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Institute of Medical Microbiology and Hygiene, Universittsklinikum Tübingen, Tübingen, Germany
Institute for Experimental Treatment of Cystic Fibrosis, HS Raffaele Scientific Institute, Milan, Italy
Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
Center for Biomedical Microbiology, BioCentrum, Danish Technical University, Lyngby, Denmark
Background.
In patients with cystic fibrosis (CF), lung infection with mucoid Pseudomonas aeruginosa strains overexpressing the exopolysaccaride alginate is preceded by colonization with nonmucoid strains. We investigated the kinetics, impact of environmental signals, and genetics of P. aeruginosa alginate expression in a mouse model and in patients with CF.
Methods.
Using indirect immunofluorescence, microarray technology and real-time reverse-transcription polymerase chain reaction, we assessed alginate gene expression during aerobic and anaerobic growth of the nonmucoid strain PAO1 in vitro, in a mouse lung-infection model and in sputum specimens from patients with CF infected with nonmucoid or mucoid P. aeruginosa strains.
Results.
Anaerobic conditions increased the transcription of alginate genes in vitro and in murine lungs within 24 h. Alginate production by PAO1 in murine lungs and by nonmucoid P. aeruginosa strains in patients with CF was reversible after in vitro culture under aerobic conditions. A subpopulation of P. aeruginosa clones revealing stable alginate production was detected in murine lungs 2 weeks after infection.
Conclusions.
Anaerobiosis and lung infection rapidly induce alginate production by gene regulation in nonmucoid P. aeruginosa. This trait may contribute to early persistence, leading to chronic P. aeruginosa infection once stable mucoid strains are generated.
Optimal survival of organisms under changing environmental conditions demands adaptation, which results in the emergence of new phenotypes [1]. In bacteria, signal-transduction systems by which the organisms sense the environment and change their phenotype accordingly have been described [2]. The presence of intricate global gene-regulation mechanisms that preserve broad cell versatility under many environmental conditions is thought to be one reason for the exceptional environmental and evolutionary success of microorganisms. Indeed, organisms with larger genomes, such as Pseudomonas aeruginosa, have a wealth of genes involved in transcriptional regulation [3]. The emergence of mutated subpopulations is thought to permit the persistence of bacterial species in a given ecological niche [47].
The airways of patients with the hereditary disease cystic fibrosis (CF) [8] may be regarded as an ecosystem in which P. aeruginosa must adapt to challenges from factors of the innate immune system, antibiotics, and growth in an microanaerobic/anaerobic environment [5, 9]. In response to anaerobic growth, P. aeruginosa can change its phenotype [9]; this process is characterized by the production of increased amounts of the extracellular polysaccharide alginate [1013]. The overexpression of alginate by P. aeruginosa leads to mucoid colony formation, which is a key factor in the organisms' persistence in the respiratory tracts of patients with CF.
Mucoid, alginate-overexpressing P. aeruginosa variants have recently been detected, 10.9 years after the acquisition of nonmucoid P. aeruginosa in patients with CF [12]; however, this may also occur earlier. Alginate overproduction has been linked to mutations in a gene cluster designated as the mucABCD genes, which encode proteins that inhibit the activity of the alternative -factor AlgU [14]. AlgU acts on the key alginate biosynthesis gene algD, which encodes a guanosine diphosphate mannose dehydrogenase, and on algR, a response regulator gene that increases alginate production [15]. Mutations in the negative regulators mucA, mucB, and mucD lead to alginate overproduction and conversion to a stable mucoid phenotype in P. aeruginosa [14, 1618]. However, the contribution of alginate gene regulation, as opposed to adaptive mutations, as a key factor in controlling the ability of P. aeruginosa to both establish and chronically infect the lungs of patients with CF in the infection process is still unclear, and this has not been investigated in vivo.
The objective of the present study was to investigate whether changes in gene regulation lead to an initial increase in alginate production that precedes the adaptive mutation causing full-fledged alginate overproduction in mucoid P. aeruginosa. To achieve this aim, we assessed alginate gene expression during aerobic and anaerobic growth of the nonmucoid strain PAO1 in vitro, in a mouse lung-infection model and in sputum specimens from patients with CF infected with nonmucoid or mucoid P. aeruginosa strains. Our data suggest that alginate gene regulation is a key factor in the process of establishing infection shortly after colonization by P. aeruginosa nonmucoid strains and before the mucoid phenotype becomes fully detectable on bacterial cells cultured in vitro.
PATIENTS, MATERIALS, AND METHODS
Bacterial strains, growth conditions, and patients with CF.
