The Pseudomonas aeruginosa AlgZR two-component system coordinates multiple phenotypes

Okkotsu, Yuta; Little, Alexander S.; Schurr, Michael J.

doi:10.3389/fcimb.2014.00082

Cited by 63 publications

(73 citation statements)

References 253 publications

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“…*, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. minants is especially tightly controlled to prevent unnecessary expression of metabolically burdening systems, such as the exopolysaccharide alginate (54)(55)(56)(57). 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).…”

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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Section: Figmentioning

confidence: 99%

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

et al. 2015

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“…The 12-days biofilm grown on glass was in a singular secondary branch. This fact suggests that after 5 days of maturation, the biofilms display specific phenotypes according to the type of surface where they are attached to, a reasonable assumption, considering how surface interactions affect molecular expressions in P. aeruginosa [19,20].…”

Section: Discussionmentioning

confidence: 98%

Use of MALDI-TOF mass spectrometry to analyze the molecular profile of Pseudomonas aeruginosa biofilms grown on glass and plastic surfaces

Pereira

Bonatto

Lopes

et al. 2015

Microbial Pathogenesis

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Biofilms are microbial sessile communities attached to surfaces that are known for causing many medical problems. A bacterial biofilm of clinical relevance is formed by the gram-negative bacteria Pseudomonas aeruginosa. During the formation of a biofilm, the initial adhesion of the cells is of crucial importance, and the characteristics of the contact surface have great influence on this step. In the present study, we aimed to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling as a new methodology to monitor P. aeruginosa biofilm development. Biofilms were grown within polypropylene tubes containing a glass slide, and were harvested after 3, 5, 7, 9, or 12 days of inoculation. Planktonic cells were obtained separately by centrifugation as control. Two independent MALDI-TOF experiments were performed, one by collecting biofilms from both the glass slide and the polypropylene tube internal surface, and the other by acquiring biofilms from these surfaces separately. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to evaluate the morphological progression of the biofilm. The molecular results showed that MALDI profiling is able not only to distinguish between different biofilm stages, but it is also appropriate to indicate when the biofilm cells are released at the dispersion stage, which occurred first on polypropylene surface. Finally, the present study pointed out that MALDI profiling may emerge as a promising tool for the clinical diagnostic and prognostic workup of biofilms formation and control.

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“…The rsmATF1-lacZ fusion was analyzed in the wild-type strain P. aeruginosa PAO1, ΔalgR and algZ mutants, and in the corresponding mucA22 mutants. The algZ mutant has a mutation in the conserved histidine residue, which prevents AlgZ-mediated phosphorylation of AlgR (19,32) but does not disrupt the internal algR promoter (33). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…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%

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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The Pseudomonas aeruginosa AlgZR two-component system coordinates multiple phenotypes

Cited by 63 publications

References 253 publications

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

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

Use of MALDI-TOF mass spectrometry to analyze the molecular profile of Pseudomonas aeruginosa biofilms grown on glass and plastic surfaces

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

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