Regulation of Membrane Permeability by a Two-Component Regulatory System in Pseudomonas aeruginosa
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610}l, http://www.100md.com
Received 7 June 2002/ Returned for modification 19 August 2002/ Accepted 10 October 2002}l, http://www.100md.com
ABSTRACT}l, http://www.100md.com
Membrane impermeability is the major contributing factor to multidrug resistance in clinical isolates of Pseudomonas aeruginosa. By using laboratory strain PAK, a spontaneous P. aeruginosa mutant (mutant PAK1-3) whose membrane had reduced permeability and which displayed increased levels of resistance to various antibiotics, especially aminoglycosides, was isolated. By complementation of the mutant with a genomic clone library derived from wild-type strain PAK, a novel two-component regulatory system (PprA and PprB) was identified and was found to be able to increase the permeability of the bacterial membrane and render PAK1-3 sensitive to antibiotics. Furthermore, specific phosphorylation of the response regulator (PprB) by histidine kinase (PprA) was observed in vitro, demonstrating that they are cognate two-component regulatory genes. Introduction of a plasmid expressing the pprB gene into randomly chosen clinical isolates (n = 17) resulted in increased sensitivity to aminoglycosides in the majority of isolates (n = 13) tested. This is the first demonstration that P. aeruginosa membrane permeability can be regulated, providing an important clue in the understanding of the mechanism of membrane impermeability-mediated multidrug resistance in P. aeruginosa.
INTRODUCTIONr7, http://www.100md.com
Pseudomonas aeruginosa is an opportunistic pathogen which poses a major threat to immunocompromised patients, burn victims, and cystic fibrosis (CF) patients (2, 6, 8, 31). Individuals with CF acquire P. aeruginosa infection from the environment at an early age, and repeated treatment with various antibiotics selects for multidrug-resistant strains that may eventually cause patient death (1). More than 90% of the deaths among CF patients are caused by lung infection with P. aeruginosa (18, 27, 28).r7, http://www.100md.com
Specific mechanisms of resistance include enzymatic inactivation of antibiotics, alterations of the target sites of antimicrobial agents, development of bypass pathways around antimicrobial targets, and reductions in cell wall membrane permeability (3, 23, 24, 25, 35). Although most resistance mechanisms are antibiotic specific, membrane impermeability-mediated resistance usually results in a multidrug resistance phenotype. Such impermeability mutants are common among clinical isolates of P. aeruginosa, especially in those individuals suffering from chronic infections. Previous studies have shown that as many as 90% of CF isolates have reduced cell wall permeability, thus presenting with a multidrug resistance phenotype (5, 28). Although the mechanisms of resistance to specific antibiotics are well known, the molecular mechanism of multidrug resistance is poorly understood.
Aminoglycosides, including kanamycin, streptomycin, gentamicin, amikacin, and tobramycin, exert their bactericidal activities by inhibiting protein synthesis by binding to the 30S ribosomal subunit. Due to their efficient bactericidal activities, aminoglycosides are widely used in the treatment of various bacterial infections. However, P. aeruginosa isolates are intrinsically less sensitive to many aminoglycosides primarily due to their outer membrane impermeability and easily become resistant to high levels of aminoglycosides. Resistance is also attributed to an efflux pump, MexXY, that is specific for aminoglycosides, although overexpression of MexYY alone does not confer resistance to aminoglycosides (11, 14, 15). More recently, a phenomenon called "adaptive resistance to aminoglycosides" has been described in P. aeruginosa both in vitro and in vivo (9, 37), in which the level of bacterial resistance to aminoglycosides gradually increases following pretreatment with aminoglycosides or other agents. The molecular mechanism for this desensitization is totally unknown.
In this report, we describe a novel two-component regulatory system that up-regulates the permeability of the membrane to various antibiotics, presumably by turning on the synthesis of membrane proteins that mediate antibiotic transport or turning off efflux systems. Most strikingly, overexpression of the response regulator gene, pprB, in clinical isolates resulted in increased sensitivities to aminoglycosides in the majority of those isolates tested. Elevated levels of expression of the two-component regulatory genes increased the outer membrane permeability of P. aeruginosa to para-nitrophenyl phosphate (pNPP), which is a chromogenic substrate for alkaline phosphatase. The pprA-pprB regulatory system may represent a new mechanism for the control of membrane impermeability in P. aeruginosa.m74}jg&, http://www.100md.com
MATERIALS AND METHODSm74}jg&, http://www.100md.com
Strains and plasmids. The strains and plasmids used in this study are listed in Table 1. To construct a clone bank of strain PAK genomic DNA, PAK chromosomal DNA was partially digested with Sau3A and fragments ranging from 3 to 5 kb were gel purified and ligated into the BamHI site of pUCP19. The ligation mixture was transformed into Escherichia coli DH5{alpha} , and 5 x 104 colonies were pooled; of these colonies, at least 20% had an insert; thus, the bank consists of at least 1 x 104 independent clones with DNA inserts.
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TABLE 1. Bacterial strains and plasmids#:.c@, 百拇医药
To generate pWC005, a 0.5-kb NdeI-XmnI DNA fragment from pUCP19 that encodes the promoter and the N-terminal signal sequence of the ampicillin resistance gene was ligated into the SmaI site of pSOP3, which is located upstream of a signal sequenceless alkaline phosphatase structural gene (23). The resulting plasmid, pWC005, encodes a translational fusion between the bla signal sequence and alkaline phosphatase.#:.c@, 百拇医药
The pprB gene was amplified from the PAK chromosome by PCR with the oligonucleotides 5'-GGC TAA TAC CAT TCG GTA TGG CTG CAT TCC-3' and 5'-CTT GGA TAC ATC GAC GCG GAG ATT CTG AAG-3'. The PCR product was cloned into pCR2.1-TOPO, resulting in pCR-RR, in which the pprB gene is in the opposite direction relative to that of the lac promoter. The pprB gene was then subcloned as an EcoRI fragment into the same site of pUCP19, with insertion of the pprB gene in the same or opposite direction relative to that of the lac promoter on the vector; these plasmids were designated pZLRR-{alpha} and pZLRR-ß, respectively. A tetracycline resistance gene cassette was isolated from pBR322 as an EcoRI-AvaI fragment, blunt ended, and inserted into the SspI and ScaI sites of pUCP19 to generate pUCPT19. Then, the EcoRI fragment from pCR-RR, which contains the pprB gene, was inserted into the same site of pUCPT19 to generate pRRBTc, in which pprB is in the same direction as the lac promoter.
Overexpression and purification of PprA and PprB. To overproduce the PprA and PprB proteins in E. coli, the coding regions of the two open reading frames were amplified by PCR with primer sets consisting of primers 5'-GTG AGC TGG TCG GAT CCT CGA ATT TTC CCG-3' and 5'-GGT TGA AAA AAC GCA AGC TTG TCC GCA GGC-3' and primers 5'-GTA TGA CCA TGG ATC CAA CCG GCC TCG C-3' and 5'-GGC AGG GCG GAA GCT TGC TGA TCT AGC A-3'. The primer sequences were based on the genome sequence of strain PAO1. The PCR products were first cloned into pCR2.1-TOPO, with the plasmids designated pYW017 and pYW020, respectively. Subsequently, a 2.8-kb pprA fragment from pYW017 and an 862-bp pprB fragment from pYW020 were isolated and subcloned into BamHI-HindIII sites of pQE32 and pQE31, resulting in pYW021 and pYW024, respectively.., 百拇医药
To purify the His-tagged PprA and PprB proteins, E. coli strain M15 harboring pREP4 and pYW021 or pYW024 was induced with 1 mM isopropyl-ß-D-thiogalactopyranoside for 6 h at 28°C. Overproduced six-His-tagged PprA and six-His-tagged PprB proteins were affinity purified from the soluble fraction of the cell extracts with a nickel-agarose column by the protocol provided by Qiagen, Inc. Purified proteins were dialyzed against storage buffer (50 mM Tris-HCl [pH 8.0], 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 50% glycerol) overnight and stored at -20°C until use.
