exoY Genes Research Articles

Molecular investigation of exoU and exoY virulence genes in Pseudomonas aeruginosa collected from hospitalized patients in North of Iran: A descriptive-analytical study

Objective: To investigate the frequency of exoU and exoY genes in patients with Pseudomonas aeruginosa infection. Methods: In this study, 100 clinical isolates of Pseudomonas aeruginosa were collected from patients hospitalized in educational- therapeutic hospitals and were identified using standard microbiological tests. Then, the antibiotic resistance pattern of the isolates was determined by the disk agar diffusion method. The bacterial DNAs were extracted by the alkaline lysis method. Finally, the presence of exoU and exoY genes was evaluated by the PCR test. Results: In this study, 47%, 72%, 29%, 39%, 40%, and 44% of the isolates were non-susceptible to piperacillin, aztreonam, ceftazidime, imipenem, tobramycin, and ciprofloxacin, respectively. In addition, 95% and 93% of the clinical isolates carried the exoU and exoY genes. Blood and fecal isolates had both virulence genes, while only one wound isolate had neither genes. Meanwhile, all urinary isolates contained the exoY gene and only one isolate lacked the exoU gene. Also, 88 isolates simultaneously had both exoU and exoY genes. Conclusions: High prevalence of exoU and exoY genes in this region indicates a significant role of type III secretion system in pathogenesis of Pseudomonas aeruginosa. The type III secretion system may be a suitable target to reduce the pathogenicity of this bacterium.

Endemic High-Risk Clone ST277 Is Related to the Spread of SPM-1-Producing Pseudomonas aeruginosa during the COVID-19 Pandemic Period in Northern Brazil.

Pseudomonas aeruginosa is a high-priority bacterial agent that causes healthcare-acquired infections (HAIs), which often leads to serious infections and poor prognosis in vulnerable patients. Its increasing resistance to antimicrobials, associated with SPM production, is a case of public health concern. Therefore, this study aims to determine the antimicrobial resistance, virulence, and genotyping features of P. aeruginosa strains producing SPM-1 in the Northern region of Brazil. To determine the presence of virulence and resistance genes, the PCR technique was used. For the susceptibility profile of antimicrobials, the Kirby-Bauer disk diffusion method was performed on Mueller-Hinton agar. The MLST technique was used to define the ST of the isolates. The exoS+/exoU- virulotype was standard for all strains, with the aprA, lasA, toxA, exoS, exoT, and exoY genes as the most prevalent. All the isolates showed an MDR or XDR profile against the six classes of antimicrobials tested. HRC ST277 played a major role in spreading the SPM-1-producing P. aeruginosa strains.

Characterization of Uropathogenic Pseudomonas aeruginosa: Serotypes, Resistance Phenotypes, and Virulence Genotypes

Pseudomonas aeruginosa is a major cause of urinary tract infections. This organism has extended resistance to antimicrobials along with multiple virulence factors, making it difficult to treat. In this study, 49 isolates from urine samples were identified as P. aeruginosa and serotyped by the slide agglutination method. The sensitivity of isolates against 10 antipseudomonal drugs was determined. Phenotypically, lipase, protease, hemolysin, and biofilm production were detected. Genes for the type III secretion system, elastase B, and exotoxin A were detected by PCR. Serotype O11 was the most predominant serotype among test isolates. High levels of resistance were observed against ceftazidime, cefepime, piperacillin, and piperacillin/tazobactam while 10.2% of isolates were resistant to amikacin. MDR was detected in 20.4% of the isolates and was significantly associated with strong biofilm producers. About 95.9% and 63.3% of P. aeruginosa isolates had proteolytic and lipolytic activity, respectively. Among the genes detected, the exoY gene was the most prevalent gene (79.6%), while the exoU gene was the least frequent one (10.2%). toxA and lasB genes were amplified in 63.27% and 75.5% of the isolates, respectively. In addition, the exoU gene was significantly associated with MDR isolates. The high incidence of exoS, exoT, exoY, lasB, and toxA genes in uropathogenic P. aeruginosa implies that these genes can be considered markers for virulent isolates. Furthermore, the coexistence of exoU and exoS genes, even in 6% of isolates, poses a significant treatment challenge because those isolates possess both the invasive and cytotoxic properties of both effector proteins.

Phenotypic and molecular characterization of fluoroquinolone resistant Pseudomonas aeruginosa isolates in Palestine.

