Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

Okkotsu, Yuta; Tieku, Prince; Fitzsimmons, Liam F.; Churchill, Mair E. A.; Schurr, Michael J.

doi:10.1128/jb.00726-13

Cited by 44 publications

(52 citation statements)

References 90 publications

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“…In addition, it has also recently been suggested that BfmRS is involved the development of virulence during bacterial adaptation to the CF lung (43,44). Interestingly, AlgR (another key P. aeruginosa transcriptional response regulator) also appears to play an essential role in bacterial virulence and motility (45).…”

Section: Discussionmentioning

confidence: 99%

Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa

Jack

Khan

Powell

et al. 2018

Antimicrob Agents Chemother

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plays a major role in many chronic infections. Its ability to readily form biofilms contributes to its success as an opportunistic pathogen and its resistance/tolerance to antimicrobial/antibiotic therapy. A low-molecular-weight alginate oligomer (OligoG CF-5/20) derived from marine algae has previously been shown to impair motility in biofilms and disrupt pseudomonal biofilm assembly. As these bacterial phenotypes are regulated by quorum sensing (QS), we hypothesized that OligoG CF-5/20 may induce alterations in QS signaling in QS regulation was studied by using CV026 biosensor assays that showed a significant reduction in acyl homoserine lactone (AHL) production following OligoG CF-5/20 treatment (≥2%; < 0.05). This effect was confirmed by liquid chromatography-mass spectrometry analysis of C-AHL and 3-oxo-C-AHL production (≥2%; < 0.05). Moreover, quantitative PCR showed that reduced expression of both the and systems was induced following 24 h of treatment with OligoG CF-5/20 (≥0.2%; < 0.05). Circular dichroism spectroscopy indicated that these alterations were not due to steric interaction between the AHL and OligoG CF-5/20. Confocal laser scanning microscopy (CLSM) and COMSTAT image analysis demonstrated that OligoG CF-5/20-treated biofilms had a dose-dependent decrease in biomass that was associated with inhibition of extracellular DNA synthesis (≥0.5%; < 0.05). These changes correlated with alterations in the extracellular production of the pseudomonal virulence factors pyocyanin, rhamnolipids, elastase, and total protease ( < 0.05). The ability of OligoG CF-5/20 to modify QS signaling in PAO1 may influence critical downstream functions such as virulence factor production and biofilm formation.

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Section: Discussionmentioning

confidence: 99%

Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa

Jack

Khan

Powell

et al. 2018

Antimicrob Agents Chemother

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“…One of the transcriptional regulators that controls alginate overproduction as well as other virulence factors, such as type IV pili, the type III secretion system, and rhamnolipid and cyanide production, is the response regulator AlgR (58). The proposed histidine kinase for AlgR, AlgZ (FimS), is required for type IV pili (25) function but also modulates alginate and rhamnolipid production by two-to threefold (24,27). Consequently, determining the transcriptional regulators that control the AlgZR two-component regulatory system provides insights into the functions of many different P. aeruginosa virulence determinants.…”

Section: Figmentioning

confidence: 99%

Expression Analysis of the Pseudomonas aeruginosa AlgZR Two-Component Regulatory System

et al. 2015

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cPseudomonas aeruginosa virulence components are subject to complex regulatory control primarily through two-component regulatory systems that allow for sensing and responding to environmental stimuli. In this study, the expression and regulation of the P. aeruginosa AlgZR two-component regulatory system were examined. Primer extension and S1 nuclease protection assays were used to identify two transcriptional initiation sites for algR within the algZ coding region, and two additional start sites were identified upstream of the algZ coding region. The two algR transcriptional start sites, RT1 and RT2, are directly regulated by AlgU, consistent with previous reports of increased algR expression in mucoid backgrounds, and RpoS additionally plays a role in algR transcription. The expression of the first algZ promoter, ZT1, is entirely dependent upon Vfr for expression, whereas Vfr, RpoS, or AlgU does not regulate the second algZ promoter, ZT2. Western blot, real-time quantitative PCR (RTqPCR), and transcriptional fusion analyses show that algZR expression is Vfr dependent. The algZ and algR genes also are cotranscribed in both nonmucoid and mucoid backgrounds. Furthermore, algZR was found to be cotranscribed with hemCD by RT-PCR. RT-qPCR confirmed that hemC transcription in the PAO1 ⌬algZ mutant was 40% of the level of the wild-type strain. Taken together, these results indicate that algZR transcription involves multiple factors at multiple start sites that control individual gene expression as well as coexpression of this two-component system with heme biosynthetic genes. Pseudomonas aeruginosa is a human opportunistic pathogen capable of causing fatal infections in individuals with compromised innate immunity, such as those undergoing cancer treatment or those with severe burn wounds or cystic fibrosis (CF) (1-3). P. aeruginosa is one of the most clinically relevant organisms in CF patients due to its ability to establish chronic infections, characterized by the overproduction of an exopolysaccharide called alginate. Alginate overproduction (a phenotype called mucoidy) increases P. aeruginosa resistance against antimicrobials and phagocytosis and is a hallmark of clinical decline and worsening prognosis in patients with CF.Alginate production is regulated in a complex manner by several transcriptional regulators, including the extracytoplasmic sigma factor AlgU (AlgT) (4-6). The activity of AlgU is increased in mucoid P. aeruginosa due to mutations in the mucA anti-sigma factor gene that releases AlgU from the inner membrane (6-8). The enzymes encoded by the algC gene and the algD operon are responsible for alginate biosynthesis, modification, and export. The AlgR transcriptional regulator is an essential activator for the algD operon and the algC gene; deletion of algR in mucoid P. aeruginosa backgrounds results in a nonmucoid phenotype (9-11). As part of its regulon, AlgU also activates the transcription of the algR gene to increase alginate production. An AlgU-dependent algR transcriptional start site was dete...