For in vitro protein phosphorylation tests, 10 µl of reaction buffer (50 mM Tris-HCl [pH 7.4], 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol) containing 5 µCi of [-32P]ATP was mixed with 1 µg of purified protein, and the mixture was incubated at room temperature for 5 min. The reaction was terminated by the addition of 5 µl of 4x loading buffer. The reaction mixture was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie blue dye, dried, and exposed to X-ray film.v, 百拇医药
Whole-cell alkaline phosphatase assay for assessment of outer membrane permeability. An overnight culture of P. aeruginosa with proper antibiotics was washed once with fresh Luria-Bertani (LB) medium and reinoculated into 3 ml of fresh LB medium without antibiotics to an A600 of about 0.1. After culture for 7 h, the cells were washed once with ice-cold reaction buffer (0.1 M Tris-HCl [pH 8.0], 100 mM NaCl, 5 mM MgCl2, 0.1 mM ZnCl2) and resuspended in 3 ml of the same buffer, and the final cell density (A600) was measured. The reaction was started by adding 0.1 ml of the cell suspension to a reaction mixture containing 0.8 ml of reaction buffer and 0.1 ml of pNPP (0.08 mg/ml in 1 M Tris-HCl [pH 8.0]). All reactions were carried out in a standard plastic spectrophotometer cuvette at room temperature. The samples were allowed to react for 2 min, and 0.1 ml of 1 M KH2PO4 was added to terminate the reaction. The cells were centrifuged, and the A410 of the supernatant was measured. Samples with no substrate were used as negative controls. An index of outer membrane permeability was calculated as A410/A600[S], where [S] is the pNPP concentration.
Miscellaneous assays. The MICs of the various antibiotics tested were determined by standard protocols (2, 19), and assays for ß-galactosidase activity were performed by a previously described protocol (16). The outer membrane proteins of P. aeruginosa were isolated by the protein fractionation protocol described before (21).cmnlq, http://www.100md.com
RESULTScmnlq, http://www.100md.com
Characterization of a spontaneous mutant of P. aeruginosa with decreased sensitivities to antibiotics. To understand the mechanism that results in a high frequency of multidrug resistance in P. aeruginosa, we selected spontaneous neomycin-resistant mutants of PAK that were able to grow on Luria agar (L-agar) medium containing 200 µg of neomycin per ml. A multidrug-resistant isolate, designated PAK1-3, was identified among the initial isolates through further screening of the resistant mutants. PAK1-3 is not only resistant to neomycin, but the MICs of all the aminoglycosides tested were also increased, including those of kanamycin, streptomycin, gentamicin, amikacin, and tobramycin, as well as those of tetracycline, ciprofloxacin, and erythromycin (Table 2), suggesting that it has a permeability defect. Consistent with this prediction, PAK1-3 has a slower growth rate compared to that of its parent strain, PAK, as seen from the smaller colony sizes and the delayed increase in cell density in liquid cultures (data not shown). However, its sensitivity to carbenicillin is not significantly changed, indicating that it has a limited substrate specificity (Table 2).
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TABLE 2. MICs for PAK and PAK1-3 derivatives(], 百拇医药
A novel two-component regulatory system controls antibiotic sensitivity in PAK1-3. With the assumption that PAK1-3 is an impermeable mutant as a result of the loss of a gene function, we attempted to complement the mutant with wild-type PAK chromosomal DNA clones. A clone bank of 3 to 5 kb of PAK chromosomal DNA in pUCP19 was electroporated into PAK1-3. Transformants were first selected on L agar containing carbenicillin (150 µg/ml) and were then replica plated on L agar containing carbenicillin (150 µg/ml) plus neomycin (200 µg/ml) to screen for isolates that are sensitive to aminoglycosides. A total of 5,000 colonies were screened, of which 3 colonies were sensitive to the neomycin (Fig. 1). To confirm that this sensitivity phenotype is due to the presence of a plasmid and is not due to a reversion or a secondary mutation, plasmids were purified from the three isolates and retransformed into fresh PAK1-3 cells. Two of the three plasmids indeed conferred to PAK1-3 sensitivity to the aminoglycosides, whereas one of them failed to do so. The effective two plasmids were identical by DNA sequence analysis, and the plasmid was designated pUCP19B.
fig.ommittedpf*, http://www.100md.com
FIG. 1. Conversion of neomycin resistance phenotype by introduction of a plasmid expressing pprA or pprB. Bacterial cells from single colonies were streaked onto an L-agar plate containing carbenicillin (150 µg/ml) alone or carbenicillin (150 µg/ml) plus neomycin (200 µg/ml) and were grown overnight at 37°C.pf*, http://www.100md.com
pUCP19B was found to contain a 3,280-bp DNA insert encoding a 2,768-bp putative histidine kinase homolog, identical to PA4293 of the Pseudomonas genome sequence designated pprA, which is transcriptionally fused under the lac promoter of the vector. Tests of subclones derived from the 3.3-kb insert as well as insertional inactivation of the histidine kinase structural gene in pUCP19B demonstrated that lac promoter-driven expression of the pprA gene is essential to confer aminoglycoside sensitivity to strain PAK1-3 (data not shown). Unlike the majority of the known sensor molecules, the PprA protein lacks a signal sequence or transmembrane segments; thus, it is likely a cytoplasmic protein. The N terminus of the PprA protein has PAS and PAC motifs that are known to be involved in various signal-sensing functions (38). Therefore, it is likely that in the cytoplasm PprA senses external signals through either binding to a diffusible molecule(s) or interacting with another sensory protein(s).
PprA and PprB form a functional two-component regulatory system. On the basis of the genome sequence of P. aeruginosa, a 827-bp putative response regulator , which we named pprB, resides 1,184 bp upstream of the pprA gene and is transcribed in the opposite direction from that of pprA. To see whether PprB is a cognate response regulator of PprA, we tested whether increased expression of pprB can also confer to PAK1-3 sensitivity to aminoglycosides. The pprB gene was amplified from the PAK chromosome by PCR and cloned into pUCP19, with the insert placed in the same or opposite direction relative to that of the lac promoter on the vector; these plasmids were designated pZLRR-{alpha} and pZLRR-ß, respectively. The pprB gene sequence of PAK differs from that of PAO1 by only 2 bases, without changes in amino acid sequences. When these two plasmids were transformed into the PAK1-3 background, only pZLRR-{alpha} and not pZLRR-ß was able to confer sensitivity to aminoglycosides, suggesting that pprA and pprB likely function as a pair of two-component regulatory genes. Further evidence supporting this possibility was that fact that the effect of PprB overexpression was much more dramatic than that of PprA overexpression on the change in the MIC for PAK1-3 (Table 2).
