| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Institute of International Health, Immunology and Microbiology, University of Copenhagen, Rigshospitalet, Copenhagen, Denmark
2 Department of Clinical Microbiology, University Hospital, Rigshospitalet, Copenhagen, Denmark
3 Uppsala CF Center, Department of Women's and Children's Health, Uppsala University, Uppsala University Hospital, SE-75185 Uppsala, Sweden
4 Department of Microbiology, Ullevaal University Hospital, Oslo, Norway
5 Lund CF Center, Children Hospital Lund, 221 85 Lund, Sweden
Correspondence
Oana Ciofu
o.ciofu{at}immi.ku.dk
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Høiby, 1977). This stage is temporary and represents a window of opportunity for the aggressive treatment of the infection (Frederiksen et al., 1997; Høiby et al., 2005). Untreated, the infection will eventually become chronic, characterized by the appearance of mucoid P. aeruginosa isolates (Doggett et al., 1966; Høiby, 1977; Pedersen, 1992) and an inflammatory response dominated by polymorphonuclear leukocytes (PMNs) (Høiby et al., 1993). It has been shown that the reactive oxygen species liberated by PMNs can cause conversion of P. aeruginosa from non-mucoid to mucoid (Mathee et al., 1999). Besides the oxygen radicals, various other stress conditions occur in the CF lung environment, such as high osmolarity, dehydration, nutrient limitation and antibiotics, all of which may affect alginate production (Bayer et al., 1990; Berry et al., 1989; DeVault et al., 1990; Speert et al., 1990; Terry et al., 1991; Bagge et al., 2004).
Once the infecting bacteria become mucoid, the infection is very difficult to eradicate due to the biofilm mode of growth (Høiby et al., 2005). Clinically, the presence of mucoid variants is associated with poor prognosis, deterioration of the lung function and increased tissue damage (Pedersen, 1992). The main characteristic of the mucoid phenotype is the production of a thick mucopolysaccharide layer consisting of the exopolysaccharide alginate (Evans & Linker, 1973). Great phenotypic heterogeneity has been shown among mucoid and non-mucoid CF isolates, including colony morphology, biofilm formation properties, antibiotic resistance and media-dependent alginate production (Fyfe & Govan, 1980; Govan et al., 1983; Pugashetti et al., 1982; Drenkard & Ausubel, 2002; Haussler, 2004; Lee et al., 2005).
The alginate biosynthesis operon (algD–algA, PA3540–PA3551) is under the control of the algD promoter. A key element in algD gene regulation is the alternative sigma factor AlgT (also termed AlgU or 
22), which induces the expression of algD and increases the expression of regulatory proteins that enhance algD transcription (Govan & Deretic, 1996). The algT gene belongs to an operon with four other genes, mucA-mucB-mucC-mucD.
Mutations in either mucA, mucB or mucD can lead to conversion to mucoidy (Martin et al., 1993a, b; Goldberg et al., 1993; Wood & Ohman, 2006), suggesting that the products of these three genes have negative regulator effects on algT. While mucA mutants are highly mucoid, mucB mutants have a slightly mucoid phenotype (Martin et al., 1993a; Mathee et al., 1997), suggesting that MucA is the primary control element of AlgT activity. Mutations in P. aeruginosa mucD confer alginate production during growth on certain media (e.g. Pseudomonas isolation agar), suggesting that MucD can also act as a negative effector of AlgT activity (Boucher et al., 1996).
MucA acts as an anti-sigma factor that binds and sequesters AlgT, thus affecting its ability to transcribe (Schurr et al., 1996). MucB is also a negative regulator that binds to MucA and forms a signal transduction complex that normally keeps the AlgT transcriptional activity low (Schurr et al., 1996; Mathee et al., 1997; Rowen & Deretic, 2000). The function of MucC is generally unknown but it has been reported as being both a positive and a negative regulator (Boucher et al., 1997a). MucD is a close homologue of Escherichia coli HtrA (DegP), a periplasmic serine protease that is involved in the proteolysis of abnormal proteins and is required for resistance to oxidative and heat stress (Boucher et al., 1997b). It has been shown that the proteolytic motif of MucD is important for its regulation of alginate production as well as temperature resistance (Wood & Ohman, 2006). The mucD gene also has a secondary promoter, independent of AlgT, within the algT operon (Wood & Ohman, 2006).
Activation from a distance is a unique feature of the algD promoter. Besides the control exerted by AlgT, MucA and MucB, the response regulators AlgR (Deretic et al., 1989; Nikolskaya & Galperin, 2002), AlgB (Wozniak & Ohman, 1991; Goldberg & Dahnke, 1992) and the DNA-binding protein AlgZ (Baynham et al., 1999) also exert control of algD transcription.
