The Pseudomonas aeruginosa reference strain PA14 displays increased virulence due to a mutation in ladS.
ABSTRACT: Pseudomonas aeruginosa is a pathogen that causes acute and chronic infections in a variety of hosts. The pathogenic potential of P. aeruginosa is strain-dependent. PA14 is a highly virulent strain that causes disease in a wide range of organisms, whereas PAO1 is moderately virulent. Although PA14 carries pathogenicity islands that are absent in PAO1, the presence or absence of specific gene clusters is not predictive of virulence. Here, we show that the virulent strain PA14 has an acquired mutation in the ladS gene. This mutation has a deleterious impact on biofilm, while it results in elevated type III secretion system (T3SS) activity and increased cytotoxicity towards mammalian cells. These phenotypes can be reverted by repairing the ladS mutation on the PA14 genome. The RetS/LadS/GacS signaling cascade is associated with virulence and the switch between acute and chronic infections. RetS is a sensor that down-regulates biofilm formation and up-regulates the T3SS. Mutations in retS are acquired in strains isolated from chronically infected cystic fibrosis patients and lead to hyperbiofilm formation and reduced cytotoxicity. Conversely, the LadS sensor promotes biofilm formation and represses the T3SS. We conclude that the ladS mutation is partly responsible for the high cytotoxicity of PA14, and our findings corroborate the central role of RetS and LadS in the switch between acute and chronic infections. Given the extensive use of the reference strain PA14 in infection and virulence models, the bias caused by the ladS mutation on the observed phenotypes will be crucial to consider in future research.
Project description:The opportunistic pathogen Pseudomonas aeruginosa may cause both acute and chronic-persistent infections in predisposed individuals. Acute infections require the presence of a functional type III secretion system (T3SS), whereas chronic P. aeruginosa infections are characterized by the formation of drug-resistant biofilms. The T3SS and biofilm formation are reciprocally regulated by the signaling kinases LadS, RetS, and GacS. RetS downregulates biofilm formation and upregulates expression of the T3SS through a unique mechanism. RetS forms a heterodimeric complex with GacS and thus prevents GacS autophosphorylation and downstream signaling. The signals that regulate RetS are not known but RetS possesses a distinctive periplasmic sensor domain that is believed to serve as receptor for the regulatory ligand. We have determined the crystal structure of the RetS sensory domain at 2.0 A resolution. The structure closely resembles those of carbohydrate binding modules of other proteins, suggesting that the elusive ligands are likely carbohydrate moieties. In addition to the conserved beta-sandwich structure, the sensory domain features two alpha helices which create a unique surface topology. Protein-protein crosslinking and fluorescence energy transfer experiments also revealed that the sensory domain dimerizes with a dissociation constant of K(d) = 580 +/- 50 nM, a result with interesting implications for our understanding of the underlying signaling mechanism.
Project description:Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in a wide range of hosts. Expression of the type III secretion system (T3SS) proteins is correlated with virulence in models of acute infection, while downregulation of the T3SS and upregulation of genes important for biofilm formation are observed during chronic infections. RetS, a hybrid sensor kinase-response regulator protein of P. aeruginosa, plays a key role in the reciprocal regulation of virulence factors required for acute versus chronic infection and is postulated to act in concert with two other sensor kinase-response regulator hybrids, GacS and LadS. This work examines the roles of the putative sensing and signal transduction domains of RetS in induction of the T3SS in vitro and in a murine model of acute pneumonia. We identify distinct signaling roles for the tandem receiver domains of RetS and present evidence suggesting that RetS may serve as a substrate for another sensor kinase. Phenotypes associated with RetS alleles lacking periplasmic and/or transmembrane domains further indicate that the periplasmic domain of RetS may transmit a signal that inhibits RetS activity during acute infections.