To further confirm the relationship between PprA and PprB, an in vitro phosphorylation test was performed. Like other two-component regulatory genes, both PprA and PprB contained conserved phosphorylation sites (histidine and aspartate, respectively). Both the PprA and PprB proteins were overproduced in E. coli by fusion to N-terminal His tags that are driven by tac promoters (see Materials and Methods). Overproduced proteins were purified from soluble fractions with nickel-agarose affinity columns and subjected to the phosphorylation test by using [-32P]ATP (see Materials and Methods). As shown in Fig. 2, PprA alone underwent active autophosphorylation, while PprB did not. However, in the presence of PprA, PprB is highly phosphorylated; thus, PprA functions as a specific kinase for PprB. Combined with the antibiotic sensitivity-conferring abilities of PprA and PprB, the phosphorylation results strongly suggest a sensor-response regulator relationship between PprA and PprB.qa, 百拇医药
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FIG. 2. (A) Purification of PprA and PprB proteins from E. coli overexpressing the pprA and pprB genes, respectively. Lane 1, total cellular protein of M15/pREP4/pYW021; lane 2, purified six-His-tagged PprA fusion protein; lane 3, total cellular protein of M15/pREP4/pYW024; lane 4, purified six-His-tagged PprB fusion protein; lane M, molecular size marker. (B) Coomassie blue stain of an SDS-polyacrylamide gel showing the PprA and PprB proteins used in the phosphorylation study. (C) X-ray film after exposure to the SDS-polyacrylamide gel shown in panel B.:o1m5a%, http://www.100md.com
PprA-PprB regulates membrane permeability. To test outer membrane permeability, a whole-cell alkaline phosphatase assay was conducted. The whole-cell alkaline-phosphatase assay was based on the membrane-diffusion barrier model described by Martinez et al. (12, 13). At low substrate concentrations, the high enzyme concentration and the low substrate level in the bacterial periplasm ensure that every substrate molecule entering the periplasm is converted into a product; therefore, the bacterial outer membrane functions as a diffusion barrier and the speed-limiting factor; thus, the reaction velocity increases in direct proportion to the substrate concentration.
Growth of PAK and PAK1-3 in L broth produced undetectable levels of endogenous alkaline phosphatase activity, whereas growth in medium with a low phosphate concentration resulted in high levels of alkaline phosphatase activity. However, under conditions with low phosphate concentrations, a high degree of cell lysis which interfered with the assay was observed. To overcome this, a plasmid that constitutively expresses the alkaline phosphatase gene, pWC005, was introduced into both PAK and PAK1-3. Using a method similar to that previously described for E. coli (12, 13), we have determined that the alkaline phosphatase activity of PAK/pWC005 is directly proportional to the pNPP concentration when the pNPP concentration is <1 mg/ml.#y, http://www.100md.com
PAK/pWC005 and PAK1-3/pWC005 were grown in L broth and incubated with 0.08 mg of pNPP per ml. As shown in Fig. 3, the outer membrane permeability of PAK1-3 is at least 30% lower than that of wild-type PAK, agreeing well with the earlier prediction based on the antibiotic resistance and the phenotype of slower growth of PAK1-3. When the plasmid expressing pprB, pZLRR-{alpha} , was introduced into PAK1-3, the resulting strain restored not only the membrane permeability (Fig. 3) but also the growth rate to the levels for wild-type PAK, demonstrating that PprA and PprB regulate bacterial membrane permeability, likely through regulation of the downstream genes directly involved in the membrane transport function.
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FIG. 3. . Comparison of bacterial outer membrane permeabilities. The relative permeabilities of the outer membranes (outer membrane permeability index = A410/A600[S]), determined by a whole-cell alkaline phosphatase assay with low substrate concentrations, are shown for indicated strains.)^.ev'^, 百拇医药
In support of the conclusion stated above, differences in a few outer membrane protein bands between PAK and PAK1-3 were clearly visible (Fig. 4), especially a 36-kDa protein band which is highly overexpressed in the PAK1-3 background. The 36-kDa protein was determined to be the major outer membrane porin, OprF, by mass spectrometric analysis. Interestingly, upon introduction of pZLRR-{alpha} into the PAK1-3 background, the outer membrane profile returned to that of the wild type, demonstrating that PprB influences the expression and/or outer membrane localization of a number of proteins, including the F porin. The contribution of OprF to aminoglycoside resistance is not known.
fig.ommitted+/, 百拇医药
FIG. 4. . Comparison of bacterial outer membrane protein profiles. Overnight cultures of the bacterial cells were fractionated into cytosol, inner membrane, and outer membrane proteins. Outer membrane proteins were separated by SDS-PAGE on 12% polyacrylamide gels and stained with Coomassie blue. Lane 1, PAK; lane 2, PAK1-3, lane 3, PAK1-3/pZLPPRB{alpha} ; lane M, molecular size marker. Arrows indicate obvious band differences between PAK and PAK1-3.+/, 百拇医药
Overexpression of the pprB gene in clinical isolates renders increased sensitivities to aminoglycosides. To test whether increased levels of expression of the pprA-pprB two-component regulatory genes can render higher levels of membrane permeability among clinical isolates of P. aeruginosa, a pprB-overexpressing construct was introduced into clinical isolates. Eighteen isolates from patients with CF and 18 isolates from the blood of individuals without CF that show various degrees of resistance to gentamicin were randomly chosen for the test. Since the majority of those strains were resistant to carbenicillin (150 µg/ml) but were inhibited by tetracycline (100 µg/ml), we have used a tetracycline resistance construct that expresses the pprB gene under the lac promoter, pRRBTc. We were able to introduce the plasmid into 17 isolates by electroporation, and in the presence of pRRBTc, the aminoglycoside MICs for 13 of those transformants decreased at least twofold compared to those for isolates containing the pUCPT19 vector alone, while the gentamicin MIC for the remaining 4 transformants did not change at all (Table 3). These moderate reductions in the MICs are the result of a mere increase in the PprB level by the introduction of pRRBTc. Active signaling through the PprA-PprB two-component system might be required for the maximum effect on the MIC; nevertheless, an elevated level of expression of the pprB gene was sufficient to confer increased sensitivity to aminoglycosides to the majority of the isolates; thus, it is consistent with the prediction that PprA and PprB regulate genes that increase membrane permeability and transport, resulting in increased bacterial sensitivities to antibiotics.
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TABLE 3. Effect of elevated levels of pprB expression on MICs for clinical isolates6.u^^$), http://www.100md.com
DISCUSSION6.u^^$), http://www.100md.com
Up to 90% of clinical isolates of P. aeruginosa appear to carry the impermeability type of resistance (18, 28). It has been demonstrated that this type of resistance arises from the combination of an unusually restricted outer membrane permeability through mutation or changes in porin and lipopolysaccharide (LPS) structures and secondary resistance mechanisms such as energy-dependent multidrug efflux and the actions of antibiotic-modifying enzymes (3, 4, 17, 20, 29, 35). P. aeruginosa strains that are sensitive to aminoglycosides can acquire such resistance at a high frequency. Under standard laboratory conditions, a laboratory strain of P. aeruginosa, PAK, shows a frequency of resistance to neomycin at 200 µg/ml as high as 10-6. This is much higher than the standard spontaneous mutational rate of 10-8 per cell per generation in E. coli. This high frequency of conversion to aminoglycoside resistance could be a result of either the hypermutational rate of P. aeruginosa (e.g., due to defective DNA repair), as has been described within the lung environment of patients with CF (22), or a simple on-off phase switch to a state of a low level of cell permeability. Preliminary analysis of the pprA mutant indicates that there was no alteration in the antibiotic resistance pattern, suggesting the existence of multiple pathways controlling membrane permeability. Therefore, strain PAK1-3 may have a defect(s) in a further upstream regulatory gene that affects the expression and function of pprA and pprB as well as other regulatory pathways. We are in the process of dissecting the regulatory pathways.
