is responsible for chronic infections in the airways of cystic fibrosis (CF) patients (13
). During the course of chronic infection, P. aeruginosa
forms biofilms, which are thought to promote persistence by protecting the bacterium from antibiotics and host clearance. P. aeruginosa
also undergoes phenotypic and genotypic diversification. A manifestation of this diversification is the appearance of colony morphology variants among CF sputum sample isolates. One clear example of this phenomenon, which has been termed “dissociative” behavior (42
), is the appearance of mucoid colonies. Mucoidy is characterized by overproduction of the exopolysaccharide (EPS) alginate, a polymer of 1,4-β-linked mannuronic acid and its epimer, guluronic acid (13
). The appearance of mucoid colonies is thought to correlate with a downturn in the patient's prognosis and the onset of chronic colonization (12
). Besides mucoid colonies, rugose small-colony variants (RSCVs) have been isolated from CF sputum. RSCVs are selected for during the course of chronic infection, which suggests that they may play a role in pathogenesis (36
). RSCVs autoaggregate and are hyperadherent (10
), and recent studies have linked their appearance to prolonged antibiotic treatment (10
biofilms grown in the laboratory also produce colony morphology variants, including RSCVs (5
). Like CF sputum-derived RCSVs, laboratory-derived RSCVs autoaggregate in liquid culture and hyperadhere to surfaces. We have characterized biofilm-derived RSCVs and found that at least one EPS biosynthetic locus, the psl
gene cluster, contributes to the autoaggregation and hyperadherence phenotypes (21
). The psl
locus (PA2231 to PA2245) is responsible for the production of a mannose-rich EPS (11
). Cyclic di-GMP (c-di-GMP) signaling has also been implicated in the RSCV phenotype (17
). For example, mutations in wspF
, which result in constitutive activation of the diguanylate cyclase, WspR, result in elevated intracellular c-di-GMP levels. A wspF
mutation is capable of converting a wild-type smooth strain into an RSCV, and subsequent depletion of c-di-GMP in a wspF
mutant strain can convert the morphology back to a wild-type morphology (7
The relationship between biofilm-derived and CF-derived RSCVs is unclear. Although these variants share some common phenotypes, such as hyperadherence and autoaggregation in liquid culture, some interesting phenotypic differences have been reported. For example, von Gotz et al. performed a transcriptional analysis of a clinical RSCV and found that type III secretion genes were induced in RSCVs (41
). These investigators also reported that the RSCV cells were hypermotile and more cytotoxic to macrophages. The motility of these cells is in stark contrast to the reduced motility reported for biofilm and other clinical RSCVs (17
). It is difficult to know with certainty whether the motility phenotypes of clinical isolates were RSCV specific because the phenotypes of the wild-type parents of the clinical RSCV isolates were unknown.
Here, we report isolation and characterization of clonally related wild-type and RSCV strains from the same CF sputum sample. Our results show that the transcriptional profiles of the clinical and laboratory RSCVs are very similar. We demonstrated that the pel and psl EPS loci contributed to the clinical RSCV phenotype. Also, all clinical and laboratory RSCVs examined in this study require the intracellular signaling molecule c-di-GMP for the RSCV phenotype. Due to the importance of c-di-GMP for this phenotype, we determined the transcriptional profile of an RSCV strain (with an elevated c-di-GMP level) and compared it to that of a psl pel mutant derivative that has an elevated intracellular c-di-GMP level but does not autoaggregate. We identified two subsets of differentially expressed genes. One subset appears to be differentially expressed as a consequence of liquid culture autoaggregation. The second, smaller subset is differentially expressed in response to elevated intracellular c-di-GMP levels. One set of regulated functions suggests that there is increased expression of factors involved in dampening the host immune response. Compared with the progenitor wild-type strains, both clinical and biofilm RSCVs elicited a reduced chemokine response from polarized airway epithelium. Our study suggests that RSCVs represent an adaptation that allows P. aeruginosa to persist in CF biofilm communities.
MATERIALS AND METHODS
Strains and growth media.
