scholarly journals The histone-like protein AlgP regulon is distinct in mucoid and nonmucoid Pseudomonas aeruginosa and does not include alginate biosynthesis genes

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 861-866 ◽  
Author(s):  
Ashley R. Cross ◽  
Erika E. Csatary ◽  
Vishnu Raghuram ◽  
Frances L. Diggle ◽  
Marvin Whiteley ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Species ◽  
Bacterial Pathogen ◽  
Nitrous Oxide Reductase ◽  
Chronic Infections ◽  
Strain Pao1 ◽  
Content Type ◽  
Link Type ◽  
Alginate Production ◽  
Lung Infections

The opportunistic bacterial pathogen Pseudomonas aeruginosa causes acute and chronic infections that are notoriously difficult to treat. In people with cystic fibrosis, P. aeruginosa can cause lifelong lung infections, and isolation of mucoid P. aeruginosa , resulting from the overproduction of alginate, is associated with chronic infection. The histone-like protein AlgP has previously been implicated in the control of alginate gene expression in mucoid strains, but this regulation is unclear. To explore AlgP in further detail, we deleted algP in mucoid strains and demonstrated that the deletion of algP did not result in a nonmucoid phenotype or a decrease in alginate production. We showed that the algP promoter is expressed by both the nonmucoid strain PAO1 and the isogenic mucoid strain PDO300, suggesting that there may be genes that are differentially regulated between these strains. In support of this, using RNA sequencing, we identified a small AlgP regulon that has no significant overlap between PAO1 and PDO300 and established that alginate genes were not differentially regulated by the deletion of algP. Of note, we found that deleting algP in PAO1 increased expression of the nitric oxide operon norCBD and the nitrous oxide reductase genes nosRZ and subsequently promoted growth of PAO1 under anaerobic conditions. Altogether, we have defined a narrow regulon of genes controlled by AlgP and provided evidence that alginate production is not greatly affected by AlgP, countering the long-standing premise in the field.

scholarly journals From genotype to phenotype: adaptations of Pseudomonas aeruginosa to the cystic fibrosis environment

2021 ◽  
Vol 7 (3) ◽  
Author(s):  
Laura Camus ◽  
François Vandenesch ◽  
Karen Moreau
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Persistent Infection ◽  
Chronic Infection ◽  
Respiratory Function ◽  
Type Species ◽  
Genetic Mutations ◽  
Chronic Infections ◽  
Content Type ◽  
Link Type

Pseudomonas aeruginosa is one of the main microbial species colonizing the lungs of cystic fibrosis patients and is responsible for the decline in respiratory function. Despite the hostile pulmonary environment, P. aeruginosa is able to establish chronic infections thanks to its strong adaptive capacity. Various longitudinal studies have attempted to compare the strains of early infection with the adapted strains of chronic infection. Thanks to new ‘-omics’ techniques, convergent genetic mutations, as well as transcriptomic and proteomic dysregulations have been identified. As a consequence of this evolution, the adapted strains of P. aeruginosa have particular phenotypes that promote persistent infection.

scholarly journals The Pseudomonas aeruginosa Sensor Kinase KinB Negatively Controls Alginate Production through AlgW-Dependent MucA Proteolysis

2009 ◽  
Vol 191 (7) ◽  
pp. 2285-2295 ◽  
Author(s):  
F. Heath Damron ◽  
Dongru Qiu ◽  
Hongwei D. Yu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Cystic Fibrosis Mutation ◽  
Strain Pao1 ◽  
Alginate Production ◽  
Mucoid Conversion ◽  
Σ Factor ◽  
Lung Infections

ABSTRACT Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-σ factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P algU and P algD . Deletion of alternative σ factor RpoN (σ54) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.

scholarly journals Overproduction of the AlgT Sigma Factor Is Lethal to Mucoid Pseudomonas aeruginosa

2020 ◽  
Vol 202 (20) ◽  
Author(s):  
Ashley R. Cross ◽  
Vishnu Raghuram ◽  
Zihuan Wang ◽  
Debayan Dey ◽  
Joanna B. Goldberg
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sigma Factor ◽  
Continuous Production ◽  
Common Mutation ◽  
Wild Type ◽  
Chronic Infections ◽  
Truncated Form ◽  
Content Type ◽  
Lung Infections

