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Pseudomonas donghuensis HYS virulence towards Caenorhabditis elegans is regulated by the Cbr/Crc system.

ABSTRACT: Pseudomonas donghuensis HYS is the type strain of a recently identified species, P. donghuensis, which has pathogenic potential with an unclear virulence mechanism. In this study, we used Caenorhabditis elegans as a host to explore the virulence mechanism of P. donghuensis HYS. Based on a correlation between P. donghuensis HYS virulence and its repellence property, we identified 68 potential virulence-related genes, among them the Cbr/Crc system, which regulates the virulence of prokaryotic microorganisms. Slow-killing assays indicated that cbrA, cbrB, or specific sRNA-encoding genes all affected P. donghuensis virulence positively, whereas crc affected it negatively. Transcriptome analyses demonstrated that the Cbr/Crc system played an important role in the pathogenesis of P. donghuensis. In addition, experiments using the worm mutant KU25 pmk-1(km25) showed a correlation between P. donghuensis HYS virulence and the PMK-1/p38 MAPK pathway in C. elegans. In conclusion, our data show that Crc plays a novel role in the Cbr/Crc system, and the P. donghuensis virulence phenotype therefore differs from that of P. aeruginosa. This process also involves C. elegans innate immunity. These findings significantly increase the available information about Cbr/Crc-based virulence mechanisms in the genus Pseudomonas.

SUBMITTER: Xie G

PROVIDER: S-EPMC6584532 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Pseudomonas donghuensis HYS virulence towards Caenorhabditis elegans is regulated by the Cbr/Crc system.

Xie Guanfang G Zeng Man M You Jia J Xie Zhixiong Z

Scientific reports 20190619 1

Pseudomonas donghuensis HYS is the type strain of a recently identified species, P. donghuensis, which has pathogenic potential with an unclear virulence mechanism. In this study, we used Caenorhabditis elegans as a host to explore the virulence mechanism of P. donghuensis HYS. Based on a correlation between P. donghuensis HYS virulence and its repellence property, we identified 68 potential virulence-related genes, among them the Cbr/Crc system, which regulates the virulence of prokaryotic micr ...[more]

PMID: 31217473

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Project description:Pseudomonas donghuensis HYS is more virulent than P. aeruginosa toward Caenorhabditis elegans but the mechanism underlying virulence is unclear. This study is the first to report that the specific gene cluster gtrA/B/II in P. donghuensis HYS is involved in the virulence of this strain toward C. elegans, and there are no reports of GtrA, GtrB and GtrII in any Pseudomonas species. The pathogenicity of P. donghuensis HYS was evaluated using C. elegans as a host. Based on the prediction of virulence factors and comparative genomic analysis of P. donghuensis HYS, we identified 42 specific virulence genes in P. donghuensis HYS. Slow-killing assays of these genes showed that the gtrAB mutation had the greatest effect on the virulence of P. donghuensis HYS, and GtrA, GtrB and GtrII all positively affected P. donghuensis HYS virulence. Two critical GtrII residues (Glu47 and Lys480) were identified in P. donghuensis HYS. Transmission electron microscopy (TEM) showed that GtrA, GtrB and GtrII were involved in the glucosylation of lipopolysaccharide (LPS) O-antigen in P. donghuensis HYS. Furthermore, colony-forming unit (CFU) assays showed that GtrA, GtrB and GtrII significantly enhanced P. donghuensis HYS colonization in the gut of C. elegans, and glucosylation of LPS O-antigen and colonization in the host intestine contributed to the pathogenicity of P. donghuensis HYS. In addition, experiments using the worm mutants ZD101, KU4 and KU25 revealed a correlation between P. donghuensis HYS virulence and the TIR-1/SEK-1/PMK-1 pathways of the innate immune p38 MAPK pathway in C. elegans. In conclusion, these results reveal that the specific virulence gene cluster gtrA/B/II contributes to the unique pathogenicity of HYS compared with other pathogenic Pseudomonas, and that this process also involves C. elegans innate immunity. These findings significantly increase the available information about GtrA/GtrB/GtrII-based virulence mechanisms in the genus Pseudomonas.

