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1 Department of Biological Sciences, Auburn University, AL 36849, USA
2 Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, AB T2N 4N1, Canada
Correspondence
Laura Silo-Suh
suhlaur{at}auburn.edu
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Isolates that undergo this conversion are typically maintained within the lungs of the patient for the patient's entire life span (Mahenthiralingam et al., 1996; Struelens et al., 1993).
Typical P. aeruginosa isolates from the environment that infect CF patients produce numerous virulence determinants that contribute to establishing infections in animal model systems (Cox, 1982; Meyer et al., 1996; Nicas & Iglewski, 1985; Ostroff et al., 1989b; Wilderman et al., 2001). However, P. aeruginosa recovered from the lungs of chronically infected CF patients typically lack some of these virulence determinants, suggesting that these products are dispensable for long-term maintenance of P. aeruginosa in vivo (Dacheux et al., 2001; Luzar & Montie, 1985; Smith et al., 2006; Woods et al., 1986). Conversely, P. aeruginosa gains an additional virulence determinant by overproducing the exopolysaccharide alginate within the CF lung. Alginate appears to form a protective barrier around the bacterium and limits exposure to oxidative radicals, antibiotics, opsonizing antibodies and phagocytes (Hatch & Schiller, 1998; Oliver & Weir, 1985; Pedersen et al., 1990; Simpson et al., 1989). In addition, P. aeruginosa forms microcolonies encapsulated in alginate and mucus within the CF lung (Worlitzsch et al., 2002). This biofilm mode of growth limits the access of various immune molecules and antibiotics to the bacterium. Biofilm growth may also aid colonization by promoting genetic diversity within the bacterial population (Ehrlich et al., 2005) and by allowing a subset of bacteria to exist in a reduced metabolic state and/or exist under anaerobic conditions, both of which can provide protection from elements of the immune system and antibiotics (Fux et al., 2005).
It is becoming increasingly clear that microbial pathogens use different sets of virulence determinants and strategies for maintaining chronic infections versus establishing acute infections (Costerton et al., 2003; Hong et al., 2000; Young et al., 2002). Both Mycobacterium tuberculosis and P. aeruginosa can establish decade-long infections in the lungs of humans and these pathogens appear to utilize similar strategies. For example, M. tuberculosis resides in granulomas during chronic lung infections, where it appears to exist in a reduced metabolic state under anaerobic conditions (Young et al., 2002), similar to the existence of P. aeruginosa in biofilms in the CF lung. In addition, both P. aeruginosa and M. tuberculosis appear to alter their carbon metabolic activities while in the human lung (Honer zu Bentrup & Russell, 2001; Silo-Suh et al., 2005). Identification of additional virulence determinants that are active in P. aeruginosa within the CF lung and characterization of the role they play in pathogenesis or survival may provide strategies for treating these and other chronic infections.
To identify chronic virulence determinants, several research groups rely upon chronic animal model systems of infection (Fang et al., 2005; McKinney et al., 2000). We suggest that an alternative approach is to study bacterial isolates that have adapted to a chronic infection lifestyle, such as P. aeruginosa isolates adapted to the CF lung. Some of these adaptations manifest in P. aeruginosa under laboratory conditions because of DNA alterations induced by and selected within the CF lung. Likewise, those virulence determinants that no longer provide an advantage, such as some acute virulence determinants, appear to be readily lost as P. aeruginosa adapts to the CF lung. Therefore, the virulence determinants that are maintained, acquired or altered in P. aeruginosa following adaptation to the CF lung are potential chronic virulence determinants.
We previously determined that FRD1, a typical P. aeruginosa CF isolate, uses novel infection mechanisms compared to the wound isolate PAO1 to infect alfalfa seedlings (Silo-Suh et al., 2002). We suggest that some of the novel virulence mechanisms employed by FRD1 to infect alfalfa may be the same mechanisms it relies upon to persist within the CF lung.
