T3SS Master Regulator Research Articles

Plant-exuded chemical signals induce surface attachment of the bacterial pathogen Pseudomonas syringae.

Many plant pathogenic bacteria suppress host defenses by secreting small molecule toxins or immune-suppressing proteins into host cells, processes that likely require close physical contact between pathogen and host. Yet, in most cases, little is known about whether phytopathogenic bacteria physically attach to host surfaces during infection. Here we report that Pseudomonas syringae pv. tomato strain DC3000, a Gram-negative bacterial pathogen of tomato and Arabidopsis, attaches to polystyrene and glass surfaces in response to chemical signals exuded from Arabidopsis seedlings and tomato leaves. We characterized the molecular nature of these attachment-inducing signals and discovered that multiple hydrophilic metabolites found in plant exudates, including citric acid, glutamic acid, and aspartic acid, are potent inducers of surface attachment. These same compounds were previously identified as inducers of P. syringae genes encoding a type III secretion system (T3SS), indicating that both attachment and T3SS deployment are induced by the same plant signals. To test if surface attachment and T3SS are regulated by the same signaling pathways, we assessed the attachment phenotypes of several previously characterized DC3000 mutants, and found that the T3SS master regulator HrpL was partially required for maximal levels of surface attachment, whereas the response regulator GacA, a negative regulator of T3SS, negatively regulated DC3000 surface attachment. Together, our data indicate that T3SS deployment and surface attachment by P. syringae may be co-regulated by the same host signals during infection, possibly to ensure close contact necessary to facilitate delivery of T3SS effectors into host cells.

Catheter-associated urinary tract infection by Pseudomonas aeruginosa progresses through acute and chronic phases of infection

Healthcare-associated infections are major causes of complications that lead to extended hospital stays and significant medical costs. The use of medical devices, including catheters, increases the risk of bacterial colonization and infection through the presence of a foreign surface. Two outcomes are observed for catheterized patients: catheter-associated asymptomatic bacteriuria and catheter-associated urinary tract infection (CAUTI). However, the relationship between these two events remains unclear. To understand this relationship, we studied a murine model of Pseudomonas aeruginosa CAUTI. In this model, we also observe two outcomes in infected animals: acute symptoms that is associated with CAUTI and chronic colonization that is associated with asymptomatic bacteriuria. The timing of the acute outcome takes place in the first week of infection, whereas chronic colonization occurs in the second week of infection. We further showed that mutants lacking genes encoding type III secretion system (T3SS), T3SS effector proteins, T3SS injection pore, or T3SS transcriptional activation all fail to cause acute symptoms of CAUTI. Nonetheless, all mutants defective for T3SS colonized the catheter and bladders at levels similar to the parental strain. In contrast, through induction of the T3SS master regulator ExsA, all infected animals showed acute phenotypes with bacteremia. Our results demonstrated that the acute symptoms, which are analogous to CAUTI, and chronic colonization, which is analogous to asymptomatic bacteriuria, are independent events that require distinct bacterial virulence factors. Experimental delineation of asymptomatic bacteriuria and CAUTI informs different strategies for the treatment and intervention of device-associated infections.

TRNA modification enzyme MiaB connects environmental cues to activation of Pseudomonas aeruginosa type III secretion system.

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.

Repression by the H-NS/YmoA histone-like protein complex enables IscR dependent regulation of the Yersinia T3SS.

The type III secretion system (T3SS) is an appendage used by many bacterial pathogens, such as pathogenic Yersinia, to subvert host defenses. However, because the T3SS is energetically costly and immunogenic, it must be tightly regulated in response to environmental cues to enable survival in the host. Here we show that expression of the Yersinia Ysc T3SS master regulator, LcrF, is orchestrated by the opposing activities of the repressive H-NS/YmoA histone-like protein complex and induction by the iron and oxygen-regulated IscR transcription factor. While deletion of iscR or ymoA has been shown to decrease and increase LcrF expression and type III secretion, respectively, the role of H-NS in this system has not been definitively established because hns is an essential gene in Yersinia. Using CRISPRi knockdown of hns, we show that hns depletion causes derepression of lcrF. Furthermore, we find that while YmoA is dispensable for H-NS binding to the lcrF promoter, YmoA binding to H-NS is important for H-NS repressive activity. We bioinformatically identified three H-NS binding regions within the lcrF promoter and demonstrate binding of H-NS to these sites in vivo using chromatin immunoprecipitation. Using promoter truncation and binding site mutation analysis, we show that two of these H-NS binding regions are important for H-NS/YmoA-mediated repression of the lcrF promoter. Surprisingly, we find that IscR is dispensable for lcrF transcription in the absence of H-NS/YmoA. Indeed, IscR-dependent regulation of LcrF and type III secretion in response to changes in oxygen, such as those Yersinia is predicted to experience during host infection, only occurs in the presence of an H-NS/YmoA complex. These data suggest that, in the presence of host tissue cues that drive sufficient IscR expression, IscR can act as a roadblock to H-NS/YmoA-dependent repression of RNA polymerase at the lcrF promoter to turn on T3SS expression.

