Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, USA1
Department of Microbiology, University of Guelph, Guelph, Ontario, Canada2
Department of Biological Sciences, University of Calgary, Calgary, Canada3
Author for correspondence: Shouguang Jin. Tel: +1 352 392 8323. Fax: +1 352 392 3133. e-mail: sjin{at}mgm.ufl.edu
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ABSTRACT |
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Keywords: migA, mucus, LPS and quorum response
Abbreviations: CF, cystic fibrosis; C12-HSL, 3-oxo-dodecanoyl homoserine lactone; C4-HSL, butanoyl homoserine lactone
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INTRODUCTION |
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As an opportunistic pathogen, P. aeruginosa relies on a combination of virulence factors to infect its host. Production and secretion of many of these virulence factors are increased at high cell density through a quorum sensing mechanism. Two such regulatory systems in P. aeruginosa, las and rhl, have been well characterized (Latifi et al., 1996 , 1995
; Pearson et al., 1994
, 1995
; Pesci et al., 1997
; Pesci & Iglewski, 1997
; Winson et al., 1995
). Each is composed of two components, an inducer locus, lasI or rhlI, that controls the synthesis of diffusible autoinducer molecule 3-oxo-dodecanoyl homoserine lactone (C12-HSL; PAI-1) or butanoyl homoserine lactone (C4-HSL; PAI-2), respectively, and a response locus, lasR or rhlR, that encodes transcriptional factors which become active upon binding to the respective autoinducers (Passador et al., 1993
). The PAI-1-bound form of LasR enhances the expression of genes encoding secreted proteases, siderophore, ADP-ribosylating enzymes and genes that control secretion of virulence factors (Passador et al. 1993
; Seed et al., 1995
; Toder et al., 1994
, 1991
; Winson et al., 1995
). PAI-2-bound RhlR controls some genes affected by LasR and, in addition, activates the expression of loci responsible for the production of toxic chemicals, including hydrogen cyanide, rhamnolipids and phenazines (Latifi et al., 1996
, 1995
; Ochsner & Reiser, 1995
; Ochsner et al., 1994
; Pearson et al., 1997
).
Chronic CF lung infections by P. aeruginosa start from acute infections early in the life of CF patients. The chronic infection has interspersed episodes of acute lower respiratory tract symptoms. As the chronic stage of the infection develops, there is a gradual transition of the P. aeruginosa isolates from a predominately non-mucoid, smooth LPS, piliated and motile phenotype to a predominately mucoid, rough LPS, non-piliated and non-motile phenotype (Govan, 1988 ; Luzar & Montie, 1985
; Mahenthiralingam et al., 1994
; Hancock et al., 1983
; Woods et al., 1986
). These morphological changes reflect bacterial adaptation to the CF lung environment, resulting from changes in gene regulation as well as mutation and selection. The mechanism for conversion to the mucoid phenotype was shown to be due to mutations in the anti-
factor gene, mucA, which alleviate repression of a 22 kDa
factor encoded by algU (or algT) that is required for the expression of the alginate biosynthetic pathway genes, in combination with environmental signals that activate genes under the control of two transcriptional regulators, AlgR and AlgB (Deretic et al., 1995
; Govan & Deretic, 1996
; Ma et al., 1998
). Alginate is the predominant exopolysaccharide moiety of the mucoid substance in CF isolates. In the case of pili and motility losses, mutation in the rpoN gene, which is required for pilus and flagellum assembly, accounts for the majority of the cases (Mahenthiralingam et al., 1994
). However, the molecular mechanism for the conversion from smooth LPS to rough LPS is not as clear. P. aeruginosa LPS consists of distinct A and B bands; the high-molecular-mass B-band determines the O-antigenic specificity of the bacterium, whereas the antigenically conserved A-band is a common antigen consisting of
-D-rhamnose trisaccharide repeating units. Wild-type P. aeruginosa strains express a proportion of their LPS as long-chain molecules, containing up to 50 repeating units (smooth LPS). In addition, these strains will have LPS that is devoid of O-polysaccharide (rough LPS) and LPS containing only one O chain repeating unit (core-plus-one LPS) (Rocchetta et al., 1999
).
