The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections,P. aeruginosaoften encounters environments where the levels of calcium (Ca2+) are elevated. Previously, we showed thatP. aeruginosaresponds to externally added Ca2+through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca2+level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca2+-induced virulence factor production and Ca2+homeostasis are not known. Here, we characterized the genome-wide transcriptional response ofP. aeruginosato elevated [Ca2+] in both planktonic cultures and biofilms. Among the genes induced by CaCl2in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here calledcarSandcarR), while the closely related two-component regulatorsphoPQandpmrABwere repressed by CaCl2addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant ofcarRand performed transcriptome analysis of the mutant strain at low and high [Ca2+]. Among the genes regulated by CarSR in response to CaCl2are the predicted periplasmic OB-fold protein, PA0320 (here calledcarO), and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here calledcarP). Mutations in bothcarOandcarPaffected Ca2+homeostasis, reducing the ability ofP. aeruginosato export excess Ca2+. In addition, a mutation incarPhad a pleotropic effect in a Ca2+-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca2+and responding through its regulatory targets that modulate Ca2+homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin.IMPORTANCEDuring infectious disease,Pseudomonas aeruginosaencounters environments with high calcium (Ca2+) concentrations, yet the cells maintain intracellular Ca2+at levels that are orders of magnitude less than that of the external environment. In addition, Ca2+signalsP. aeruginosato induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca2+homeostasis or how Ca2+acts as a signal. In this study, we identified a two-component regulatory system inP. aeruginosaPAO1, termed CarRS, that is induced at elevated Ca2+levels. CarRS modulates Ca2+signaling and Ca2+homeostasis through its regulatory targets, CarO and CarP. The results demonstrate thatP. aeruginosauses a two-component regulatory system to sense external Ca2+and relays that information for Ca2+-dependent cellular processes.

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Indole Inhibits ToxR Regulon Expression inVibrio cholerae

Infection and Immunity ◽

10.1128/iai.00776-18 ◽

2019 ◽

Vol 87 (3) ◽

Cited By ~ 7

Author(s):

Mondraya F. Howard ◽

X. Renee Bina ◽

James E. Bina

Keyword(s):

Virulence Factor ◽

Virulence Gene ◽

Dependent Manner ◽

Content Type ◽

Virulence Gene Expression ◽

Type Vi Secretion ◽

Biofilm Production ◽

Factor Production ◽

Periplasmic Domain ◽

Virulence Factor Production

ABSTRACTIndole is a degradation product of tryptophan that functions as a signaling molecule in many bacteria. This includesVibrio cholerae, where indole was shown to regulate biofilm and type VI secretion in nontoxigenic environmental isolates. Indole is also produced by toxigenicV. choleraestrains in the human intestine, but its significance in the host is unknown. We investigated the effects of indole on toxigenicV. choleraeO1 El Tor during growth under virulence inducing conditions. The indole transcriptome was defined by RNA sequencing and showed widespread changes in the expression of genes involved in metabolism, biofilm production, and virulence factor production. In contrast, genes involved in type VI secretion were not affected by indole. We subsequently found that indole repressed genes involved inV. choleraepathogenesis, including the ToxR virulence regulon. Consistent with this, indole inhibited cholera toxin and toxin-coregulated pilus production in a dose-dependent manner. The effects of indole on virulence factor production and biofilm were linked to ToxR and the ToxR-dependent regulator LeuO. The expression ofleuOwas increased by exogenous indole and linked to repression of the ToxR virulence regulon. This process was dependent on the ToxR periplasmic domain, suggesting that indole was a ToxR agonist. This conclusion was further supported by results showing that the ToxR periplasmic domain contributed to indole-mediated increased biofilm production. Collectively, our results suggest that indole may be a niche-specific cue that can function as a ToxR agonist to modulate virulence gene expression and biofilm production inV. cholerae.