P. aeruginosa has an extraordinary ability to adapt to various growth conditions, enabling its survival under a variety of conditions ranging from different natural environments to various human tissues (10). To do so, it is equipped with a large number of regulatory genes including more than 90 two-component regulatory genes (30). It is possible that there are multiple regulons that are each capable of mediating the uptake of different classes of antibiotics. Indeed, the genome of strain PAO1 contains a total of 568 putative small-molecule transporters and 685 putative membrane proteins possibly involved in a complicated regulatory network. Comparison of the outer membrane protein profiles of PAK and PAK1-3 indeed shows significant differences, especially in major outer membrane porin F (Fig. 4). Furthermore, the LPS patterns of PAK and PAK1-3 are significantly different, consistent with earlier observations that a low level of permeability is associated with an altered LPS pattern (4, 17, 29).
A whole-cell alkaline phosphatase assay was used to evaluate outer membrane permeability. The whole-cell alkaline phosphatase assay was based on a membrane-diffusion barrier model described by Martinez et al. (12, 13). In that model, alkaline phosphatase displays two types of kinetics: at high substrate concentrations (>1 mg/ml), equilibrium of the substrate between the solution outside the membrane and the periplasm was achieved and the reaction has Michaelis-Menten kinetics, while at low substrate concentrations (<1 mg/ml), the high enzyme concentration and the low substrate level in the bacterial periplasm ensure that every substrate molecule entering the periplasm is converted into a product; therefore, the bacterial outer membrane functions as a diffusion barrier and the speed-limiting factor. Thus, the reaction velocity increases in direct proportion to the substrate concentration. It is the latter type of kinetics that makes it possible to use this assay as a convenient tool to assess bacterial outer membrane permeability. Our data have demonstrated a positive correlation between the outer membrane permeability index and bacterial sensitivity to aminoglycosides. However, it is not a linear correlation, which might reflect the structural differences between pNPP and aminoglycosides.
On the basis of the working model of two-component regulatory systems, it is likely that PprA and PprB regulate the expression of downstream genes in response to a certain environmental cue(s) which in turn affects membrane permeability. In the absence of such an environmental signal, a simple increased level of expression of the response regulator alone resulted in higher membrane permeability and increased sensitivity to antibiotics, presumably through increased basal levels of expression of the downstream genes. Once stimulated by the natural environmental cue(s), the effect of PprA and PprB is expected to be much greater. Identification of downstream genes regulated by PprA and PprB will provide clues for the natural signals to which this system responds. Most interestingly, the pprB gene was recently reported to be suppressed during the formation of a biofilm (36), a structure in which bacteria are highly resistant to various antimicrobials, including aminoglycosides. The relevance of pprA and pprB to biofilm formation is not known at this point.
Although the mechanisms of resistance among the clinical isolates that we have tested are not known, introduction of the pprB gene was sufficient to confer increased sensitivities to aminoglycosides to a majority of them; thus, those isolates are likely to have permeability defects, whereas the remaining four isolates on which pprB had no effect might also harbor other mechanisms of resistance, such as aminoglycoside-modifying enzymes and/or the MexXY efflux system. A more definitive answer to the effectiveness of pprA and pprB on aminoglycoside uptake relies on surveys of clinical isolates whose resistance mechanisms are well defined.9&4^8u], 百拇医药
An understanding of the mechanisms of antibiotic resistance is indispensable to the discovery of new agents that inhibit bacterial resistance. Well-studied examples are inhibitors of ß-lactamases, clavulanic acid, sulbactam, and tazobactam, which are used in combination with ß-lactams to treat infections caused by bacterial pathogens that harbor ß-lactamase enzymes (26). The pprA-pprB system could be used to screen for chemicals that specifically increase bacterial cell membrane permeability, which can be used together with aminoglycosides to fight infections caused by multidrug-resistant P. aeruginosa isolates.
ACKNOWLEDGMENTS{@3)}, http://www.100md.com
We thank Reuben Ramphal for providing clinical isolates of P. aeruginosa.{@3)}, http://www.100md.com
Work in the authors' laboratory was supported by NIH, the American Cancer Society, and the Cystic Fibrosis Foundation.{@3)}, http://www.100md.com
REFERENCES{@3)}, http://www.100md.com
Alonso, A., E. Campanario, and J. L. Martinez. 1999. Emergence of multidrug-resistant mutants is increased under antibiotic selective pressure in Pseudomonas aeruginosa. Microbiology 145(Pt 10):2857-2862.{@3)}, http://www.100md.com
Bodey, G. P., R. Bolivar, V. Fainstein, and L. Jadeja. 1983. Infections caused by Pseudomonas aeruginosa. Rev. Infect. Dis. 5:279-313.{@3)}, http://www.100md.com
Bryan, L. E. 1988. General mechanisms of resistance to antibiotics. J. Antimicrob. Chemother. 22(Suppl. A):1-15.{@3)}, http://www.100md.com
Bryan, L. E., K. O'Hara, and S. Wong. 1984. Lipopolysaccharide changes in impermeability-type aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 26:250-255.
Burns, J. L., J. M. Van Dalfsen, R. M. Shawar, K. L. Otto, R. L. Garber, J. M. Quan, A. B. Montgomery, G. M. Albers, B. W. Ramsey, and A. L. Smith. 1999. Effect of chronic intermittent administration of inhaled tobramycin on respiratory microbial flora in patients with cystic fibrosis. J. Infect. Dis. 179:1190-1196.x\, 百拇医药
Gilligan, P. H. 1991. Microbiology of airway disease in patients with cystic fibrosis. Clin. Microbiol. Rev. 4:35-51.x\, 百拇医药
Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580.x\, 百拇医药
Holder, I. A. 1993. Pseudomonas aeruginosa virulence-associated factors and their role in burn wound infections, p. 235-245. In R. B. Fick, Jr. (ed.), Pseudomonas aeruginosa, the opportunist pathogenesis and disease. CRC Press, Inc., Boca Raton, Fla.x\, 百拇医药
Karlowsky, J. A., S. A. Zelenitsky, and G. G. Zhanel. 1997. Aminoglycoside adaptive resistance. Pharmacotherapy 17:549-555.x\, 百拇医药
LeClerc, J. E., and T. A. Cebula. 2000. Pseudomonas survival strategies in cystic fibrosis. Science 289:391-392.
Mao, W., M. S. Warren, A. Lee, A. Mistry, and O. Lomovskaya. 2001. MexXY-OprM efflux pump is required for antagonism of aminoglycosides by divalent cations in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 45:2001-2007..|np, http://www.100md.com
Martinez, M. B., M. C. Flickinger, and G. L. Nelsestuen. 1996. Accurate kinetic modeling of alkaline phosphatase in the Escherichia coli periplasm: implications for enzyme properties and substrate diffusion. Biochemistry 35:1179-1186..|np, http://www.100md.com
Martinez, M. B., F. J. Schendel, M. C. Flickinger, and G. L. Nelsestuen. 1992. Kinetic properties of enzyme populations in vivo: alkaline phosphatase of the Escherichia coli periplasm. Biochemistry 31:11500-11509..|np, http://www.100md.com
Masuda, N., E. Sakagawa, S. Ohya, N. Gotoh, H. Tsujimoto, and T. Nishino. 2000. Contribution of the MexX-MexY-OprM efflux system to intrinsic resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44:2242-2246..|np, http://www.100md.com
Masuda, N., E. Sakagawa, S. Ohya, N. Gotoh, H. Tsujimoto, and T. Nishino. 2000. Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux pumps in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44:3322-3327.
Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.^t5], http://www.100md.com
Moore, R. A., and R. E. Hancock. 1986. Involvement of outer membrane of Pseudomonas cepacia in aminoglycoside and polymyxin resistance. Antimicrob. Agents Chemother. 30:923-926.^t5], http://www.100md.com
Mouton, J. W., J. G. den Hollander, and A. M. Horrevorts. 1993. Emergence of antibiotic resistance amongst Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J. Antimicrob. Chemother. 31:919-926.^t5], http://www.100md.com
National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. National Committee for Clinical Laboratory Standards Wayne, Pa.^t5], http://www.100md.com
Nikaido, H. 1994. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264:382-388.^t5], http://www.100md.com
Nunn, D. N., and S. Lory. 1991. Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase. Proc. Natl. Acad. Sci. USA 88:3281-3285.
Oliver, A., R. Canton, P. Campo, F. Baquero, and J. Blazquez. 2000. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288:1251-1254.+, 百拇医药
Ouchane, S., and S. Kaplan. 1999. Topological analysis of the membrane-localized redox-responsive sensor kinase PpprB from Rhodobacter sphaeroides 2.4.1. J. Biol. Chem. 274:17290-17296.+, 百拇医药
Poole, K., and R. Srikumar. 2001. Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance. Curr. Top. Med. Chem. 1:59-71.+, 百拇医药
Putman, M., H. W. van Veen, and W. N. Konings. 2000. Molecular properties of bacterial multidrug transporters. Microbiol. Mol. Biol. Rev. 64:672-693.+, 百拇医药
Rolinson, G. N. 1991. Evolution of beta-lactamase inhibitors. Rev. Infect. Dis. 13(Suppl. 9):S727-S732.+, 百拇医药
Saiman, L., F. Mehar, W. W. Niu, H. C. Neu, K. J. Shaw, G. Miller, and A. Prince. 1996. Antibiotic susceptibility of multiply resistant Pseudomonas aeruginosa isolated from patients with cystic fibrosis, including candidates for transplantation. Clin. Infect. Dis. 23:532-537.
Shawar, R. M., D. L. MacLeod, R. L. Garber, J. L. Burns, J. R. Stapp, C. R. Clausen, and S. K. Tanaka. 1999. Activities of tobramycin and six other antibiotics against Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob. Agents Chemother. 43:2877-2880.?;a, 百拇医药
Shearer, B. G., and N. J. Legakis. 1985. Pseudomonas aeruginosa: evidence for the involvement of lipopolysaccharide in determining outer membrane permeability to carbenicillin and gentamicin. J. Infect. Dis. 152:351-355.?;a, 百拇医药
Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, and I. T. Paulsen. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959-964.?;a, 百拇医药
Tatterson, L. E., J. F. Poschet, A. Firoved, J. Skidmore, and V. Deretic. 2001. CFTR and Pseudomonas infections in cystic fibrosis. Front Biosci. 6:D890-D897.
Totten, P. A., J. C. Lara, and S. Lory. 1990. The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. J. Bacteriol. 172:389-396.m'vs7.i, http://www.100md.com
Totten, P. A., and S. Lory. 1990. Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J. Bacteriol. 172:7188-7199.m'vs7.i, http://www.100md.com
West, S. E., H. P. Schweizer, C. Dall, A. K. Sample, and L. J. Runyen-Janecky. 1994. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81-86.m'vs7.i, http://www.100md.com
Westbrock-Wadman, S., D. R. Sherman, M. J. Hickey, S. N. Coulter, Y. Q. Zhu, P. Warrener, L. Y. Nguyen, R. M. Shawar, K. R. Folger, and C. K. Stover. 1999. Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability. Antimicrob. Agents Chemother. 43:2975-2983.m'vs7.i, http://www.100md.com
Whiteley, M., M. G. Bangera, R. E. Bumgarner, M. R. Parsek, G. M. Teitzel, S. Lory, and E. P. Greenberg. 2001. Gene expression in Pseudomonas aeruginosa biofilms. Nature 413:860-864.m'vs7.i, http://www.100md.com
Xiong, Y. Q., J. Caillon, M. F. Kergueris, H. Drugeon, D. Baron, G. Potel, and A. S. Bayer. 1997. Adaptive resistance of Pseudomonas aeruginosa induced by aminoglycosides and killing kinetics in a rabbit endocarditis model. Antimicrob. Agents Chemother. 41:823-826.m'vs7.i, http://www.100md.com
Zhulin, I. B., B. L. Taylor, and R. Dixon. 1997. PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox. Trends Biochem. Sci. 22:331-333.(Yanping Wang Unhwan Ha Lin Zeng and Shouguang Jin)
Received 7 June 2002/ Returned for modification 19 August 2002/ Accepted 10 October 2002}l, http://www.100md.com
ABSTRACT}l, http://www.100md.com
Membrane impermeability is the major contributing factor to multidrug resistance in clinical isolates of Pseudomonas aeruginosa. By using laboratory strain PAK, a spontaneous P. aeruginosa mutant (mutant PAK1-3) whose membrane had reduced permeability and which displayed increased levels of resistance to various antibiotics, especially aminoglycosides, was isolated. By complementation of the mutant with a genomic clone library derived from wild-type strain PAK, a novel two-component regulatory system (PprA and PprB) was identified and was found to be able to increase the permeability of the bacterial membrane and render PAK1-3 sensitive to antibiotics. Furthermore, specific phosphorylation of the response regulator (PprB) by histidine kinase (PprA) was observed in vitro, demonstrating that they are cognate two-component regulatory genes. Introduction of a plasmid expressing the pprB gene into randomly chosen clinical isolates (n = 17) resulted in increased sensitivity to aminoglycosides in the majority of isolates (n = 13) tested. This is the first demonstration that P. aeruginosa membrane permeability can be regulated, providing an important clue in the understanding of the mechanism of membrane impermeability-mediated multidrug resistance in P. aeruginosa.
INTRODUCTIONr7, http://www.100md.com
Pseudomonas aeruginosa is an opportunistic pathogen which poses a major threat to immunocompromised patients, burn victims, and cystic fibrosis (CF) patients (2, 6, 8, 31). Individuals with CF acquire P. aeruginosa infection from the environment at an early age, and repeated treatment with various antibiotics selects for multidrug-resistant strains that may eventually cause patient death (1). More than 90% of the deaths among CF patients are caused by lung infection with P. aeruginosa (18, 27, 28).r7, http://www.100md.com
Specific mechanisms of resistance include enzymatic inactivation of antibiotics, alterations of the target sites of antimicrobial agents, development of bypass pathways around antimicrobial targets, and reductions in cell wall membrane permeability (3, 23, 24, 25, 35). Although most resistance mechanisms are antibiotic specific, membrane impermeability-mediated resistance usually results in a multidrug resistance phenotype. Such impermeability mutants are common among clinical isolates of P. aeruginosa, especially in those individuals suffering from chronic infections. Previous studies have shown that as many as 90% of CF isolates have reduced cell wall permeability, thus presenting with a multidrug resistance phenotype (5, 28). Although the mechanisms of resistance to specific antibiotics are well known, the molecular mechanism of multidrug resistance is poorly understood.
Aminoglycosides, including kanamycin, streptomycin, gentamicin, amikacin, and tobramycin, exert their bactericidal activities by inhibiting protein synthesis by binding to the 30S ribosomal subunit. Due to their efficient bactericidal activities, aminoglycosides are widely used in the treatment of various bacterial infections. However, P. aeruginosa isolates are intrinsically less sensitive to many aminoglycosides primarily due to their outer membrane impermeability and easily become resistant to high levels of aminoglycosides. Resistance is also attributed to an efflux pump, MexXY, that is specific for aminoglycosides, although overexpression of MexYY alone does not confer resistance to aminoglycosides (11, 14, 15). More recently, a phenomenon called "adaptive resistance to aminoglycosides" has been described in P. aeruginosa both in vitro and in vivo (9, 37), in which the level of bacterial resistance to aminoglycosides gradually increases following pretreatment with aminoglycosides or other agents. The molecular mechanism for this desensitization is totally unknown.