The nonmucoid laboratory strain P. aeruginosa PAO1, its mucoid isogenic variant PDO300 [3, 19], and Bacteroides fragilis (ATCC 25285) were used for in vitro experiments and mouse infection studies. Twenty-five environmental P. aeruginosa strains were isolated from water and salad sources, and 40 clinical strains were obtained from sputum or throat swabs from patients with CF. P. aeruginosa was cultured in trypticase soy broth (TSB) or was plated onto Columbia blood agar plates or Pseudomonas isolation agar (PIA; Heipha) for 24 h at 37°C. B. fragilis was grown in chopped meat carbohydrate medium [20]. For anaerobic growth, strain PAO1 was cultivated aerobically on solid medium for 12 h and then incubated anaerobically for an additional 12 h in anaerobic jars with Anaerocult A (Merck). For microarray experiments, strain PAO1 was grown in TSB/PBS medium (pH 7.4) overnight, adjusted to a starting OD600 of 0.05, and grown aerobically and anaerobically. For fermenter experiments, overnight cultures of PAO1 were adjusted to an OD600 of 0.1 in modified basic medium (10 g of casein hydrolysate, 5 g of yeast extract, 2.5 g of NaCl, 10 g of disodium hydrogenphosphatetrihydrate, and 10 L of antifoam solution [pH 7.25], supplemented with 0.5% glucose) and cultured to the midlog (4 h) and stationary (8 h) phases in a fermenter (Biostat Q-Fermenter; Braun-Biotech) under aerobic and anaerobic conditions. Anaerobiosis was created by bubbling filtered helium gas (150 mL/min) through the medium, and aerobiosis was created by use of filtered oxygen gas (150 mL/min).
Microbiological cultures of biological specimens from 24 patients revealed only nonmucoid strains of P. aeruginosa, whereas 16 patients carried at least 1 mucoid colony, as detected by use of a specific antialginate antibody (see below). The nonmucoid phenotype of P. aeruginosa was defined by the appearance of single colonies on agar plates (figure 1A). The mucoid phenotype of P. aeruginosa was defined as a viscous, slimy colony appearing on agar plates in which single cells could not be distinguished because of the production of copious amounts of alginate (figure 1B). Nonmucoid phenotypes may be alginate negative (figure 1D) or alginate positive (figure 1F), as detected by use of a specific antialginate antibody.
Agar bead preparation and mouse model.
Agar beads containing P. aeruginosa or B. fragilis were prepared as described elsewhere [21]. Sodium deoxycholic acid was omitted in washings of B. fragilis beads. C57Bl/6 male mice (Charles-River; weight, 2426 g) were infected as described elsewhere [21, 22]. After 1, 3, 7, 14, and 28 days, mice were killed, and lungs were excised, homogenized in PBS, and plated onto PIA. Additionally, 1 loop of PAO1, grown on blood agar, was processed for immunostaining. For histopathological analysis, lungs were removed en bloc and fixed in 4% paraformaldehyde/PBS for 24 h at 4°C and processed for paraffin embedding. Longitudinal sections of 5 m, collected at regular intervals, were obtained by use of a microtome from the proximal, medial, and distal lung regions. Sections were stained with hematoxylin-eosin (HE).
P. aeruginosa alginate gene expression.
To assess how alginate gene expression responds to a change from an aerobic to an anaerobic environment, P. aeruginosa Affymetrix GeneChips and LightCycler reverse-transcription polymerase chain reaction (RT-PCR) were used. For microarray experiments, RNA was isolated at 2 h from aerobically grown cultures and on days 1, 2, 3, and 4 for anaerobically grown cultures by use of Trizol (Gibco). Cell walls were disrupted with FastRNA tubes blue and FastPrepFP120 (Bio101). Contaminating DNA was digested by use of DNaseI (Ambion). Purified RNA was used for microarray experiments according to the manufacturer's procedure for use of the P. aeruginosa GeneChip (Affymetrix). Data analysis was performed by use of the Affymetrix Microarray Suite (version 5.0; Affymetrix UK) and Data Mining Tool (version 3.0; Affymetrix UK) software. Student's t test was applied for statistical analysis.