All relevant P. aeruginosa
strains and plasmids are described in Table 1
. Three separate samples from a longitudinal CF collection (6
), samples 009 37 to 009 39, produced two distinct colony morphologies on agar plates. The two colony morphotypes were separated and designated CF37wt to CF39wt for colonies resembling PAO1 colonies and CF37s to CF39s for colonies similar to colonies of laboratory-derived RSCV strains. Cultures were routinely grown on LB agar or in LB broth. Antibiotic selection was performed with 1.0 μg/ml carbenicillin and 1.5 μg/ml of gentamicin for clinical strains CF37 to CF39 and with 300 μg/ml carbenicillin and 100 μg/ml gentamicin for all other P. aeruginosa
strains. Plasmids were maintained in Escherichia coli
using 15 μg/ml gentamicin and 100 μg/ml ampicillin.
To determine doubling times, the appropriate strains were inoculated into separate 250-ml baffled Erlenmeyer flasks containing 50 ml LB broth and a stir bar. The flasks were incubated at 37°C with shaking, and samples were removed periodically over a 3-h period. Each sample was divided into aliquots to measure growth in two different ways: optical density at 600 nm (OD600) and protein content. The OD600 sample aliquots were vortexed for 10 s, and the OD600 was measured in duplicate. The cells from the protein content sample aliquot were pelleted, washed, and resuspended in 100 mM Tris-HCl (pH 7.2). Three aliquots of the resuspended cells were placed in a microtiter plate, and the cells were lysed with a microplate horn sonicator (20 kHz; XL-2020; Misonix, Inc., Farmingdale, NY). The cells were sonicated for 1 min at an output of 7.5, followed by a 1-min rest period; this cycle was repeated a total of 12 times. The lysed cells were subjected to a standard microplate protein assay (Coomassie Plus protein assay reagent kit; Pierce Biotechnology, Inc., Rockford, IL).
PCR was used to confirm the presence of psl
DNA in the clinical RSCV backgrounds. Mutants were generated by allelic exchange (18
mutants were constructed using a previously described allelic replacement construct, pMPSL-KO1(21
). A pelA
allelic replacement construct (pMPELA) was generated by first amplifying a 5-kb DNA fragment encompassing PA3064 and cloning it into suicide vector pEX18.Ap. A blunt-ended SacI fragment from pPS858 was then inserted into the blunt-ended SmaI and XbaI sites. The resulting plasmid, pMPELA, was mated into P. aeruginosa
strains, and mutants were selected on Pseudomonas
isolation agar containing gentamicin. Double-recombinant mutants were selected on LB agar plates containing 5% sucrose and confirmed by PCR. A PA1169 mutant strain was generated by amplifying a 3.4-kb segment of genomic DNA encompassing PA1169 and cloning it into pEX18.Ap via SacI and HindIII sites incorporated into the primers. Internal ClaI and BamHI sites were used to replace most of PA1169 with the blunt-ended SacI fragment from pPS858, resulting in plasmid pM1169. Mutant strains were generated as described above. To create a cupA3
mutant, primers cupA3
were used to amplify a 3.6-kb sequence including cupA3
, which was cloned into pEX18.Ap using SacI and HindIII. The resistance cassette from pPS858 was inserted via blunt-ended EcoRI and SalI sites found in the cupA3
gene, creating plasmid pMCUPA3. Following allelic replacement of the cupA3
gene, the resistance cassette was excised using pFLP2, and colonies were selected for sucrose resistance and carbenicillin sensitivity (18
Cultures were grown in 250-ml baffled flasks in LB broth supplemented with 100 mM morpholinepropanesulfonic acid (MOPS) (pH 7). Stir bars were added to reduce clumping, and cultures were shaken at 37°C. Log-phase cells at an OD 600
of 0.25 were harvested in RNA Protect (Qiagen, Inc., Valencia, CA). RNA and cDNA were prepared as described by Schuster et al. (33
). Samples were hybridized to Affymetrix P. aeruginosa
GeneChips at the University of Iowa DNA Facility. Replicates from biologically distinct experiments were analyzed for each strain. Data were analyzed using GeneSpring software and the online program Cyber-T (http://visitor.ics.uci.edu/genex/cybert/index.html
Reverse transcription (RT)-quantitative PCR (qPCR).
