated bacterial infections. Diffusible signal factor (DSF) is widely conserved in many Gram-negative bacterial pathogens. In this study, we developed an efficient method for screening of highly active DSF degradation microorganisms. Among them, Pseudomonas sp. strain HS-18 showed a superior DSF degradation activity. Bioinformatics and genetic analyses showed that at least 4 genes, designated digA to digD, encoding fatty acyl coenzyme A ligase homologues, are responsible for DSF signal degradation. Interestingly, all 4 dig genes were induced by exogenous DSF, with digA being the most significantly induced. Expression of the dig genes in Xanthomonas campestris pv. campestris markedly reduced the accumulation of endogenous DSF, decreased production of virulence factors, and attenuated bacterial virulence on host plants. Similarly, application of strain HS-18 as a biocontrol agent could substantially reduce the disease severity caused by X. campestris pv. campestris These results unveil the molecular basis of a highly efficient DSF degradation bacterial isolate and present useful genes and biocontrol agents for control of the infectious diseases caused by DSF-dependent bacterial pathogens.IMPORTANCE Diffusible signal factor (DSF) represents a family of widely conserved quorum sensing signals involved in the regulation of virulence factor production in many Gram-negative bacterial pathogens. In this study, we developed a novel and efficient method for screening highly active DSF degradation microorganisms. With this method, we identified a bacterial isolate, Pseudomonas sp. strain HS-18, with a superb DSF degradation activity. We further found that strain HS-18 contains 4 genes responsible for DSF signal degradation, and significantly, these were induced by exogenous DSF molecules. These findings unveil the molecular basis of a highly efficient DSF degradation bacterial isolate and present useful methods, genes, and agents for control of the infectious diseases caused by DSF-dependent bacterial pathogens.
Asunto(s)
Genes Bacterianos/fisiología , Microorganismos Modificados Genéticamente/metabolismo , Pseudomonas/crecimiento & desarrollo , Percepción de Quorum , Xanthomonas campestris/metabolismo , Biología Computacional , Expresión Génica , Pseudomonas/metabolismoRESUMEN
Pseudomonas aeruginosa, an opportunistic life-threatening human bacterial pathogen, employs quorum-sensing (QS) signal molecules to modulate virulence gene expression. 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) is a recently identified QS signal that integrates the canonical lasR-type QS of P. aeruginosa and host phosphate stress response to fine-tune its virulence production for a successful infection. To address the role of IQS in pathogen-host interaction, we here present that IQS inhibits host cell growth and stimulates apoptosis in a dosage-dependent manner. By downregulating the telomere-protecting protein POT1 in host cells, IQS activates CHK1, CHK2, and p53 in an Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR)-dependent manner and induces DNA damage response. Overexpression of POT1 in host cells presents a resistance to IQS treatment. These results suggest a pivotal role of IQS in host apoptosis, highlighting the complexity of pathogenesis mechanisms developed by P. aeruginosa during infection.
Asunto(s)
Apoptosis/efectos de los fármacos , Fenoles/farmacología , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/patogenicidad , Proteínas de Unión a Telómeros/metabolismo , Tiazoles/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Células A549 , Animales , Apoptosis/genética , Proteínas Bacterianas/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Quinasa de Punto de Control 2/genética , Quinasa de Punto de Control 2/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Humanos , Ratones , Fenoles/química , Proteolisis , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/química , Percepción de Quorum , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Tiazoles/química , Proteína p53 Supresora de Tumor/genética , Virulencia/genéticaRESUMEN
Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia, the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis-2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria.