ABSTRACT Pseudomonas aeruginosa isolates from chronic lung infections often overproduce alginate, giving rise to the mucoid phenotype. Isolation of mucoid strains from chronic lung infections correlates with a poor patient outcome. The most common mutation that causes the mucoid phenotype is called mucA22 and results in a truncated form of the anti-sigma factor MucA that is continuously subjected to proteolysis. When a functional MucA is absent, the cognate sigma factor, AlgT, is no longer sequestered and continuously transcribes the alginate biosynthesis operon, leading to alginate overproduction. In this work, we report that in the absence of wild-type MucA, providing exogenous AlgT is toxic. This is intriguing, since mucoid strains endogenously possess high levels of AlgT. Furthermore, we show that suppressors of toxic AlgT production have mutations in mucP, a protease involved in MucA degradation, and provide the first atomistic model of MucP. Based on our findings, we speculate that mutations in mucP stabilize the truncated form of MucA22, rendering it functional and therefore able to reduce toxicity by properly sequestering AlgT. IMPORTANCE Pseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing chronic lung infections. Phenotypes important for the long-term persistence and adaption to this unique lung ecosystem are largely regulated by the AlgT sigma factor. Chronic infection isolates often contain mutations in the anti-sigma factor mucA, resulting in uncontrolled AlgT and continuous production of alginate in addition to the expression of ∼300 additional genes. Here, we report that in the absence of wild-type MucA, AlgT overproduction is lethal and that suppressors of toxic AlgT production have mutations in the MucA protease, MucP. Since AlgT contributes to the establishment of chronic infections, understanding how AlgT is regulated will provide vital information on how P. aeruginosa is capable of causing long-term infections.

scholarly journals Characterization of the φCTX-like Pseudomonas aeruginosa phage Dobby isolated from the kidney stone microbiota

2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Genevieve Johnson ◽  
Alan J. Wolfe ◽  
Catherine Putonti
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Urinary Tract ◽  
Human Body ◽  
Kidney Stone ◽  
Type Species ◽  
Phage Group ◽  
Content Type ◽  
Link Type ◽  
Abundance And Diversity ◽  
Cultured Bacteria

Bacteriophages (phages) are vital members of the human microbiota. They are abundant even within low biomass niches of the human body, including the lower urinary tract. While several prior studies have cultured bacteria from kidney stones, this is the first study to explore phages within the kidney stone microbiota. Here we report Dobby, a temperate phage isolated from a strain of Pseudomonas aeruginosa cultured from a kidney stone. Dobby is capable of lysing clinical P. aeruginosa strains within our collection from the urinary tract. Sequencing was performed producing a 37 152 bp genome that closely resembles the temperate P. aeruginosa phage φCTX, a member of the P2 phage group. Dobby does not, however, encode for the cytotoxin CTX. Dobby’s genome was queried against publicly available bacterial sequences identifying 44 other φCTX-like prophages. These prophages are integrated within the genomes of P. aeruginosa strains from a variety of environments, including strains isolated from urine samples and other niches of the human body. Phylogenetic analysis suggests that the temperate φCTX phage species is widespread. With the isolation of Dobby, we now have evidence that phages are members of the kidney stone microbiota. Further investigation, however, is needed to determine their abundance and diversity within these communities.

scholarly journals An antipathogenic compound that targets the OxyR peroxide sensor in Pseudomonas aeruginosa

2021 ◽  
Vol 70 (4) ◽  
Author(s):  
Hyo-Young Oh ◽  
Shivakumar S. Jalde ◽  
In-Young Chung ◽  
Yeon-Ji Yoo ◽  
Hye-Jeong Jang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Resistance ◽  
Stress Responses ◽  
Type Species ◽  
Reduced Form ◽  
Infection Model ◽  
Redox Cycle ◽  
Content Type ◽  
Link Type ◽  
Computer Based