Cultures were grown as described above and harvested during logarithmic growth. RNA was isolated using reagents from a RiboPure bacterial kit (Ambion, Austin, TX). Cells were pelleted and resuspended in 700 μl RNAwiz. The cell suspension was vortexed for 20 s, sonicated for 5 min, and passed through a 19.5-gauge syringe needle 10 times. Cell suspensions from duplicate cultures were transferred to a single 2-ml RNase-free microcentrifuge tube with 500 μl of 100-μm zirconia beads and bead beaten for 10 min. The remainder of the RNA purification procedure was performed according to the manufacturer's instructions. RNA was eluted with two 50-μl aliquots of elution solution. Residual DNA was removed by DNase I treatment as described in the RiboPure bacterial kit instruction manual. DNase-treated RNA was ethanol precipitated, and the pellet was resuspended in RiboPure bacterial elution solution.
Duplicate cDNA synthesis reactions were performed with random hexamer primers and avian myeloblastosis virus reverse transcriptase (Roche, Germany). Four micrograms of RNA was mixed with 2 μl of primer (10 μM) and nuclease-free water (Ambion, Austin, TX) to obtain a final volume of 10 μl. The RNA mixture was denatured at 70°C for 5 min and placed on ice. A 4-μl aliquot of 5× first-strand buffer, 2 μl of a deoxynucleoside triphosphate mixture (10 mM of each nucleoside; New England Biolabs, Beverly, MA), 2 μl of water, and 2 μl of avian myeloblastosis virus reverse transcriptase were added to each reaction mixture on ice. Each tube was incubated at 42°C for 1.5 h in a Primus 25 personal cycler with a heated lid (MWG Biotech, High Point, NC). At the end of the first-strand synthesis, the reaction mixtures were placed on ice. Negative controls for RT-qPCR were prepared by omitting the reverse transcriptase during the cDNA synthesis reactions, and these controls were used to verify that significant amounts of contaminating genomic DNA were not present in the total RNA preparations. Duplicate cDNA reaction mixtures were pooled and used to perform triplicate real-time qPCRs. qPCRs were performed with an Applied Biosystems 7900HT real-time PCR system (Applied Biosystems, Foster City, CA) using DyNAmo HS SYBR green qPCR kit reagents (Finnzymes, Finland) and following the manufacturer's instructions. Primers are listed in Table 1
. The final primer concentration was 0.3 μM for all reactions. The following thermocycler program was used: 95°C for 15 min, followed by 40 cycles of 94°C for 10 s, 60°C for 30 s, and 72°C for 30 s. Fivefold serial dilutions of cDNA prepared from a mixture containing equal aliquots of cDNA from the strains were used to construct a relative standard curve. The cycle number at which the fluorescence crossed a selected threshold value during linear amplification was correlated to a relative quantity. Real-time PCR product dissociation curves were used to verify the specificity of the amplified product. Relative quantities of PA3064 and PA1169 were normalized to relative quantities of PA4946 and PA1615.
wspF and PA2133 complementation.
To express wspF in RSCV backgrounds, plasmid pSP5 was generated by cloning wspF plus 18 bp upstream of the start codon into the EcoRI and XbaI sites of pUCP18. To express an EAL domain protein in RSCV backgrounds, plasmid pSP6 was generated by cloning PA2133 plus 22 bp upstream of the start codon into the EcoRI and XbaI sites of pUCP18. Constructs were electroporated into P. aeruginosa strains.
Procedures for two-dimensional (2D) thin-layer chromatography (TLC) were adapted from procedures described previously (3
). Bacteria were grown overnight in MOPS minimal medium with 30 mM succinate and 0.15 mM KH2
and then subcultured in the same medium to an OD600
of 0.05. After one or two doublings, 0.1 mCi of [32
P]orthophosphate (Perkin-Elmer) was added to 1 ml of culture and incubation was continued overnight (12 h), after which 10 μl of 1 M cold formic acid was added to 100 μl of the labeled cell culture.