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Staphylococcal Virulence Factors on the Skin of Atopic Dermatitis Patients

mSphere ◽

10.1128/msphere.00616-19 ◽

2019 ◽

Vol 4 (6) ◽

Author(s):

Mary C. Moran ◽

Michael P. Cahill ◽

Matthew G. Brewer ◽

Takeshi Yoshida ◽

Sara Knowlden ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Atopic Dermatitis ◽

Virulence Factors ◽

Virulence Factor ◽

Protein Level ◽

Content Type ◽

Factor Production ◽

First Time ◽

Virulence Factor Production ◽

Specific Virulence

ABSTRACT Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, infective endocarditis, osteoarticular, pleuropulmonary, and device-related infections. Virulence factors secreted by S. aureus, including superantigens and cytotoxins, play significant roles in driving disease. The ability to identify virulence factors present at the site of infection will be an important tool in better identifying and understanding how specific virulence factors contribute to disease. Previously, virulence factor production has been determined by culturing S. aureus isolates and detecting the mRNA of specific virulence factors. We demonstrated for the first time that virulence factors can be directly detected at the protein level from human samples, removing the need to first culture isolated bacteria. Superantigens and cytotoxins were detected and quantified with a Western dot blot assay by using reconstituted skin swabs obtained from patients with atopic dermatitis. This methodology will significantly enhance our ability to investigate the complex host-microbe environment and the effects various therapies have on virulence factor production. Overall, the ability to directly quantify virulence factors present at the site of infection or colonization will enhance our understanding of S. aureus-related diseases and help identify optimal treatments. IMPORTANCE For the first time, we show that secreted staphylococcal virulence factors can be quantified at the protein level directly from skin swabs obtained from the skin of atopic dermatitis patients. This technique eliminates the need to culture Staphylococcus aureus and then test the strain’s potential to produce secreted virulence factors. Our methodology shows that secreted virulence factors are present on the skin of atopic patients and provides a more accurate means of evaluating the physiological impact of S. aureus in inflammatory diseases such as atopic dermatitis.

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A Novel Use of Allopurinol as A Quorum-Sensing Inhibitor in Pseudomonas aeruginosa

Antibiotics ◽

10.3390/antibiotics10111385 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1385

Author(s):

Ahmed Al Saqr ◽

Mohammed F. Aldawsari ◽

El-Sayed Khafagy ◽

Moataz A. Shaldam ◽

Wael A. H. Hegazy ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Virulence Factors ◽

Virulence Factor ◽

Cell Communication ◽

Factor Production ◽

In Silico Study ◽

Liver And Kidney ◽

Healthcare Associated ◽

Virulence Factor Production

Pseudomonas aeruginosa can cause a variety of healthcare-associated infections by its arsenal of virulence factors. Virulence factor production is largely controlled by the cell-to-cell communication system termed quorum sensing (QS). Targeting QS may be a good approach to inhibit the production of virulence factors and attenuate pathogenicity without exerting selective stress on bacterial growth. This will greatly reduce the emergence of resistant mutants. In this work, we investigated the anti-virulence and anti-QS activities of the FDA-approved drug allopurinol against the P. aeruginosa PAO1 strain. Allopurinol at 200 µg/mL (1/10 MIC) significantly decreased the production of the QS-controlled Chromobacterium violaceum CV026 violet pigment violacein and other P. aeruginosa QS-controlled virulence factors phenotypically. Furthermore, allopurinol reduced the infiltration of P. aeruginosa and leucocytes and diminished the congestion in the liver and kidney tissues of infected mice. In silico study showed that allopurinol could compete with the autoinducers on binding to the receptors LasR and RhlR by hydrogen bonding. On the molecular level, qRT-PCR proved that allopurinol showed a significant downregulating effect on all tested QS-encoding genes that regulate virulence factor production. In summary, allopurinol is a promising QS inhibitor that may be useful in the future treatment of P. aeruginosa infection.

