Search Results (5)

Iron is essential for vital biological processes, with its metabolism closely linked to host–pathogen interactions. Pseudomonas donghuensis HYS, with its superior iron uptake capacity, demonstrates pronounced virulence toward Caenorhabditis elegans. However, the virulence mechanisms remain unexplored. Ferric uptake regulator (Fur) regulates iron homeostasis and pathogenicity in bacteria, yet its role in HYS-mediated C. elegans pathogenesis requires systematic investigation. In this study, comparing the pathogenic processes of HYS and P. aeruginosa PA14 revealed that HYS causes stronger, irreversible toxicity via distinct mechanisms. Transcriptomics revealed that HYS infection disrupts C. elegans iron metabolism pathways, specifically iron transport, and iron–sulfur cluster utilization. Fur was identified as a pivotal regulator in HYS virulence and was indispensable for its colonization. Specifically, Fur was critical for disrupting nematode iron metabolism, as fur deletion eliminated this effect. While Fur regulated two HYS siderophores, neither of them mediated in the iron metabolism disruption of C. elegans. Screening identified Fur-regulated virulence factors to further investigate the function of Fur in HYS virulence, particularly alkaline proteases, and type II secretion system components. This study highlight that HYS can disrupt the iron metabolism pathway in C. elegans; Fur serves as a pivotal positive regulator in HYS-induced damage, particularly in disrupting iron metabolism through a siderophore-independent pathway. These findings expand the understanding of Pseudomonas pathogenicity and Fur-mediated virulence regulation. Full article

Compared to pathogens Pseudomonas aeruginosa and P. putida, P. donghuensis HYS has stronger virulence towards Caenorhabditis elegans. However, the underlying mechanisms haven’t been fully understood. The heme synthesis system is essential for Pseudomonas virulence, and former studies of HemN have focused on the synthesis of heme, while the relationship between HemN and Pseudomonas virulence were barely pursued. In this study, we hypothesized that hemN2 deficiency affected 7-hydroxytropolone (7-HT) biosynthesis and redox levels, thereby reducing bacterial virulence. There are four hemN genes in P. donghuensis HYS, and we reported for the first time that deletion of hemN2 significantly reduced the virulence of HYS towards C. elegans, whereas the reduction in virulence by the other three genes was not significant. Interestingly, hemN2 deletion significantly reduced colonization of P. donghuensis HYS in the gut of C. elegans. Further studies showed that HemN2 was regulated by GacS and participated in the virulence of P. donghuensis HYS towards C. elegans by mediating the synthesis of the virulence factor 7-HT. In addition, HemN2 and GacS regulated the virulence of P. donghuensis HYS by affecting antioxidant capacity and nitrative stress. In short, the findings that HemN2 was regulated by the Gac system and that it was involved in bacterial virulence via regulating 7-HT synthesis and redox levels were reported for the first time. These insights may enlighten further understanding of HemN-based virulence in the genus Pseudomonas. Full article

The newly discovered iron scavenger 7-hydroxytropolone (7-HT) is secreted by Pseudomonas donghuensis HYS. In addition to possessing an iron-chelating ability, 7-HT has various other biological activities. However, 7-HT’s biosynthetic pathway remains unclear. This study was the first to report that the phenylacetic acid (PAA) catabolon genes in cluster 2 are involved in the biosynthesis of 7-HT and that two genes, paaZ (orf13) and ech, are synergistically involved in the biosynthesis of 7-HT in P. donghuensis HYS. Firstly, gene knockout and a sole carbon experiment indicated that the genes orf17–21 (paaEDCBA) and orf26 (paaG) were involved in the biosynthesis of 7-HT and participated in the PAA catabolon pathway in P. donghuensis HYS; these genes were arranged in gene cluster 2 in P. donghuensis HYS. Interestingly, ORF13 was a homologous protein of PaaZ, but orf13 (paaZ) was not essential for the biosynthesis of 7-HT in P. donghuensis HYS. A genome-wide BLASTP search, including gene knockout, complemented assays, and site mutation, showed that the gene ech homologous to the ECH domain of orf13 (paaZ) is essential for the biosynthesis of 7-HT. Three key conserved residues of ech (Asp39, His44, and Gly62) were identified in P. donghuensis HYS. Furthermore, orf13 (paaZ) could not complement the role of ech in the production of 7-HT, and the single carbon experiment indicated that paaZ mainly participates in PAA catabolism. Overall, this study reveals a natural association between PAA catabolon and the biosynthesis of 7-HT in P. donghuensis HYS. These two genes have a synergistic effect and different functions: paaZ is mainly involved in the degradation of PAA, while ech is mainly related to the biosynthesis of 7-HT in P. donghuensis HYS. These findings complement our understanding of the mechanism of the biosynthesis of 7-HT in the genus Pseudomonas. Full article

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. Full article

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. Full article