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METHODS |
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. Unless otherwise indicated, bacteria were cultured in L-broth (LB) or on L-agar. No-carbon E minimal medium (NCE) supplemented with 0.1 % (w/v) Casamino acids and with succinate (20 mM) or acetate (20 mM) as a carbon source was used for growth on minimal media (Davis et al., 1980). Data for growth curves were obtained with a Synergy HT plate reader (Bio-Tek Instruments). UV-visible absorption spectra were recorded on a Shimadzu UV-1601 spectrophotometer using 1 cm path-length cells. P. aeruginosa was grown in King's B medium for pyoverdine assays and in 1 % (w/v) peptone broth supplemented with 1 % (w/v) NaCl and 1 % (w/v) glycerol for phospholipase C assays and pyocyanin assays as previously described (Essar et al., 1990). A 1 : 1 mixture of L-agar and Pseudomonas Isolation Agar (PIA) was used to select for P. aeruginosa transconjugants and to counterselect for Escherichia coli following triparental mating. Media were solidified with 1.5 % (w/v) Bacto Agar (Difco; Becton Dickinson). Antibiotics were purchased from Sigma-Aldrich and used at the following concentrations: 100 µg ampicillin ml–1 for E. coli; 180 µg carbenicillin ml–1 for P. aeruginosa; 20 µg gentamicin ml–1 for E. coli and 200 µg ml–1 for P. aeruginosa.
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). The transposon insertion library was generated in the P. aeruginosa CF isolate FRD1: pAG408 was introduced into P. aeruginosa via triparental mating as previously described (Suh et al., 1999). The conjugation mixtures were plated onto PIA containing gentamicin (180 µg ml–1) to select for P. aeruginosa containing transposon insertions. Colonies that grew on gentamicin were replicated onto L-agar supplemented with carbenicillin to identify clones with pAG408 insertions. The clones resistant to both gentamicin and carbenicillin were discarded while the gentamicin-resistant carbenicillin-sensitive clones were characterized further.
Alfalfa seedling infection assay.
Approximately 1700 independent FRD1 transposon insertion mutants were screened for virulence in the alfalfa seedling infection assay. Seeds of alfalfa variety 57Q77, a wild-type strain not bred for pest resistance, were provided by Pioneer Hi-Bred International. The alfalfa assay was conducted as previously described (Silo-Suh et al., 2002) with the following modifications: FRD1 and derivatives were inoculated onto wounded alfalfa seedlings using 
105 c.f.u. per seedling while PAO1 and derivatives were inoculated using 104 c.f.u. per seedling. Water agar plates containing inoculated seedlings were sealed with Parafilm and placed in a 30 °C incubator without light. Disease symptoms were scored 6–7 days following inoculation by visual inspection. Seedlings with maceration symptoms were scored positive for infection. Each transposon insertion mutant was initially screened with 10 alfalfa seedlings. Putative mutants that were reduced for virulence on alfalfa by twofold or more compared to the parental strain were rescreened in subsequent rounds of 20 and 40 seedlings. PAO1 and PAO1aceA were inoculated on 60 seedlings for each experiment. Data were expressed as the mean±standard deviation and analysed for significance using an ANOVA (InStat; Graph Pad Software). A value of P<0.05 was considered significant.
DNA manipulations, transformations and conjugations.
E. coli strain DH10B was routinely used as a host strain for cloning. DNA was introduced into E. coli by electroporation and into P. aeruginosa by conjugation as previously described (Suh et al., 1999). Plasmids were purified with QIAprep Spin Miniprep columns (Qiagen). DNA fragments were excised from agarose gels and purified using the Qiaex II DNA gel extraction kit (Qiagen) according to the manufacturer's instructions. Restriction enzymes and DNA modification enzymes were purchased from New England Biolabs. Either Pfu from Stratagene or Taq from New England Biolabs was used for PCR amplification of DNA. Oligonucleotides were purchased from Integrated DNA Technologies.
Southern blot analysis.
Genomic DNA was extracted from FRD1 and derivatives using the Wizard Genomic Extraction kit (Promega), digested with the appropriate restriction enzymes and electrophoretically separated on a 1 % agarose gel. The DNA was transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech) via capillary blotting and fixed by baking for 2 h at 80 °C. The blot was probed with a 800 bp DNA fragment corresponding to the gentamicin resistance gene, which was biotinylated using the Psoralen-biotin kit (Ambion). Detection of the probe was performed using the BrightStar BioDetect kit (Ambion).