Characterization of the binding motif for the T3SS master regulator LcrF in Yersinia pseudotuberculosis

LcrF is the master regulator that positively regulates the Ysc type III secretion system (T3SS) in Yersinia and shares a high similarity with the DNA-binding domain of the T3SS master regulator ExsA in Pseudomonas aeruginosa. Based on these features, bioinformatics analysis has predicted a putative LcrF-binding site in its target promoters. Here, we experimentally characterized its binding motif. An adenine-rich LcrF-binding region in the lcrG promoter sequence, a typical regulatory target of LcrF, was first confirmed. To obtain detailed information, this binding region was cloned into a synthetized promoter and mutations in this region were further constructed. We demonstrated that the 5'-AAAAA-n5-GnCT-3' sequence is required for LcrF regulation and this motif is strictly located 4-bp upstream of a noncanonical promoter, in which the -35 and -10 elements are separated by a 21-bp spacer. Consistently, the putative binding motif was found in promoters of nine T3SS related operons or genes positively regulated by LcrF. Transcriptome analysis further confirmed that LcrF specifically activates T3SS genes in Yersinia. Collectively, our data suggest that LcrF has evolved to be a specific T3SS activator with a stringent sequence requirement for transcriptional regulation.

Crystal structure of the nucleoid-associated protein Fis (PA4853) from Pseudomonas aeruginosa.

Factor for inversion stimulation (Fis) is a versatile bacterial nucleoid-associated protein that can directly bind and bend DNA to influence DNA topology. It also plays crucial roles in regulating bacterial virulence factors and in optimizing bacterial adaptation to various environments. Fis from Pseudomonas aeruginosa (PA4853, referred to as PaFis) has recently been found to be required for virulence by regulating the expression of type III secretion system (T3SS) genes. PaFis can specifically bind to the promoter region of exsA, which functions as a T3SS master regulator, to regulate its expression and plays an essential role in transcription elongation from exsB to exsA. Here, the crystal structure of PaFis, which is composed of a four-helix bundle and forms a homodimer, is reported. PaFis shows remarkable structural similarities to the well studied Escherichia coli Fis (EcFis), including an N-terminal flexible loop and a C-terminal helix-turn-helix (HTH) motif. However, the critical residues for Hin-catalyzed DNA inversion in the N-terminal loop of EcFis are not conserved in PaFis and further studies are required to investigate its exact role. A gel-electrophoresis mobility-shift assay showed that PaFis can efficiently bind to the promoter region of exsA. Structure-based mutagenesis revealed that several conserved basic residues in the HTH motif play essential roles in DNA binding. These structural and biochemical studies may help in understanding the role of PaFis in the regulation of T3SS expression and in virulence.

A DeoR-Type Transcription Regulator Is Required for Sugar-Induced Expression of Type III Secretion-Encoding Genes in Pseudomonas syringae pv. tomato DC3000.

The type III secretion system (T3SS) of plant-pathogenic Pseudomonas syringae is essential for virulence. Genes encoding the T3SS are not constitutively expressed and must be induced upon infection. Plant-derived metabolites, including sugars such as fructose and sucrose, are inducers of T3SS-encoding genes, yet the molecular mechanisms underlying perception of these host signals by P. syringae are unknown. Here, we report that sugar-induced expression of type III secretion A (setA), predicted to encode a DeoR-type transcription factor, is required for maximal sugar-induced expression of T3SS-associated genes in P. syringae DC3000. From a Tn5 transposon mutagenesis screen, we identified two independent mutants with insertions in setA. When both setA::Tn5 mutants were cultured in minimal medium containing fructose, genes encoding the T3SS master regulator HrpL and effector AvrRpm1 were expressed at lower levels relative to that of a wild-type strain. Decreased hrpL and avrRpm1 expression also occurred in a setA::Tn5 mutant in response to glucose, sucrose, galactose, and mannitol, demonstrating that setA is genetically required for T3SS induction by many different sugars. Expression of upstream regulators hrpR/S and rpoN was not altered in setA::Tn5, indicating that SetA positively regulates hrpL expression independently of increased transcription of these genes. In addition to decreased response to defined sugar signals, a setA::Tn5 mutant had decreased T3SS deployment during infection and was compromised in its ability to grow in planta and cause disease. These data suggest that SetA is necessary for P. syringae to effectively respond to T3SS-inducing sugar signals encountered during infection.