In this report, we describe a novel two-component regulatory system that up-regulates the permeability of the membrane to various antibiotics, presumably by turning on the synthesis of membrane proteins that mediate antibiotic transport or turning off efflux systems. Most strikingly, overexpression of the response regulator gene, pprB, in clinical isolates resulted in increased sensitivities to aminoglycosides in the majority of those isolates tested. Elevated levels of expression of the two-component regulatory genes increased the outer membrane permeability of P. aeruginosa to para-nitrophenyl phosphate (pNPP), which is a chromogenic substrate for alkaline phosphatase. The pprA-pprB regulatory system may represent a new mechanism for the control of membrane impermeability in P. aeruginosa.m74}jg&, http://www.100md.com
MATERIALS AND METHODSm74}jg&, http://www.100md.com
Strains and plasmids. The strains and plasmids used in this study are listed in Table 1. To construct a clone bank of strain PAK genomic DNA, PAK chromosomal DNA was partially digested with Sau3A and fragments ranging from 3 to 5 kb were gel purified and ligated into the BamHI site of pUCP19. The ligation mixture was transformed into Escherichia coli DH5{alpha} , and 5 x 104 colonies were pooled; of these colonies, at least 20% had an insert; thus, the bank consists of at least 1 x 104 independent clones with DNA inserts.
fig.ommitted#:.c@, 百拇医药
TABLE 1. Bacterial strains and plasmids#:.c@, 百拇医药
To generate pWC005, a 0.5-kb NdeI-XmnI DNA fragment from pUCP19 that encodes the promoter and the N-terminal signal sequence of the ampicillin resistance gene was ligated into the SmaI site of pSOP3, which is located upstream of a signal sequenceless alkaline phosphatase structural gene (23). The resulting plasmid, pWC005, encodes a translational fusion between the bla signal sequence and alkaline phosphatase.#:.c@, 百拇医药
The pprB gene was amplified from the PAK chromosome by PCR with the oligonucleotides 5'-GGC TAA TAC CAT TCG GTA TGG CTG CAT TCC-3' and 5'-CTT GGA TAC ATC GAC GCG GAG ATT CTG AAG-3'. The PCR product was cloned into pCR2.1-TOPO, resulting in pCR-RR, in which the pprB gene is in the opposite direction relative to that of the lac promoter. The pprB gene was then subcloned as an EcoRI fragment into the same site of pUCP19, with insertion of the pprB gene in the same or opposite direction relative to that of the lac promoter on the vector; these plasmids were designated pZLRR-{alpha} and pZLRR-ß, respectively. A tetracycline resistance gene cassette was isolated from pBR322 as an EcoRI-AvaI fragment, blunt ended, and inserted into the SspI and ScaI sites of pUCP19 to generate pUCPT19. Then, the EcoRI fragment from pCR-RR, which contains the pprB gene, was inserted into the same site of pUCPT19 to generate pRRBTc, in which pprB is in the same direction as the lac promoter.
Overexpression and purification of PprA and PprB. To overproduce the PprA and PprB proteins in E. coli, the coding regions of the two open reading frames were amplified by PCR with primer sets consisting of primers 5'-GTG AGC TGG TCG GAT CCT CGA ATT TTC CCG-3' and 5'-GGT TGA AAA AAC GCA AGC TTG TCC GCA GGC-3' and primers 5'-GTA TGA CCA TGG ATC CAA CCG GCC TCG C-3' and 5'-GGC AGG GCG GAA GCT TGC TGA TCT AGC A-3'. The primer sequences were based on the genome sequence of strain PAO1. The PCR products were first cloned into pCR2.1-TOPO, with the plasmids designated pYW017 and pYW020, respectively. Subsequently, a 2.8-kb pprA fragment from pYW017 and an 862-bp pprB fragment from pYW020 were isolated and subcloned into BamHI-HindIII sites of pQE32 and pQE31, resulting in pYW021 and pYW024, respectively.., 百拇医药
To purify the His-tagged PprA and PprB proteins, E. coli strain M15 harboring pREP4 and pYW021 or pYW024 was induced with 1 mM isopropyl-ß-D-thiogalactopyranoside for 6 h at 28°C. Overproduced six-His-tagged PprA and six-His-tagged PprB proteins were affinity purified from the soluble fraction of the cell extracts with a nickel-agarose column by the protocol provided by Qiagen, Inc. Purified proteins were dialyzed against storage buffer (50 mM Tris-HCl [pH 8.0], 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 50% glycerol) overnight and stored at -20°C until use.
For in vitro protein phosphorylation tests, 10 µl of reaction buffer (50 mM Tris-HCl [pH 7.4], 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol) containing 5 µCi of [-32P]ATP was mixed with 1 µg of purified protein, and the mixture was incubated at room temperature for 5 min. The reaction was terminated by the addition of 5 µl of 4x loading buffer. The reaction mixture was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with Coomassie blue dye, dried, and exposed to X-ray film.v, 百拇医药
Whole-cell alkaline phosphatase assay for assessment of outer membrane permeability. An overnight culture of P. aeruginosa with proper antibiotics was washed once with fresh Luria-Bertani (LB) medium and reinoculated into 3 ml of fresh LB medium without antibiotics to an A600 of about 0.1. After culture for 7 h, the cells were washed once with ice-cold reaction buffer (0.1 M Tris-HCl [pH 8.0], 100 mM NaCl, 5 mM MgCl2, 0.1 mM ZnCl2) and resuspended in 3 ml of the same buffer, and the final cell density (A600) was measured. The reaction was started by adding 0.1 ml of the cell suspension to a reaction mixture containing 0.8 ml of reaction buffer and 0.1 ml of pNPP (0.08 mg/ml in 1 M Tris-HCl [pH 8.0]). All reactions were carried out in a standard plastic spectrophotometer cuvette at room temperature. The samples were allowed to react for 2 min, and 0.1 ml of 1 M KH2PO4 was added to terminate the reaction. The cells were centrifuged, and the A410 of the supernatant was measured. Samples with no substrate were used as negative controls. An index of outer membrane permeability was calculated as A410/A600[S], where [S] is the pNPP concentration.
Miscellaneous assays. The MICs of the various antibiotics tested were determined by standard protocols (2, 19), and assays for ß-galactosidase activity were performed by a previously described protocol (16). The outer membrane proteins of P. aeruginosa were isolated by the protein fractionation protocol described before (21).cmnlq, http://www.100md.com
RESULTScmnlq, http://www.100md.com
Characterization of a spontaneous mutant of P. aeruginosa with decreased sensitivities to antibiotics. To understand the mechanism that results in a high frequency of multidrug resistance in P. aeruginosa, we selected spontaneous neomycin-resistant mutants of PAK that were able to grow on Luria agar (L-agar) medium containing 200 µg of neomycin per ml. A multidrug-resistant isolate, designated PAK1-3, was identified among the initial isolates through further screening of the resistant mutants. PAK1-3 is not only resistant to neomycin, but the MICs of all the aminoglycosides tested were also increased, including those of kanamycin, streptomycin, gentamicin, amikacin, and tobramycin, as well as those of tetracycline, ciprofloxacin, and erythromycin (Table 2), suggesting that it has a permeability defect. Consistent with this prediction, PAK1-3 has a slower growth rate compared to that of its parent strain, PAK, as seen from the smaller colony sizes and the delayed increase in cell density in liquid cultures (data not shown). However, its sensitivity to carbenicillin is not significantly changed, indicating that it has a limited substrate specificity (Table 2).
