PrtR Homeostasis Contributes to Pseudomonas aeruginosa Pathogenesis and Resistance against Ciprofloxacin

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced byP. aeruginosathat are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component ofP. aeruginosa. In this study, we demonstrate that mutation inprtRresults in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by atacpromoter repressed the endogenousprtRpromoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

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Pseudomonas aeruginosa las and rhl quorum-sensing systems are important for infection and inflammation in a rat prostatitis model

Microbiology ◽

10.1099/mic.0.028464-0 ◽

2009 ◽

Vol 155 (8) ◽

pp. 2612-2619 ◽

Cited By ~ 21

Author(s):

Lisa K. Nelson ◽

Genevieve H. D'Amours ◽

Kimberley M. Sproule-Willoughby ◽

Douglas W. Morck ◽

Howard Ceri

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Tissue Damage ◽

Inflammatory Markers ◽

Bacterial Colonization ◽

Opportunistic Pathogen ◽

Infection Model ◽

Chronic Infections ◽

Strain Pao1 ◽

Rat Prostate

Pseudomonas aeruginosa frequently acts as an opportunistic pathogen of mucosal surfaces; yet, despite causing aggressive prostatitis in some men, its role as a pathogen in the prostate has not been investigated. Consequently, we developed a Ps. aeruginosa infection model in the rat prostate by instilling wild-type (WT) Ps. aeruginosa strain PAO1 into the rat prostate. It was found that Ps. aeruginosa produced acute and chronic infections in this mucosal tissue as determined by bacterial colonization, gross morphology, tissue damage and inflammatory markers. WT strain PAO1 and its isogenic mutant PAO-JP2, in which both the lasI and rhlI quorum-sensing signal systems have been silenced, were compared during both acute and chronic prostate infections. In acute infections, bacterial numbers and inflammatory markers were comparable between WT PA01 and PAO-JP2; however, considerably less tissue damage occurred in infections with PAO-JP2. Chronic infections with PAO-JP2 resulted in reduced bacterial colonization, tissue damage and inflammation as compared to WT PAO1 infections. Therefore, the quorum-sensing lasI and rhlI genes in Ps. aeruginosa affect acute prostate infections, but play a considerably more important role in maintaining chronic infections. We have thus developed a highly reproducible model for the study of Ps. aeruginosa virulence in the prostate.

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Fis Contributes to Resistance of Pseudomonas aeruginosa to Ciprofloxacin by Regulating Pyocin Synthesis

Journal of Bacteriology ◽

10.1128/jb.00064-20 ◽

2020 ◽

Vol 202 (11) ◽

Cited By ~ 1

Author(s):

Yuqing Long ◽

Weixin Fu ◽

Su Wang ◽

Xuan Deng ◽

Yongxin Jin ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Bacterial Resistance ◽

Global Gene Expression ◽

Chronic Infections ◽

Mobility Shift ◽

Content Type ◽

Regulatory Pathways ◽

Opportunistic Pathogenic

ABSTRACT Factor for inversion stimulation (Fis) is a versatile DNA binding protein that plays an important role in coordinating bacterial global gene expression in response to growth phases and environmental stresses. Previously, we demonstrated that Fis regulates the type III secretion system (T3SS) in Pseudomonas aeruginosa. In this study, we explored the role of Fis in the antibiotic resistance of P. aeruginosa and found that mutation of the fis gene increases the bacterial susceptibility to ciprofloxacin. We further demonstrated that genes related to pyocin biosynthesis are upregulated in the fis mutant. The pyocins are produced in response to genotoxic agents, including ciprofloxacin, and the release of pyocins results in lysis of the producer cell. Thus, pyocin biosynthesis genes sensitize P. aeruginosa to ciprofloxacin. We found that PrtN, the positive regulator of the pyocin biosynthesis genes, is upregulated in the fis mutant. Genetic experiments and electrophoretic mobility shift assays revealed that Fis directly binds to the promoter region of prtN and represses its expression. Therefore, our results revealed novel Fis-mediated regulation on pyocin production and bacterial resistance to ciprofloxacin in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an important opportunistic pathogenic bacterium that causes various acute and chronic infections in human, especially in patients with compromised immunity, cystic fibrosis (CF), and/or severe burn wounds. About 60% of cystic fibrosis patients have a chronic respiratory infection caused by P. aeruginosa. The bacterium is intrinsically highly resistant to antibiotics, which greatly increases difficulties in clinical treatment. Therefore, it is critical to understand the mechanisms and the regulatory pathways that are involved in antibiotic resistance. In this study, we elucidated a novel regulatory pathway that controls the bacterial resistance to fluoroquinolone antibiotics, which enhances our understanding of how P. aeruginosa responds to ciprofloxacin.