Iron availability and oxygen tension regulate the Yersinia Ysc type III secretion system to enable disseminated infection

The enteropathogen Yersinia pseudotuberculosis and the related plague agent Y. pestis require the Ysc type III secretion system (T3SS) to subvert phagocyte defense mechanisms and cause disease. Yet type III secretion (T3S) in Yersinia induces growth arrest and innate immune recognition, necessitating tight regulation of the T3SS. Here we show that Y. pseudotuberculosis T3SS expression is kept low under anaerobic, iron-rich conditions, such as those found in the intestinal lumen where the Yersinia T3SS is not required for growth. In contrast, the Yersinia T3SS is expressed under aerobic or anaerobic, iron-poor conditions, such as those encountered by Yersinia once they cross the epithelial barrier and encounter phagocytic cells. We further show that the [2Fe-2S] containing transcription factor, IscR, mediates this oxygen and iron regulation of the T3SS by controlling transcription of the T3SS master regulator LcrF. IscR binds directly to the lcrF promoter and, importantly, a mutation that prevents this binding leads to decreased disseminated infection of Y. pseudotuberculosis but does not perturb intestinal colonization. Similar to E. coli, Y. pseudotuberculosis uses the Fe-S cluster occupancy of IscR as a readout of oxygen and iron conditions that impact cellular Fe-S cluster homeostasis. We propose that Y. pseudotuberculosis has coopted this system to sense entry into deeper tissues and induce T3S where it is required for virulence. The IscR binding site in the lcrF promoter is completely conserved between Y. pseudotuberculosis and Y. pestis. Deletion of iscR in Y. pestis leads to drastic disruption of T3S, suggesting that IscR control of the T3SS evolved before Y. pestis split from Y. pseudotuberculosis.

Cross-Talk between the Aeromonas hydrophila Type III Secretion System and Lateral Flagella System

Aeromonas hydrophila is responsible for aeromonad septicaemia in fish, and gastroenteritis and wound infections in humans. The type III secretion system (T3SS) is utilized by aeromonads to inject protein effectors directly into host cells. One of the major genetic regulators of the T3SS in several bacterial species is the AraC-like protein ExsA. Previous studies have suggested a link between T3SS regulation and lateral flagella expression. The aim of this study was to determine the genetic regulation of the T3SS and its potential interaction with the lateral flagella system in A. hydrophila. To investigate the genes encoding the T3SS regulatory components exsA, exsD, exsC, and exsE were mutated and the activities of the T3SS promoters were measured in wild type and mutant backgrounds demonstrating a regulatory network. The Exs proteins were shown to interact with each other by BACTH assay and Far-Western Blot. The findings suggested a regulatory cascade in which ExsE was bound to the chaperone protein ExsC. When ExsC was free it sequestered the anti-activator ExsD thus stopping the inhibition of the T3SS master regulator ExsA allowing T3SS expression. The T3SS regulatory components were also shown to affect the expression of the lateral flagella system. The activities of the lateral flagella promoters were shown to be repressed by the absence of ExsD and ExsE, suggesting that the T3SS master regulator ExsA was a negative regulator of the lateral flagella system.

Hereditary Hemochromatosis Predisposes Mice to Yersinia pseudotuberculosis Infection Even in the Absence of the Type III Secretion System.