Polyethyleneimine-cellulose plates (Selecto Scientific) were washed in 0.5 M LiCl and then in distilled H2O and allowed to air dry. Radioactivity in formic acid extracts was measured using a scintillation counter. Equal amounts of total radioactivity from each extract were spotted on a TLC plate. Plates were soaked in methanol for 5 min, air dried, and developed in 0.2 M NH4HCO3 (pH 7.8) in the first dimension. The plates were then soaked in methanol for 15 min, dried, and developed in the second dimension with 1.5 M KH2PO4 (pH 3.65). Plates were soaked again in methanol for 15 min, air dried, and exposed to a phosphorimager screen overnight. The data were collected with a Storm 860 phosphorimager and analyzed using ImageQuant software (Molecular Dynamics).
Phenotype MicroArray analysis.
Phenotype MicroArray analysis (Biolog, Hayward, CA) was performed in duplicate according to the manufacturer's instructions. P. aeruginosa
strains were streaked on plates containing LB agar diluted 1:5 (2 g tryptone per liter, 1 g yeast extract per liter, 2 g NaCl per liter, 15 g agar per liter). Colonies were scraped from the plates and suspended in IF-0 inoculating fluid to obtain 85% transmittance. Cells were diluted 1:200 in IF-0 minimal medium containing Biolog redox dye mixture A, and 100-μl aliquots were added to carbon source plates (PM1 and PM2). For the remaining metabolic plates (PM3 to PM8), inocula were supplemented with 20 mM sodium succinate and 2 μM ferric citrate. The plates were incubated at 37°C in an OmniLog plate reader, and growth was measured kinetically by determining the colorimetric reduction of a tetrazolium dye. Data were analyzed with the Biolog Kinetic and Parametric software (version 1.20.02). The keys for determining the test conditions for each well in Fig. 8
and Fig. S6 in the supplemental material are available at http://www.biolog.com/pmMicrobialCells.html
Polarized epithelium chemokine signaling assays. (i) Preparation of bacterial cultures.
P. aeruginosa cultures grown for 16 h in LB broth were vortexed for 30 s to disperse aggregates. Cells were harvested, washed with phosphate-buffered saline (PBS), and diluted in PBS to obtain a final concentration of 106 CFU per 50 μl.
(ii) Airway epithelial cell culture.
Calu-3 cells (ATCC HTB-55) were prepared and grown at the air-liquid interface with medium containing Ultroser-G (Biosepra, France) as previously described (20
). Penicillin, streptomycin, gentamicin, and fluconazole used for initial establishment of epithelial cultures were removed by repeated apical and basolateral washing with 1:1 Dulbecco's modified Eagle's medium—Ham's F-12 medium (Gibco, Carlsbad, CA) and antibiotic-free medium for 4 to 5 days prior to apical stimulation with bacteria. Additionally, to decrease the stimulatory effects of serum products, Ultroser-G was omitted from the medium starting 3 days prior to interleukin-8 (IL-8) or NF-κB experiments.
(iii) Airway epithelial NF-κB signaling.
Airway epithelial cultures were initially prepared as described above. Cell cultures were basolaterally infected with a replication-incompetent adenoviral vector (multiplicity of infection, 10) containing NF-κB response elements driving a luciferase reporter as previously described (32
). One day after transduction, epithelia were apically inoculated with 1 × 106
CFU of bacteria suspended in 50 μl of PBS using the following bacteria (the numbers of replicate independent experiments are indicated in parentheses): PAO1 (10), MJK8 (10), MJK8PA1169
(6), CF39wt (4), CF39s (4), and the PA1169 mutant strain (4). As a negative control, epithelia were incubated with 50 μl of PBS apically and 500 μl of medium basolaterally, and as a positive control, apical PBS and basolateral medium contained 100 ng/ml recombinant human IL-1β (Sigma, St. Louis, MO). Four hours following stimulation with PBS, IL-1β, or bacteria, cells were lysed, and luciferase activity was measured (luciferase assay system; Promega Madison, WI) according to the manufacturer's recommendations using a Monolight 3010 luminometer (Pharmogen, San Diego, CA).
(iv) Basolateral chemokines and cytokine abundance.