Besides controlling alginate production, AlgT is the P. aeruginosa equivalent of the extreme heat-shock sigma factor E of E. coli (Yu et al., 1995). Congruent with the functions played by the E in E. coli, AlgT is involved in the regulation of a number of other systems in P. aeruginosa, such as heat shock, osmotic protection and protection against reactive oxygen species (Martin et al., 1994; Yu et al., 1995; Firoved et al., 2002). It has also been shown that AlgT represses flagellum biosynthesis in P. aeruginosa (Tart et al., 2006). Thus, the presence of uncontrolled AlgT in mucoid isolates interferes with the normal physiology of the cell and therefore the selective advantage of mucoid forms must be critical for the ability of this organism to persistently colonize and chronically infect CF patients (Schurr et al., 1994). Besides, bacteria use a lot of energy to produce alginate and therefore it is not surprising that the mucoid phenotype is highly unstable in the absence of the selective pressure, e.g. once the strains are taken out from the CF lung (Govan et al., 1979; Ohman, 1981).
Non-mucoid isolates are often isolated from the CF sputum samples with or without simultaneous isolation of the mucoid isolates (Doggett, 1969; Høiby, 1975). These non-mucoid isolates are either wild-type P. aeruginosa strains or revertants from the mucoid isolates (Schurr et al., 1994). The revertants might occur by repair of the mutation in mucA, or by secondary-site mutations in algT or other alginate regulatory genes (DeVries & Ohman, 1994). Inactivation of algT is the most frequent mechanism that has been demonstrated for revertants obtained in vitro (DeVries & Ohman, 1994; Schurr et al., 1994). However, this is not the only mechanism of conversion to the non-mucoid phenotype; several alternative pathways have been suggested for the reversion to the non-mucoid state (Schurr et al., 1994). It has been shown that the non-mucoid revertants due to mutations in algT become motile through upregulation of the flagella and this was considered an adaptive mechanism allowing P. aeruginosa to swim towards areas of higher oxygen concentration (Wyckoff et al., 2002; Tart et al., 2006). However, the nature of the non-mucoid isolates has not previously been investigated on a large number of P. aeruginosa isolates collected from the sputum of CF patients.
In a recent study of 37 sequential isolates from 10 CF patients attending the Hannover CF Clinic, Germany, mucA mutations were found in both mucoid and non-mucoid isolates but the occurrence of secondary-site mutations responsible for the reversion to the non-mucoid phenotype was not investigated (Bragonzi et al., 2006).
The purpose of the present study was to analyse the mutations in the algTmucABD operon, which encodes the most important regulators of alginate production. This was performed in a large collection of mucoid and non-mucoid isolates from the sputum of Scandinavian CF patients with chronic or intermittent P. aeruginosa infection as well as in in vitro-obtained non-mucoid revertants. The correlation between the genotype of the alginate regulatory genes and the alginate morphotypes is discussed.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
In addition, non-mucoid isolates from 24 early intermittently colonized Danish CF patients were included in this study.
The CF patients were considered chronically infected when P. aeruginosa was cultured in the sputum for six consecutive months or serum contained
2 precipitating antibodies to P. aeruginosa by crossed-immunoelectrophoresis (Høiby, 1977).
Sputum samples obtained by expectoration or endolaryngeal suction were Gram-stained and examined under the microscope to confirm the origin from the lower airways, with the exception of the samples from Norway. The sputum samples of the 83 Scandinavian CF patients were plated on Blue agar plates (a modified Conrad Drigalski medium selective for Gram-negative rods, Statens Serum Institute, Copenhagen, Denmark, containing: peptone 10 g, yeast extract 5 g, NaCl 5 g, agar 11 g, detergent 0.05 g, sodium thiosulphate 1 g, bromthymolblue 0.1 g, lactose 9 g and glucose 0.4 g).
One mucoid and one non-mucoid P. aeruginosa isolate, which were simultaneously present in the sputum samples of the 83 Scandinavian patients, were collected from the Blue agar plate and considered a pair of isolates.
The sputum samples of the eight Danish CF patients who were registered in the records as presenting only non-mucoid isolates were plated on PIA (Pseudomonas isolation agar, Difco, containing peptone 20 g, magnesium chloride 1.4 g, potassium sulphate 10 g, Irgasan 25 mg and agar 13.6 mg), LB and Blue agar plates and incubated for at least 7 days. Colonies with different morphotypes were observed and a total of 20 colonies were isolated (one and up to four different colonies per sputum sample).
From the sputum samples of CF patients with intermittent colonization only non-mucoid isolates were cultured and one isolate from each patient was collected. All isolates were stored at –80 °C in broth supplemented with 10 % glycerol.
Spontaneous non-mucoid revertants isolated in vitro.
Nine different mucoid CF isolates and PAO1 mucA22 (Mathee et al., 1999) were grown for 4–7 days in continuous culture in flow-cell biofilms as previously described (Lee et al., 2005). The bacteria recovered from biofilm reverted to the non-mucoid phenotype in a proportion of 80 %, and 17 different non-mucoid revertants were isolated.
The nine different mucoid CF isolates and PAO1 mucA22 were also grown in shaking aerobic conditions for 4 or 7 days and 12 different spontaneous non-mucoid revertants were isolated.
In addition, 60 separate shaking cultures, 30 in aerobic and 30 in anaerobic conditions, of one CF mucoid isolate were performed. One non-mucoid revertant from each of the 60 cultures was isolated.