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The Pseudomonas aeruginosa PrrF Small RNAs Regulate Iron Homeostasis during Acute Murine Lung Infection

Infection and Immunity ◽

10.1128/iai.00764-16 ◽

2017 ◽

Vol 85 (5) ◽

Cited By ~ 21

Author(s):

Alexandria A. Reinhart ◽

Angela T. Nguyen ◽

Luke K. Brewer ◽

Justin Bevere ◽

Jace W. Jones ◽

...

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Small Rnas ◽

Iron Homeostasis ◽

Critical Role ◽

Opportunistic Pathogen ◽

Lung Infection ◽

Content Type ◽

The Individual

ABSTRACT Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrHIG sequence). Previous studies showed that the prrF locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrHIG sequence [prrF(ΔHIG)]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa. Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo. Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.

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Genome Sequences of Two Pseudomonas aeruginosa Isolates with Defects in Type III Secretion System Gene Expression from a Chronic Ankle Wound Infection

Microbiology Spectrum ◽

10.1128/spectrum.00340-21 ◽

2021 ◽

Author(s):

Sardar Karash ◽

Robert Nordell ◽

Egon A. Ozer ◽

Timothy L. Yahr

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Biofilm Formation ◽

Secretion System ◽

Chronic Infections ◽

Genome Sequences ◽

Content Type ◽

Chronic Colonization ◽

Host Tolerance

A common feature of microorganisms that cause chronic infections is a stealthy lifestyle that promotes immune avoidance and host tolerance. During chronic colonization of cystic fibrosis (CF) patients, Pseudomonas aeruginosa acquires numerous adaptations that include reduced expression of some factors, such as motility, O antigen, and the T3SS, and increased expression of other traits, such as biofilm formation.

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SOS-Independent Pyocin Production in P. aeruginosa Is Induced by XerC Recombinase Deficiency

mBio ◽

10.1128/mbio.02893-21 ◽

2021 ◽

Author(s):

Nina S. Baggett ◽

Adam S. Bronson ◽

Matthew T. Cabeen

Keyword(s):

Pseudomonas Aeruginosa ◽

Dna Damage ◽

Sos Response ◽

Opportunistic Pathogen ◽

Content Type

Pseudomonas aeruginosa is a versatile and ubiquitous bacterium that frequently infects humans as an opportunistic pathogen. P. aeruginosa competes with other strains within the species by producing killing complexes termed pyocins, which are only known to be induced by cells experiencing DNA damage and the subsequent SOS response. Here, we discovered that strains lacking a recombinase enzyme called XerC strongly produce pyocins independently of the SOS response.