fig.ommitted(], 百拇医药
TABLE 2. MICs for PAK and PAK1-3 derivatives(], 百拇医药
A novel two-component regulatory system controls antibiotic sensitivity in PAK1-3. With the assumption that PAK1-3 is an impermeable mutant as a result of the loss of a gene function, we attempted to complement the mutant with wild-type PAK chromosomal DNA clones. A clone bank of 3 to 5 kb of PAK chromosomal DNA in pUCP19 was electroporated into PAK1-3. Transformants were first selected on L agar containing carbenicillin (150 µg/ml) and were then replica plated on L agar containing carbenicillin (150 µg/ml) plus neomycin (200 µg/ml) to screen for isolates that are sensitive to aminoglycosides. A total of 5,000 colonies were screened, of which 3 colonies were sensitive to the neomycin (Fig. 1). To confirm that this sensitivity phenotype is due to the presence of a plasmid and is not due to a reversion or a secondary mutation, plasmids were purified from the three isolates and retransformed into fresh PAK1-3 cells. Two of the three plasmids indeed conferred to PAK1-3 sensitivity to the aminoglycosides, whereas one of them failed to do so. The effective two plasmids were identical by DNA sequence analysis, and the plasmid was designated pUCP19B.
fig.ommittedpf*, http://www.100md.com
FIG. 1. Conversion of neomycin resistance phenotype by introduction of a plasmid expressing pprA or pprB. Bacterial cells from single colonies were streaked onto an L-agar plate containing carbenicillin (150 µg/ml) alone or carbenicillin (150 µg/ml) plus neomycin (200 µg/ml) and were grown overnight at 37°C.pf*, http://www.100md.com
pUCP19B was found to contain a 3,280-bp DNA insert encoding a 2,768-bp putative histidine kinase homolog, identical to PA4293 of the Pseudomonas genome sequence designated pprA, which is transcriptionally fused under the lac promoter of the vector. Tests of subclones derived from the 3.3-kb insert as well as insertional inactivation of the histidine kinase structural gene in pUCP19B demonstrated that lac promoter-driven expression of the pprA gene is essential to confer aminoglycoside sensitivity to strain PAK1-3 (data not shown). Unlike the majority of the known sensor molecules, the PprA protein lacks a signal sequence or transmembrane segments; thus, it is likely a cytoplasmic protein. The N terminus of the PprA protein has PAS and PAC motifs that are known to be involved in various signal-sensing functions (38). Therefore, it is likely that in the cytoplasm PprA senses external signals through either binding to a diffusible molecule(s) or interacting with another sensory protein(s).
PprA and PprB form a functional two-component regulatory system. On the basis of the genome sequence of P. aeruginosa, a 827-bp putative response regulator , which we named pprB, resides 1,184 bp upstream of the pprA gene and is transcribed in the opposite direction from that of pprA. To see whether PprB is a cognate response regulator of PprA, we tested whether increased expression of pprB can also confer to PAK1-3 sensitivity to aminoglycosides. The pprB gene was amplified from the PAK chromosome by PCR and cloned into pUCP19, with the insert placed in the same or opposite direction relative to that of the lac promoter on the vector; these plasmids were designated pZLRR-{alpha} and pZLRR-ß, respectively. The pprB gene sequence of PAK differs from that of PAO1 by only 2 bases, without changes in amino acid sequences. When these two plasmids were transformed into the PAK1-3 background, only pZLRR-{alpha} and not pZLRR-ß was able to confer sensitivity to aminoglycosides, suggesting that pprA and pprB likely function as a pair of two-component regulatory genes. Further evidence supporting this possibility was that fact that the effect of PprB overexpression was much more dramatic than that of PprA overexpression on the change in the MIC for PAK1-3 (Table 2).
To further confirm the relationship between PprA and PprB, an in vitro phosphorylation test was performed. Like other two-component regulatory genes, both PprA and PprB contained conserved phosphorylation sites (histidine and aspartate, respectively). Both the PprA and PprB proteins were overproduced in E. coli by fusion to N-terminal His tags that are driven by tac promoters (see Materials and Methods). Overproduced proteins were purified from soluble fractions with nickel-agarose affinity columns and subjected to the phosphorylation test by using [-32P]ATP (see Materials and Methods). As shown in Fig. 2, PprA alone underwent active autophosphorylation, while PprB did not. However, in the presence of PprA, PprB is highly phosphorylated; thus, PprA functions as a specific kinase for PprB. Combined with the antibiotic sensitivity-conferring abilities of PprA and PprB, the phosphorylation results strongly suggest a sensor-response regulator relationship between PprA and PprB.qa, 百拇医药
fig.ommitted:o1m5a%, http://www.100md.com
FIG. 2. (A) Purification of PprA and PprB proteins from E. coli overexpressing the pprA and pprB genes, respectively. Lane 1, total cellular protein of M15/pREP4/pYW021; lane 2, purified six-His-tagged PprA fusion protein; lane 3, total cellular protein of M15/pREP4/pYW024; lane 4, purified six-His-tagged PprB fusion protein; lane M, molecular size marker. (B) Coomassie blue stain of an SDS-polyacrylamide gel showing the PprA and PprB proteins used in the phosphorylation study. (C) X-ray film after exposure to the SDS-polyacrylamide gel shown in panel B.:o1m5a%, http://www.100md.com
PprA-PprB regulates membrane permeability. To test outer membrane permeability, a whole-cell alkaline phosphatase assay was conducted. The whole-cell alkaline-phosphatase assay was based on the membrane-diffusion barrier model described by Martinez et al. (12, 13). At low substrate concentrations, the high enzyme concentration and the low substrate level in the bacterial periplasm ensure that every substrate molecule entering the periplasm is converted into a product; therefore, the bacterial outer membrane functions as a diffusion barrier and the speed-limiting factor; thus, the reaction velocity increases in direct proportion to the substrate concentration.
Growth of PAK and PAK1-3 in L broth produced undetectable levels of endogenous alkaline phosphatase activity, whereas growth in medium with a low phosphate concentration resulted in high levels of alkaline phosphatase activity. However, under conditions with low phosphate concentrations, a high degree of cell lysis which interfered with the assay was observed. To overcome this, a plasmid that constitutively expresses the alkaline phosphatase gene, pWC005, was introduced into both PAK and PAK1-3. Using a method similar to that previously described for E. coli (12, 13), we have determined that the alkaline phosphatase activity of PAK/pWC005 is directly proportional to the pNPP concentration when the pNPP concentration is <1 mg/ml.#y, http://www.100md.com
PAK/pWC005 and PAK1-3/pWC005 were grown in L broth and incubated with 0.08 mg of pNPP per ml. As shown in Fig. 3, the outer membrane permeability of PAK1-3 is at least 30% lower than that of wild-type PAK, agreeing well with the earlier prediction based on the antibiotic resistance and the phenotype of slower growth of PAK1-3. When the plasmid expressing pprB, pZLRR-{alpha} , was introduced into PAK1-3, the resulting strain restored not only the membrane permeability (Fig. 3) but also the growth rate to the levels for wild-type PAK, demonstrating that PprA and PprB regulate bacterial membrane permeability, likely through regulation of the downstream genes directly involved in the membrane transport function.
