Characterization of the SPI-1 Type III Secretion System in Pseudomonas fluorescens 2P24.
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ABSTRACT:
SUBMITTER: Wang J
PROVIDER: S-EPMC8490769 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The type III secretion system (T3SS) of plant and animal bacterial pathogens directs the secretion and injection of proteins into host cells. Some homologous genes of T3SS were found also in non-pathogenic bacteria, but the organization of its machinery and basic function are still unknown. In this study, we identified a T3SS gene cluster from the plant growth-promoting Pseudomonas fluorescens 2P24 and isolated the corresponding T3SS apparatus. The T3SS gene cluster of strain 2P24 is similar organizationally to that of pathogenic P. syringae, except that it lacks the regulator hrpR and the hrpK1 and hrpH genes, which are involved in translocation of proteins. Electron microscopy revealed that the T3SS supramolecular structure of strain 2P24 was comprised of two distinctive substructures: a long extracellular, filamentous pilus, and a membrane-embedded base. We show that strain 2P24 deploys a harpin homolog protein, RspZ1, to elicit a hypersensitive response when infiltrated into Nicotiana tabacum cv. xanthi leaves with protein that is partially purified, and by complementing the hrpZ1 mutation of pHIR11. The T3SS of strain 2P24 retained ability to secrete effectors, whereas its effector translocation activity appeared to be excessively lost. Mutation of the rscC gene from 2P24 T3SS abolished the secretion of effectors, but the general biocontrol properties were unaffected. Remarkably, strain 2P24 induced functional MAMP-triggered immunity that included a burst of reactive oxygen species, strong suppression of challenge cell death, and disease expansion, while it was not associated with the secretion functional T3SS.
| S-EPMC4700148 | biostudies-literature
Project description:Pseudomonas fluorescens Q8r1-96 represents a group of rhizosphere strains responsible for the suppressiveness of agricultural soils to take-all disease of wheat. It produces the antibiotic 2,4-diacetylphloroglucinol and aggressively colonizes the roots of cereal crops. In this study, we analyzed the genome of Q8r1-96 and identified a type III protein secretion system (T3SS) gene cluster that has overall organization similar to that of the T3SS gene cluster of the plant pathogen Pseudomonas syringae. We also screened a collection of 30 closely related P. fluorescens strains and detected the T3SS genes in all but one of them. The Q8r1-96 genome contained ropAA and ropM type III effector genes, which are orthologs of the P. syringae effector genes hopAA1-1 and hopM1, as well as a novel type III effector gene designated ropB. These type III effector genes encoded proteins that were secreted in culture and injected into plant cells by both P. syringae and Q8r1-96 T3SSs. The Q8r1-96 T3SS was expressed in the rhizosphere, but mutants lacking a functional T3SS were not altered in their rhizosphere competence. The Q8r1-96 type III effectors RopAA, RopB, and RopM were capable of suppressing the hypersensitive response and production of reactive oxygen species, two plant immune responses.
| S-EPMC3019950 | biostudies-literature
Project description:Intracellular replication of Salmonella enterica occurs in membrane-bound compartments, called Salmonella-containing vacuoles (SCVs). Following invasion of epithelial cells, most SCVs migrate to a perinuclear region and replicate in close association with the Golgi network. The association of SCVs with the Golgi is dependent on the Salmonella-pathogenicity island-2 (SPI-2) type III secretion system (T3SS) effectors SseG, SseF and SifA. However, little is known about the dynamics of SCV movement. Here, we show that in epithelial cells, 2 h were required for migration of the majority of SCVs to within 5 microm from the microtubule organizing centre (MTOC), which is located in the same subcellular region as the Golgi network. This initial SCV migration was saltatory, bidirectional and microtubule-dependent. An intact Golgi, SseG and SPI-2 T3SS were dispensable for SCV migration to the MTOC, but were essential for maintenance of SCVs in that region. Live-cell imaging between 4 and 8 h post invasion revealed that the majority of wild-type SCVs displaced less than 2 microm in 20 min from their initial starting positions. In contrast, between 6 and 8 h post invasion the majority of vacuoles containing sseG, sseF or ssaV mutant bacteria displaced more than 2 microm in 20 min from their initial starting positions, with some undergoing large and dramatic movements. Further analysis of the movement of SCVs revealed that large displacements were a result of increased SCV speed rather than a change in their directionality, and that SseG influences SCV motility by restricting vacuole speed within the MTOC/Golgi region. SseG might function by tethering SCVs to Golgi-associated molecules, or by controlling microtubule motors, for example by inhibiting kinesin recruitment or promoting dynein recruitment.