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Pseudomonas aeruginosa ExsA Regulates a Metalloprotease, ImpA, That Inhibits Phagocytosis of Macrophages

Infection and Immunity ◽

10.1128/iai.00695-19 ◽

2019 ◽

Vol 87 (12) ◽

Cited By ~ 3

Author(s):

Zhenyang Tian ◽

Sen Cheng ◽

Bin Xia ◽

Yongxin Jin ◽

Fang Bai ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Critical Role ◽

Surface Protein ◽

Host Cells ◽

Bacterial Cells ◽

Mobility Shift ◽

Content Type ◽

Direct Binding ◽

Mobility Shift Assay ◽

Opportunistic Pathogenic

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogenic bacterium whose type III secretion system (T3SS) plays a critical role in acute infections. Translocation of the T3SS effectors into host cells induces cytotoxicity. In addition, the T3SS promotes the intracellular growth of P. aeruginosa during host infections. The T3SS regulon genes are regulated by an AraC-type regulator, ExsA. In this study, we found that an extracellular metalloprotease encoded by impA (PA0572) is under the regulation of ExsA. An ExsA consensus binding sequence was identified upstream of the impA gene, and direct binding of the site by ExsA was demonstrated via an electrophoretic mobility shift assay. We further demonstrate that secreted ImpA cleaves the macrophage surface protein CD44, which inhibits the phagocytosis of the bacterial cells by macrophages. Combined, our results reveal a novel ExsA-regulated virulence factor that cooperatively inhibits the functions of macrophages with the T3SS.

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The Two-Component Sensor KinB Acts as a Phosphatase To Regulate Pseudomonas aeruginosa Virulence

Journal of Bacteriology ◽

10.1128/jb.01168-12 ◽

2012 ◽

Vol 194 (23) ◽

pp. 6537-6547 ◽

Cited By ~ 14

Author(s):

Nikhilesh S. Chand ◽

Anne E. Clatworthy ◽

Deborah T. Hung

Keyword(s):

Pseudomonas Aeruginosa ◽

Response Regulator ◽

Opportunistic Pathogen ◽

Regulatory Control ◽

Primary Role ◽

Chronic Infections ◽

Content Type ◽

Two Component ◽

Sense And Respond ◽

Cognate Response Regulator

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that is capable of causing both acute and chronic infections.P. aeruginosavirulence is subject to sophisticated regulatory control by two-component systems that enable it to sense and respond to environmental stimuli. We recently reported that the two-component sensor KinB regulates virulence in acuteP. aeruginosainfection. Furthermore, it regulates acute-virulence-associated phenotypes such as pyocyanin production, elastase production, and motility in a manner independent of its kinase activity. Here we show that KinB regulates virulence through the global sigma factor AlgU, which plays a key role in repressingP. aeruginosaacute-virulence factors, and through its cognate response regulator AlgB. However, we show that rather than phosphorylating AlgB, KinB's primary role in the regulation of virulence is to act as a phosphatase to dephosphorylate AlgB and alleviate phosphorylated AlgB's repression of acute virulence.

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A Large-Scale Whole-Genome Comparison Shows that Experimental Evolution in Response to Antibiotics Predicts Changes in Naturally Evolved Clinical Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01619-19 ◽

2019 ◽

Vol 63 (12) ◽

Cited By ~ 6

Author(s):

Samuel J. T. Wardell ◽

Attika Rehman ◽

Lois W. Martin ◽

Craig Winstanley ◽

Wayne M. Patrick ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Experimental Evolution ◽

Large Scale ◽

Opportunistic Pathogen ◽

Clinical Isolates ◽

Genome Comparison ◽

Chronic Infections ◽

Content Type ◽

Large Deletions ◽

Wide Range

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have mutations that make them antibiotic resistant, making treatment difficult. To identify resistance-associated mutations, we experimentally evolved the antibiotic-sensitive strain P. aeruginosa PAO1 to become resistant to three widely used antipseudomonal antibiotics, namely, ciprofloxacin, meropenem, and tobramycin. Mutants could tolerate up to 2,048-fold higher concentrations of antibiotics than strain PAO1. Genome sequences were determined for 13 mutants for each antibiotic. Each mutant had between 2 and 8 mutations. For each antibiotic, at least 8 genes were mutated in multiple mutants, demonstrating the genetic complexity of resistance. For all three antibiotics, mutations arose in genes known to be associated with resistance but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in this study, we analyzed the corresponding genes in 558 isolates of P. aeruginosa from patients with chronic lung disease and in 172 isolates from the general environment. Many genes identified through experimental evolution had predicted function-altering changes in clinical isolates but not in environmental isolates, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. Additionally, large deletions of up to 479 kb arose in experimentally evolved meropenem-resistant mutants, and large deletions were present in 87 of the clinical isolates. These findings significantly advance understanding of antibiotic resistance in P. aeruginosa and demonstrate the validity of experimental evolution in identifying clinically relevant resistance-associated mutations.