Airway epithelial cultures were prepared and grown in antibiotic- and serum-free conditions as described above. Cultures were inoculated with bacteria as described above for NF-κB signaling. We measured IL-8 protein concentrations in basolateral medium using IL-8 (human IL-8 OptEIA set; BD Biosciences, San Jose, CA) enzyme-linked immunosorbent assay kits according to the manufacturer's instructions. All enzyme-linked immunosorbent assays were performed in duplicate to ensure reproducibility. With one exception, experiments were performed with matching cultures for NF-κB experiments but without adenoviral reporter vector infection. The numbers of replicate-independent experiments for the bacterial strains were as follows: PAO1, nine; MJK8, nine; MJK8PA1169, five; MJK8ΔpslBCDpelA, four; CF39wt, five; CF39s, five; and PA1169, five.
Previous studies have reported isolation of P. aeruginosa RSCVs, and there is evidence that these variants play a role in disease pathogenesis. In this study, we report isolation and characterization of clonally related RSCV and wild-type isolates from a CF sputum sample. We demonstrated that our clinical RSCV displayed many of the same characteristics as laboratory biofilm RSCV isolates. One such characteristic is the contribution of the Pel and Psl EPS to the phenotype. Another similarity is the elevated c-di-GMP levels in the clinical RSCV compared to the clinical wild-type strain. Overexpression of a phosphodiesterase that degrades intracellular c-di-GMP eliminated the autoaggregation and hyper-biofilm-formation phenotypes in all the RSCV strains tested. This suggests that c-di-GMP plays a central role in modulating the transition between wild-type and RSCV phenotypes.
Our transcriptome and Biolog profiling data suggest that the clinical and laboratory-derived RSCVs that we examined are physiologically similar. The autoaggregation of RSCV cells in liquid culture affects the expression of a large subset of genes, including some genes that impact metabolism. For example, RSCV strains were characterized by increased expression of denitrification genes. The expression of these genes likely occurred in response to anaerobic pockets that developed in aggregates of cells as they consumed the available oxygen. Biolog phenotyping data suggest that autoaggregation has other effects on metabolism. RSCV cells were deficient in growth on several carbon sources (Fig. 8
). This also appeared to be a consequence of autoaggregation, since the wspF pelA pslBDCD
triple mutant (which exhibited no autoaggregation but had elevated c-di-GMP levels) had wild-type carbon utilization patterns. This suggests that in laboratory biofilms and CF airways, the carbon sources that RSCVs can utilize may be limited. Interestingly, we found that the type VI secretion locus HSI-I was upregulated in the clinical RSCV. Antibodies to Hcp, the secreted effector, have been used to detect Hcp in sputum from a CF patient infected for a long time; thus, type VI secretion may be another hallmark of chronic infections (28
The differential expression of a second, smaller subset of genes in RSCVs is a consequence of elevated cyclic-di-GMP levels. It appears that motility and EPS production are major functions that are transcriptionally controlled by c-di-GMP. Some of the differentially expressed genes are known to be controlled by FleQ, a cyclic-di-GMP-responsive transcriptional repressor (16
). However, some genes whose expression is impacted by elevated cyclic-di-GMP levels (e.g., PA0169 to PA0172) are not regulated by FleQ. This suggests that there may be other cyclic-di-GMP-sensitive transcriptional regulators.