| S-EPMC2062534 | biostudies-literature
Project description:Protein secretion systems are crucial mediators of bacterial interactions with other organisms. Among them, the type VI secretion system (T6SS) is widespread in Gram-negative bacteria and appears to inject toxins into competitor bacteria and/or eukaryotic cells. Major human pathogens, such as Vibrio cholerae, Burkholderia and Pseudomonas aeruginosa, express T6SSs. Bacteria prevent self-intoxication by their own T6SS toxins by producing immunity proteins, which interact with the cognate toxins. We describe here an environmental P. fluorescens strain, MFE01, displaying an uncommon oversecretion of Hcp (hemolysin-coregulated protein) and VgrG (valine-glycine repeat protein G) into the culture medium. These proteins are characteristic components of a functional T6SS. The aim of this study was to attribute a role to this energy-consuming overexpression of the T6SS. The genome of MFE01 contains at least two hcp genes (hcp1 and hcp2), suggesting that there may be two putative T6SS clusters. Phenotypic studies have shown that MFE01 is avirulent against various eukaryotic cell models (amebas, plant or animal cell models), but has antibacterial activity against a wide range of competitor bacteria, including rhizobacteria and clinical bacteria. Depending on the prey cell, mutagenesis of the hcp2 gene in MFE01 abolishes or reduces this antibacterial killing activity. Moreover, the introduction of T6SS immunity proteins from S. marcescens, which is not killed by MFE01, protects E. coli against MFE01 killing. These findings suggest that the protein encoded by hcp2 is involved in the killing activity of MFE01 mediated by effectors of the T6SS targeting the peptidoglycan of Gram-negative bacteria. Our results indicate that MFE01 can protect potato tubers against Pectobacterium atrosepticum, which causes tuber soft rot. Pseudomonas fluorescens is often described as a major PGPR (plant growth-promoting rhizobacterium), and our results suggest that there may be a connection between the T6SS and the PGPR properties of this bacterium.
| S-EPMC3925238 | biostudies-literature
Project description:BACKGROUND: Type III secretion systems (TTSS) are employed by numerous pathogenic and symbiotic bacteria to inject a cocktail of different "effector proteins" into host cells. These effectors subvert host cell signaling to establish symbiosis or disease. METHODOLOGY/PRINCIPAL FINDINGS: We have studied the injection of SipA and SptP, two effector proteins of the invasion-associated Salmonella type III secretion system (TTSS-1). SipA and SptP trigger different host cell responses. SipA contributes to triggering actin rearrangements and invasion while SptP reverses the actin rearrangements after the invasion has been completed. Nevertheless, SipA and SptP were both pre-formed and stored in the bacterial cytosol before host cell encounter. By time lapse microscopy, we observed that SipA was injected earlier than SptP. Computer modeling revealed that two assumptions were sufficient to explain this injection hierarchy: a large number of SipA and SptP molecules compete for transport via a limiting number of TTSS; and the TTSS recognize SipA more efficiently than SptP. CONCLUSIONS/SIGNIFICANCE: This novel mechanism of hierarchical effector protein injection may serve to avoid functional interference between SipA and SptP. An injection hierarchy of this type may be of general importance, allowing bacteria to precisely time the host cell manipulation by type III effectors.
| S-EPMC2364654 | biostudies-literature
Project description:Type VI secretion systems (T6SSs) are widespread in Gram-negative bacteria, including Pseudomonas. These macromolecular machineries inject toxins directly into prokaryotic or eukaryotic prey cells. Hcp proteins are structural components of the extracellular part of this machinery. We recently reported that MFE01, an avirulent strain of Pseudomonas fluorescens, possesses at least two hcp genes, hcp1 and hcp2, encoding proteins playing important roles in interbacterial interactions. Indeed, P. fluorescens MFE01 can immobilise and kill diverse bacteria of various origins through the action of the Hcp1 or Hcp2 proteins of the T6SS. We show here that another Hcp protein, Hcp3, is involved in killing prey cells during co-culture on solid medium. Even after the mutation of hcp1, hcp2, or hcp3, MFE01 impaired biofilm formation by MFP05, a P. fluorescens strain isolated from human skin. These mutations did not reduce P. fluorescens MFE01 biofilm formation, but the three Hcp proteins were required for the completion of biofilm maturation. Moreover, a mutant with a disruption of one of the unique core component genes, MFE01ΔtssC, was unable to produce its own biofilm or inhibit MFP05 biofilm formation. Finally, MFE01 did not produce detectable N-acyl-homoserine lactones for quorum sensing, a phenomenon reported for many other P. fluorescens strains. Our results suggest a role for the T6SS in communication between bacterial cells, in this strain, under biofilm conditions.
| S-EPMC5256989 | biostudies-literature
Project description:BackgroundPseudomonas fluorescens 2P24 is a rhizosphere bacterium that produces 2,4-diacetyphloroglucinol (2,4-DAPG) as the decisive secondary metabolite to suppress soilborne plant diseases. The biosynthesis of 2,4-DAPG is strictly regulated by the RsmA family proteins RsmA and RsmE. However, mutation of both of rsmA and rsmE genes results in reduced bacterial growth.ResultsIn this study, we showed that overproduction of 2,4-DAPG in the rsmA rsmE double mutant influenced the growth of strain 2P24. This delay of growth could be partially reversal when the phlD gene was deleted or overexpression of the phlG gene encoding the 2,4-DAPG hydrolase in the rsmA rsmE double mutant. RNA-seq analysis of the rsmA rsmE double mutant revealed that a substantial portion of the P. fluorescens genome was regulated by RsmA family proteins. These genes are involved in the regulation of 2,4-DAPG production, cell motility, carbon metabolism, and type six secretion system.ConclusionsThese results suggest that RsmA and RsmE are the important regulators of genes involved in the plant-associated strain 2P24 ecologic fitness and operate a sophisticated mechanism for fine-tuning the concentration of 2,4-DAPG in the cells.