Identification of transposon insertion sites.
Genomic DNA was prepared from each of the transposon insertion mutants using Wizard Genomic (Promega), digested with the appropriate enzymes, cloned into pBluescript K(+) and electroporated into E. coli DH10B. The gentamicin-resistant colonies recovered from the transformation were verified for the presence of transposon sequences by PCR and then sequenced using primer LSP368 (cgagcgcgtcaattcgagggc). The plasmids containing cloned transposon and flanking P. aeruginosa sequences were sequenced by the Auburn University Research and Instrumentation Facility.
Growth competition.
FRD1 and FRD1 pqsE were inoculated into 12 ml fresh L-broth using 20 µl of an overnight culture. The mixed culture was sampled for c.f.u. on L-agar and L-agar plates containing gentamicin periodically over 24 h.
Construction of P. aeruginosa aceA and phzS mutants.
To generate aceA mutants of P. aeruginosa, the suicide plasmid pLS1536 was constructed: a DNA sequence containing 400 bp upstream and 430 bp downstream of the aceA coding sequence was PCR amplified from FRD1 cells with Pfu and cloned into the SmaI site of pBluescript K(+). The resulting plasmid was digested with SphI and the internal 1.3 kb fragment of the aceA coding sequence was removed and replaced with the aacC1 gene encoding gentamicin resistance as a SmaI fragment (Schweizer, 1993). This was followed by introduction of an origin of transfer (moriT) of RP4 on a 230 bp HindIII fragment (Suh et al., 2004). pLS1536 was introduced into P. aeruginosa strains FRD1 and PAO1 by triparental mating, and potential aceA mutants were isolated as gentamicin-resistant carbenicillin-sensitive clones, indicating a double crossover event. Replacement of the wild-type aceA gene with the aceA101 : : aacC1 allele was verified by PCR analysis.
To construct P. aeruginosa phzS mutants, the phzS coding sequence along with 345 bp upstream and 150 bp downstream sequences was PCR amplified from FRD1 cells with Pfu and cloned into the SmaI site of pBluescript K(+). An aacC1 gene carried on a SmaI fragment was cloned into the unique ScaI site internal to the phzS coding sequence and then oriT was added as an 230 bp HindIII fragment. The resulting plasmid, pLS1552, was conjugated into strain FRD1 as described above and potential mutant alleles were isolated as gentamicin-resistant carbenicillin-sensitive clones. Replacement of the wild-type phzS gene with the phzS101 : : aacC1 allele was verified by PCR.
Construction of aceA transcriptional fusion and complemented strains.
To construct the aceA : : lacZ transcriptional fusion, the PCR fragment containing the aceA gene from FRD1 described above was digested with EcoRV and cloned into the SmaI site of pSS223 (Suh et al., 2004). The construct containing the aceA promoter and the 5' coding sequence in the proper orientation was verified by PCR and restriction digest before it was conjugated into P. aeruginosa.
To complement the aceA mutation in cis, aceA was PCR amplified from FRD1 with Pfu and cloned into pBluescript K(+) as a 2.07 kb EcoRI–SmaI fragment. The resulting plasmid, pJH109, was converted to a mobilizable plasmid, pJH122, by the addition of moriT to the HindIII site. pJH122 was introduced into P. aeruginosa via triparental mating. The complemented FRD1aceA and PAO1aceA mutants were designated FRD1aceA+ (JH168) and PAO1aceA+ (JH166) respectively.
Biochemical assays.
Alginate was isolated from P. aeruginosa culture supernatants that were dialysed against distilled water as previously described (Suh et al., 1999), and the alginate level (i.e. uronic acid) was quantified by the carbazole method (Knutson & Jeanes, 1968) using Macrocystis pyrifera alginate (Sigma-Aldrich) as a standard. Pyocyanin was purified and measured from 20 h cultures as described by Essar et al. (1990). Degradation of p-nitrophenylphosphorylcholine was used to measure phospholipase C activity as previously described, using 1 mg protein from filtered supernatant (Suh et al., 1999). Phospholipase C activity was measured as the increase in A405 min–1 (mg protein)–1. Elastase and pyoverdine were measured as previously described (Suh et al., 1999). β-Galactosidase assays were performed as described by Miller (1972). Isocitrate lyase (ICL) was measured according to the Sigma protocol EC 4.1.3.1 with minor modifications. P. aeruginosa cells were harvested from stationary cultures, resuspended in TE pH 6.8 and sonicated. Following centrifugation, the quantity of total proteins in the cell-free extracts was determined by the Bradford method (Bio-Rad). Each assay was conducted with 50 µg protein in a final volume of 1 ml.