The iron overload disorder hereditary hemochromatosis (HH) predisposes humans to serious disseminated infection with pathogenic Yersinia as well as several other pathogens. Recently, we showed that the iron-sulfur cluster coordinating transcription factor IscR is required for type III secretion in Y. pseudotuberculosis by direct control of the T3SS master regulator LcrF. In E. coli and Yersinia, IscR levels are predicted to be regulated by iron bioavailability, oxygen tension, and oxidative stress, such that iron depletion should lead to increased IscR levels. To investigate how host iron overload influences Y. pseudotuberculosis virulence and the requirement for the Ysc type III secretion system (T3SS), we utilized two distinct murine models of HH: hemojuvelin knockout mice that mimic severe, early-onset HH as well as mice with the HfeC282Y∕C282Y mutation carried by 10% of people of Northern European descent, associated with adult-onset HH. Hjv−∕− and HfeC282Y∕C282Y transgenic mice displayed enhanced colonization of deep tissues by Y. pseudotuberculosis following oral inoculation, recapitulating enhanced susceptibility of humans with HH to disseminated infection with enteropathogenic Yersinia. Importantly, HH mice orally infected with Y. pseudotuberculosis lacking the T3SS-encoding virulence plasmid, pYV, displayed increased deep tissue colonization relative to wildtype mice. Consistent with previous reports using monocytes from HH vs. healthy donors, macrophages isolated from HfeC282Y∕C282Y mice were defective in Yersinia uptake compared to wildtype macrophages, indicating that the anti-phagocytic property of the Yersinia T3SS plays a less important role in HH animals. These data suggest that Yersinia may rely on distinct virulence factors to cause disease in healthy vs. HH hosts.

Modulation of Type III Secretion System in Pseudomonas aeruginosa: Involvement of the PA4857 Gene Product.

Pseudomonas aeruginosa is an opportunistic pathogen that causes serious acute or chronic infections in humans. Acute infections typically involve the type III secretion systems (T3SSs) and bacterial motility, whereas chronic infections are often associated with biofilm formation and the type VI secretion system. To identify new genes required for pathogenesis, a transposon mutagenesis library was constructed and the gene PA4857, named tspR, was found to modulate T3SS gene expression. Deletion of P. aeruginosa tspR reduced the virulence in a mouse acute lung infection model and diminished cytotoxicity. Suppression of T3SS gene expression in the tspR mutant resulted from compromised translation of the T3SS master regulator ExsA. TspR negatively regulated two small RNAs, RsmY and RsmZ, which control RsmA. Our data demonstrated that defects in T3SS expression and biofilm formation in retS mutant could be partially restored by overexpression of tspR. Taken together, our results demonstrated that the newly identified retS-tspR pathway is coordinated with the retS-gacS system, which regulates the genes associated with acute and chronic infections and controls the lifestyle choice of P. aeruginosa.

RcsB positively regulates the Yersinia Ysc-Yop type III secretion system by activating expression of the master transcriptional regulator LcrF.

The Rcs phosphorelay is a complex signaling pathway used by the family Enterobacteriaceae to sense, respond and adapt to environmental changes during free-living or host-associated lifestyles. In this study, we show that the Rcs phosphorelay pathway positively regulates the virulence plasmid encoded Ysc-Yop type III secretion system (T3SS) in the enteropathogen Yesinia pseudotuberculosis. Both the overexpression of the wild-type Rcs regulator RcsB or the constitutive active RscB(D56E) variant triggered more abundant Ysc-Yop synthesis and secretion, whereas the non-phosphorylatable mutant RcsB(D56Q) negated this. Congruently, enhanced Yops expression and secretion occurred in an in cis rscB(D56E) mutant but not in an isogenic rscB(D56Q) mutant. Screening for regulatory targets of RcsB identified the virG-lcrF operon that encodes for LcrF, the Ysc-Yop T3SS master regulator. Protein-DNA binding assays confirmed that RcsB directly bound to this operon promoter, which subsequently caused stimulated lcrF transcription. Moreover, active RcsB enhanced the ability of bacteria to deliver Yop effectors into immune cells during cell contact, and this promoted an increase in bacterial viability. Taken together, our study demonstrates the role of the Rcs system in regulating the Ysc-Yop T3SS in Yersinia and reports on RcsB being the first transcriptional activator known to directly control lcrF transcription.

IscR Is Essential for Yersinia pseudotuberculosis Type III Secretion and Virulence

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.

SuhB Is a Regulator of Multiple Virulence Genes and Essential for Pathogenesis of Pseudomonas aeruginosa

ABSTRACTDuring initial colonization and chronic infection, pathogenic bacteria encounter distinct host environments. Adjusting gene expression accordingly is essential for the pathogenesis. Pseudomonas aeruginosa has evolved complicated regulatory networks to regulate different sets of virulence factors to facilitate colonization and persistence. The type III secretion system (T3SS) and motility are associated with acute infections, while biofilm formation and the type VI secretion system (T6SS) are associated with chronic persistence. To identify novel regulatory genes required for pathogenesis, we screened a P. aeruginosa transposon (Tn) insertion library and found suhB to be an essential gene for the T3SS gene expression. The expression of suhB was upregulated in a mouse acute lung infection model, and loss of suhB resulted in avirulence. Suppression of T3SS gene expression in the suhB mutant is linked to a defective translation of the T3SS master regulator, ExsA. Further studies demonstrated that suhB mutation led to the upregulation of GacA and its downstream small RNAs, RsmY and RsmZ, triggering T6SS expression and biofilm formation while inhibiting the T3SS. Our results demonstrate that an in vivo-inducible gene, suhB, reciprocally regulates genes associated with acute and chronic infections and plays an essential role in the pathogenesis of P. aeruginosa.