| S-EPMC7331252 | biostudies-literature
Project description:Pseudomonas fluorescens 2P24 is a beneficial plant root-associated microorganism capable of suppressing several soilborne plant diseases. The capacity of P. fluorescens to aggressively colonize the rhizosphere is an important requirement for its biocontrol trait. We previously found that the PcoI/PcoR quorum-sensing system (QS) is involved in regulating the rhizosphere colonization of P. fluorescens. Here, we revealed a sophisticated regulatory network that connects PcoR, RsaL, and MvaT proteins to fine-tune the PcoI/PcoR QS system. Our data showed that PcoR could directly bind to the promoter region of pcoI thereby inducing the PcoI/PcoR QS system, whereas RsaL binds simultaneously with PcoR to the promoter region of pcoI and represses the PcoR-dependent activation of pcoI gene. In addition, RsaL indirectly downregulates the expression of pcoR. Furthermore, we showed that disruption of mvaT enhanced the expression of pcoI, pcoR, and rsaL, whereas MvaT controls the PcoI/PcoR QS in a RsaL-independent manner. Overall, this study elucidates that PcoR, RsaL, and MvaT regulate the PcoI/PcoR QS through a multi-tiered regulatory mechanism and that PcoR is necessary in the RsaL- and MvaT-mediated repression on the expression of pcoI. IMPORTANCE The PcoI/PcoR quorum-sensing system of Pseudomonas fluorescens 2P24 is important for its effective colonization in the plant rhizosphere. Many regulatory elements appear to directly or indirectly influence the QS system. Here, we found a complex regulatory network employing transcriptional factors PcoR, RsaL, and MvaT to influence the expression of the PcoI/PcoR QS in P. fluorescens 2P24. Our results indicate that PcoR and RsaL directly bind to the promoter region of pcoI and then positively and negatively regulate the expression of pcoI, respectively. Furthermore, the H-NS family protein MvaT negatively controls the PcoI/PcoR QS in a RsaL-independent manner. Taken together, our data provide new insights into the interplays between different regulatory elements that fine-tune the QS system of P. fluorescens.
| S-EPMC9275216 | biostudies-literature
Project description:IntroductionPseudomonas fluorescens 2P24 was isolated from soil of natural decay associated with wheat take-all and it can effectively control soil-borne diseases caused by a variety of plant pathogens. 2,4-diacetylphloroglucinol (2,4-DAPG), is produced by P. fluorescens 2P24 and plays an important role in the prevention and control of plant diseases. To understand the resistant mechanism, in this study, we conducted experiments to explore the regulation role of rpoZ in the synthesis of the antibiotic 2,4-DAPG and regulation of QS system.MethodsA random mini-Tn5 mutagenesis procedure was used to screen regulators for phlA transcription in stain PM901, which containing a phlA∷lacZ transcriptional fusion reporter plasmid. We identified 12 insertion mutants could significantly change phlA gene expression. By analyzing the amino acid sequences of the interrupted gene, we obtained a mutant strain Aa4-29 destroyed the rpoZ gene, which encodes the omiga subunit. We constructed the plasmid of rpoZ mutant (pBBR-△rpoZ) transformed into competent cells of P. fluorescens 2P24 by electro-transformation assay. The strains of P. fluorescens 2P24/pBBR, 2P24-△rpoZ/pBBR, 2P24-△rpoZ/pBBR-rpoZ were used to evaluate the regulation role of rpoZ in 2,4-DAPG production and quorum sensing system.ResultsAccording to β-galactosidase activity, we found that rpoZ positively regulated the expression of phlA (a synthesis gene of 2,4-DAPG) and PcoI (a synthesis gene of PcoI/PcoR QS signal system) at the transcriptional level. The production of 2,4-DAPG antibiotic and signal molecule AHL was influenced by rpoZ. Further, rpoZ was involved in regulating rsmA expression. RpoZ also has a certain regulatory effect on rpoS transcription, but no effect on the transcription of phlF, emhABC and emhR. According to the biocontrol assay, P. fluorescens 2P24 strains with rpoZ showed obvious antagonism ability against the Rhizoctonia solani in cotton, while the mutant strain of rpoZ lost the biocontrol effect. RpoZ had a significant effect on the swimming and biofilm formation in P. fluorescens 2P24.ConclusionOur data showed that rpoZ was an important regulator of QS system, 2,4-DAPG in P. fluorescens 2P24. This may imply that P. fluorescens 2P24 has evolved different regulatory features to adapt to different environmental threats.
| S-EPMC10213339 | biostudies-literature
Project description:UnlabelledThe opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa.ImportanceThe inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.
| S-EPMC4438214 | biostudies-literature