The Phenomenon of T3SS Inactivation for Pseudomonas aeruginosa Strains from a Chronic Infection Locus: Do Mutations in T3SS-Regulators Matter?
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ABSTRACT:
SUBMITTER: Savinova T
PROVIDER: S-EPMC9241816 | biostudies-literature | 2022 Jun
REPOSITORIES: biostudies-literature
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The Phenomenon of T3SS Inactivation for <i>Pseudomonas aeruginosa</i> Strains from a Chronic Infection Locus: Do Mutations in T3SS-Regulators Matter?
Savinova T T Bocharova Y Y Mayanskiy N N Chebotar I I
Microbiology spectrum 20220516 3
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Project description:Diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Bacterial infection is recognized as an important factor associated with impaired healing in diabetic ulcers. Pseudomonas aeruginosa is the most frequently detected Gram-negative pathogen in diabetic ulcers. P. aeruginosa infection has been shown to impair healing in diabetic wounds in a manner that correlates with its ability to form biofilm. While the majority of infections in diabetic ulcers are biofilm associated, 33% of infections are nonbiofilm in nature. P. aeruginosa is the most prevalent Gram-negative pathogen in all diabetic wound types, which suggests that the deleterious impact of P. aeruginosa on healing in diabetic wounds goes beyond its ability to form biofilm and likely involves other factors. The Type III Secretion System (T3SS) virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers. We evaluated the role of T3SS in mediating P. aeruginosa-induced tissue damage in the wounds of diabetic mice. Our data demonstrate that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its T3SS.
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Project description:We determined the sequences of the quinolone resistance-determining regions of gyrA, gyrB, and parC genes for 30 clinical strains of Pseudomonas aeruginosa resistant to ciprofloxacin that were previously complemented by wild-type gyrA and gyrB plasmid-borne alleles and studied for their coresistance to imipenem (E. Cambau, E. Perani, C. Dib, C. Petinon, J. Trias, and V. Jarlier, Antimicrob. Agents Chemother. 39:2248-2252, 1995). In the present study, we found mutations in type II topoisomerase genes for all strains. Twenty-eight strains had a missense mutation in gyrA (codon 83 or 87). Ten of them had an additional mutation in parC (codon 80 or 84), including a novel mutation of Ser-80 to Trp, but all were fully complemented by a plasmid-borne wild-type gyrA allele. The remaining two strains harbored the first gyrB mutation described in P. aeruginosa, leading to the substitution of phenylalanine for serine 464. The strains which had two mutations in type II topoisomerase genes (i.e., gyrA and parC) were significantly more resistant to fluoroquinolones than those with a single mutation in gyrA or gyrB (geometric mean MICs of ciprofloxacin, 39.4 versus 10.9 microg/ml, P < 0.01; geometric mean MICs of sparfloxacin, 64.0 versus 22.6, P < 0. 01). No mutant with a parC mutation alone was observed, which favors DNA gyrase being the primary target for fluoroquinolones. These results demonstrate that gyrA mutations are the major mechanism of resistance to fluoroquinolones for clinical strains of P. aeruginosa and that additional mutations in parC lead to a higher level of quinolone resistance.
| S-EPMC89021 | biostudies-literature
Project description:Pseudomonas aeruginosa is a vital opportunistic human bacterial pathogen that causes acute and chronic infections. In this study, we set to determine whether the endogenous spermidine biosynthesis plays a role in regulation of type III secretion system (T3SS). The results showed that deletion of speA and speC, which encode putrescine biosynthesis, did not seem to affect cellular spermidine level and the T3SS gene expression. In contrast, mutation of speD and speE encoding spermidine biosynthesis led to significantly decreased spermidine production and expression of T3SS genes. We also showed that endogenous spermidine could auto-induce the transcriptional expression of speE and its full functionality required the transporter SpuDEFGH. Cytotoxicity analysis showed that mutants ΔspeE and ΔspuE were substantially attenuated in virulence compared with their wild-type strain PAO1. Our data imply a possibility that spermidine biosynthesis in P. aeruginosa may not use putrescine as a substrate, and that spermidine signaling pathway may interact with other two T3SS regulatory mechanisms in certain degree, i.e., cAMP-Vfr and GacS/GacA signaling systems. Taken together, these results specify the role of endogenous spermidine in regulation of T3SS in P. aeruginosa and provide useful clues for design and development antimicrobial therapies. IMPORTANCE Type III secretion system (T3SS) is one of the pivotal virulence factors of Pseudomonas aeruginosa responsible for evading phagocytosis, and secreting and translocating effectors into host cells. Previous studies underline the complicated and elaborate regulatory mechanisms of T3SS for the accurate, fast, and malicious pathogenicity of P. aeruginosa. Among these regulatory mechanisms, our previous study indicated that the spermidine from the host was vital to the host-pathogen interaction. However, the role of endogenous spermidine synthesized by P. aeruginosa on the regulation of T3SS expression is largely unknown. Here we reveal the role and regulatory network of endogenous spermidine synthesis in regulation of T3SS and bacterial virulence, showing that the spermidine is an important interspecies signal for modulating the virulence of P. aeruginosa through regulating T3SS expression.