In all in vitro experiments the growth medium was FB minimal medium [1 mM MgCl2, 0.1 mM CaCl2, non-chelated trace elements, 2 g (NH4)2SO4 l–1, 6 g Na2HPO4 . 2H2O l–1, 3 g KH2PO4 l–1, 3 g NaCl l–1] supplemented with 0.2 % Casamino acids and 0.2 % glucose. Anaerobic growth was performed in the presence of 0.2 % KNO3 in an anaerobic chamber (85 % N2, 5 % CO2, 10 % H2; Don Whitley Scientific).
Investigation of the alginate morphotypes on PIA and LB media.
Different alginate morphotypes were classified according to Schurr et al. (1994) on the basis of their phenotypes on two media, PIA and LB (Fig. 1). Type I was mucoid on PIA and LB; type II was mucoid on PIA but lost its mucoid appearance on LB; type III was non-mucoid on both PIA and LB; and type IV displayed very slight but detectable mucoidy on both media after an incubation period of at least 4 days (Boucher et al., 1996; Schurr et al., 1994). In order to classify the 83 pairs of mucoid and non-mucoid isolates into the different phenotypes, the isolates were subsequently grown on Blue agar plates, PIA plates and LB plates and followed for 7 days. The 20 isolates of the eight CF patients with chronic non-mucoid P. aeruginosa were recovered after prolonged growth of the sputum samples on the three different media.
|
; Römling & Tümmler, 2000). After PFGE, the band patterns were visualized by ethidium bromide staining and then photographed (GelDoc imaging system, Bio-Rad). The patterns were analysed by Fingerprinting II software (Bio-Rad). The clonal relatedness of the individual pairs of mucoid and non-mucoid P. aeruginosa was confirmed according to Tenover et al. (1995). Isolates with PFGE patterns that differed from each other by two to three bands were considered clonally related, as this pattern is consistent with a single genetic event, a point mutation or an insertion or deletion of the DNA. Isolates with PFGE patterns that differed by more than three bands were considered to belong to different strains.
Sequence analysis of algTmucABD operon.
DNA extraction was carried out according to the manufacturer using a commercial DNA isolation kit (Puregene, Gentra Systems).
For PCR amplification and sequencing of algT, mucA, mucB and mucD, the primers shown in Table 1 were used. PCR amplification of the entire algTmucABD operon was carried out using DYNAzyme EXT DNA polymerase (FINNZYMES).
|
Complementation analysis.
Triparental mating was used to mobilize the algT-containing recombinant plasmid pCD100 from E. coli JM109 to non-mucoid P. aeruginosa isolates showing a mucA algT mutant genotype with the conjugation helper plasmid pRK600 (Kessler et al., 1992), as previously described (Hoffmann et al., 2005). Transconjugants of P. aeruginosa were selected on PIA agar containing 80 µg tetracycline ml–1, and complementation of algT mutations was scored by the mucoid phenotype observed on PIA plates after incubation at 37 °C to confirm that the mutations found in algT were responsible for the non-mucoid phenotype.
Alginate measurement.
Alginate production was measured using a borate/carbazol method (Knutson & Jeanes, 1968) on overnight cultures in ox broth of the 83 pairs of mucoid and non-mucoid P. aeruginosa isolates from chronically infected CF patients. D-Mannuronate lactone (Sigma) was used to calibrate a standard curve, and PAO1 and PAO1 mucA22 were used as control strains. The results are presented as medians (range) and the unpaired t-test (Statview 4.5 software, Abacus Concepts) was used to compare the different alginate morphotype groups. Our group has previously shown that this method is specific for alginate (Pedersen et al., 1989).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Jelsbak et al., 2007). Among the 26 Swedish patients, three different clones were found among patients attending the Lund CF centre: L1 (three patients), L2 and L3 (each infecting two patients). One patient from Lund and one patient from Århus harboured the same clone. Among the 19 Norwegian patients, one dominant clone was found, clone N (13 patients). The presence of this dominant clone among Norwegian CF patients was published previously (Fluge et al., 2001). Unique clones among P. aeruginosa isolates from Denmark (Ud), Sweden (Us) and Norway (Un) were found in the rest of the patients. Among the 24 CF patients with intermittent colonization, three clones, each harboured by two patients, were found, while the rest of the patients harboured unique clones.
The genotyping analysis showed that with the exception of one patient from Sweden and one from Denmark who shared the same clone, no cross-infection occurred among the Danish, Swedish and Norwegian CF patients attending the six different clinics in Scandinavia. Patients who moved to a newly established clinic in Denmark harboured some of the dominant clones from the original centre.
Distribution of the different alginate morphotypes
Four alginate morphotypes could be distinguished, in agreement with Schurr et al. (1994): see Fig. 1
.
The alginate phenotype of the mucoid and non-mucoid isolates from the 83 chronically infected CF patients was initially determined on Blue agar plates. Subsequent culture on PIA and LB plates showed that 87 % of the non-mucoid and 89 % of the mucoid maintained their phenotype on PIA and LB. This means that 87 % of the non-mucoid isolates showed a type III alginate production and 89 % of the mucoid isolates a type I alginate production.
However, six non-mucoid and eight mucoid isolates showed a phenotype of Alg+ on PIA and Alg– on LB, corresponding to a type II alginate morphotype, and five non-mucoid isolates showed slightly increased alginate production on PIA after 7 days incubation, corresponding to type IV alginate production.