Biofilm growth.
Measurement of static biofilm activity was performed as described by Head & Yu (2004). Briefly, P. aeruginosa was grown overnight in L-broth, diluted and adjusted to OD600 0.5, from which 5 µl was inoculated into 125 µl fresh L-broth in a 96-well microtitre plate. The plate was incubated overnight at 37 °C for 15 h prior to staining with crystal violet and optical density readings.
Rat chronic lung infections.
P. aeruginosa strains were tested for their ability to cause respiratory infections in the agar bead model in rats as described by Cash et al. (1979). For each P. aeruginosa strain tested, eight male Sprague–Dawley rats weighing 200–220 g (Charles River Breeding Laboratories) were tracheostomized under anaesthesia and inoculated with 104 c.f.u. bacteria embedded in agar beads. On day 14 post-infection, the lungs were aseptically removed. For each set of strains tested, lungs from four animals were homogenized in PBS (Polytron homogenizer, Brinkmann Instruments) and serial dilutions were plated onto trypticase soy agar to determine bacterial counts. The remaining lungs were fixed in 10 % formalin and examined for quantitative histopathological changes as previously described (Bernier et al., 2003). The lung sections were scanned using an Epson 1650 scanner. Areas of inflammation, characterized by cellular infiltration, were identified and digitized with Scion Image software and reported as the percentage of the total area of the lung section that was covered by inflammatory exudates.
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RESULTS |
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1 %, indicating random insertion of the mini-Tn5 in FRD1. The non-mucoid and auxotrophic mutants were discarded. Non-mucoid mutants were likely to contain transposon insertions in the alginate biosynthetic operon or in algT, which encodes the alternative sigma factor, sigma-22. Both classes of non-mucoid mutants have previously been tested for virulence in the alfalfa assay (Silo-Suh et al., 2002).
Of 1700 FRD1 transposon insertion mutants screened in the alfalfa assay, 53 exhibited reduced virulence on alfalfa seedlings. The 21 mutants most severely affected for virulence on alfalfa seedlings were characterized for this investigation (Table 2). With the exception of the FRD1 pqsB mutant, all mutants showed a more than twofold reduction in virulence on alfalfa compared to the parental strain. In addition, none of the mutants showed a growth defect in minimal medium with succinate as the carbon source compared to FRD1 (data not shown).
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). A comparison of the DNA sequences indicates that two groupings of mutants can be distinguished based on functionality. The first group contains transposon insertions in pqs genes, which encode one of three quorum-sensing systems in P. aeruginosa. Three of the mutants contained transposon insertions in pqsE, while one mutant each contained insertions in pqsB and pqsC. All the pqsE insertion mutations mapped to an identical location, suggesting that there is an insertion hot-spot for this gene or that the mutants are genetic siblings. Alternatively, the pqsE mutant might have a growth advantage over the parental strain. However, a competitive growth assay did not support this possibility (data not shown). All three FRD1 pqsE mutants were similarly reduced for virulence on alfalfa (Table 2), indicating that the alfalfa seedling assay is highly reproducible.