| S-EPMC9241758 | biostudies-literature
Project description:Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that possesses a type III secretion system (T3SS) critical for evading innate immunity and establishing acute infections in compromised patients. Our research has focused on the structure-activity relationships of ExoU, the most toxic and destructive type III effector produced by P. aeruginosa. ExoU possesses phospholipase activity, which is detectable in vitro only when a eukaryotic cofactor is provided with membrane substrates. We report here that a subpopulation of ubiquitylated yeast SOD1 and other ubiquitylated mammalian proteins activate ExoU. Phospholipase activity was detected using purified ubiquitin of various chain lengths and linkage types; however, free monoubiquitin is sufficient in a genetically engineered dual expression system. The use of ubiquitin by a bacterial enzyme as an activator is unprecedented and represents a new aspect in the manipulation of the eukaryotic ubiquitin system to facilitate bacterial replication and dissemination.
| S-EPMC3237844 | biostudies-literature
Project description:In this study, we report the insertion sequence ISPpu21 in the oprD porin gene of carbapenem-resistant Pseudomonas aeruginosa isolates from burn patients in Tehran, Iran. Antibiotic susceptibility tests for P. aeruginosa isolates were determined. Production of metallo-β-lactamases (MBLs) and carbapenemase was evaluated and the β-lactamase-encoding and aminoglycoside-modifying enzyme genes were investigated by PCR and sequencing methods. The mRNA transcription level of oprD and mex efflux pump genes were evaluated by real-time PCR. The outer membrane protein profile was determined by SDS-PAGE. The genetic relationship between the P. aeruginosa isolates was assessed by random amplified polymorphic DNA PCR. In all, 10.52% (10/95) of clinical isolates of P. aeruginosa harboured the ISPpu21 insertion element in the oprD gene. The extended-spectrum β-lactamase-encoding gene in ISPpu21-carrying isolates was blaTEM. PCR assays targeting MBL and carbapenemase-encoding genes were also negative in all ten isolates. The rmtA, aadA, aadB and armA genes were positive in all ISPpu21 harbouring isolates. The relative expression levels of the mexX, mexB, mexT and mexD genes in ten isolates ranged from 0.1- to 1.4-fold, 1.1- to 3.68-fold, 0.3- to 8.22-fold and 1.7- to 35.17-fold, respectively. The relative expression levels of the oprD in ten isolates ranged from 0.57- to 35.01-fold, which was much higher than those in the control strain P. aeruginosa PAO1. Evaluation of the outer membrane protein by SDS-PAGE suggested that oprD was produced at very low levels by all isolates. Using random amplified polymorphic DNA PCR genotyping, eight of the ten isolates containing ISPpu21 were shown to be clonally related. The present study describes a novel molecular mechanism, ISPpu21 insertion of the oprD gene, associated with carbapenem resistance in clinical P. aeruginosa isolates.
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Project description:The opportunistic pathogen Pseudomonas aeruginosa is a significant cause of infection in immunocompromised individuals, cystic fibrosis patients, and burn victims. To benefit its survival, the bacterium adapt to either a motile or sessile lifestyle when infecting the host. The motile bacterium has an often activated type III secretion system (T3SS), which is virulent to the host, whereas the sessile bacterium harbors an active T6SS and lives in biofilms. Regulatory pathways involving Gac-Rsm or secondary messengers such as c-di-GMP determine which lifestyle is favorable for P. aeruginosa. Here, we introduce the RNA binding protein RtcB as a modulator of the switch between motile and sessile bacterial lifestyles. Using the wild-type P. aeruginosa PAO1, and a retS mutant PAO1(∆retS) in which T3SS is repressed and T6SS active, we show that deleting rtcB led to simultaneous expression of T3SS and T6SS in both PAO1(∆rtcB) and PAO1(∆rtcB∆retS). The deletion of rtcB also increased biofilm formation in PAO1(∆rtcB) and restored the motility of PAO1(∆rtcB∆retS). RNA-sequencing data suggested RtcB as a global modulator affecting multiple virulence factors, including bacterial secretion systems. Competitive killing and infection assays showed that the three T6SS systems (H1, H2, and H3) in PAO1(∆rtcB) were activated into a functional syringe, and could compete with Escherichia coli and effectively infect lettuce. Western blotting and RT-PCR results showed that RtcB probably exerted its function through RsmA in PAO1(∆rtcB∆retS). Quantification of c-di-GMP showed an elevated intracellular levels in PAO1(∆rtcB), which likely drove the switch between T6SS and T3SS, and contributed to the altered phenotypes and characteristics observed. Our data demonstrate a pivotal role of RtcB in the virulence of P. aeruginosa by controlling multiple virulence determinants, such as biofilm formation, motility, pyocyanin production, T3SS, and T6SS secretion systems towards eukaryotic and prokaryotic cells. These findings suggest RtcB as a potential target for controlling P. aeruginosa colonization, establishment, and pathogenicity.
