Identification of quorum‐sensing regulated proteins in the opportunistic pathogen <i>Pseudomonas aeruginosa</i> by proteomics

Arevalo-Ferro, Catalina; Hentzer, Morten; Reil, Gerold; Görg, Angelika; Kjelleberg, Staffan; Givskov, Michael; Riedel, Katharina; Eberl, Leo

doi:10.1046/j.1462-2920.2003.00532.x

Cited by 149 publications

(146 citation statements)

References 69 publications

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“…By replacing the defective lasRI genes, we demonstrated that protease IV secretion can be restored to the normal levels. Restoration of protease IV secretion was also observed in clinical isolates that were known to be defective in lasRI genes (Paer1 and Paer3), suggesting that protease IV production is also controlled, at least in part, through quorum-sensing regulation; these findings are in agreement with the work of Arevalo-Ferro et al (2003). However, control may not be direct as there is no evidence of a lux box (Bleves et al, 2005) within any of the gene sequences.…”

Section: Discussionsupporting

confidence: 74%

Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates

Conibear

Willcox

Flanagan

et al. 2012

Journal of Medical Microbiology

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Expression of protease IV by Pseudomonas aeruginosa during ocular infections contributes significantly to tissue damage. However, several P. aeruginosa strains isolated from ocular infections or inflammatory events produce very low levels of protease IV. The aim of the present study was to characterize, genetically and phenotypically, the presence and expression of the protease IV gene in a group of clinical isolates that cause adverse ocular events of varying degrees, and to elucidate the possible control mechanisms of expression associated with this virulence factor. Protease IV gene sequences from seven clinical isolates of P. aeruginosa were determined and compared to P. aeruginosa strains PAO1 and PA103-29. Production and enzyme activity of protease IV were measured in test strains and compared to that of quorumsensing gene (lasRI) mutants and the expression of other virulence factors. Protease IV gene sequence similarities between the isolates were 97.5-99.5 %. The strains were classified into two distinct phylogenetic groups that correlated with the presence of exo-enzymes from type three secretion systems (TTSS). Protease IV concentrations produced by PAODlasRI mutants and the two clinical isolates with a lasRI gene deficiency were restored to levels comparable to strain PAO1 following complementation of the quorum-sensing gene deficiencies. The protease IV gene is highly conserved in P. aeruginosa clinical isolates that cause a range of adverse ocular events. Observed variations within the gene sequence appear to correlate with presence of specific TTSS genes. Protease IV expression was shown to be regulated by the Las quorum-sensing system. INTRODUCTIONInflammation and infection in the cornea (keratitis) arising from Pseudomonas aeruginosa infection can lead rapidly to scarring and loss of sight unless prompt and targeted treatment is initiated (Sankaridurg et al., 2000). P. aeruginosa virulence is attributed, in part, to the production of several destructive proteins and stimulation of host immune response in the ocular tissues (Hazlett, 2002). The virulence factors most commonly associated with P. aeruginosa-induced ocular damage are exo-enzymes S (exoS) and U (exoU) (Fleiszig et al., 1997), elastase (lasB) (Kessler & Blumberg, 1987), alkaline protease (aprA) (Twining et al., 1993) and protease IV (piv) (O'Callaghan et al., 1996). Another protease, P. aeruginosa small protease (PASP), can also cause rapid corneal damage (Marquart et al., 2005). Other important pathogenic mechanisms include the genes involved in translocation and activation of virulence proteins via the type II (xcp) and type III secretory pathways (Sandkvist, 2001;Yahr et al., 1997).Protease IV is a lysine-specific endoprotease and a significant virulence factor for pathogenesis of P. aeruginosa in the eye and lung (Caballero et al., 2004; Engel et al., 1997;Wilderman et al., 2001). Mature protease IV degrades important host immunological proteins such as complement and IgG (Engel et al., 1998). Protease IV also compromises the inte...

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Section: Discussionsupporting

confidence: 74%

Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates

Conibear

Willcox

Flanagan

et al. 2012

Journal of Medical Microbiology

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“…This is in contrast to the higher protein abundance of HasAp and the ferripyoverdin receptor protein FpvA found in SCV 20265 during exponential growth, as shown by proteome analysis (50) and an approximately threefold-larger amount of pyoverdin accumulating in the supernatant of SCV 20265 in comparison to the wild type during growth in the stationary phase (data not shown). Interestingly, a posttranscriptional regulation of FpvA and HasAp by quorum sensing has recently been proposed (1). The global iron uptake regulator fur (17) was slightly upregulated in SCV 20265 in comparison to the revertant, but we did not find differential expression between SCV 20265 and the wild type.…”

Section: Resultscontrasting

confidence: 44%

Expression Analysis of a Highly Adherent and Cytotoxic Small Colony Variant of Pseudomonas aeruginosa Isolated from a Lung of a Patient with Cystic Fibrosis

Götz

Häußler²,

Jordan³

et al. 2004

J Bacteriol

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The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms. In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P. aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population. Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins. This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type. The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS. Thus, the prevailing assumption that P. aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants.

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“…Several large-scale transcriptomic and proteomic studies of QS and iron limitation in P. aeruginosa have been published recently Arevalo-Ferro et al, 2003;Nouwens et al, 2002Nouwens et al, , 2003Palma et al, 2003;Wagner et al, 2003;Schuster et al, 2003Schuster et al, , 2004Juhas et al, 2004;Wolfgang et al, 2004;Cornelis & Aendekerk, 2004). We compared the regulation of secreted proteins detected in our work by proteomic analysis with those from the transcriptomic and proteomic studies reported in the literature (Table 1).…”

Section: Discussionmentioning

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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Identification of quorum‐sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics

Cited by 149 publications

References 69 publications

Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates

Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates

Expression Analysis of a Highly Adherent and Cytotoxic Small Colony Variant of Pseudomonas aeruginosa Isolated from a Lung of a Patient with Cystic Fibrosis

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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