Project description:Pseudomonas aeruginosa is an opportunistic human pathogen and a common cause of chronic infections in individuals with cystic fibrosis (CF). Oxygen limitation was recently reported to regulate the expression of a major virulence determinant in P. aeruginosa, the type III secretion system (T3SS). Here, we show that expression of the T3SS in oxygen-limited growth conditions is strongly dependent on the glyoxylate shunt enzyme, isocitrate lyase (ICL; encoded by aceA), which was previously shown to be highly expressed in CF isolates. ICL-dependent regulation of the T3SS did not alter the expression level of the master transcriptional regulator, ExsA, but did affect expression of the T3 structural proteins, effectors and regulators (ExsC, ExsD and ExsE). An aceA mutant displayed enhanced biofilm formation during anaerobic growth, which suggested that AceA-dependent modulation of type III secretion might impinge upon the RetS/LadS signalling pathways. Indeed, our data suggest that RetS is able to mediate some of its effects through AceA, as expression of aceA in trans partially restored T3SS expression in a retS mutant. Our findings indicate that AceA is a key player in the metabolic regulation of T3SS expression during oxygen-limited growth of P. aeruginosa. To the best of our knowledge, this is the first demonstration that the T3SS can be regulated by factors that do not affect ExsA expression levels.
Project description:Pseudomonas aeruginosa chronic lung infections are the major cause of morbidity and mortality in cystic fibrosis (CF) patients. The P. aeruginosa strains PAO1 and PA14 were compared with the Liverpool epidemic strain LESB58 to assess in vivo growth, infection kinetics, and bacterial persistence and localization within tissues in a rat model of chronic lung infection. The three P. aeruginosa strains demonstrated similar growth curves in vivo but differences in tissue distribution. The LESB58 strain persisted in the bronchial lumen, while the PAO1 and PA14 strains were found localized in the alveolar regions and grew as macrocolonies after day 7 postinfection. Bacterial strains were compared for swimming and twitching motility and for the production of biofilm. The P. aeruginosa LESB58 strain produced more biofilm than PAO1 and PA14. Competitive index (CI) analysis of PAO1, PA14, and LESB58 in vivo indicated CI values of 0.002, 0.0002, and 0.14 between PAO1-PA14, PAO1-LESB58, and LESB58-PA14, respectively. CI analysis comparing the in vivo growth of the PAO1 DeltaPA5441 mutant and four PA14 surface attachment-defective (sad) mutants gave CI values 10 to 1,000 times lower in competitions with their respective wild-type strains PAO1 and PA14. P. aeruginosa strains studied in the rat model of chronic lung infection demonstrated similar in vivo growth but differences in virulence as shown with a competitive in vivo assay. These differences were further confirmed with biofilm and motility in vitro assays, where strain LESB58 produced more biofilm but had less capacity for motility than PAO1 and PA14.
Project description:Microarray analysis of Pseudomonas aeruginosa mRNA transcripts expressed in vivo during animal infection has not been previously used to investigate potential virulence factors needed in this setting. We compared mRNA expression in bacterial cells recovered from the gastrointestinal (GI) tracts of P. aeruginosa-colonized mice to that of P. aeruginosa in the drinking water used to colonize the mice. Genes associated with biofilm formation and type III secretion (T3SS) had markedly increased expression in the GI tract. A non-redundant transposon library in P. aeruginosa strain PA14 was used to test mutants in genes identified as having increased transcription during in vivo colonization. All of the Tn-library mutants in biofilm-associated genes had an attenuated ability to form biofilms in vitro, but there were no significant differences in GI colonization and dissemination between these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI colonization among these mutant strains and their WT counterparts, whereas rates of survival following dissemination were significantly decreased in mice infected by the T3SS mutant strains. However, there was a variable, strain-dependent effect on overall survival between parental and T3SS mutants. Thus, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia.
Project description:In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS?D1LadS?H2GacS?D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.