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Pseudomonas aeruginosa Forms Biofilms in Acute Infection Independent of Cell-to-Cell Signaling

Infection and Immunity ◽

10.1128/iai.00586-07 ◽

2007 ◽

Vol 75 (8) ◽

pp. 3715-3721 ◽

Cited By ~ 108

Author(s):

J. Andy Schaber ◽

W. Jeffrey Triffo ◽

Sang Jin Suh ◽

Jeffrey W. Oliver ◽

Mary Catherine Hastert ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bacterial Colonization ◽

Acute Infection ◽

Opportunistic Pathogen ◽

Confocal Scanning Laser Microscopy ◽

Chronic Infections ◽

Signaling Mechanism ◽

Confocal Scanning Laser

ABSTRACT Biofilms are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 h of infection in thermally injured mice, demonstrating that biofilms contribute to bacterial colonization in acute infections as well. Using light, electron, and confocal scanning laser microscopy, P. aeruginosa biofilms were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates differentiation of biofilms in vitro, wild-type and QS-deficient P. aeruginosa strains formed similar biofilms in vivo. Our findings demonstrate that P. aeruginosa forms biofilms on specific host tissues independently of QS.

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The Pseudomonas aeruginosa Periplasmic Protease CtpA Can Affect Systems That Impact Its Ability To Mount Both Acute and Chronic Infections

Infection and Immunity ◽

10.1128/iai.01035-13 ◽

2013 ◽

Vol 81 (12) ◽

pp. 4561-4570 ◽

Cited By ~ 21

Author(s):

Jin Seo ◽

Andrew J. Darwin

Keyword(s):

Pseudomonas Aeruginosa ◽

Sigma Factor ◽

Cell Envelope ◽

Virulence Gene ◽

Normal Function ◽

Host Cells ◽

Tissue Destruction ◽

Chronic Infections ◽

Content Type ◽

Virulence Gene Expression

ABSTRACTProteases play important roles in the virulence ofPseudomonas aeruginosa. Some are exported to act on host targets and facilitate tissue destruction and bacterial dissemination. Others work within the bacterial cell to process virulence factors and regulate virulence gene expression. Relatively little is known about the role of one class of bacterial serine proteases known as the carboxyl-terminal processing proteases (CTPs). TheP. aeruginosagenome encodes two CTPs annotated as PA3257/Prc and PA5134/CtpA in strain PAO1. Prc degrades mutant forms of the anti-sigma factor MucA to promote mucoidy in some cystic fibrosis lung isolates. However, nothing is known about the role or importance of CtpA. We have now found that endogenous CtpA is a soluble periplasmic protein and that actpAnull mutant has specific phenotypes consistent with an altered cell envelope. Although actpAnull mutation has no major effect on bacterial growth in the laboratory, CtpA is essential for the normal function of the type 3 secretion system (T3SS), for cytotoxicity toward host cells, and for virulence in a mouse model of acute pneumonia. Conversely, increasing the amount of CtpA above its endogenous level induces an uncharacterized extracytoplasmic function sigma factor regulon, an event that has been reported to attenuateP. aeruginosain a rat model of chronic lung infection. Therefore, a normal level of CtpA activity is critical for T3SS function and acute virulence, whereas too much activity can trigger an apparent stress response that is detrimental to chronic virulence.

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