From the eight CF patients with chronic infection with non-mucoid P. aeruginosa, five isolates showed type IV alginate production, starting to produce increased amounts of alginate after prolonged incubation, while the rest of the 15 non-mucoid isolates maintained their phenotype and showed type III alginate production.
When the phenotypic characterization of alginate production (types I, II, III and IV) as judged by the colony morphology on PIA and LB plates was compared to the level of alginate production measured on overnight culture in ox broth, a good correlation was found. The median amount of alginate measured in isolates that showed a type I, II, III and IV morphotype was 180.69 (16.8–757.9), 79.6 (1.07–449), 0 (0–27.9) and 2 (0–496) mg l–1, respectively. The differences between the different groups were statistically significant. This shows that colony morphology on PIA plates correlates with the amount of alginate produced by the isolates in a rich medium such as ox broth.
Mutations in mucA, mucB and mucD in non-mucoid and mucoid P. aeruginosa isolates from chronically infected patients
The DNA sequence analysis of mucA carried out on non-mucoid (103) and mucoid (83) P. aeruginosa isolates from chronically infected Scandinavian CF patients showed that 71 (69 %) of the non-mucoid isolates and 77 (93 %) of the mucoid isolates had mutations in mucA. No mutations in mucA were found in the non-mucoid isolates from 24 intermittently colonized patients. The types of mucA mutations in Scandinavian mucoid and non-mucoid isolates are presented in Table 2.
|
An association between the dominant clones and the types of mucA mutation was observed: clone Dk1 and T349C, clone Dk2 and mucA22, and clone N and T352C. These associations provide an explanation for the high frequency with which these types of mucA mutations were found among the Scandinavian CF isolates. However, the same types of mutations were also found in unrelated clones from different geographical locations in Scandinavia, suggesting that these loci represent hot-spot regions for mutation in mucA. In addition, the mucA22 mutation was reported previously in several mucoid isolates from the USA, Europe and Australia (Boucher et al., 1997b; Bragonzi et al., 2006; Anthony et al., 2002). Isolates belonging to the Dk1, Dk2 and N clones showed several other types of mutations.
The sequence alterations found in mucB and mucD in mucoid and non-mucoid isolates of the chronically infected CF patients are presented in Table 3. For mucB we identified several insertions and deletions, leading to frameshifts and premature stop codons. We also identified a T deletion at position 41 of mucD leading to a premature stop codon. These types of gene alterations probably result in loss of function of their gene products and have apparently not been reported before. Some isolates had several point mutations leading to multiple amino acid changes in MucB or MucD. However, in a large number of the CF isolates simultaneous mutations in mucA and mucB or mucD were identified (results not shown). Therefore, the alginate regulator roles played by mucB and mucD are difficult to assess from our data. We identified five isolates with sequence alterations in mucB or mucD and wild-type mucA. One isolate showed a mucoid phenotype only on PIA (type II) and four isolates showed a mucoid phenotype after prolonged incubation (type IV) (Table 3). The isolates with type II alginate production had a 4 bp insertion at position 808 in mucB, resulting in a frameshift and a premature stop codon at position 855 (mucB consists of 951 bp). Two of the isolates with type IV alginate production harboured a 
162 bp at 235 and an 8 bp insertion at 466, resulting in premature stop codons in mucB. The other two isolates with type IV alginate production showed a T41, resulting in a stop codon in mucD (mucD consists of 1425 bp), and several point mutations in mucD (A409G, C673G, G1321A), resulting in amino acid changes (Table 3). These findings suggest that these sequence alterations might result in loss of function of the negative regulators MucB or MucD, leading to increased transcription of the alginate operon when growing on PIA plates or after prolonged incubation.
|
) and might suggest that these are hot-spot regions for mutations in mucB and mucD.
Mutations in the algT gene in non-mucoid P. aeruginosa isolates
Of the 71 non-mucoid isolates with a mucA mutant genotype, only 37 (52 %) had sequence alterations in algT. Moreover, some algT mutations, such as A55G resulting in a change of Lys to Glu (13 non-mucoid isolates), C115T resulting in a change of Arg to Trp (one non-mucoid isolate), C389T resulting in a change from Ala to Val (one non-mucoid isolate) and C430T resulting in a change of Arg to Cys (one non-mucoid isolate), were also found in the algT sequence of the paired mucoid isolates, suggesting that these mutations do not affect the functionality of AlgT.
