Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in<i>Pseudomonas aeruginosa</i>

Wilderman, Paula J.; Vasil, Adriana I.; Johnson, Zaiga; Wilson, Megan J.; Cunliffe, Heather E.; Lamont, Iain L.; Vasil, Michael L.

doi:10.1128/iai.69.9.5385-5394.2001

Cited by 178 publications

(186 citation statements)

References 52 publications

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“…As previously reported by Purschke et al (2012), we observed a significant increase in the production of iron-regulated proteases by P. aeruginosa in coculture supernatants, including PrpL, PasP, AprA and a hypothetical protein zinc-protease, PA0572 (Supplementary Table S3) (Wilderman et al, 2001;Marquart et al, 2005). These proteins are known for their strong proteolytic activities, which are responsible for rupturing tight junctions in the epithelium, leading to tissue invasion and bacterial spreading (Azghani et al, 1993).…”

Section: Interspecies Competition Triggers Virulencesupporting

confidence: 61%

Interspecies competition triggers virulence and mutability in Candida albicans–Pseudomonas aeruginosa mixed biofilms

et al. 2014

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Inter-kingdom and interspecies interactions are ubiquitous in nature and are important for the survival of species and ecological balance. The investigation of microbe-microbe interactions is essential for understanding the in vivo activities of commensal and pathogenic microorganisms. Candida albicans, a polymorphic fungus, and Pseudomonas aeruginosa, a Gram-negative bacterium, are two opportunistic pathogens that interact in various polymicrobial infections in humans. To determine how P. aeruginosa affects the physiology of C. albicans and vice versa, we compared the proteomes of each species in mixed biofilms versus single-species biofilms. In addition, extracellular proteins were analyzed. We observed that, in mixed biofilms, both species showed differential expression of virulence proteins, multidrug resistance-associated proteins, proteases and cell defense, stress and iron-regulated proteins. Furthermore, in mixed biofilms, both species displayed an increase in mutability compared with monospecific biofilms. This characteristic was correlated with the downregulation of enzymes conferring protection against DNA oxidation. In mixed biofilms, P. aeruginosa regulates its production of various molecules involved in quorum sensing and induces the production of virulence factors (pyoverdine, rhamnolipids and pyocyanin), which are major contributors to the ability of this bacterium to cause disease. Overall, our results indicate that interspecies competition between these opportunistic pathogens enhances the production of virulence factors and increases mutability and thus can alter the course of hostpathogen interactions in polymicrobial infections.

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Section: Interspecies Competition Triggers Virulencesupporting

confidence: 61%

Interspecies competition triggers virulence and mutability in Candida albicans–Pseudomonas aeruginosa mixed biofilms

et al. 2014

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“…We excised the bands and identified the proteins by N-terminal sequence analysis. The 49-kDa band was identified as alkaline metalloprotease AprA (24), the 33-kDa band was identified as elastase LasB (4,31), and the 27-kDa band is the PrpL protease (65). To ensure that the increased amounts of proteases were due to Ca 2ϩ addition and not due to PO 4 3Ϫ limitation (by possible PO 4 3Ϫ precipitation as calcium phosphate), experiments were performed with various concentrations of Ca 2ϩ and PO 4 3Ϫ as described in Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of MucoidPseudomonas aeruginosaBiofilms

Sarkisova¹,

Patrauchan

Berglund³

et al. 2005

J Bacteriol

199

173

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Pseudomonas aeruginosa colonizes the pulmonary tissue of patients with cystic fibrosis (CF), leading to biofilm-associated infections. The pulmonary fluid of CF patients usually contains elevated concentrations of cations and may contain the P. aeruginosa redox-active pigment pyocyanin, which is known to disrupt calcium homeostasis of host cells. Since divalent cations are important bridging ions for bacterial polysaccharides and since they may play regulatory roles in bacterial gene expression, we investigated the effect of calcium ions on the extracellular matrix constituents of P. aeruginosa biofilms. For mucoid strain P. aeruginosa FRD1, calcium addition (1.0 and 10 mM as CaCl 2 ) resulted in biofilms that were at least 10-fold thicker than biofilms without added calcium. Scanning confocal laser microscopy showed increased spacing between cells for the thick biofilms, and Fourier transform infrared spectroscopy revealed that the material between cells is primarily alginate. An algD transcriptional reporter demonstrated that calcium addition caused an eightfold increase in alg gene expression in FRD1 biofilms. Calcium addition also resulted in increased amounts of three extracellular proteases (AprA, LasB, and PrpL). Immunoblots of the biofilm extracellular material established that AprA was harbored within the biofilm extracellular matrix. An aprA deletion mutation and a mutation in gene for a putative P. aeruginosa calmodulin-like protein did not significantly affect calcium-induced biofilm structure. Two-dimensional gel electrophoresis showed increased amounts of phenazine biosynthetic proteins in FRD1 biofilms and in calcium-amended planktonic cultures. Spectrochemical analyses showed that the calcium addition causes a three-to fivefold increase in pyocyanin production. These results demonstrate that calcium addition affects the structure and extracellular matrix composition of mucoid P. aeruginosa biofilms, through increased expression and stability of bacterial extracellular products. The calcium-induced extracellular matrix of mucoid P. aeruginosa consists primarily of the virulence factor alginate and also harbors extracellular proteases and perhaps pyocyanin, a biomolecule that may further disrupt cellular calcium levels.