Introduction. Antipathogenic or antivirulence strategy is to target a virulence pathway that is dispensable for growth, in the hope to mitigate the selection for drug resistance. Hypothesis/Gap Statment. Peroxide stress responses are one of the conserved virulence pathways in bacterial pathogens and thus good targets for antipathogenic strategy. Aim. This study aims to identify a new chemical compound that targets OxyR, the peroxide sensor required for the full virulence of the opportunistic human pathogen, Pseudomonas aeruginosa . Methodology. Computer-based virtual screening under consideration of the ‘eNTRy’ rules and molecular docking were conducted on the reduced form of the OxyR regulatory domain (RD). Selected hits were validated by their ability to phenocopy the oxyR null mutant and modulate the redox cycle of OxyR. Results. We first isolated three robust chemical hits that inhibit OxyR without affecting prototrophic growth or viability. One (compound 1) of those affected the redox cycle of OxyR in response to H2O2 treatment, in a way to impair its function. Compound 1 displayed selective antibacterial efficacy against P. aeruginosa in Drosophila infection model, without antibacterial activity against Staphylococcus aureus . Conclusion. These results suggest that compound 1 could be an antipathogenic hit inhibiting the P. aeruginosa OxyR. More importantly, our study provides an insight into the computer-based discovery of new-paradigm selective antibacterials to treat Gram-negative bacterial infections presumably with few concerns of drug resistance.

scholarly journals Study of quorum-sensing LasR and RhlR genes and their dependent virulence factors in Pseudomonas aeruginosa isolates from infected burn wounds

2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Aya Ahmad Elnegery ◽  
Wafaa Kamel Mowafy ◽  
Tarek Ahmed Zahra ◽  
Noha Tharwat Abou El-Khier
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Proteolytic Activity ◽  
Virulence Factors ◽  
Biofilm Formation ◽  
Type Species ◽  
Assay Method ◽  
Content Type ◽  
Burn Wounds ◽  
Link Type

Background. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen responsible for burn-wound infection. High incidence, infection severity and increasing resistance characterize P. aeruginosa -induced burn infection. Purpose. To estimate quorum-sensing (QS)-dependent virulence factors of P. aeruginosa isolates from burn wounds and correlate it to the presence of QS genes. Methods. A cross-sectional descriptive study included 50 P . aeruginosa isolates from burn patients in Mansoura University Plastic and Burn Hospital, Egypt. Antibiotic sensitivity tests were done. All isolates were tested for their ability to produce biofilm using a micro-titration assay method. Protease, pyocyanin and rhamnolipid virulence factors were determined using skimmed milk agar, King’s A medium and CTAB agar test, respectively. The identity of QS lasR and rhlR genes was confirmed using PCR. Results. In total, 86 % of isolates had proteolytic activity. Production of pyocyanin pigment was manifested in 66 % of isolates. Altogether, 76 % of isolates were rhamnolipid producers. Biofilm formation was detected in 96 % of isolates. QS lasR and rhlR genes were harboured by nearly all isolates except three isolates were negative for both lasR and rhlR genes and two isolates were positive for lasR gene and negative for rhlR gene. Forty-nine isolates were considered as extremely QS-proficient strains as they produced QS-dependent virulence factors. In contrast, one isolate was a QS deficient strain. Conclusions. QS affects P. aeruginosa virulence-factor production and biofilm in burn wounds. Isolates containing lasR and rhlR seem to be a crucial regulator of virulence factors and biofilm formation in P. aeruginosa whereas the lasR gene positively regulates biofilm formation, proteolytic activity, pyocyanin production and rhamnolipid biosurfactant synthesis. The QS regulatory RhlR gene affects protease and rhamnolipid production positively.

scholarly journals Overproduction of the AlgT sigma factor is lethal to mucoid Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Ashley R. Cross ◽  
Vishnu Raghuram ◽  
Zihuan Wang ◽  
Debayan Dey ◽  
Joanna B. Goldberg
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Sigma Factor ◽  
Continuous Production ◽  
Bacterial Pathogen ◽  
Common Mutation ◽  
Wild Type ◽  
Chronic Infections ◽  
Truncated Form ◽  
Lung Infections