For real-time RT-PCR, fermenter-cultured PAO1 cells were incubated with RNA Protect Bacteria Reagent (Qiagen) and disrupted with lysozyme (1 mg/mL). RNA was isolated by use of the RNeasy Mini kit (Qiagen) and treated with DNaseI. This method was also used for the isolation of RNA from homogenized murine lungs and sputum specimens from patients with CF. Real-time RT-PCR was performed by use of a LightCycler (Roche) and the LightCycler-RNA amplification kit SYBR Green I (Roche). The following primers were used: algD, 5-TGTCGCGCTACTACATGCGTC-3 and 5-GTGTCGTGGCTGGTGATGAGA-3; and gyrA, 5-TGTGCTTTATGCCATGAGCGA-3 and 5-TCCACCGAACCGAAGTTGC-3. After RT for 20 min at 50°C, the following temperature profile was used: denaturation for 1 cycle at 95°C for 30 s; 45 cycles at 95°C for 1 s, 62°C58°C for 10 s, and 72°C for 13 s; and fluorescence acquisition at 62°C58°C in single mode. Melting curves were obtained from 45°C to 96°C by stepwise fluorescence acquisition.
Alginate determination.
P. aeruginosa alginate gene expression was quantified by use of the carbazole assay [23] and was determined by indirect immunofluorescence that used a rabbit antiserum specific for P. aeruginosa alginate [24]. The secondary antibody was indocarbocyanin-3 (Cy3) or Texas Redlabeled goat antirabbit IgG (Molecular Probes). For the in vivo experiments, 38 single clones and 4 mixtures of different clones from mice infected for 7 days and 101 single clones and 10 mixtures of different clones from mice infected for 14 days were randomly picked and analyzed for alginate production by use of the alginate-specific antiserum. Positive clones were subcultured twice for analysis of the stability of alginate gene expression. Because of the presence of a heterogeneous population of bacterial cells, clones were considered to be positive when at least 50% of the cells revealed positive staining with the alginate antiserum.
RESULTS
To determine whether such a phenotypical change is a general phenomenon, we tested 25 environmental P. aeruginosa strains for alginate production using the carbazol assay. Only traces of alginate were produced by the strains grown aerobically (mean ± SD, 0.022 ± 0.004 g of alginate/g of protein), whereas a mean (±SD) of 0.191 ± 0.037 g of alginate/g of protein was produced under anaerobic growth conditions. Similarly, under aerobic growth conditions, these strains were all found to be negative for alginate by immunofluorescence, whereas, under anaerobic growth conditions, they were found to be positive (data not shown). We therefore conclude that increased alginate production during anaerobic growth is a general phenomenon of P. aeruginosa strains.
When we used P. aeruginosa Affymetrix GeneChips to assess differential gene expression under anaerobic versus aerobic conditions, the expression of the positive alginate regulator algU increased 10-fold during days 14 of anaerobic growth, compared with that during aerobic growth (figure 2). Additionally, algR and algD transcription was also increased under anaerobic conditions. We also noted increased transcription of negative regulatory genes in the muc cluster, which may have served as a counterbalance to the increased transcription of algR and algD, resulting in increased alginate production without the transition to the full mucoid phenotype. Transcription of the napEFDABC and arcDABC clusters was increased, reflecting anaerobically increased nitrate and arginine turnover, whereas transcription of the housekeeping gene gyrA was not affected (figure 2).
These results were confirmed by real-time RT-PCR when strain PAO1 was grown in a fermenter under anaerobic and aerobic growth conditions for 4 and 8 h. Although, at 4 h, no difference was observed in algD expression, a 4-fold increase in algD expression was detected at 8 h under anaerobic versus aerobic growth conditions (figure 3).
Induction of anaerobic growth conditions for bacteria by agar beads leading to alginate gene expression in a mouse lung-infection model of P. aeruginosa.
We next monitored the kinetics of alginate production in vitro using the agar bead model of chronic P. aeruginosa lung infection [21]. We asked to what extent does this model mimic the microanaerobic growth conditions of P. aeruginosa in the CF lung [9]. The obligate anaerobe B. fragilis, embedded into agar beads, was incubated aerobically and anaerobically in vitro for 24 h at 37°C, and survival was quantified by plating serial dilutions on blood agar plates under anaerobic conditions. No statistical difference was observed in the survival of B. fragilis in beads incubated either aerobically or anaerobically (mean ± SD; aerobic, 0.92 × 109 ± 6.8 × 108 cfu/mL; anaerobic, 1.3 × 109 ± 9.5 × 108 cfu/mL; P = .455, Student's t test). When B. fragilis was grown in the absence of beads, growth was observed under only anaerobic conditions. HE staining of freshly prepared bead sections revealed low numbers of B. fragilis cells, whereas, 24 h after incubation, in the absence or presence of oxygen, there was bacterial growth inside the beads (data not shown). When B. fragilis, embedded in beads that contained 2 × 106 bacteria, was inoculated into the lungs of C57Bl/6 mice, 7.5 × 105 cfu of B. fragilis/lung were recovered after 24 h. The decrease in counts in vivo was not unexpected, because B. fragilis may escape from the anaerobic bead niche and subsequently be cleared by host defenses.