The second group of mutants is represented by insertions in seven different genes, all of which potentially play a role in carbon metabolism (Fig. 1). ICL (PA2634), encoded by aceA, is specific to the glyoxylate shunt pathway and allows a variety of organisms to grow on fatty acids or acetate as a sole carbon source. PA0794 encodes an uncharacterized aconitate hydratase that may substitute for AcnA or AcnB in the tricarboxylic acid (TCA) pathway or may function in propionate metabolism as suggested by Grimek & Escalante-Semerena (2004). L-Asparaginase I (PA2253) converts asparagine into aspartate, which is one step away from conversion to oxaloacetate. PA3588 encodes OpdR, a porin of the OpdK subfamily, and is proposed to be involved in transport of phenylacetate, a short-chain fatty acid (Tamber et al., 2006). PA1066 is predicted to encode a short-chain alcohol dehydrogenase involved in fatty acid β-oxidation. The gene nuoA (PA2637) encodes chain A of the proton-pumping NADH dehydrogenase I. Mutations in the nuo operon in E. coli inhibit citrate synthase and malate dehydrogenase of the TCA cycle (Pruss et al., 1994). PA4466 encodes a gene with homology to npr, postulated to link carbon and nitrogen assimilation in E. coli (Powell et al., 1995). Most of the genes in this group affect other cellular functions, including roles in nitrogen metabolism (ansA and npr) and iron homeostasis (npr and aconitase). Further testing will distinguish which of these functions are required for virulence of P. aeruginosa on alfalfa seedlings.
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We first tested for elastase and pyocyanin production because these virulence determinants play a role in animal lung infection models (Blackwood et al., 1983; Elsheikh et al., 1987). Three of the 18 mutations that affected virulence of FRD1 on alfalfa also produced less elastase than FRD1 (Fig. 2a), including the pqsE mutant as expected (P<0.001) (Coin et al., 1997; Diggle et al., 2003). Unexpectedly, neither the FRD1 pqsB nor pqsC mutants showed reduced elastase production, suggesting that the transposon insertions in these genes are not polar on the downstream pqsE gene. In addition, there appears to be a differential requirement for Pseudomonas quinolone signal (PQS) biosynthesis (pqsB and pqsC) versus response to PQS (pqsE) for elastase production.
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). Pyocyanin production was variable in many of the FRD1 mutants reduced for virulence on alfalfa (Fig. 2b). Disruption of the pqs operon, nuoA, PA0428 (putative RNA helicase) and PA0794 (putative aconitase) led to reduced pyocyanin production by FRD1, while disruptions in opdR, PA4466 and sbcB resulted in increased pyocyanin production. These ambiguous results led us to test whether pyocyanin is essential for FRD1 virulence on alfalfa. Disruption of phzS, a gene required for pyocyanin biosynthesis, did not affect the ability of FRD1 to infect alfalfa (data not shown), indicating that lack of pyocyanin production by several of the target mutants was not the cause for loss of virulence.
Phospholipase C cleaves membrane phospholipids and is essential for P. aeruginosa virulence on a variety of host organisms (Berk et al., 1987; Ostroff et al., 1989a; Rahme et al., 1995; Wiener-Kronish et al., 1993). Given that several of the target mutants are possibly defective in fatty acid catabolism, we predicted a role for phospholipase C in liberating fatty acids for P. aeruginosa catabolism during infection. However, only one of the FRD1 targets, PA0428 (RNA helicase), in this study was defective for phospholipase C activity compared to the parental strain (Fig. 3a). Verification of a role for phospholipases in the alfalfa assay will require the generation and testing of mutants that are specifically affected in phospholipase production.
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). The mechanisms underlying the loss of pyoverdine production by CF isolates of P. aeruginosa are unknown. The data presented here suggest that FRD1 is impaired in regulation of pyoverdine production and not biosynthesis. In addition, the uncharacterized aconitase encoded by PA0794 appears to play a role in iron metabolism because disruption of this gene resulted in increased pyoverdine production.
Although all the targets selected for this study were visibly mucoid on solid media, we observed substantial differences in the viscosity of liquid-grown cultures, suggesting alterations in alginate production. As shown in Fig. 3(c), disruption of the PQS biosynthesis genes pqsB and pqsC reduced alginate production by FRD1, suggesting a relationship between quinolone signalling and alginate biosynthesis. Several genes not previously linked with alginate production were also identified here and include opdR, nuoA and aceA, all three of which are associated with carbon metabolism as indicated above.