Project description:Two clinical isolates of the opportunist pathogen Pseudomonas aeruginosa named PAO1 and PA14 are commonly studied in research laboratories. Despite the isolates being closely related, PA14 exhibits increased virulence compared to that of PAO1 in various models. To determine which players are responsible for the hypervirulence phenotype of the PA14 strain, we elected a transcriptomic approach through RNA sequencing. We found 2,029 genes that are differentially expressed between the two strains, including several genes that are involved with or regulated by quorum sensing (QS), known to control most of the virulence factors in P. aeruginosa Among them, we chose to focus our study on QslA, an antiactivator of QS whose expression was barely detectable in the PA14 strain according our data. We hypothesized that lack of expression of qslA in PA14 could be responsible for higher QS expression in the PA14 strain, possibly explaining its hypervirulence phenotype. After confirming that QslA protein was highly produced in PAO1 but not in the PA14 strain, we obtained evidence showing that a PAO1 deletion strain of qslA has faster QS gene expression kinetics than PA14. Moreover, known virulence factors activated by QS, such as (i) pyocyanin production, (ii) H2-T6SS (type VI secretion system) gene expression, and (iii) Xcp-T2SS (type II secretion system) machinery production and secretion, were all lower in PAO1 than in PA14, due to higher qslA expression. However, biofilm formation and cytotoxicity toward macrophages, although increased in PA14 compared to PAO1, were independent of QslA control. Together, our findings implicated differential qslA expression as a major determinant of virulence factor expression in P. aeruginosa strains PAO1 and PA14.IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen responsible for acute nosocomial infections and chronic pulmonary infections. P. aeruginosa strain PA14 is known to be hypervirulent in different hosts. Despite several studies in the field, the underlining molecular mechanisms sustaining this phenotype remain enigmatic. Here we provide evidence that the PA14 strain has faster quorum sensing (QS) kinetics than the PAO1 strain, due to the lack of QslA expression, an antiactivator of QS. QS is a major regulator of virulence factors in P. aeruginosa; therefore, we propose that the hypervirulent phenotype of the PA14 strain is, at least partially, due to the lack of QslA expression. This mechanism could be of great importance, as it could be conserved among other P. aeruginosa isolates.
Project description: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.
Project description:The opportunistic pathogen Pseudomonas aeruginosa is responsible for a wide range of acute and chronic infections. The transition to chronic infections is accompanied by physiological changes in the bacteria favoring formation of biofilm communities. Here we report the identification of LadS, a hybrid sensor kinase that controls the reciprocal expression of genes for type III secretion and biofilm-promoting polysaccharides. Domain organization of LadS and the range of LadS-controlled genes suggest that it counteracts the activities of another sensor kinase, RetS. These two pathways converge by controlling the transcription of a small regulatory RNA, RsmZ. This work identifies a previously undescribed signal transduction network in which the activities of signal-receiving sensor kinases LadS, RetS, and GacS regulate expression of virulence genes associated with acute or chronic infection by transcriptional and posttranscriptional mechanisms.
Project description:Pseudomonas syringae pv. syringae B728a is a resident on leaves of common bean, where it utilizes several well-studied virulence factors, including secreted effectors and toxins, to develop a pathogenic interaction with its host. The B728a genome was recently sequenced, revealing the presence of 1,297 genes with unknown function. This study demonstrates that a 29.9-kb cluster of genes in the B728a genome shares homology to the novel type VI secretion system (T6SS) locus recently described for other gram-negative bacteria. Western blot analyses showed that B728a secretes Hcp, a T6SS protein, in culture and that this secretion is dependent on clpV, a gene that likely encodes an AAA(+) ATPase. In addition, we have identified two B728a sensor kinases that have homology to the P. aeruginosa proteins RetS and LadS. We demonstrate that B728a RetS and LadS reciprocally regulate the T6SS and collectively modulate several virulence-related activities. Quantitative PCR analyses indicated that RetS and LadS regulate genes associated with the type III secretion system and that LadS controls the expression of genes involved in the production of the exopolysaccharides alginate and levan. These analyses also revealed that LadS and the hybrid sensor kinase GacS positively regulate the expression of a putative novel exopolysaccharide called Psl. Plate assays demonstrated that RetS negatively controls mucoidy, while LadS negatively regulates swarming motility. A mutation in retS affected B728a population levels on the surfaces of bean leaves. A model for the LadS and RetS control of B728a virulence activities is proposed.