Next, we infected C57Bl/6 mice intratracheally with strain PAO1 embedded in beads. A total dose of 106107 cfu/mouse was found to cause only low mortality during the first 3 days of infection (figure 4), whereas higher doses resulted in 100% mortality, and lower doses did not cause a chronic infection (data not shown). Numbers of bacteria increased 1 day after infection, from 106107 to 6.3 × 109 cfu/lung, and decreased thereafter, to a mean of 2.3 × 104 cfu/lung at day 7 (figure 4). In parallel with the decrease in numbers of bacteria, the number of infected mice decreased after day 3, to 27.5% ± 3.5% (mean ± SD) on day 7. Thereafter, there were no significant differences in the percentage of infected mice (P = .75, 2 test) or the bacterial number of colony-forming units per lung during the next month (P = .55, Kruskal-Wallis test). The data suggest that a subpopulation of PAO1 that persists for at least 7 days is selected in vivo in approximately one-fourth of the mice.
To assess whether microaerobic or anaerobic growth conditions in agar beads would lead to a switch from a nonalginate-expressing to an alginate-expressing P. aeruginosa phenotype, as occurs in the airways of patients with CF [10, 12, 13], we analyzed infected murine lungs histologically and in situ bacteria by indirect immuofluorescence. One hour after infection, beads deposited in the bronchial lumen contained bacterial cells negative for alginate (figure 5A and 5B). However, 1 day after infection, bacteria formed alginate-expressing microcolonies (figure 5D and 5E). Real-time RT-PCR was used to measure algD gene expression in murine lungs. One day after infection, algD gene expression of strain PAO1 increased 4-fold, compared with that 1 h after infection (P < .05, Student's t test) (figure 3). Three days after infection, microscopic examination of HE-stained lung-tissue sections revealed that the number of beads per lung had decreased significantly (mean ± SD; day 1, 7.1 ± 3.5 beads/lobe; day 3, 0.3 ± 0.5 beads/lobe; P < .01, Student's t test), which suggests that the persisting alginate-positive PAO1 cells were protected from the murine respiratory defense system by the alginate coat and not by the beads.
Stability of alginate gene expression during chronic lung infection.
We next monitored the phenotype of the selected population of P. aeruginosa during the establishment of chronic airway colonization. PAO1 was positive for alginate in all lung homogenates obtained without subculturing from mice infected for 114 days (figure 6AC). P. aeruginosa recovered from murine lungs up to 7 days after infection reverted to a nonalginate-producing phenotype when they were cultured aerobically in vitro (figure 6D and 6E). However, later (weeks 24), many of the recovered colonies stably produced alginate aerobically in vitro (figure 6F). After 14 days of infection, 30.6% of the clones stably produced alginate in vitro. When selected alginate-positive clones were further subcultured aerobically (either once or twice), 67% and 60% of the clones, respectively, stained positive for alginate, which suggests a heterogeneous population among which the majority were stably producing alginate after 14 days in murine lungs. However, none of the colonies revealed the classic alginate-overexpressing mucoid CF phenotype.
Alginate gene expression of phenotypically nonmucoid and mucoid P. aeruginosa in sputum specimens from patients with CF.
To determine whether the increased production of alginate early after infection of mice with P. aeruginosa was representative of CF, we analyzed sputum specimens from patients with CF. As expected, sputum specimens from patients whose respiratory cultures grew mucoid P. aeruginosa when they were cultured aerobically (figure 7A) stained positive for alginate (figure 7B). However, sputum specimens from a patient with CF whose P. aeruginosa strain grew exclusively nonmucoid colonies on blood agar (figure 7D) also stained positive for alginate (figure 7E), which suggests a gene-regulation mechanism for alginate gene expression in the lungs of patients with CF similar to that in murine lungs. We used real-time RT-PCR to confirm the immunofluorescence data by measuring algD gene expression directly in sputum specimens from patients with CF. Compared with strain PAO1 expression, algD expression was increased by 5-fold in the sputum from a patient with CF whose respiratory cultures grew only nonmucoid P. aeruginosa (figure 3). A 9-fold increase in algD expression was detectable in sputum from patients with CF whose respiratory cultures grew mucoid P. aeruginosa (figure 3). No significant differences were detected in algD expression between the 2 patient groups (P = .3, Student's t test). The data demonstrate, for the first time, that P. aeruginosa that reveals a nonmucoid, alginate-negative phenotype when subcultured in vitro produces alginate within the CF lung habitat.