FRD1 virulence mutants are defective for biofilm formation
P. aeruginosa is reported to form biofilms within the lungs of CF patients and this mode of growth likely facilitates persistence of the bacterium within this niche (Werner et al., 2004; Worlitzsch et al., 2002). Although CF isolates can be highly variable with respect to biofilm formation, strain FRD1 can form more robust biofilms than PAO1 under certain conditions (Lee et al., 2005; O'Toole & Kolter, 1998; Pham et al., 2004). We observed increased biofilm formation by FRD1 compared to PAO1 using the biostatic biofilm assay developed by O'Toole & Kolter (1998). Disruptions in pqsE, PA0428 (RNA helicase), opdR, nuoA and sbcB resulted in poor biofilm formation by FRD1, while a disruption of pqsC enhanced biofilm formation (Fig. 4). Several of the targets described here have not previously been associated with biofilm formation in P. aeruginosa and therefore may be specific for the ability of P. aeruginosa CF isolates to form biofilms.
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; McKinney et al., 2000). To determine whether aceA is required for virulence and persistence of P. aeruginosa, we generated a mutation in this gene in PAO1, an isolate known to persist in the rat chronic lung infection model. We previously determined that FRD1 is unable to establish an infection in this model system (Silo-Suh et al., 2002). We verified that the disruption of aceA rendered PAO1 unable to grow on acetate as a sole carbon source as previously described (data not shown and Diaz-Perez et al., 2007). As shown in Table 3, the PAO1 aceA mutant was significantly reduced for virulence on alfalfa seedlings. More importantly, disruption of aceA in PAO1 led to a fourfold reduction in histopathology in rat lungs, indicating that ICL is required for optimal virulence of P. aeruginosa in mammals (Table 3, Fig. 5).
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). The low ICL activity observed for PAO1 growing in L-broth is consistent with published results that aceA is specifically induced by carbon sources that utilize the glyoxylate pathway, such as fatty acids and acetate (Diaz-Perez et al., 2007; Honer Zu Bentrup et al., 1999). As expected, the aceA mutants of each parental strain were deficient for ICL and this phenotype was complemented in cis by the introduction of plasmid pJH122 containing a wild-type copy of aceA derived from FRD1 (Fig. 6a). The FRD1 and PAO1 aceA mutants were restored to approximately wild-type activity, indicating that the FRD1 aceA gene functioned comparably in each background.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In this study we identified genes required for FRD1, a CF isolate of P. aeruginosa, to infect alfalfa seedlings. On the basis of our earlier observations that FRD1 invokes novel strategies to infect alfalfa seedlings compared to the non-CF isolate PAO1, we believe some of these strategies to be important for the persistence of P. aeruginosa within the CF lung. Although our mutagenesis and screening strategy did not reach saturation of the P. aeruginosa genome, it does provide a glimpse of some of the virulence mechanisms functioning in FRD1. These include the glyoxylate pathway, biofilm activity and quorum sensing mediated by PQS.
We are especially intrigued by the large number of mutations affecting carbon metabolism because of the implications regarding P. aeruginosa's metabolism in the host and the potential avenues for therapeutic intervention. The requirement for ICL by FRD1 and PAO1 to infect alfalfa seedlings and by PAO1 to infect rat lungs indicates that the glyoxylate pathway plays an important role during P. aeruginosa pathogenesis. In a recent publication, lung surfactant lipids and amino acids were identified as carbon sources utilized by P. aeruginosa within the CF lung as indicated by high in vivo expression of the genes encoding catabolism or uptake of these compounds (Son et al., 2007). While considerable data previously supported that peptides and amino acids fuelled the growth of P. aeruginosa within the CF lung (Barth & Pitt, 1996; Palmer et al., 2005), our study supports Son et al. (2007) in that fatty acids are also important carbon sources for P. aeruginosa during chronic infection. The CF lung is rich in various fatty acids, some of which play important roles in lung function, including prostaglandins and phosphatidylcholine. Catabolism of such compounds might facilitate colonization of the CF lung by P. aeruginosa by impairing the functions of these compounds.