Thus, algT mutations present only in isolates with a non-mucoid phenotype were found in 21 isolates representing 30 % of the mucA mutant non-mucoid revertants (Table 4). With the exception of C58T (one isolate), C496T (one isolate), C499T (seven isolates) and a T deletion at position 97 (one isolate), all the other mutations could be complemented by pCD100, encoding the wild-type algT (Table 4). Although we could not confirm by complementation the effect of the T deletion at position 97, this mutation very probably causes a loss of function of the gene product, as it leads to a frameshift and a stop codon at position 106 bp in algT (algT consists of 582 bp). Even though we did not succeed in the complementation of all point mutations, we cannot exclude that the functionality of the algT gene product is affected by these point mutations (Table 4). In contrast to the low percentage of CF non-mucoid revertants that had changes in algT, up to 83 % (73 out of 88) of the spontaneous non-mucoid revertants obtained in vitro showed mutations in algT (Table 5).
|
|
The types of algT mutations found in the 21 clinical non-mucoid revertants were different from the types of mutations found in the 88 in vitro-obtained isolates. However, a 9 bp insertion found in 3 out of 21 CF isolates was frequently observed in the in vitro isolates, especially after aerobic growth. Interestingly, a mutation at codon 29 leading to a change of Tyr to Cys previously found by DeVries & Ohman (1994) in non-mucoid revertants was also present among the in vitro revertants obtained in this study.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and two out of three (Schurr et al., 1994) spontaneous revertants had mutations in algT. These data show that although algT mutants can grow and survive in laboratory conditions, they are less proficient in the CF lung environment due to the role played by AlgT in adaptation to stress. The large variety of the algT mutations found in the present and previous studies might suggest that algT contains no hot-spot regions for mutations as is the case for its negative regulator, mucA.
The role played by AlgT in stress adaptations has been established by several studies.
Transcriptomic analysis revealed differential AlgT-dependent expression of the osmC and osmE genes, which potentially play a role in stress defence (Palma et al., 2004). Therefore, algT mutants would be less proficient in adapting to osmotic stress. It has also been shown that algT mutants of P. aeruginosa showed increased susceptibility to reactive oxygen intermediates (Martin et al., 1994; Yu et al., 1996), indicating that even though algT mutants occur frequently in vitro (DeVries & Ohman, 1994; Schurr et al., 1994), they are probably destroyed by the host response in vivo. Finally, in a recent paper (Wood et al., 2006) algT was identified together with algB and algR as an important regulator of the alginate induction by environmental factors such as cell-wall-inhibitory antibiotics. If algT activation is involved in the response to cell-wall stress, then an algT mutant could be more susceptible to cell-wall antibiotics and therefore easily destroyed in the lung by the antibiotic treatment of the chronic lung infection. If this is true, we can not exclude a selection bias towards non-mucoid revertants with wild-type algT, which might be more resistant to antibiotics. Another possible explanation could be that mutations that arise during the strong selection pressure in the CF lung are different from the mutations that arise during non-selective growth in the laboratory. Interestingly, in vitro spontaneous non-mucoid isolates with wild-type algT were recovered after anaerobic or biofilm growth conditions, suggesting that the non-mucoid CF isolates might occur in the anaerobic conditions encountered in the mucus plugs present in the conductive airways of the CF lungs.
Although the existence of alternative pathways that cause revertance to the non-mucoid phenotype has been postulated, the site of these mutations is not yet known. However, the investigations of the alginate morphotypes on LB and PIA agar might suggest that mutations in the regulatory genes algB and algR could be candidates for the secondary mutations responsible for in vivo revertance. PIA medium contains Irgasan, the trade name for triclosan, which has been shown to be a strong alginate inducer by a mechanism dependent on algT, algB and algR (Wood et al., 2006). Thus, lack of inducibility of alginate production on PIA agar (type III alginate production) should characterize mutants in one of the three genes. Our study shows that the majority of the CF non-mucoid revertants showing a type III morphotype had no mutations in algT and therefore mutations in algB and algR could explain this morphotype.
The mucA mutations identified in non-mucoid CF isolates were also found in the paired mucoid isolates. In agreement with previous studies (Boucher et al., 1997b; Yoon et al., 2006; Bragonzi et al., 2006), mutations in mucA were found to be the main mechanism of conversion to mucoidy. However, we were also able to identify loss-of-function mutations in mucB or mucD in some non-mucoid isolates with increased alginate production on PIA plates (type II) or after prolonged incubation (type IV). As these non-mucoid isolates with attenuated alginate production in vitro have mutations in the alginate regulatory genes, they might have the potential for increased alginate production in vivo in the CF lung. In some CF patients with chronic P. aeruginosa infections no mucoid isolates are cultured. We suggest that these P. aeruginosa isolates have mucB or mucD mutations and might be misinterpreted as non-mucoid in the clinical microbiological laboratory.
Mucoid isolates with a wild-type mucA allele were found in 7 % of the isolates, showing, in agreement with previous studies (Martin et al., 1993b), that other loci are involved in the conversion to mucoidy. However, this seems to be a rare event among Scandinavian CF isolates compared to mucoid isolates from CF clinics in the USA and Canada, where, according to Yoon et al. (2006), approximately 13 % of the mucoid isolates had a wild-type mucA.
As expected, 100 % of the non-mucoid strains isolated from intermittently colonized patients showed wild-type mucA, confirming that the initial colonization of the CF patients occurs with environmental wild-type isolates that convert to alginate overproducers under the selection pressure in the CF lung. The demographic data showed that the intermittently colonized patients were much younger than the chronically colonized patients. These intermittently colonized patients are in the window of opportunity for the treatment of the infection before mutations in mucA, responsible for conversion to mucoidy, occur.