Fluoroquinolones are important antimicrobial agents for the treatment of Pseudomonas infections. A total of 11 isolates of P. aeruginosa were collected from different clinical samples from different medical centers in the North West Bank-Palestine during 2017. In this study, resistance to fluoroquinolones and secretions of β-lactamases were detected by phenotypic methods, while presence of β-lactamase gene sequences and other virulence factors were detected by PCR technique. PCR product for gyrA, parC and parE genes were sequenced for further analyses. The phylogenetic analyses, population diversity indices and haplotypes determination were conducted using computer programs MEGA version 6, DnaSP 5.1001 and median-joining algorithm in the program Network 5, respectively. Results of this study showed that the MIC for ciprofloxacin and norfloxacin had a range of 32-256 µg/ml. In addition, all isolates carried either exoT or exoT and exoY genes, different β-lactamase genes and 82% of these isolates harbored class 1 integrons. Analyses of the gyrA, parC and parE sequences were found to be polymorphic, had high haplotype diversity (0.945-0.982), low nucleotide diversity (0.01225-0.02001) and number of haplotypes were 9 for each gyrA and parE genes and 10 haplotypes for parC gene. The founder haplotypes being Hap-1 (18%), Hap-2 (27.3%) and Hap-6 (9.1%) for gyrA, parC and parE genes, respectively. Two of ParE haplotypes were detected as indel haplotypes. The Median-joining- (MJ) networks constructed from haplotypes of these genes showed a star-like expansion. The neutrality tests (Tajima's D test and Fu's Fs test) for these genes showed negative values. Palestinian fluoroquinolone resistant P. aeruginosa strains showed high MIC level for fluoroquinolones, β-lactamase producers, carried type III secretion exotoxin-encoding genes, most of them had integrase I gene and had high level of mutations in QRDR regions in gyrA, parC and parE genes. All these factors may play an important role in the invasiveness of these strains and make them difficult to treat. Isolation of these strains from different medical centers, indicate the need for a strict application of infection control measures in Medical centers in the North West Bank-Palestine that aim to reduce expense and damage caused by P. aeruginosa infections. Molecular analyses showed that Palestinian fluoroquinolone resistant P. aeruginosa haplotypes are not genetically differentiated; however, more mutations may exist in these strains.

Prevalence of ExoY Activity in Pseudomonas aeruginosa Reference Panel Strains and Impact on Cytotoxicity in Epithelial Cells.

ExoY is among the effectors that are injected by the type III secretion system (T3SS) of Pseudomonas aeruginosa into host cells. Inside eukaryotic cells, ExoY interacts with F-actin, which stimulates its potent nucleotidyl cyclase activity to produce cyclic nucleotide monophosphates (cNMPs). ExoY has broad substrate specificity with GTP as a preferential substrate in vitro. How ExoY contributes to the virulence of P. aeruginosa remains largely unknown. Here, we examined the prevalence of active ExoY among strains from the international P. aeruginosa reference panel, a collection of strains that includes environmental and clinical isolates, commonly used laboratory strains, and sequential clonal isolates from cystic fibrosis (CF) patients and thus represents the large diversity of this bacterial species. The ability to secrete active ExoY was determined by measuring the F-actin stimulated guanylate cyclase (GC) activity in bacterial culture supernatants. We found an overall ExoY activity prevalence of about 60% among the 40 examined strains with no significant difference between CF and non-CF isolates. In parallel, we used cellular infection models of human lung epithelial cells to compare the cytotoxic effects of isogenic reference strains expressing active ExoY or lacking the exoY gene. We found that P. aeruginosa strains lacking ExoY were in fact more cytotoxic to the epithelial cells than those secreting active ExoY. This suggests that under certain conditions, ExoY might partly alleviate the cytotoxic effects of other virulence factors of P. aeruginosa.

High mortality by nosocomial infections caused by carbapenem-resistant P. aeruginosa in a referral hospital in Brazil: facing the perfect storm.