Also in agreement with Son et al. (2007), we report that aceA is more highly expressed in CF P. aeruginosa compared to non-CF isolates. However, our current data suggest that overexpression of aceA in FRD1 is due to aberrant regulation, most likely due to loss of a negative regulator. Consistent with this hypothesis is the observation that the FRD1 aceA gene complemented PAO1aceA to wild-type levels of ICL, suggesting that the FRD1 aceA gene is unaltered. Increased expression of aceA in FRD1 is a likely consequence of adaptation to the CF lung and, as such, expected to facilitate P. aeruginosa's ability to maintain infection within the CF lung. Our current studies raise the possibility of controlling P. aeruginosa infections within the CF lung with the use of drugs that inhibit ICL. The apparent absence of this enzyme in humans makes it an attractive therapeutic target.
Interestingly, several other genes we report here to be required for FRD1 virulence on alfalfa were shown to be either upregulated in P. aeruginosa within the CF lung, or constitutively expressed in P. aeruginosa CF isolates by Son et al. (2007). These genes include nuoA and PA4466 (npr). Therefore, the results of the alfalfa assay agrees with the conclusion of Son et al. (2007) that some highly expressed genes within P. aeruginosa CF isolates play important roles during infection.
The second recognized grouping of mutants reduced for virulence on alfalfa seedlings was impaired in PQS biosynthesis and response. Because PQS positively regulates the production of several virulence determinants, including pyocyanin, rhamnolipid, elastase and PA-IL lectin in PAO1, we considered the possibility that the reduced virulence observed for this class of mutants is related to the loss of these standard virulence determinants (Diggle et al., 2003). Not surprisingly, FRD1 was reduced for the production of elastase, phospholipase C and pyoverdine compared to the non-CF isolate PAO1. Loss or reduction of acute virulence determinants is a common feature in P. aeruginosa isolates adapted to the CF lung, which suggests that these products do not play a role in maintaining a chronic infection or are required in reduced quantities. Unexpectedly, disruptions in pqsB, pqsC and pqsE differentially affected several FRD1 phenotypes analysed in this report. The differential effects may be partially explained by the functions encoded by these genes. While PqsB and PqsC are predicted to play a role in synthesis of the quinolone, PqsE appears to be required for the cellular response to PQS (Diggle et al., 2006). We are interested in this class of mutants because the PQS signalling system is suspected of facilitating adaptation of P. aeruginosa to the CF lung environment during early infection (Guina et al., 2003). Furthermore, exogenously added PQS can affect P. aeruginosa biofilm development and PQS was recently demonstrated to regulate iron homeostasis and oxidative stress resistance in P. aeruginosa (Bredenbruch et al., 2006; De Kievit et al., 2001).
In addition to the two functional groupings of mutants recognized here, several of the mutants reduced for virulence on alfalfa have transposon insertions in genes that affect the envelope and outer-membrane properties of P. aeruginosa. These targets include two genes predicted to encode multidrug efflux proteins (PA3521 and PA4374) and PA0011, which is predicted to encode a lauroyl transferase with homology to HtrB. This protein functions in lipid A biosynthesis and is required for virulence by several human pathogens, including Haemophilus influenzae and Salmonella enterica serovar Typhimurium (DeMaria et al., 1997; Jones et al., 1997). Included in this group are the two mutants with transposon insertions in mdoH and mdoG, which encode membrane-derived oligosaccharides (MDOs). MDOs are branched substituted β-glucan chains present in the periplasm of various Gram-negative bacteria and their expression increases in low-osmolarity medium (Geiger et al., 1992). The MDO mutants tend to suffer pleiotropic effects, most likely due to altered membrane properties, which may account for the requirement for MDOs by a variety of plant and animal pathogens for virulence (Bhagwat et al., 2004; Page et al., 2001). Loss of MDOs can impair growth in hypo-osmotic media, alter chemotaxis and motility, alter porin composition and increase sensitivity to biliary salts (Fiedler & Rotering, 1988).
Six of the FRD1 transposon mutants reduced for virulence on alfalfa were affected in their ability to form static biofilms. Such a large grouping of mutants suggests a continuing role for biofilm formation, or maintenance, in chronic P. aeruginosa infections. Continued screening for chronic virulence determinants from CF isolates of P. aeruginosa will likely identify additional genes with important roles in persistence of this bacterium in eukaryotic hosts.
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ACKNOWLEDGEMENTS |
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Edited by: M. S. Ullrich
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Received 29 October 2007;
revised 29 January 2008;
accepted 3 March 2008.
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