ABSTRACTPseudomonas aeruginosa isolates from chronic lung infections often overproduce alginate, giving rise to the mucoid phenotype. Isolation of mucoid strains from chronic lung infections correlates with a poor patient outcome. The most common mutation that causes the mucoid phenotype is called mucA22 and results in a truncated form of the anti-sigma factor MucA that is continuously subjected to proteolysis. When a functional MucA is absent, the cognate sigma factor, AlgT, is no longer sequestered and continuously transcribes the alginate biosynthesis operon leading to alginate overproduction. In this work, we report that in the absence of wild-type MucA, providing exogenous AlgT is toxic. This is intriguing since mucoid strains endogenously possess high levels of AlgT. Furthermore, we show that suppressors of toxic AlgT production have mutations in mucP, a protease involved in MucA degradation, and provide the first atomistic model of MucP. Our findings support a model where mutations in mucP stabilize the truncated form of MucA22 rendering it functional and therefore able to reduce toxicity by properly sequestering AlgT.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing chronic lung infections. Phenotypes important for the long-term persistence and adaption to this unique lung ecosystem are largely regulated by the AlgT sigma factor. Chronic infection isolates often contain mutations in the anti-sigma factor mucA resulting in uncontrolled AlgT and continuous production of alginate, in addition to the expression of ~300 additional genes including algT itself. Here we report that in the absence of wild-type MucA, AlgT overproduction is lethal and that suppressors of toxic AlgT production have mutations in the MucA protease, MucP. Since AlgT contributes to the establishment of chronic infections, understanding how AlgT is regulated will provide vital information on how P. aeruginosa is capable of causing long-term infections.

scholarly journals Prevalence of target anaerobes associated with chronic periodontitis

2020 ◽  
Vol 2 (12) ◽  
Author(s):  
Ameerah M. Alazemi ◽  
W. Jamal ◽  
A. Al Khabbaz ◽  
V. O. Rotimi
Keyword(s):  
Quantitative Pcr ◽  
Chronic Periodontitis ◽  
Type Species ◽  
Periodontal Diseases ◽  
Chronic Infections ◽  
Content Type ◽  
Link Type ◽  
Significant Difference ◽  
Pcr Assays ◽  
Semi Quantitative Pcr

Introduction. Periodontal diseases are a group of chronic infections that destroy tissues surrounding and supporting the teeth. Data on the anaerobes associated with periodontal infections in Kuwait is lacking. Aim. To investigate the target anaerobes associated with chronic periodontitis (CP) in patients admitted to Dental Clinics in Kuwait University Health Sciences Center, Kuwait. Methodology. Patients with CP (severe and moderate) were recruited into this study during a period of 15 months. Samples were collected directly from inside the gingival pockets and subjected to semi-quantitative PCR assays. Results. A total of 30 patients, stratified into moderate and severe CP and 31 healthy individuals, used as controls, were studied. Nine (30 %) of the 30 patients were in the 50–59-year age group. The detection rate of Aggregatibacter actinomycetemcomitans between the patients (9 : 30 %) versus the controls (5 : 16.1 %) was non-significant (P >0.05). Fusobacterium spp., were detected in all patients versus 29 (93.1 %) controls, (P >0.05). However, four target anaerobes were significantly associated with CP patients; Porphyromonas gingivalis was detected in ten (33.3 %) patients versus two (6.4 %) controls (P <0.0001); Tannerella forsythia 25 (83.3 %) versus 16 (51.6 %) controls (P <0.0001); Parvimonas micra 27 (90 %) versus 16 (51.6 %) controls (P <0.0001) and Treponema denticola, 18 (60 %) versus nine (29 %) controls (P <0.0001), respectively. Prevotella spp. were detected in 27 (90 %) patients and 30 (96.7 %) controls (P>0.5). There was no significant difference in the burden of Prevotella spp. between patients and controls determined by semi-quantitative PCR assays. Conclusion. Some (4/7) of the target anaerobes were significantly associated with CP in our study. P. gingivalis was the most strongly associated anaerobe with CP, although not the keystone bacteria, while Prevotella spp. was similar to the healthy controls.

scholarly journals Evaluation of WGS performance for bacterial pathogen characterization with the Illumina technology optimized for time-critical situations

