Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

Nouwens, Amanda; Beatson, Scott A.; Whitchurch, Cynthia B.; Walsh, Bradley J.; Schweizer, Herbert P.; Mattick, John S.; Cordwell, Stuart J.

doi:10.1099/mic.0.25967-0

Cited by 134 publications

(170 citation statements)

References 66 publications

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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: 84%

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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

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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Section: Discussionmentioning

confidence: 84%

“…References: (1), Nouwens et al (2003); (2) Table 1. Overall, the proteomic analysis also strongly supports the notion that iron availability can affect the regulation of QS-related genes.…”

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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“…Another explanation for the absence of biofilm in SG81lasB+ could be the degradation or modification of extracellular proteins by the activity of the proteolytic LasB. Proteome analysis has shown that extracellular proteins undergo a substantial post-translational degradation during biofilm maturation (Nouwens et al, 2003). LasB has been identified as one of the enzymes involved.…”

Section: Discussionmentioning

confidence: 99%

Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa

et al. 2010

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Pseudomonas aeruginosa secretes a variety of hydrolases, many of which contribute to virulence or are thought to play a role in the nutrition of the bacterium. As most studies concerning extracellular enzymes have been performed on planktonic cultures of non-mucoid P. aeruginosa strains, knowledge of the potential role of these enzymes in biofilm formation in mucoid (alginateproducing) P. aeruginosa remains limited. Here we show that mucoid P. aeruginosa produces extracellular hydrolases during biofilm growth. Overexpression of the extracellular lipases LipA and LipC, the esterase EstA and the proteolytic elastase LasB from plasmids revealed that some of these hydrolases affected the composition and physicochemical properties of the extracellular polymeric substances (EPS). While no influence of LipA was observed, the overexpression of estA and lasB led to increased concentrations of extracellular rhamnolipids with enhanced levels of mono-rhamnolipids, elevated amounts of total carbohydrates and decreased alginate concentrations, resulting in increased EPS hydrophobicity and viscosity. Moreover, we observed an influence of the enzymes on cellular motility. Overexpression of estA resulted in a loss of twitching motility, although it enhanced the ability to swim and swarm. The lasB-overexpression strain showed an overall enhanced motility compared with the parent strain. Moreover, the EstAand LasB-overproduction strains completely lost the ability to form 3D biofilms, whereas the overproduction of LipC increased cell aggregation and the heterogeneity of the biofilms formed. Overall, these findings indicate that directly or indirectly, the secreted enzymes EstA, LasB and LipC can influence the formation and architecture of mucoid P. aeruginosa biofilms as a result of changes in EPS composition and properties, as well as the motility of the cells. INTRODUCTIONPseudomonas aeruginosa is a common environmental bacterium and represents an increasingly prevalent opportunistic human pathogen whose ecological success is based on a remarkable degree of genomic flexibility and phenotypic adaptation (Wehmhöner et al., 2003). P. aeruginosa successfully colonizes a wide range of habitats, including natural soil and aquatic environments as well as artificial water systems (Botzenhart & Döring, 1993). It is involved in acute and chronic diseases, for example in infections of surgery and burn patients, in urogenital tract and wound infections, chronic lung infections of cystic fibrosis patients, and nosocomial pneumonia in intubated and mechanically ventilated patients (Drenkard, 2003;Singh et al., 2000).The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa under varying environmental conditions in nature, as well as in technical systems and the clinical setting. P. aeruginosa is now regarded as one of the medically most relevant biofilmforming bacterial species. Therefore, it has become one of the best-studied model organisms for biofilm formation (McDougald et al., 2008). Biofilm forma...

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“…However, the situation is not as clear for many LasR-controlled genes, for which it has not been possible to define a single consensus LasR binding site sequence in the promoter region, suggesting that some of these genes are activated indirectly (Schuster et al, 2004;Schuster & Greenberg, 2007). Actually, most QS-regulated factors are more or less influenced by both LasR and RhlR, as is the case for the proteases LasA (staphylolytic protease) and LasB (elastase) (Freck-O'Donnell & Darzins, 1993;Hentzer et al, 2003;Nouwens et al, 2003;Schuster et al, 2003;Toder et al, 1994;Wagner et al, 2004). Thus QS plays a predominant role in the regulation of virulence determinants in P. aeruginosa.…”

Section: Introductionmentioning

confidence: 99%

Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors

2009

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Pseudomonas aeruginosa uses the two major quorum-sensing (QS) regulatory systems las and rhl to modulate the expression of many of its virulence factors. The las system is considered to stand at the top of the QS hierarchy. However, some virulence factors such as pyocyanin have been reported to still be produced in lasR mutants under certain conditions. Interestingly, such mutants arise spontaneously under various conditions, including in the airways of cystic fibrosis patients. Using transcriptional lacZ reporters, LC/MS quantification and phenotypic assays, we have investigated the regulation of QS-controlled factors by the las system. Our results show that activity of the rhl system is only delayed in a lasR mutant, thus allowing the expression of multiple virulence determinants such as pyocyanin, rhamnolipids and C 4 -homoserine lactone (HSL) during the late stationary phase. Moreover, at this stage, RhlR is able to overcome the absence of the las system by activating specific LasR-controlled functions, including production of 3-oxo-C 12 -HSL and Pseudomonas quinolone signal (PQS). P. aeruginosa is thus able to circumvent the deficiency of one of its QS systems by allowing the other to take over. This work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system.

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Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1

Cited by 134 publications

References 66 publications

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

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

Extracellular enzymes affect biofilm formation of mucoid Pseudomonas aeruginosa

Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors

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