Introduction. Carbapenem-resistant Pseudomonas aeruginosa is responsible for increased patient mortality.Gap Statement. Five and 30 day in-hospital all-cause mortality in patients with P. aeruginosa infections were assessed, followed by evaluations concerning potential correlations between the type III secretion system (TTSS) genotype and the production of metallo-β-lactamase (MBL).Methodology. This assessment comprised a retrospective cohort study including consecutive patients with carbapenem-resistant infections hospitalized in Brazil from January 2009 to June 2019. PCR analyses were performed to determine the presence of TTSS-encoding genes and MBL genes.Results. The 30-day and 5-day mortality rates for 262 patients were 36.6 and 17.9 %, respectively. The unadjusted survival probabilities for up to 5 days were 70.55 % for patients presenting exoU-positive isolates and 86 % for those presenting exo-negative isolates. The use of urinary catheters, as well as the presence of comorbidity conditions, secondary bacteremia related to the respiratory tract, were independently associated with death at 5 and 30 days. The exoS gene was detected in 64.8 % of the isolates, the presence of the exoT and exoY genes varied and exoU genes occurred in 19.3 % of the isolates. The exoU genotype was significantly more frequent among multiresistant strains. MBL genes were not detected in 92 % of the isolates.Conclusions. Inappropriate therapy is a crucial factor regarding the worse prognosis among patients with infections caused by multiresistant P. aeruginosa, especially those who died within 5 days of diagnosis, regardless of the genotype associated with TTSS virulence.

Molecular detection of type III secretory toxins in Pseudomonas aeruginosa isolates

Pseudomonas aeruginosa has been known as a common unscrupulous pathogen that reasons cause nosocomial infections in patients with immunocompromise. Infection with multi-drug resistant Pseudomonas aeruginosa infection in many patients is a public health problem. The bacterium causes urinary tract infections, respiratory tract infections, skin inflammation and inflammation, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and various systemic infections, especially in patients with severe burns, cancer and AIDS, whose immune systems are suppressed. Among diverse virulence factors, the type III secretion system is known as a significant agent in virulence and development of antimicrobial resistance in P. aeruginosa. A total of 50 isolates of P. aeruginosa were gathered from burn wound and milk specimens. Documentation and antimicrobial susceptibility evidence were performed using the VITEK 2 system. Multiplex PCR was done to detect the secretion toxins-encoding genes. Out of 50 samples: 45/225 (20%) burn wound and 6/120 (5%) raw milk samples were found positive for P. aeruginosa. The multiplex PCR analysis of ExoT and ExoY genes showed that all P. aeruginosa 50 (100%) were positive. The occurrence of the ExoS and ExoU genes was 97.7% and 86.6% among clinical isolates while none of the raw milk isolates harbored the ExoU gene and 60% of them carried the ExoS gene. The results found 20 (40%) of isolates were multidrug resistance and the most effective antibiotics against clinical isolates were Ciprofloxacin and Meropenem. The aim of this study was to prevalence the exotoxin genes encoded type III secretion system and pattern of antimicrobial susceptibility of P. aeruginosa isolated from clinical and raw milk specimens.

High frequency of the exoU+/exoS+ genotype associated with multidrug-resistant \u201chigh-risk clones\u201d of Pseudomonas aeruginosa clinical isolates from Peruvian hospitals

The type III secretion system of Pseudomonas aeruginosa is an important virulence factor contributing to the cytotoxicity and the invasion process of this microorganism. The current study aimed to determine the presence of the exoU+/exoS+ genotype in P. aeruginosa clinical isolates. The presence of exoS, exoT, exoU and exoY was determined in 189 P. aeruginosa by PCR, and the presence/absence of exoU was analysed according to source infection, clonal relationships, biofilm formation, motility and antimicrobial susceptibility. The gyrA, parC, oprD, efflux pump regulators and β-lactamases genes were also analysed by PCR/sequencing. The exoS, exoT and exoY genes were found in 100% of the isolates. Meanwhile, exoU was present in 43/189 (22.8%) of the isolates, being significantly associated with multidrug resistance, extensively drug resistance as well as with higher level quinolone resistance. However, the presence of β-lactamases, mutations in gyrA and parC, and relevant modifications in efflux pumps and OprD were not significantly associated with exoU+ isolates. MLST analysis of a subset of 25 isolates showed 8 different STs displaying the exoU+/exoS+ genotype. The MDR basis of the exoU+ isolates remain to be elucidated. Furthermore, the clinical implications and spread of exoU+/exoS+ P. aeruginosa isolates need to be established.