2021 ◽  
Vol 7 (11) ◽  
Author(s):  
Bert Bogaerts ◽  
Raf Winand ◽  
Julien Van Braekel ◽  
Stefan Hoffman ◽  
Nancy H. C. Roosens ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Type Species ◽  
Bacterial Pathogen ◽  
Read Length ◽  
Time Data ◽  
Content Type ◽  
Emergency Situations ◽  
Link Type ◽  
Pathogen Characterization ◽  
Time Critical

Whole genome sequencing (WGS) has become the reference standard for bacterial outbreak investigation and pathogen typing, providing a resolution unattainable with conventional molecular methods. Data generated with Illumina sequencers can however only be analysed after the sequencing run has finished, thereby losing valuable time during emergency situations. We evaluated both the effect of decreasing overall run time, and also a protocol to transfer and convert intermediary files generated by Illumina sequencers enabling real-time data analysis for multiple samples part of the same ongoing sequencing run, as soon as the forward reads have been sequenced. To facilitate implementation for laboratories operating under strict quality systems, extensive validation of several bioinformatics assays (16S rRNA species confirmation, gene detection against virulence factor and antimicrobial resistance databases, SNP-based antimicrobial resistance detection, serotype determination, and core genome multilocus sequence typing) for three bacterial pathogens ( Mycobacterium tuberculosis , Neisseria meningitidis , and Shiga-toxin producing Escherichia coli ) was performed by evaluating performance in function of the two most critical sequencing parameters, i.e. read length and coverage. For the majority of evaluated bioinformatics assays, actionable results could be obtained between 14 and 22 h of sequencing, decreasing the overall sequencing-to-results time by more than half. This study aids in reducing the turn-around time of WGS analysis by facilitating a faster response in time-critical scenarios and provides recommendations for time-optimized WGS with respect to required read length and coverage to achieve a minimum level of performance for the considered bioinformatics assay(s), which can also be used to maximize the cost-effectiveness of routine surveillance sequencing when response time is not essential.

scholarly journals nosX is essential for whole-cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase

Microbiology ◽  
2020 ◽  
Vol 166 (10) ◽  
pp. 909-917 ◽  
Author(s):  
Sophie P. Bennett ◽  
Maria J. Torres ◽  
Manuel J. Soriano-Laguna ◽  
David J. Richardson ◽  
Andrew J. Gates ◽  
...  
Keyword(s):  
Nitrous Oxide ◽  
Type Species ◽  
Paracoccus Denitrificans ◽  
Pseudomonas Stutzeri ◽  
Reduction Reaction ◽  
Nitrous Oxide Reductase ◽  
Content Type ◽  
Link Type ◽  
Reverse Transcription Pcr

Nitrous oxide (N2O) is a potent greenhouse gas that is produced naturally as an intermediate during the process of denitrification carried out by some soil bacteria. It is consumed by nitrous oxide reductase (N2OR), the terminal enzyme of the denitrification pathway, which catalyses a reduction reaction to generate dinitrogen. N2OR contains two important copper cofactors (CuA and CuZ centres) that are essential for activity, and in copper-limited environments, N2OR fails to function, contributing to rising levels of atmospheric N2O and a major environmental challenge. Here we report studies of nosX, one of eight genes in the nos cluster of the soil dwelling α-proteobaterium Paraccocus denitrificans. A P. denitrificans ΔnosX deletion mutant failed to reduce N2O under both copper-sufficient and copper-limited conditions, demonstrating that NosX plays an essential role in N2OR activity. N2OR isolated from nosX-deficient cells was found to be unaffected in terms of the assembly of its copper cofactors, and to be active in in vitro assays, indicating that NosX is not required for the maturation of the enzyme; in particular, it plays no part in the assembly of either of the CuA and CuZ centres. Furthermore, quantitative Reverse Transcription PCR (qRT-PCR) studies showed that NosX does not significantly affect the expression of the N2OR-encoding nosZ gene. NosX is a homologue of the FAD-binding protein ApbE from Pseudomonas stutzeri , which functions in the flavinylation of another N2OR accessory protein, NosR. Thus, it is likely that NosX is a system-specific maturation factor of NosR, and so is indirectly involved in maintaining the reaction cycle of N2OR and cellular N2O reduction.

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