Non-mucoid isolates from chronically colonized CF patients with wild-type mucA allele were found in 30 % of the isolates, suggesting that these isolates might be the original wild-type isolates that infected the patient. However, some of these isolates showed algT mutations (data not shown), raising the possibility that acquisition of a wild-type mucA allele had occurred by horizontal gene transfer.
In conclusion, most of the non-mucoid CF isolates are revertants from mucoid isolates and the mechanism of revertance is algT-independent; this is in contrast to the in vitro-obtained isolates, which frequently show mutations in algT. Investigations are continuing in our laboratory to find out which genes might be responsible for the conversion to non-mucoidy in vivo.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: P. Cornelis
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bagge, N., Schuster, M., Hentzer, M., Ciofu, O., Givskov, M., Greenberg, E. P. & Høiby, N. (2004). Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production. Antimicrob Agents Chemother 48, 1175–1187.
Bayer, A. S., Eftekhar, F., Tu, J., Nast, C. C. & Speert, D. P. (1990). Oxygen-dependent up-regulation of mucoid exopolysaccharide (alginate) production in Pseudomonas aeruginosa. Infect Immun 58, 1344–1349.
Baynham, P. J., Brown, A. L., Hall, L. L. & Wozniak, D. J. (1999). Pseudomonas aeruginosa AlgZ, a ribbon-helix-helix DNA-binding protein, is essential for alginate synthesis and algD transcriptional activation. Mol Microbiol 33, 1069–1080.[CrossRef][Medline]
Berry, A., DeVault, J. D. & Chakrabarty, A. M. (1989). High osmolarity is a signal for enhanced algD transcription in mucoid and nonmucoid Pseudomonas aeruginosa strains. J Bacteriol 171, 2312–2317.
Boucher, J. C., Martinez-Salazar, J., Schurr, M. J., Mudd, M. H., Yu, H. & Deretic, V. (1996). Two distinct loci affecting conversion to mucoidy in Pseudomonas aeruginosa in cystic fibrosis encode homologs of the serine protease HtrA. J Bacteriol 178, 511–523.
Boucher, J. C., Schurr, M. J., Yu, H., Rowen, D. W. & Deretic, V. (1997a). Pseudomonas aeruginosa in cystic fibrosis: role of mucC in the regulation of alginate production and stress sensitivity. Microbiology 143, 3473–3480.
Boucher, J. C., Yu, H., Mudd, M. H. & Deretic, V. (1997b). Mucoid Pseudomonas aeruginosa in cystic fibrosis: characterization of muc mutations in clinical isolates and analysis of clearance in a mouse model of respiratory infection. Infect Immun 65, 3838–3846.[Abstract]
Bragonzi, A., Wiehlmann, L., Klockgether, J., Cramer, N., Worlitzsch, D., Doring, G. & Tummler, B. (2006). Sequence diversity of the mucABD locus in Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Microbiology 152, 3261–3269.
Deretic, V., Dikshit, R., Konyecsni, W. M., Chakrabarty, A. M. & Misra, T. K. (1989). The algR gene, which regulates mucoidy in Pseudomonas aeruginosa, belongs to a class of environmentally responsive genes. J Bacteriol 171, 1278–1283.
DeVault, J. D., Kimbara, K. & Chakrabarty, A. M. (1990). Pulmonary dehydration and infection in cystic fibrosis: evidence that ethanol activates alginate gene expression and induction of mucoidy in Pseudomonas aeruginosa. Mol Microbiol 4, 737–745.[CrossRef][Medline]
DeVries, C. A. & Ohman, D. E. (1994). Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation. J Bacteriol 176, 6677–6687.
Doggett, R. G. (1969). Incidence of mucoid Pseudomonas aeruginosa from clinical sources. Appl Microbiol 18, 936–937.[Medline]
Doggett, R. G., Harrison, G. M., Stillwell, R. N. & Wallis, E. S. (1966). An atypical Pseudomonas aeruginosa associated with cystic fibrosis of the pancreas. J Pediatr 68, 215–221.[CrossRef]
Drenkard, E. & Ausubel, F. M. (2002). Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation. Nature 416, 740–743.[CrossRef][Medline]
Evans, L. R. & Linker, A. (1973). Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacteriol 116, 915–924.
Firoved, A. M., Boucher, J. C. & Deretic, V. (2002). Global genomic analysis of AlgU (E)-dependent promoters (sigmulon) in Pseudomonas aeruginosa and implications for inflammatory processes in cystic fibrosis. J Bacteriol 184, 1057–1064.
Fluge, G., Ojeniyi, B., Høiby, N., Digranes, A., Ciofu, O., Hunstad, E., Haanaes, O. C. & Storrosten, O. T. (2001). Typing of Pseudomonas aeruginosa strains in Norwegian cystic fibrosis patients. Clin Microbiol Infect 7, 238–243.[CrossRef][Medline]
Frederiksen, B., Koch, C. & Høiby, N. (1997). Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis. Pediatr Pulmonol 23, 330–335.[CrossRef][Medline]
Fyfe, J. A. & Govan, J. R. (1980). Alginate synthesis in mucoid Pseudomonas aeruginosa: a chromosomal locus involved in control. J Gen Microbiol 119, 443–450.[Medline]
Goldberg, J. B. & Dahnke, T. (1992). Pseudomonas aeruginosa AlgB, which modulates the expression of alginate, is a member of the NtrC subclass of prokaryotic regulators. Mol Microbiol 6, 59–66.[CrossRef][Medline]
Goldberg, J. B., Gorman, W. L., Flynn, J. L. & Ohman, D. E. (1993). A mutation in algN permits trans activation of alginate production by algT in Pseudomonas species. J Bacteriol 175, 1303–1308.