Project description:7-Hydroxytropolone (7-HT) is a symmetrical seven-membered heteroatomic ring with a carboxyl group and two hydroxyl groups and was recently reported to be an iron scavenger of Pseudomonas donghuensis HYS. Cluster 1 includes 12 genes related to the synthesis of 7-HT; among these genes, those for two regulators, Orf1 and Orf12, were predicted to regulate 7-HT biosynthesis and to be LysR-type transcriptional regulators (LTTRs) and TetR/AcrR family transcriptional regulators, respectively. Data from real-time quantitative PCR and β-galactosidase and classical siderophore assays indicated that the transcription levels of orf1 and orf12, as well as those of crucial genes orf6 to orf9, were repressed under high-iron conditions. The deletion of orf1 and orf12 led to an absence of 7-HT and a decrease in orf6-orf9 expression. Orf1 and Orf12 were essential for the production of 7-HT through orf6-orf9 These two regulators are regulated by the Gac/Rsm system; Orf1 facilitates the expression of Orf12, and Orf12 concomitantly stimulates the expression of orf6-orf9 to synthesize 7-HT. The overexpression of Orf12 decreased 7-HT yields, possibly through decreased orf6-orf9 expression. This work thus outlines a complex mechanism regulating the biosynthesis of the iron scavenger 7-HT in P. donghuensis HYS. The synergy between Orf1 and Orf12 ensures that 7-HT acts as an iron chelator despite being toxic to bacteria and provides new ideas for the novel regulation of dual-functional secondary metabolism and research on 7-HT and its derivates in other bacteria.IMPORTANCE A complex regulation mechanism including two regulators, LysR and TetR/AcrR, in the biosynthesis of the novel iron scavenger 7-hydroxytropolone (7-HT) was verified in Pseudomonas donghuensis HYS. The coaction of LysR Orf1 and TetR/AcrR Orf12 may balance the toxicity and iron chelation of 7-HT in P. donghuensis HYS to overcome iron deficiency, as well as improve the bacterial competitiveness under iron-scarce conditions because of the toxicity of 7-HT toward other bacteria, making the accurate regulation of 7-HT biosynthesis indispensable. This regulation mechanism may be ubiquitous in the Pseudomonas putida group but may better explain the group's strong adaptability.

Project description:7-Hydroxytropolone (7-HT) is a unique iron scavenger synthesized by Pseudomonas donghuensis HYS that has various biological activities in addition to functioning as a siderophore. P. donghuensis HYS is more pathogenic than P. aeruginosa toward Caenorhabditis elegans, an observation that is closely linked to the biosynthesis of 7-HT. The nonfluorescent siderophore (nfs) gene cluster is responsible for the orderly biosynthesis of 7-HT and represents a competitive advantage that contributes to the increased survival of P. donghuensis HYS; however, the regulatory mechanisms of 7-HT biosynthesis remain unclear. This study is the first to propose that the ECF σ factor has a regulatory effect on 7-HT biosynthesis. In total, 20 ECF σ factors were identified through genome-wide scanning, and their responses to extracellular ferrous ions were characterized. We found that SigW was both significantly upregulated under high-iron conditions and repressed by an adjacent anti-σ factor. RNA-Seq results suggest that the SigW/RsiW system is involved in iron metabolism and 7-HT biosynthesis. Combined with the siderophore phenotype, we also found that SigW could inhibit siderophore synthesis, and this inhibition can be relieved by RsiW. EMSA assays proved that SigW, when highly expressed, can directly bind to the promoter region of five operons of the nfs cluster to inhibit the transcription of the corresponding genes and consequently suppress 7-HT biosynthesis. In addition, SigW not only directly negatively regulates structural genes related to 7-HT synthesis but also inhibits the transcription of regulatory proteins, including of the Gac/Rsm cascade system. Taken together, our results highlight that the biosynthesis of 7-HT is negatively regulated by SigW and that the SigW/RsiW system is involved in mechanisms for the regulation of iron homeostasis in P. donghuensis HYS. As a result of this work, we identified a novel mechanism for the global negative regulation of 7-HT biosynthesis, complementing our understanding of the function of ECF σ factors in Pseudomonas.

Dataset Information

Pseudomonas donghuensis HYS virulence towards Caenorhabditis elegans is regulated by the Cbr/Crc system.

Publications

Pseudomonas donghuensis HYS virulence towards Caenorhabditis elegans is regulated by the Cbr/Crc system.

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