The global regulator Crc plays a multifaceted role in modulation of type III secretion system in Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa utilizes type III secretion system (T3SS) to translocate effector proteins into eukaryotic host cells that subvert normal host cell functions to the benefit of the pathogen, and results in serious infections. T3SS in P. aeruginosa is controlled by a complex system of regulatory mechanisms and signaling pathways. In this study, we described that Crc, an RNA-binding protein, exerts a positive impact on T3SS in P. aeruginosa, as evidenced by promoter activity assays of several key T3SS genes, transcriptomics, RT-PCR, and immunoblotting in crc mutant. We further demonstrated that the regulatory function of Crc on the T3SS was mediated through the T3SS master regulator ExsA and linked to the Cbr/Crc signaling system. Expression profiling of the crc mutant revealed a downregulation of flagship T3SS genes as well as 16 other genes known to regulate T3SS gene expression in P. aeruginosa. On the basis of these data, we proposed that Crc may exert multifaceted control on the T3SS through various pathways, which may serve to fine-tune this virulence mechanism in response to environmental changes and nutrient sources.

SlyA Regulates Type III Secretion System (T3SS) Genes in Parallel with the T3SS Master Regulator HrpL in Dickeya dadantii 3937

The hypersensitive response and pathogenicity (hrp) genes of Dickeya dadantii 3937 encode a type III secretion system (T3SS) which is essential for its full virulence. Previous studies of the T3SS regulation in D. dadantii 3937 revealed that the expression of the hrp genes is regulated by a master regulator, HrpL, through the HrpX-HrpY-HrpS-HrpL and GacS-GacA-rsmB-RsmA pathways. In this work, we identified a novel regulator of the SlyA/MarR family, SlyA, which regulates hrp genes of the HrpL regulon in parallel with HrpL in D. dadantii. SlyA regulates the T3SS in a two-tier manner. It negatively regulates the expression of hrpL by downregulating hrpS and upregulating rsmA. Interestingly, concomitant with its downregulation of the hrpL, SlyA positively regulates the expression of hrpA and hrpN, two hrp genes located in the HrpL regulon. In contrast to Pectobacterium carotovorum, the expression of slyA is not controlled by ExpR and ExpI in D. dadantii 3937. We further show that SlyA is involved in controlling swimming motility and pellicle formation in D. dadantii 3937.

Cross talk between type III secretion and flagellar assembly systems in Pseudomonas aeruginosa.

Pseudomonas aeruginosa cytotoxicity is linked to a type III secretion system (T3SS) that delivers effectors into the host cell. We show here that a negative cross-control exists between T3SS and flagellar assembly. We observed that, in a strain lacking flagella, T3SS gene expression, effector secretion, and cytotoxicity were increased. Conversely, we revealed that flagellar-gene expression and motility were decreased in a strain overproducing ExsA, the T3SS master regulator. Interestingly, a nonmotile strain lacking the flagellar filament (DeltafliC) presented a hyperefficient T3SS and a nonmotile strain assembling flagella (DeltamotAB) did not. More intriguingly, a strain lacking motCD genes is a flagellated strain with a slight defect in swimming. However, in this strain, T3SS gene expression was up-regulated. These results suggest that flagellar assembly and/or mobility antagonizes the T3SS and that a negative cross talk exists between these two systems. An illustration of this is the visualization by electron microscopy of T3SS needles in a nonmotile P. aeruginosa strain, needles which otherwise are not detected. The molecular basis of the cross talk is complex and remains to be elucidated, but proteins like MotCD might have a crucial role in signaling between the two processes. In addition, we found that the GacA response regulator negatively affects the T3SS. In a gacA mutant, the T3SS effector ExoS is hypersecreted. Strikingly, GacA was previously reported as a positive regulator for motility. Globally, our data document the idea that some virulence factors are coordinately but inversely regulated, depending on the bacterial colonization phase and infection types.