Asunto(s)
Proteínas Bacterianas/genética , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/patogenicidad , GMP Cíclico/análogos & derivados , Ácidos Grasos Monoinsaturados/metabolismo , Regulación Bacteriana de la Expresión Génica , Percepción de Quorum/genética , Animales , Carga Bacteriana , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Infecciones por Burkholderia/microbiología , Infecciones por Burkholderia/patología , Burkholderia cenocepacia/crecimiento & desarrollo , GMP Cíclico/metabolismo , Ratones , Mutación , Fenotipo , Transducción de Señal , VirulenciaRESUMEN
Quorum sensing (QS) is widely utilized by bacterial pathogens to regulate biological functions and pathogenicity. Recent evidence has shown that QS is subject to regulatory cascades, especially two-component systems that often respond to environmental stimulation. At least two different types of QS systems regulate pathogenesis in Burkholderia cenocepacia. However, it remains unclear how this bacterial pathogen controls these QS systems. Here, we demonstrate a novel two-component system RqpSR (Regulating Quorum sensing and Pathogenicity), which plays an important role in modulating QS and pathogenesis in B. cenocepacia. We demonstrate strong protein-protein binding affinity between RqpS and RqpR. Mutations in rqpS and rqpR exerted overlapping effects on B. cenocepacia transcriptomes and phenotypes, including motility, biofilm formation and virulence. In trans expression of rqpR rescued the defective phenotypes in the rqpS mutant. RqpR controls target gene expression by direct binding to DNA promoters, including the cis-2-dodecenoic acid (BDSF) and N-acylhomoserine lactone (AHL) signal synthase gene promoters. These findings suggest that the RqpSR system strongly modulates physiology by forming a complicated hierarchy with QS systems. This type of two-component system appears to be widely distributed and coexists with the BDSF QS system in various bacterial species.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/patogenicidad , Percepción de Quorum , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Biopelículas , Burkholderia cenocepacia/genética , Movimiento Celular , Regiones Promotoras Genéticas/genética , Unión Proteica , Eliminación de Secuencia , Transcriptoma , beta-Galactosidasa/genéticaRESUMEN
The fungal pathogen Sporisorium scitamineum causes sugarcane smut disease. The formation and growth of dikaryotic hypha after sexual mating is critical for S. scitamineum pathogenicity, however regulation of S. scitimineum mating has not been studied in detail. We identified and characterized the core components of the conserved cAMP/PKA pathway in S. scitamineum by reverse genetics. Our results showed that cAMP/PKA signalling pathway is essential for proper mating and filamentation, and thus critical for S. scitamineum virulence. We further demonstrated that an elevated intracellular ROS (reactive oxygen species) level promotes S. scitamineum mating-filamentation, via transcriptional regulation of ROS catabolic enzymes, and is under regulation of the cAMP/PKA signalling pathway. Furthermore, we found that fungal cAMP/PKA signalling pathway is also involved in regulation of host ROS response. Overall, our work displayed a positive role of elevated intracellular ROS in fungal differentiation and virulence.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Enfermedades de las Plantas/microbiología , Saccharum/microbiología , Ustilaginales/fisiología , Homeostasis , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Ustilaginales/patogenicidad , VirulenciaRESUMEN
The initiative strategy for the development of novel anti-microbial agents usually uses the virulence factors of bacteria as a target, without affecting their growth and survival. The type III secretion system (T3SS), one of the essential virulence factors in most Gram-negative pathogenic bacteria because of its highly conserved construct, has been regarded as an effective target that developed new anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) causes leaf blight diseases and is one of the most important pathogens on rice. To find potential anti-virulence agents against this pathogen, a number of natural compounds were screened for their effects on the T3SS of Xoo. Three of 34 compounds significantly inhibited the promoter activity of the harpin gene, hpa1, and were further checked for their impact on bacterial growth and on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results indicated that treatment of Xoo with CZ-1, CZ-4 and CZ-9 resulted in an obviously attenuated HR without affecting bacterial growth and survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Xoo T3SS was suppressed by treatment with the three inhibitors. The mRNA levels of representative genes in the hypersensitive response and pathogenicity (hrp) cluster, as well as the regulatory genes hrpG and hrpX, were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of Xoo on the rice cultivar (Oryza sativa) IR24.