Ml proteins from Mesorhizobium loti and MucR from Brucella abortus: an AT-rich core DNA-target site and oligomerization ability

Mesorhizobium loti contains ten genes coding for proteins sharing high amino acid sequence identity with members of the Ros/MucR transcription factor family. Five of these Ros/MucR family members from Mesorhizobium loti (Ml proteins) have been recently structurally and functionally characterized demonstrating that Ml proteins are DNA-binding proteins. However, the DNA-binding studies were performed using the Ros DNA-binding site with the Ml proteins. Currently, there is no evidence as to when the Ml proteins are expressed during the Mesorhizobium loti life cycle as well as no information concerning their natural DNA-binding site. In this study, we examine the ml genes expression profile in Mesorhizobium loti and show that ml1, ml2, ml3 and ml5 are expressed during planktonic growth and in biofilms. DNA-binding experiments show that the Ml proteins studied bind a conserved AT-rich site in the promoter region of the exoY gene from Mesorhizobium loti and that the proteins make important contacts with the minor groove of DNA. Moreover, we demonstrate that the Ml proteins studied form higher-order oligomers through their N-terminal region and that the same AT-rich site is recognized by MucR from Brucella abortus using a similar mechanism involving contacts with the minor groove of DNA and oligomerization.

Influence of virulence genotype and resistance profile in the mortality of Pseudomonas aeruginosa bloodstream infections.

The type III secretion system (TTSS) is a major virulence determinant of Pseudomonas aeruginosa. The objective of this study was to determine whether the TTSS genotype is a useful prognostic marker of P. aeruginosa bacteremia mortality. We also studied the potential association between TTSS genotypes and multidrug-resistant (MDR) profiles, and how this interaction impacts the outcome of bloodstream infections. We performed a post hoc analysis of a published prospective multicenter cohort of P. aeruginosa bloodstream infections. The impact in mortality of TTSS genotypes (exoS, exoT, exoU, and exoY genes) and resistance profiles was investigated. Cox regression analysis was used to control for confounding variables. Among 590 patients, the 30-day mortality rate was 30% (175 patients), and 53% of them died in the first 5 days (early mortality). The unadjusted probabilities of survival until 5 days was 31.4% (95% confidence interval [CI], 17.4%-49.4%) for the patients with exoU-positive isolates and 53.2% (95% CI, 44.6%-61.5%) for exoU-negative isolates (log rank P = .005). After adjustment for confounders, exoU genotype (adjusted hazard ratio [aHR], 1.90 [95% CI, 1.15-3.14]; P = .01) showed association with early mortality. In contrast, late (30-day) mortality was not influenced by TTSS genotype but was independently associated with MDR profiles (aHR,1.40 [95% CI, 1.01-1.94]; P = .04). Moreover, the exoU genotype (21% of all isolates) was significantly less frequent (13%) among MDR strains (particularly among extensively drug-resistant isolates, 5%), but was positively linked to moderately resistant (1-2 antipseudomonals) phenotypes (34%). Our results indicate that the exoU genotype, which is associated with specific susceptibility profiles, is a relevant independent marker of early mortality in P. aeruginosa bacteremia.

Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa.

The type III secretion system of Pseudomonas aeruginosa transports four known effector proteins: ExoS, ExoT, ExoU and ExoY. However, the prevalence of the type III secretion system genes or the effector-encoding genes in clinical and environmental isolates of P. aeruginosa has not been well studied. Southern hybridization analyses and PCR were performed on over 100 P. aeruginosa isolates to determine the distribution of these genes. Clinical isolates were obtained from urine, endotracheal, blood and wound specimens, from the sputum of cystic fibrosis (CF) patients, and from non-hospital environmental sites. The popB gene was used as a marker for the presence of the large chromosomal locus encoding the type III secretion machinery proteins. Each isolate contained the popB gene, indicating that at least a portion of this large chromosomal locus was present in all isolates. Likewise, each isolate contained exoT-like sequences. In contrast, the exoS, exoU and exoY genes were variable traits. Overall, 72% of examined isolates contained the exoS gene, 28% contained the exoU gene, and 89% contained the exoY gene. Interestingly, an inverse correlation was noted between the presence of the exoS and exoU genes in that all isolates except two contained either exoS or exoU but not both. No significant difference in exoS, exoU or exoY prevalence was observed between clinical and environmental isolates or between isolates cultured from different disease sites except for CF respiratory isolates. CF isolates harboured the exoU gene less frequently and the exoS gene more frequently than did isolates from some of the other sites of infection, including the respiratory tract of patients without CF. These results suggest that the P. aeruginosa type III secretion system is present in nearly all clinical and environmental isolates but that individual isolates and populations of isolates from distinct disease sites differ in their effector genotypes. The ubiquity of type III secretion genes in clinical isolates is consistent with an important role for this system in human disease.

Iron-dependent transcription of the regulatory gene ros of Agrobacterium radiobacter.