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“…Because AlgR is part of a two-component system that is important for both acute and chronic infections (19,32), it was necessary to address the mechanism for AlgR activation of rsmA in order to create a framework to understand how AlgR regulation of rsmA might impact virulence gene expression. AlgR binds a consensus sequence CCGTTCGTC (21,48,49), and phosphorylation is thought to enable AlgR to bind potential binding sites that deviate from this consensus, such as the sites found in the fimU promoter (32,47). However, the rsmA promoter deviates from the AlgR-binding consensus, and AlgR phosphorylation was not required for rsmA expression.…”

Section: Discussionmentioning

confidence: 99%

“…In the case of acute infections, AlgR activates the fimU operon, enabling the production of T4P (19,21,47). In chronic infections, AlgR, AlgU, and other transcriptional regulators activate the production of alginate (22,(48)(49)(50).…”

Section: Discussionmentioning

confidence: 99%

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner

2017

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Pseudomonas aeruginosa is an important pathogen of the immunocompromised, causing both acute and chronic infections. In cystic fibrosis (CF) patients, P. aeruginosa causes chronic disease. The impressive sensory network of P. aeruginosa allows the bacterium to sense and respond to a variety of stimuli found in diverse environments. Transcriptional regulators, including alternative sigma factors and response regulators, integrate signals changing gene expression, allowing P. aeruginosa to cause infection. The two-component transcriptional regulator AlgR is important in P. aeruginosa pathogenesis in both acute and chronic infections. In chronic infections, AlgR and the alternative sigma factor AlgU activate the genes responsible for alginate production. Previous work demonstrated that AlgU controls rsmA expression. RsmA is a posttranscriptional regulator that is antagonized by two small RNAs, RsmY and RsmZ. In this work, we demonstrate that AlgR directly activates rsmA expression from the same promoter as AlgU. In addition, phosphorylation was not necessary for AlgR activation of rsmA using algR and algZ mutant strains. AlgU and AlgR appear to affect the antagonizing small RNAs rsmY and rsmZ indirectly. RsmA was active in a mucA22 mutant strain using leader fusions of two RsmA targets, tssA1 and hcnA. AlgU and AlgR were necessary for posttranscriptional regulation of tssA1 and hcnA. Altogether, our work demonstrates that the alginate regulators AlgU and AlgR are important in the control of the RsmA posttranscriptional regulatory system. These findings suggest that RsmA plays an unknown role in mucoid strains due to AlgU and AlgR activities.IMPORTANCE P. aeruginosa infections are difficult to treat and frequently cause significant mortality in CF patients. Understanding the mechanisms of persistence is important. Our work has demonstrated that the alginate regulatory system also significantly impacts the posttranscriptional regulator system RsmA/Y/Z. We demonstrate that AlgR directly activates rsmA expression, and this impacts the RsmA regulon. This leads to the possibility that the RsmA/Y/Z system plays a role in helping P. aeruginosa persist during chronic infection. In addition, this furthers our understanding of the reach of the alginate regulators AlgU and AlgR.KEYWORDS P. aeruginosa, RsmA, AlgR, mucoid, Pseudomonas aeruginosa, cystic fibrosis, mucA, two-component regulatory systems T he opportunistic pathogen Pseudomonas aeruginosa possesses multiple virulence factors for causing disease. This allows P. aeruginosa to cause both acute and chronic infections. Acute infecting strains are characterized by the presence of type IV pili (T4P), flagella, and a type III secretion system (T3SS) (1-3). In contrast, chronic infecting strains diversify (4, 5) and frequently do not express T3SS, T4P, or flagella (6, 7). Chronic infecting strains often form biofilms composed of exopolysaccharides, such as alginate, and signal a decline in lung function in CF patients (8-11). Alginate

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Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

Cited by 44 publications

References 90 publications

Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa

Alginate Oligosaccharide-Induced Modification of the lasI-lasR and rhlI-rhlR Quorum-Sensing Systems in Pseudomonas aeruginosa

Expression Analysis of the Pseudomonas aeruginosa AlgZR Two-Component Regulatory System

The Pseudomonas aeruginosa Two-Component Regulator AlgR Directly Activates rsmA Expression in a Phosphorylation-Independent Manner

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