Data presented here and elsewhere show that the pel
EPS biosynthetic loci are key contributors to the RSCV phenotype. Single mutations in either of these gene clusters have only a partial effect on RSCV phenotypes. Only pel psl
double mutations fully convert RSCVs to wild-type strains. It is not clear whether these EPS types are functionally redundant or if they have unique roles in RSCVs. Interestingly, in contrast to previous reports, the cupA
operon did not appear to contribute to the RSCV phenotype (26
). Insertion-deletion mutations in cupA3
appeared to suppress the RSCV phenotype; however, excision of the antibiotic cassette insertion resulted in reversion back to the RSCV phenotype. One potential explanation for this is that the antibiotic resistance cassette induces expression of the last gene in the operon, PA2133 encoding the c-di-GMP-degrading phophodiesterase. Our analysis of intracellular c-di-GMP levels supports this; the c-di-GMP levels were depleted in the insertion mutant, while excision of the antibiotic resistance cassette restored c-di-GMP levels (see Fig. S4 in the supplemental material). Meissner et al. reported upregulation of CupA fimbriae in RSCV backgrounds. We have evidence that liquid culture autoaggregation creates anaerobic pockets in the aggregates. Since microaerobic conditions induce cupA
expression, we think that reduced oxygen tension in RSCVs might have contributed to the cupA
expression in the study of Meissner et al. (2
The complementation analysis suggests that there are two classes of variants based on wspF
complementation. Even though these two classes appear to have many phenotypic similarities, we do not know whether there are any important phenotypic differences. Although the levels of intracellular c-di-GMP are high for both classes, the quantities may differ. This may affect Pel and Psl expression levels, which in turn have the potential to impact the degree of autoaggregation. RSCVs characterized by higher cyclic-di-GMP levels may have differences in key phenotypes, such as antimicrobial resistance. A challenge for the future is to identify the mutations that confer the RSCV phenotype for class B strains. Once this has been achieved, functional comparisons can be made among isogenic RSCV strains. von Gotz et al. reported that an RSCV-like strain isolated from a CF patient is highly motile and extremely cytotoxic and expresses high levels of type III secretion genes (41
). In our transcriptional analysis, we found that the RSCVs did not express elevated levels of type III secretion genes. The differences between the results illustrate the fact that not all RSCV strains are exactly alike (15
What are the selective forces in the CF lung that are responsible for selecting for RSCV formation? Are these selective forces the same as those that produce RSCVs in laboratory-cultured biofilms? Since chronic CF infections involve biofilms, some selective pressure present in both laboratory biofilms and CF biofilms might amplify the RSCV phenotype (35
). Previous studies have shown that RSCVs exhibit elevated tolerance to hydrogen peroxide (5
). Recent work by Boles and Singh also identified oxidative stress as a selective pressure for diversification in laboratory biofilms (4
). One stimulus for the generation of mucoid variants in the lung is thought to be reactive oxygen species (24
). Perhaps oxidative stress present in both lab and CF biofilms is a key selective pressure for RSCVs as well.
RSCV strains appear to grow relatively poorly on certain carbon and sulfur sources, including amino acids. Amino acids are suggested to be a major constituent of airway secretions, and they presumably support the growth of P. aeruginosa
and other colonizing species (29
). RSCVs may not be able to compete with wild-type strains (or other bacterial species) for amino acids, which may explain why they are never the predominant organisms in P. aeruginosa
strains isolated from a given patient. The heightened resistance of RSCVs to antimicrobials may allow them to successfully compete for growth substrates in microniches subjected to elevated antimicrobial stress, where wild-type P. aeruginosa
strains and other bacterial species would be impaired. Alternatively, RSCVs may be better adapted to use other growth substrates with which they do not have such a severe growth handicap, such as fatty acid and hydrophobic substrates. For example, RSCVs grew as well as wild-type strains on Tween substrates. The CF airways contain host-derived fatty acids and lipids. In addition, hydrophobic substrates are also present in older laboratory biofilms as cells begin to lyse. Consideration of all these points leads to the hypothesis that RSCVs may occupy a nutritional niche in these biofilm environments.
The CF host mounts a tremendous immune response to chronic infection (13
). This response probably exhibits temporal and spatial variability in the airways. Given their highly autoaggregative nature, RSCVs probably exist in the airways as distinct aggregates. The local host immune response surrounding these aggregates may be dampened due to reduced expression of flagella and an increase in expression of RSCV-induced immunomodulatory functions. Thus, RSCVs may represent a nidus of persistence that can contribute to reseeding of the airways after a course of antibiotic treatment. Precisely how the RSCV strains, of both clinical and biofilm origin, elicit a reduced inflammatory response is unknown. One observation from the array data is that flagellar genes are downregulated in RSCV strains. Since flagellar expression is known to cause Toll-like receptor 5-mediated inflammation in the host, this might be one factor contributing to the response in our assays (31
). Collectively, the hyper-biofilm-formation, increased antibiotic tolerance, and reduced inflammation traits of RSCVs suggest they are a subpopulation geared toward persistence in biofilms and the CF airway environment.