Transcription of the regulatory gene ros of Agrobacterium radiobacter requires growth in the presence of Fe although this regulation was not mediated by ros itself. The ros gene repressed its own transcription, independently of the Fe status of the growth media. It was shown that the two cysteine residues in the Ros protein were essential for the complementation of the exopolysaccharide synthesis defect of ros mutant strains. It was found that the mutation in one exo mutant strain that was complemented both by ros and by the "structural" exoY gene was not, in fact, in ros but is in some other, unknown gene. The two cysteines were also essential for the correction of this mutant. This mutant affected the expression of exoY but not of ros.

Expression of the exoY gene, required for exopolysaccharide synthesis in Agrobacterium, is activated by the regulatory ros gene.

Some mutants of Agrobacterium radiobacter, defective in exopolysaccharide synthesis, were phenotypically complemented by two different regions of cloned chromosomal DNA. One of these had been shown to contain a gene termed ros, a novel class of transcriptional regulator. The other contains a gene termed exoY which encodes a glycosyltransferase that is involved in one of the early steps in exopolysaccharide synthesis. Mutations in ros reduced the expression of exoY and a model to account for the complementation of certain exo alleles by both ros and exoY is presented. TnphoA insertions into exoY which expressed alkaline phosphatase activity were isolated and mapped, confirming the membrane location of the exoY gene product. Some of these mutations were dominant, causing merodiploids to be non-mucoid. exoY is linked to two genes, one encoding an omega-aminotransferase and the other encoding an aldehyde dehydrogenase.

A molecular characterization of the Gunnera–Nostoc symbiosis: comparison with Rhizobium– and Agrobacterium– plant interactions

summaryThe Gunnera‐Nostoc symbiosis is a N2‐fixing endosymbiosis between an angiosperm and a prokaryote, in some respects showing similarities to legume‐rhizobial symbioses as well as to pathogenic Agrobacterium‐plant interactions. We here describe the results of a molecular comparison of these systems, along with a characterization of the Gunnera mucilage which is important for Gunnera–Nostoc communication. Nostoc DNA sequences homologous to the rhizobial nodEF, nodMN, exoY genes, along with the nod box, were identified by heterologous hybridization, whereas no homologues to either nodABC, nodD1 or nodD2 were found. Nevertheless, no anti‐Nod protein antibodies recognized any Nostoc protein, either in normally grown or mucilage‐induced cells. Furthermore, probes containing the agrobactenal chvA, chvB and picA genes also hybridized with Nostoc DNA, whereas virA and virG genes did not.Using rhizobia with nod gene–lacZ fusions as reporters, Gunnera mucilage and seed rinse were shown to possess nod gene‐inducing activity.The mucilage secreted by the Gunnera stem gland, the organ susceptible to Nostoc infection, was composed of arabinose and galactose in relative molar proportions of 1.00:0.25, with an additional 0.13 molar parts of glucuronic acid. It showed a reaction on dot blot assays with the arabinogalactan protein‐specific monoclonal antibody MAC 207, indicating that it contains arabinogalactan proteins.

Genetic analysis of the Rhizobium meliloti exoYFQ operon: ExoY is homologous to sugar transferases and ExoQ represents a transmembrane protein.

The nucleotide sequence of a 4.8-kb ClaI-EcoRI DNA fragment of megaplasmid 2 of Rhizobium meliloti Rm2011 involved in succinoglucan (EPS I) synthesis and nodule infection was determined. Four open reading frames (ORFs) were identified on this fragment. A mutational analysis revealed that these ORFs represent genes that were termed exoX, exoY, exoF, and exoQ. The locations of transposon insertions in these exo genes were determined at the nucleotide level. Plasmid integration mutagenesis revealed that the genes exoY, exoF, and exoQ are organized in an operon. The exoX gene running in opposite direction forms a monocistronic transcriptional unit. The exoX gene was shown to negatively influence the amount of EPS I synthesized. The exoY gene is coding for a membrane associated protein homologous to the C-terminal part of the Xanthomonas campestris glucosyltransferase GumD and the Salmonella typhimurium galactose transferase RfbP. ExoF, a probable periplasmatic protein, is nearly identical to the protein encoded by ORF1 of Rhizobium sp. strain NGR234. ExoQ is most probably a membrane associated protein as deduced by its hydrophobic structural features. All three genes of the exoYFQ operon were shown to be essential for succinoglucan synthesis and nodule infection.