Govan, J. R. & Deretic, V. (1996). Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60, 539–574.
Govan, J. R., Fyfe, J. A. & McMillan, C. (1979). The instability of mucoid Pseudomonas aeruginosa: fluctuation test and improved stability of the mucoid form in shaken culture. J Gen Microbiol 110, 229–232.
Govan, J. R., Fyfe, J. A. & Baker, N. R. (1983). Heterogeneity and reduction in pulmonary clearance of mucoid Pseudomonas aeruginosa. Rev Infect Dis 5 (Suppl. 5), S874–S879.[Medline]
Haussler, S. (2004). Biofilm formation by the small colony variant phenotype of Pseudomonas aeruginosa. Environ Microbiol 6, 546–551.[CrossRef][Medline]
Hoffmann, N., Rasmussen, T. B., Jensen, P. O., Stub, C., Hentzer, M., Molin, S., Ciofu, O., Givskov, M., Johansen, H. K. & Høiby, N. (2005). Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis. Infect Immun 73, 2504–2514.
Høiby, N. (1975). Prevalence of mucoid strains of Pseudomonas aeruginosa in bacteriological specimens from patients with cystic fibrosis and patients with other diseases. Acta Pathol Microbiol Scand Suppl 83, 549–552.[Medline]
Høiby, N. (1977). Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis. A survey. Acta Pathol Microbiol Scand Suppl 262, 1–96.[Medline]
Høiby, N., Giwercman, B., Jensen, E. T., Johansen, H. K., Kronborg, G., Pressler, T. & Kharazmi, A. (1993). Immune response in cystic fibrosis – helpful or harmful? In 18th European CF Conference, pp. 133–139. Edited by H. Escobar, F. Baquero & L. Suarez. Madrid: Elsevier.
Høiby, N., Frederiksen, B. & Pressler, T. (2005). Eradication of early Pseudomonas aeruginosa infection. J Cyst Fibros 4 (Suppl. 2), 49–54.[Medline]
Jelsbak, L., Johansen, H. K., Frost, A. L., Thøgersen, R., Thomsen, L. E., Ciofu, O., Yang, L., Haagensen, J. A., Høiby, N. & Molin, S. (2007). Molecular epidemiology and dynamics of Pseudomonas aeruginosa populations in lungs of cystic fibrosis patients. Infect Immun 75, 2214–2224.
Kessler, B., de Lorenzo, V. & Timmis, K. N. (1992). A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol Gen Genet 233, 293–301.[CrossRef][Medline]
Knutson, C. A. & Jeanes, A. (1968). A new modification of the carbazole analysis: application to heteropolysaccharides. Anal Biochem 24, 470–481.[CrossRef][Medline]
Lee, B., Haagensen, J. A., Ciofu, O., Andersen, J. B., Høiby, N. & Molin, S. (2005). Heterogeneity of biofilms formed by nonmucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J Clin Microbiol 43, 5247–5255.
Martin, D. W., Schurr, M. J., Mudd, M. H. & Deretic, V. (1993a). Differentiation of Pseudomonas aeruginosa into the alginate-producing form: inactivation of mucB causes conversion to mucoidy. Mol Microbiol 9, 497–506.[Medline]
Martin, D. W., Schurr, M. J., Mudd, M. H., Govan, J. R., Holloway, B. W. & Deretic, V. (1993b). Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients. Proc Natl Acad Sci U S A 90, 8377–8381.
Martin, D. W., Schurr, M. J., Yu, H. & Deretic, V. (1994). Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa: relationship to E and stress response. J Bacteriol 176, 6688–6696.
Mathee, K., McPherson, C. J. & Ohman, D. E. (1997). Posttranslational control of the algT (algU)-encoded 22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN). J Bacteriol 179, 3711–3720.
Mathee, K., Ciofu, O., Sternberg, C., Lindum, P. W., Campbell, J. L., Jensen, P., Johnsen, A. H., Givskov, M., Ohman, D. E. & other authors (1999). Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145, 1349–1357.
Nikolskaya, A. N. & Galperin, M. Y. (2002). A novel type of conserved DNA-binding domain in the transcriptional regulators of the AlgR/AgrA/LytR family. Nucleic Acids Res 30, 2453–2459.
Ohman, D. E. (1981). Genetic mapping of chromosomal determinants for the production of exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun 33, 142–148.
Ojeniyi, B., Høiby, N. & Rosdahl, V. T. (1991). Genome fingerprinting as a typing method used on polyagglutinable Pseudomonas aeruginosa isolates from cystic fibrosis patients. APMIS 99, 492–498.[Medline]
Ojeniyi, B., Petersen, U. S. & Høiby, N. (1993). Comparison of genome fingerprinting with conventional typing methods used on Pseudomonas aeruginosa isolates from cystic fibrosis patients. APMIS 101, 168–175.[Medline]
Palma, M., DeLuca, D., Worgall, S. & Quadri, L. E. (2004). Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide. J Bacteriol 186, 248–252.