DISCUSSION
The exopolysaccharide alginate is regarded as an essential component of the mucoid P. aeruginosa phenotype, and it has been observed primarily on strains recovered from chronically infected airways of patients with CF [1013]. Also, phenotypically nonmucoid strains of P. aeruginosa from patients with CF have been reported to produce low levels of alginate [25, 26]. It appears that the elaboration of some alginate, even in the absence of a switch to a mucoid morphology, is critical for bacterial virulence in the setting of CF, given that an alginate-negative mutant of a phenotypically nonmucoid strain was unable to establish chronic colonization in mice with CF [27]. The isolation of mucoid strains from the airways of patients with CF indicates the onset of an increased rate of deterioration in pulmonary function [12, 28, 29]. Overall, it appears that there is a clear pathophysiological importance for alginate in chronic P. aeruginosa lung infection in patients with CF, but it is still not clear how fast alginate is produced when nonmucoid, alginate-negative P. aeruginosa strains colonize the airways of patients with CF. Our results show that nonalginate-producing P. aeruginosa rapidly expresses alginate when it is exposed to anaerobic conditions or is inoculated into murine lungs embedded in agar beads. Alginate gene expression is initially transient but becomes stable among a subset of clones after 2 weeks of animal infection.
Anaerobic conditions may be one stimulus for initiation of alginate production by P. aeruginosa. We have confirmed previous findings [9] that the microaerobic/anaerobic growth conditions present in agar beads rapidly induce a switch from a completely alginate-negative to an alginate-positive but nonmucoid phenotype in strain PAO1. As a biological marker for the degree of anaerobiosis, we used the obligate anaerobic microorganism B. fragilis. Inside beads, this bacterium grew well in the presence of air, which suggests that the beads decrease oxygen diffusion sufficiently to allow growth. The switch from a nonalginate-producing to an alginate-producing phenotype after the infection of mice with strain PAO1 was observed within 24 h. Although this model has been used by others [21, 22, 3032], changes in alginate elaboration by a nonmucoid P. aeruginosa strain has not been reported previously. Additionally, we found that, in sputum from patients with CF whose bacterial cultures grew only phenotypically nonmucoid colonies, there was detectable alginate production on a large number of bacterial cells. Our results suggest that, in agar beadinfected murine lungs as in the airways of patients with CF, alginate is elaborated by many P. aeruginosa strains shortly after infection.
The rapid switch of PAO1 to an alginate-positive, nonmucoid phenotype in the murine lung and under anaerobic growth conditions in vitro was reversible up to 7 days after infection when cells were cultured in an aerobic environment, which suggests that the initial increase in alginate production was due to changes in regulatory gene activity and not to newly acquired, stable mutations (data not shown). A similar finding has been obtained in an acute lung-infection model with strain PAO1, wherein no alginate was detectable on the surface of the infecting strain but, within 1 h of infection, bacteria in lung sections stained strongly positive for alginate gene expression [33]. Here, we have shown that specific genes involved in alginate biosynthesis and its regulation have increased transcription under anaerobic versus aerobic growth conditions, according to the results of DNA arrays and real-time RT-PCR, which corroborate data derived from indirect immunofluorescence with antialginate antibodies. These results are of clinical importance, because alginate-producing, nonmucoid P. aeruginosa variants may be present in the airways of patients with CF (but this phenotype may not by expressed by organisms cultured on agar plates in vitro) within days after colonization and may contribute to bacterial persistence [26]. Consistent with this conclusion are the results of a previous study that demonstrated the presence of serum antibodies to P. aeruginosa alginate in patients with CF harboring only nonmucoid strains, as determined by in vitro cultures from respiratory secretions [34]. Conversion to the stable, mucoid P. aeruginosa phenotype that produces high levels of alginate has been linked to mutations in the alginate repressor gene cluster mucABCD [14, 18, 35, 36]; however, whether such mutations would occur in alginate-producing, nonmucoid strains is not known and is subject to further investigations.
In summary, we found that anaerobic conditions induce a rapid production of alginate in P. aeruginosa and that this response is initially reversible but can become stable after a fairly short period (2 weeks) of chronic lung infection in mice. These findings corroborate those of an analysis of nonmucoid P. aeruginosa isolates from patients with CF, which showed that these phenotypes are also alginate positive. Because alginate gene expression by nonmucoid strains is key in the establishment and maintenance of chronic lung infection in transgenic mice with CF [27], it appears that this exopolysaccharide has to be regarded as one of the most important virulence factors of this opportunistic pathogen, at least in patients with CF.
Acknowledgments
We thank Lisa Cariani, for assistance in collecting the specimens from patients with CF; and Giliola Calori, for statistical analysis.
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