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Project description:Pseudomonas aeruginosa uses quorum sensing (QS) to coordinate the expression of multiple genes necessary for establishing and maintaining infection. It has previously been shown that lasR QS mutations frequently arise in cystic fibrosis (CF) lung infections, however, there has been far less emphasis on determining whether other QS system mutations arise during infection or in other environments. To test this, we utilized 852 publicly available sequenced P. aeruginosa genomes from the Pseudomonas International Consortium Database (IPCD) to study P. aeruginosa QS mutational signatures. To study isolates by source, we focused on a subset of 654 isolates collected from CF, wounds, and non-infection environmental isolates, where we could clearly identify their source. We also worked with a small collection of isolates in vitro to determine the impact of lasR and pqs mutations on isolate phenotypes. We found that lasR mutations are common across all environments and are not specific to infection nor a particular infection type. We also found that the pqs system proteins PqsA, PqsH, PqsL and MexT, a protein of increasing importance to the QS field, are highly variable. Conversely, RsaL, a negative transcriptional regulator of the las system, was found to be highly conserved, suggesting selective pressure to repress las system activity. Overall, our findings suggest that QS mutations in P. aeruginosa are common and not limited to the las system; however, LasR is unique in the frequency of putative loss-of-function mutations.
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Project description:Constitutive overproduction of the pump MexXY-OprM is recognized as a major cause of resistance to aminoglycosides, fluoroquinolones, and zwitterionic cephalosporins in Pseudomonas aeruginosa. In this study, 57 clonally unrelated strains recovered from non-cystic fibrosis patients were analyzed to characterize the mutations resulting in upregulation of the mexXY operon. Forty-four (77.2%) of the strains, classified as agrZ mutants were found to harbor mutations inactivating the local repressor gene (mexZ) of the mexXY operon (n = 33; 57.9%) or introducing amino acid substitutions in its product, MexZ (n = 11; 19.3%). These sequence variations, which mapped in the dimerization domain, the DNA binding domain, or the rest of the MexZ structure, mostly affected amino acid positions conserved in TetR-like regulators. The 13 remaining MexXY-OprM strains (22.8%) contained intact mexZ genes encoding wild-type MexZ proteins. Eight (14.0%) of these isolates, classified as agrW1 mutants, overexpressed the gene PA5471, which codes for the MexZ antirepressor ArmZ [corrected], with 5 strains exhibiting growth defects at 37°C and 44°C, consistent with mutations impairing ribosome activity. Interestingly, one agrW1 mutant appeared to harbor a 7-bp deletion in the coding sequence of the leader peptide, PA5471.1, involved in ribosome-dependent, translational attenuation of PA5471 expression. Finally, DNA sequencing and complementation experiments revealed that 5 (8.8%) strains, classified as agrW2 mutants, harbored single amino acid variations in the sensor histidine kinase of ParRS, a two-component system known to positively control mexXY expression. Collectively, these results demonstrate that clinical strains of P. aeruginosa exploit different regulatory circuitries to mutationally overproduce the MexXY-OprM pump and become multidrug resistant, which accounts for the high prevalence of MexXY-OprM mutants in the clinical setting.
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Project description:Pseudomonas aeruginosa is an important opportunistic human pathogen, which raises a worldwide concern for its increasing resistance. Nonthermal plasma, which is also called cold atmospheric plasma (CAP), is an alternative therapeutic approach for clinical infectious diseases. However, the bacterial factors that affect CAP treatment remain unclear. The sterilization effect of a portable CAP device on different P. aeruginosa strains was investigated in this study. Results revealed that CAP can directly or indirectly kill P. aeruginosa in a time-dependent manner. Scanning electron microscopy and transmission electron microscope showed negligible surface changes between CAP-treated and untreated P. aeruginosa cells. However, cell leakage occurred during the CAP process with increased bacterial lactate dehydrogenase release. More importantly, pigmentation of the P. aeruginosa culture was remarkably reduced after CAP treatment. Further mechanical exploration was performed by utilizing mutants with loss of functional genes involved in pyocyanin biosynthesis, including P. aeruginosa PAO1 strain-derived phzA1::Tn, phzA2::Tn, ΔphzA1/ΔphzA2, phzM::Tn and phzS::Tn, as well as corresponding gene deletion mutants based on clinical PA1 isolate. The results indicated that pyocyanin and its intermediate 5-methyl phenazine-1-carboxylic acid (5-Me-PCA) play important roles in P. aeruginosa resistance to CAP treatment. The unique enzymes, such as PhzM in the pyocyanin biosynthetic pathway, could be novel targets for the therapeutic strategy design to control the growing P. aeruginosa infections.
| S-EPMC9151332 | biostudies-literature
Project description:Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.
| S-EPMC2812968 | biostudies-literature