To understand the molecular mechanism of bacterial adaptation to the CF lung environment, we have previously isolated and characterized P. aeruginosa genes that are specifically inducible by respiratory mucus derived from CF patients (Wang et al., 1996 ). One of these encodes a putative glycosyltransferase which was predicted to be involved in LPS or exopolysaccharide modification. In this report, we show that the migA gene product indeed modifies bacterial LPS structure, predominantly the core-plus-one LPS. Furthermore, we demonstrate that the migA gene is regulated by the RhlI/RhlR quorum sensing regulatory system and is highly expressed in the CF lung environment. The significance of these observations is discussed.
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METHODS |
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To generate migA promoter deletion constructs, either available restriction sites upstream of the migA promoter or EcoRI sites generated in desired positions by site-directed mutagenesis (Kunkel et al., 1987 ) were used in combination with the BamHI site in pMS18 (Wang et al., 1996
). Isolated migA promoter fragments were fused to a promoterless lacZ by cloning into the EcoRI/BamHI sites of pDN19lac
(Totten & Lory, 1990
). To introduce an EcoRI site at positions -200, -89 and -38 relative to the ATG translational start codon of migA in pMS18, the following three oligonucleotides were used: migA-200, 5'-GGCCTACCCTCTAGAATTCCGACGGTATACTG-3'; migA-100, 5'-CCAGCCCGCGCCGAATTCCGATCGCCGGAACG-3'; and migA-50, 5'-GACGGCCCCCTGGAATTCCTACCGGGCAAGACG-3', respectively.
Sputum collection and extraction of total RNA.
Sputum samples from nine paediatric patients were utilized in this study. These patients were part of a larger cohort of patients currently being followed (Storey et al., 1992 , 1997
, 1998
). The patients in this study all attend the Alberta Childrens Hospital Cystic Fibrosis Clinic, Canada. Voluntary consent was obtained from all patients and their guardians. Ethical approval of the study design was given by the Conjoint Research Ethics Board of the University of Calgary, Canada. All samples used in this study were collected from patients who were in the moderate to severe disease categories. Also, the selected patients had acquired solely or predominantly P. aeruginosa infections.
Sputum from the CF patients was collected as described previously (Storey et al., 1998 ). RNA was extracted from the sputum samples, blotted onto Nylon membrane, hybridized and signal intensity was measured as described by Storey et al. (1992)
. A 613 bp internal HincII fragment of migA from pIL17 was used as a probe to measure migA-specific transcripts. A 700 bp internal EcoRI fragment of algD from plasmid pCC27 (Chitinis & Ohman, 1990
) and a 450 bp PstI fragment of lasA from pPS1816 (Schad & Iglewski, 1988
) were used to examine algD-specific and lasA-specific transcripts, respectively.
Primer extension.
Total bacterial RNA was isolated from strain PAK that was grown in minimal medium A with or without 10% respiratory mucus from CF patients. For primer extension experiments, an oligonucleotide, migA-down (5'-CTTCTTCCAGGTACTTTTCCGCGTTG-3') was used as primer and according to the protocol described by Maniatis et al. (1982) . The same primer was also used to sequence the migA promoter region to identify the transcription initiation site.
LPS preparation, SDS-PAGE and Western blot analysis.
LPS was prepared from whole-cell lysates of P. aeruginosa by the method of Hitchcock & Brown (1983) . LPS samples were run on standard discontinuous 12·5% glycine SDS-PAGE gels (Hancock & Carey, 1979
) or by commercially prepared 16% Tricine SDS-PAGE gels (Novex). LPS was visualized by the silver-staining methods of Dubray & Bezard (1982)
for glycine SDS-PAGE and Tsai & Frasch (1982)
for Tricine gels. For Western immunoblots, the procedure was essentially as described by de Kievit & Lam (1994)
. LPS separated by SDS-PAGE was transferred onto nitrocellulose and then blocked with 3% skim milk. For detection of P. aeruginosa LPS, blots were immersed in hybridoma-culture supernatants containing mAb O25G3D6 (specific for B-band LPS serotype O6; Emara et al., 1995
), mAb N1F10 (specific for A-band LPS; Lam et al., 1989
) or mAb 7-4 (specific for LPS inner core; de Kievit & Lam, 1994
). After incubation with the corresponding first antibodies, blots were washed and then incubated with the appropriate second antibody. The second antibody for anti-P. aeruginosa LPS mAbs was goat anti-mouse F(ab')2alkaline phosphatase conjugate (Jackson ImmunoResearch Laboratories) at the dilutions suggested by the manufacturer. Following a series of washes, the blots were developed using a substrate consisting of 30 mg nitro blue tetrazolium and 15 mg 5-bromo-4-chloro-3-indolyl phosphate (toluidine salt; Sigma) in 100 ml 0·1 M sodium bicarbonate buffer (pH 9·8).