Pedersen, S. S. (1992). Lung infection with alginate-producing, mucoid Pseudomonas aeruginosa in cystic fibrosis. APMIS Suppl 28, 1–79.[Medline]
Pedersen, S. S., Espersen, F., Høiby, N. & Shand, G. H. (1989). Purification, characterization, and immunological cross-reactivity of alginates produced by mucoid Pseudomonas aeruginosa from patients with cystic fibrosis. J Clin Microbiol 27, 691–699.
Pugashetti, B. K., Metzger, H. M., Jr, Vadas, L. & Feingold, D. S. (1982). Phenotypic differences among clinically isolated mucoid Pseudomonas aeruginosa strains. J Clin Microbiol 16, 686–691.
Römling, U. & Tümmler, B. (2000). Achieving 100 % typeability of Pseudomonas aeruginosa by pulsed-field gel electrophoresis. J Clin Microbiol 38, 464–465.
Rowen, D. W. & Deretic, V. (2000). Membrane-to-cytosol redistribution of ECF sigma factor AlgU and conversion to mucoidy in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Mol Microbiol 36, 314–327.[CrossRef][Medline]
Schurr, M. J., Martin, D. W., Mudd, M. H. & Deretic, V. (1994). Gene cluster controlling conversion to alginate-overproducing phenotype in Pseudomonas aeruginosa: functional analysis in a heterologous host and role in the instability of mucoidy. J Bacteriol 176, 3375–3382.
Schurr, M. J., Yu, H., Martinez-Salazar, J. M., Boucher, J. C. & Deretic, V. (1996). Control of AlgU, a member of the E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis. J Bacteriol 178, 4997–5004.
Speert, D. P., Farmer, S. W., Campbell, M. E., Musser, J. M., Selander, R. K. & Kuo, S. (1990). Conversion of Pseudomonas aeruginosa to the phenotype characteristic of strains from patients with cystic fibrosis. J Clin Microbiol 28, 188–194.
Tart, A. H., Blanks, M. J. & Wozniak, D. J. (2006). The AlgT-dependent transcriptional regulator AmrZ (AlgZ) inhibits flagellum biosynthesis in mucoid, nonmotile Pseudomonas aeruginosa cystic fibrosis isolates. J Bacteriol 188, 6483–6489.
Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H. & Swaminathan, B. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33, 2233–2239.[Medline]
Terry, J. M., Pina, S. E. & Mattingly, S. J. (1991). Environmental conditions which influence mucoid conversion Pseudomonas aeruginosa PAO1. Infect Immun 59, 471–477.
Wood, L. F. & Ohman, D. E. (2006). Independent regulation of MucD, an HtrA-like protease in Pseudomonas aeruginosa, and the role of its proteolytic motif in alginate gene regulation. J Bacteriol 188, 3134–3137.
Wood, L. F., Leech, A. J. & Ohman, D. E. (2006). Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa: roles of sigma (AlgT) and the AlgW and Prc proteases. Mol Microbiol 62, 412–426.[CrossRef][Medline]
Wozniak, D. J. & Ohman, D. E. (1991). Pseudomonas aeruginosa AlgB, a two-component response regulator of the NtrC family, is required for algD transcription. J Bacteriol 173, 1406–1413.
Wyckoff, T. J., Thomas, B., Hassett, D. J. & Wozniak, D. J. (2002). Static growth of mucoid Pseudomonas aeruginosa selects for non-mucoid variants that have acquired flagellum-dependent motility. Microbiology 148, 3423–3430.
Yoon, S. S., Coakley, R., Lau, G. W., Lymar, S. V., Gaston, B., Karabulut, A. C., Hennigan, R. F., Hwang, S. H., Buettner, G. & other authors (2006). Anaerobic killing of mucoid Pseudomonas aeruginosa by acidified nitrite derivatives under cystic fibrosis airway conditions. J Clin Invest 116, 436–446.[CrossRef][Medline]
Yu, H., Schurr, M. J. & Deretic, V. (1995). Functional equivalence of Escherichia coli sigma E and Pseudomonas aeruginosa AlgU: E. coli rpoE restores mucoidy and reduces sensitivity to reactive oxygen intermediates in algU mutants of P. aeruginosa. J Bacteriol 177, 3259–3268.
Yu, H., Boucher, J. C., Hibler, N. S. & Deretic, V. (1996). Virulence properties of Pseudomonas aeruginosa lacking the extreme-stress sigma factor AlgU (sigma E). Infect Immun 64, 2774–2781.[Abstract]
Received 10 July 2007;
revised 9 October 2007;
accepted 24 October 2007.
This article has been cited by other articles:
|
|
 |
|
  F. H. Damron, D. Qiu, and H. D. Yu The Pseudomonas aeruginosa Sensor Kinase KinB Negatively Controls Alginate Production through AlgW-Dependent MucA Proteolysis J. Bacteriol., April 1, 2009; 191(7): 2285 - 2295. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