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“…A similar change was also found for the secretion of PrpL, an endoprotease which is regulated by PvdS and the QS system. It can hydrolyse casein, elastin and lactoferrin (Wilderman et al, 2001;Lamont et al, 2002;Ravel & Cornelis, 2003;ArevaloFerro et al, 2003). It seems that the secretion of both AprA and PrpL is very sensitive to oxygen concentration.…”

Section: Proteomic Analysis Of Extracellular Proteinsmentioning

confidence: 99%

Expression of the quorum-sensing regulatory protein LasR is strongly affected by iron and oxygen concentrations in cultures of Pseudomonas aeruginosa irrespective of cell density

et al. 2005

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Expression of the quorum-sensing regulatory protein LasR is strongly affected by iron and oxygen concentrations in cultures of Pseudomonas aeruginosa irrespective of cell density The expression of the transcriptional regulatory protein LasR, a main component of the quorum-sensing (QS) system in Pseudomonas aeruginosa, was recently found to be sensitive to several environmental factors in addition to its dependency on cell density. However, the inherent effects of the different factors have seldom been separately demonstrated due to concurrent changes of culture conditions in typical experimental settings. Furthermore, the interplays of the different factors are unknown. In this work, the effects and interplay of iron concentration and dissolved oxygen tension (pO 2 ) on the expression of lasR in P. aeruginosa were studied in defined growth media with varied iron concentration and pO 2 values in computer-controlled batch and continuous cultures. b-Galactosidase activity in a recombinant P. aeruginosa PAO1 (NCCB 2452) strain with a lasRp-lacZ fusion was used as a reporter for lasR expression. In batch culture with a constant pO 2 <10 % air saturation, a strong correlation between the exhaustion of iron and the increase of lasR expression was observed. In continuous culture with nearly constant cell density but varied pO 2 values, lasR expression generally increased with increasing oxidative stress with the exception of growth under O 2 -limited conditions (pO 2 <0 %). Under O 2 limitation, the expression of lasR strongly depended on the concentration of iron. It showed a nearly twofold increase in cells grown under iron deprivation in comparison with cells grown in iron-replete conditions and reached the expression level seen at high oxidative stress. A preliminary proteomic analysis was carried out for extracellular proteins in samples from batch cultures grown under different iron concentrations. Several of the extracellular proteins (e.g. AprA, LasB, PrpL) which were up-regulated under iron-limited conditions were found to be QS regulated proteins. Thus, this study clearly shows the links between QS and genes involved in iron and oxygen regulation in P. aeruginosa. INTRODUCTIONPseudomonas aeruginosa, a Gram-negative opportunistic human pathogen, is often found in nosocomial infections. In particular, it can cause chronic pulmonary infection of cystic fibrosis patients, leading to a high mortality rate due to the formation of virulence factors and the inappropriate host response (Hybiske et al., 2004;Wolfgang et al., 2004;Lyczak et al., 2002). In P. aeruginosa, the formation of numerous virulence factors is controlled to a large extent by a mechanism called quorum sensing (QS) (Fuqua & Greenberg, 1998;Wagner et al., 2003;Schuster et al., 2003;Hentzer et al., 2003;Juhas et al., 2004). Two major genetic components of QS, the las and rhl systems, have been identified in P. aeruginosa. The las system consists of LasR, a transcriptional regulatory protein, and LasI, the autoinducer synthase that controls the producti...

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Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene inPseudomonas aeruginosa

Cited by 178 publications

References 52 publications

Interspecies competition triggers virulence and mutability in Candida albicans–Pseudomonas aeruginosa mixed biofilms

Interspecies competition triggers virulence and mutability in Candida albicans–Pseudomonas aeruginosa mixed biofilms

Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of MucoidPseudomonas aeruginosaBiofilms

Expression of the quorum-sensing regulatory protein LasR is strongly affected by iron and oxygen concentrations in cultures of Pseudomonas aeruginosa irrespective of cell density

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