ß-Galactosidase assay.
P. aeruginosa strains harbouring various lacZ fusion constructs were grown in MinA with or without 10% CF respiratory mucus and measured for ß-galactosidase activities as described by Wang et al. (1996) . To induce migA and lasB, 100 nM of their respective autoinducers, C12-HSL (PAI-1) or C4-HSL (PAI-2) was added at the start of culture and ß-galactosidase activities were measured at various cell densities.
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RESULTS |
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Deletion analysis of the migA promoter
To identify functional promoter elements of the migA gene, a series of promoter deletion constructs were generated using both existing restriction sites as well as sites generated by site-directed mutagenesis. The promoter deletion fragments were fused to the promoterless lacZ gene in pDN19lac, and PAK cells harbouring the resulting constructs were tested for ß-galactosidase activities with or without the addition of CF respiratory mucus. As shown in Fig. 2
, promoter deletion up to -200 (relative to the putative ATG start codon) did not affect wild-type promoter activity but deletion to -89 totally abolished the inducibility by mucus. These results indicate that important migA promoter elements reside within the 111 bp region between -200 and -89.
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migA is regulated by the RhlI/RhlR quorum sensing system
To investigate whether migA is indeed regulated by the quorum sensing system, cell-density-dependent expression of the migA::lacZ fusion was first tested in a PAK background. PAK(pSJ9729) was grown in L-broth with appropriate antibiotics at 37 °C. As shown in Fig. 4(a), ß-galactosidase activity increased proportional to cell density and more than fourfold greater ß-galactosidase activity was observed at high cell density than that at low cell density (OD600 4·5 vs 1·0), demonstrating cell-density-dependent expression, a characteristic of genes under the control of quorum sensing systems. We further examined whether migA is regulated by either one or both of the two well characterized quorum sensing systems in P. aeruginosa, lasI/R or rhlI/R. Expression of migA::lacZ in lasI and rhlI single mutants, as well as in a lasI/rhlI double mutant background was compared to that in the wild-type PAO1 background. At high cell density (OD600 4·5), migA expression levels were much lower in either the rhlI or lasI/rhlI double mutant background than in wild-type strain. However, the migA expression level in the lasI mutant background was not different from that in the wild-type PAO1 background (Fig. 4b
), suggesting that migA is regulated by the RhlI/R system. Furthermore, migA expression in a lasI/rhlI double mutant background is stimulated by the addition of C4-HSL (PAI-2) but not by C12-HSL (PAI-1). Interestingly, addition of both PAI-1 and PAI-2 did not induce migA expression (Fig. 4c
), which is similar to other RhlI/RhlR-regulated genes described by Pesci et al. (1997)
. It was postulated in these cases that PAI-1 inhibits the binding of PAI-2 to RhlR. As a control, the expression of lasB, which is known to be regulated by LasR, was stimulated by PAI-1 but not by PAI-2 in the same mutant background (Fig. 4c
).
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DISCUSSION |
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Quorum sensing gene regulation plays an important role in the pathogenesis of P. aeruginosa. In response to an increased bacterial cell density, the quorum sensing system not only mediates an increased synthesis and secretion of extracellular enzymes to meet the nutritional requirement of the pathogen, but also mediates biofilm formation to overcome adverse environmental conditions (Pesci & Iglewski, 1997 ; Fuqua & Greenberg, 1998
). Whiteley et al. (1999)
have recently reported on the identification of a large number of genes that are under the control of quorum sensing systems. By comparing their promoter sequences, a consensus las box promoter element (CT-N12-AG), potentially the LasR and or RhlR recognition sequence, has been deduced. However, the migA gene was not included in that study. Our experimental data clearly demonstrate that migA expression is dependent on the rhlI/rhlR regulatory system and its promoter also contains two las-box-like sequences (CT-N11-AG) that fall into the essential promoter region identified by the promoter deletion analysis. Whether one or both of the las-box-like sequences are required for migA expression is not known at this point.