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The Pseudomonas aeruginosa PhoP-PhoQ Two-Component Regulatory System Is Induced upon Interaction with Epithelial Cells and Controls Cytotoxicity and Inflammation

Infection and Immunity ◽

10.1128/iai.00382-12 ◽

2012 ◽

Vol 80 (9) ◽

pp. 3122-3131 ◽

Cited By ~ 35

Author(s):

Shaan L. Gellatly ◽

Brittany Needham ◽

Laurence Madera ◽

M. Stephen Trent ◽

Robert E. W. Hancock

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Lipid A ◽

Regulatory System ◽

Dependent Manner ◽

Wild Type ◽

Content Type ◽

Regulatory Systems ◽

Two Component ◽

Inflammatory Phenotype

ABSTRACTThe adaptation ofPseudomonas aeruginosato its environment, including the host, is tightly controlled by its network of regulatory systems. The two-component regulatory system PhoPQ has been shown to play a role in the virulence and polymyxin resistance ofP. aeruginosaas well as several other Gram-negative species. Dysregulation of this system has been demonstrated in clinical isolates, yet how it affects virulence ofP. aeruginosais unknown. To investigate this, an assay was used whereby bacteria were cocultured with human bronchial epithelial cells. The interaction of wild-type (WT) bacteria that had adhered to epithelial cells led to a large upregulation of the expression of theoprH-phoP-phoQoperon and its target, thearnlipopolysaccharide (LPS) modification operon, in a PhoQ-dependent manner, compared to cells in the supernatant that had failed to adhere. Relative to the wild type, aphoQmutant cocultured on epithelial cells produced less secreted protease and lipase and, like thephoQmutant,piv,lipH, andlasBmutants demonstrated reduced cytotoxicity toward epithelial cells. Mutation inphoQalso resulted in alterations to lipid A and to increased inflammatory LPS. These data indicate that mutation ofphoQresults in a phenotype that is similar to the less virulent but more inflammatory phenotype of clinical strains isolated from chronic-stage cystic fibrosis lung infections.

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Development of anEx VivoPorcine Lung Model for Studying Growth, Virulence, and Signaling of Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.01554-14 ◽

2014 ◽

Vol 82 (8) ◽

pp. 3312-3323 ◽

Cited By ~ 42

Author(s):

Freya Harrison ◽

Aneesha Muruli ◽

Steven Higgins ◽

Stephen P. Diggle

Keyword(s):

Pseudomonas Aeruginosa ◽

Virulence Factor ◽

Ex Vivo ◽

Homoserine Lactone ◽

Lung Model ◽

Content Type ◽

Evolutionary Forces ◽

Factor Production ◽

Virulence Factor Production

ABSTRACTResearch into chronic infection by bacterial pathogens, such asPseudomonas aeruginosa, uses variousin vitroand live host models. While these have increased our understanding of pathogen growth, virulence, and evolution, each model has certain limitations.In vitromodels cannot recapitulate the complex spatial structure of host organs, while experiments on live hosts are limited in terms of sample size and infection duration for ethical reasons; live mammal models also require specialized facilities which are costly to run. To address this, we have developed anex vivopig lung (EVPL) model for quantifyingPseudomonas aeruginosagrowth, quorum sensing (QS), virulence factor production, and tissue damage in an environment that mimics a chronically infected cystic fibrosis (CF) lung. In a first test of our model, we show thatlasRmutants, which do not respond to 3-oxo-C12-homoserine lactone (HSL)-mediated QS, exhibit reduced virulence factor production in EVPL. We also show thatlasRmutants grow as well as or better than a corresponding wild-type strain in EVPL.lasRmutants frequently and repeatedly arise during chronic CF lung infection, but the evolutionary forces governing their appearance and spread are not clear. Our data are not consistent with the hypothesis thatlasRmutants act as social “cheats” in the lung; rather, our results support the hypothesis thatlasRmutants are more adapted to the lung environment. More generally, this model will facilitate improved studies of microbial disease, especially studies of how cells of the same and different species interact in polymicrobial infections in a spatially structured environment.

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