Since the CF respiratory mucus that we used was derived from CF patients who had acquired P. aeruginosa infection, the mucus should contain high concentrations of autoinducers. However, the mucus was subjected to dialysis against distilled water before use and therefore it is unlikely that residual autoinducer molecules are responsible for the activation of migA gene expression. Recently, our group has observed that addition of CF respiratory mucus into minimal medium actually promotes P. aeruginosa growth which results in a higher cell density (unpublished results). It is therefore possible that this higher cell density triggers the quorum sensing response which activates migA expression. The growth promoting activity of the CF mucus is consistent with the reported observations of high bacterial cell densities in CF lungs (Potts et al., 1995 ). The exact components of the mucus that promote the growth of P. aeruginosa deserve further study, since they represent important host factors that promote P. aeruginosa colonization and the bacterial genes mediating the utilization of these mucus components could potentially serve as targets in antimicrobial strategy.
The observation that MigA shares amino acid sequence homology with glycosyltransferases, implies a potential role of this protein in LPS modification. We have shown here that migA indeed affects the modification of low-molecular-mass LPS. High-level expression of migA leads to the loss of the core-plus-one LPS band, whereas migA mutation results in an increase in this band. We hypothesize that MigA is a glycosyltransferase that transfers a sugar residue onto the core region and consequently modifies the attachment point for O antigen on the outer-core oligosaccharide. The MigA modified outer-core molecule would therefore be less able to serve as an acceptor for O antigen. A ligase enzyme, WaaL, joins the O antigen polysaccharide to the lipid A core and there is a strong evidence that the nature of the acceptor molecule is important for this ligation reaction. Evidence to support this hypothesis is as follows. (i) In P. aeruginosa, the structure of the outer-core region in a fully assembled O chain containing LPS differs from that of the O-chain-deficient rough LPS. The point of attachment of the O chain to the outer core is at the 1,3-linked rhamnose (Sadovskaya et al., 2000 ). When this residue is missing, the core molecule is less able to serve as an acceptor for O antigen attachment. Genetic evidence suggests that the rhamnose residue in the outer-core region is necessary for O antigen attachment (Rahim et al., 2000
). (ii) A rhamnosyltransfersase, required for the addition of the 1,3-linked rhamnose which serves as the attachment point for O antigen to the outer core, shares amino acid sequence homology with MigA (Rahim et al., 2000
). (iii) MigA is homologous to glycosyltransferases, including WaaV of E. coli, possessing a type R1 core (Heinrichs et al., 1998
). The proposed function of WaaV is that of a UDP-glucosyltransferase required for the addition of a 1,3-linked glucose, which is a side-branch substituent in the outer core and acts as the attachment site for long-chain O antigen to an R1-type core.
Recently, Ernst et al. (1999) have demonstrated that the lipid A portion of the P. aeruginosa LPS undergoes specific modification during chronic colonization of CF lungs. The CF-specific lipid A apparently contains palmitate and aminoarabinose which are associated with bacterial resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease. Since the migA gene is highly expressed in the CF lung environment and interferes with the formation of core-plus-one LPS, it is possible that suppression of such low-molecular-mass LPS renders P. aeruginosa a survival advantage in the CF lung environment. If indeed this is the case, a prolonged colonization in the CF lung environment could result in enrichment of adaptive mutants in which migA gene expression become constitutive, similar to the enrichment of mucoid isolates of P. aeruginosa. Our survey of CF- and non-CF-derived P. aeruginosa strains indicates that a high proportion of CF isolates lost core-plus-one LPS, whereas all of the non-CF isolates retained it (unpublished results). However, it is not clear if this is due to migA up-regulation or a defect in the ligase which mediates core-plus-one LPS synthesis. Further studies are underway to understand this as well as the role of migA up-regulation in the adaptation of P. aeruginosa to the CF lung environment.
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ACKNOWLEDGEMENTS |
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Received 2 June 2000;
revised 21 July 2000;
accepted 24 July 2000.