fig.ommitted)^.ev'^, 百拇医药
FIG. 3. . Comparison of bacterial outer membrane permeabilities. The relative permeabilities of the outer membranes (outer membrane permeability index = A410/A600[S]), determined by a whole-cell alkaline phosphatase assay with low substrate concentrations, are shown for indicated strains.)^.ev'^, 百拇医药
In support of the conclusion stated above, differences in a few outer membrane protein bands between PAK and PAK1-3 were clearly visible (Fig. 4), especially a 36-kDa protein band which is highly overexpressed in the PAK1-3 background. The 36-kDa protein was determined to be the major outer membrane porin, OprF, by mass spectrometric analysis. Interestingly, upon introduction of pZLRR-{alpha} into the PAK1-3 background, the outer membrane profile returned to that of the wild type, demonstrating that PprB influences the expression and/or outer membrane localization of a number of proteins, including the F porin. The contribution of OprF to aminoglycoside resistance is not known.
fig.ommitted+/, 百拇医药
FIG. 4. . Comparison of bacterial outer membrane protein profiles. Overnight cultures of the bacterial cells were fractionated into cytosol, inner membrane, and outer membrane proteins. Outer membrane proteins were separated by SDS-PAGE on 12% polyacrylamide gels and stained with Coomassie blue. Lane 1, PAK; lane 2, PAK1-3, lane 3, PAK1-3/pZLPPRB{alpha} ; lane M, molecular size marker. Arrows indicate obvious band differences between PAK and PAK1-3.+/, 百拇医药
Overexpression of the pprB gene in clinical isolates renders increased sensitivities to aminoglycosides. To test whether increased levels of expression of the pprA-pprB two-component regulatory genes can render higher levels of membrane permeability among clinical isolates of P. aeruginosa, a pprB-overexpressing construct was introduced into clinical isolates. Eighteen isolates from patients with CF and 18 isolates from the blood of individuals without CF that show various degrees of resistance to gentamicin were randomly chosen for the test. Since the majority of those strains were resistant to carbenicillin (150 µg/ml) but were inhibited by tetracycline (100 µg/ml), we have used a tetracycline resistance construct that expresses the pprB gene under the lac promoter, pRRBTc. We were able to introduce the plasmid into 17 isolates by electroporation, and in the presence of pRRBTc, the aminoglycoside MICs for 13 of those transformants decreased at least twofold compared to those for isolates containing the pUCPT19 vector alone, while the gentamicin MIC for the remaining 4 transformants did not change at all (Table 3). These moderate reductions in the MICs are the result of a mere increase in the PprB level by the introduction of pRRBTc. Active signaling through the PprA-PprB two-component system might be required for the maximum effect on the MIC; nevertheless, an elevated level of expression of the pprB gene was sufficient to confer increased sensitivity to aminoglycosides to the majority of the isolates; thus, it is consistent with the prediction that PprA and PprB regulate genes that increase membrane permeability and transport, resulting in increased bacterial sensitivities to antibiotics.
fig.ommitted6.u^^$), http://www.100md.com
TABLE 3. Effect of elevated levels of pprB expression on MICs for clinical isolates6.u^^$), http://www.100md.com
DISCUSSION6.u^^$), http://www.100md.com
Up to 90% of clinical isolates of P. aeruginosa appear to carry the impermeability type of resistance (18, 28). It has been demonstrated that this type of resistance arises from the combination of an unusually restricted outer membrane permeability through mutation or changes in porin and lipopolysaccharide (LPS) structures and secondary resistance mechanisms such as energy-dependent multidrug efflux and the actions of antibiotic-modifying enzymes (3, 4, 17, 20, 29, 35). P. aeruginosa strains that are sensitive to aminoglycosides can acquire such resistance at a high frequency. Under standard laboratory conditions, a laboratory strain of P. aeruginosa, PAK, shows a frequency of resistance to neomycin at 200 µg/ml as high as 10-6. This is much higher than the standard spontaneous mutational rate of 10-8 per cell per generation in E. coli. This high frequency of conversion to aminoglycoside resistance could be a result of either the hypermutational rate of P. aeruginosa (e.g., due to defective DNA repair), as has been described within the lung environment of patients with CF (22), or a simple on-off phase switch to a state of a low level of cell permeability. Preliminary analysis of the pprA mutant indicates that there was no alteration in the antibiotic resistance pattern, suggesting the existence of multiple pathways controlling membrane permeability. Therefore, strain PAK1-3 may have a defect(s) in a further upstream regulatory gene that affects the expression and function of pprA and pprB as well as other regulatory pathways. We are in the process of dissecting the regulatory pathways.
P. aeruginosa has an extraordinary ability to adapt to various growth conditions, enabling its survival under a variety of conditions ranging from different natural environments to various human tissues (10). To do so, it is equipped with a large number of regulatory genes including more than 90 two-component regulatory genes (30). It is possible that there are multiple regulons that are each capable of mediating the uptake of different classes of antibiotics. Indeed, the genome of strain PAO1 contains a total of 568 putative small-molecule transporters and 685 putative membrane proteins possibly involved in a complicated regulatory network. Comparison of the outer membrane protein profiles of PAK and PAK1-3 indeed shows significant differences, especially in major outer membrane porin F (Fig. 4). Furthermore, the LPS patterns of PAK and PAK1-3 are significantly different, consistent with earlier observations that a low level of permeability is associated with an altered LPS pattern (4, 17, 29).
A whole-cell alkaline phosphatase assay was used to evaluate outer membrane permeability. The whole-cell alkaline phosphatase assay was based on a membrane-diffusion barrier model described by Martinez et al. (12, 13). In that model, alkaline phosphatase displays two types of kinetics: at high substrate concentrations (>1 mg/ml), equilibrium of the substrate between the solution outside the membrane and the periplasm was achieved and the reaction has Michaelis-Menten kinetics, while at low substrate concentrations (<1 mg/ml), the high enzyme concentration and the low substrate level in the bacterial periplasm ensure that every substrate molecule entering the periplasm is converted into a product; therefore, the bacterial outer membrane functions as a diffusion barrier and the speed-limiting factor. Thus, the reaction velocity increases in direct proportion to the substrate concentration. It is the latter type of kinetics that makes it possible to use this assay as a convenient tool to assess bacterial outer membrane permeability. Our data have demonstrated a positive correlation between the outer membrane permeability index and bacterial sensitivity to aminoglycosides. However, it is not a linear correlation, which might reflect the structural differences between pNPP and aminoglycosides.
On the basis of the working model of two-component regulatory systems, it is likely that PprA and PprB regulate the expression of downstream genes in response to a certain environmental cue(s) which in turn affects membrane permeability. In the absence of such an environmental signal, a simple increased level of expression of the response regulator alone resulted in higher membrane permeability and increased sensitivity to antibiotics, presumably through increased basal levels of expression of the downstream genes. Once stimulated by the natural environmental cue(s), the effect of PprA and PprB is expected to be much greater. Identification of downstream genes regulated by PprA and PprB will provide clues for the natural signals to which this system responds. Most interestingly, the pprB gene was recently reported to be suppressed during the formation of a biofilm (36), a structure in which bacteria are highly resistant to various antimicrobials, including aminoglycosides. The relevance of pprA and pprB to biofilm formation is not known at this point.
Although the mechanisms of resistance among the clinical isolates that we have tested are not known, introduction of the pprB gene was sufficient to confer increased sensitivities to aminoglycosides to a majority of them; thus, those isolates are likely to have permeability defects, whereas the remaining four isolates on which pprB had no effect might also harbor other mechanisms of resistance, such as aminoglycoside-modifying enzymes and/or the MexXY efflux system. A more definitive answer to the effectiveness of pprA and pprB on aminoglycoside uptake relies on surveys of clinical isolates whose resistance mechanisms are well defined.9&4^8u], 百拇医药
An understanding of the mechanisms of antibiotic resistance is indispensable to the discovery of new agents that inhibit bacterial resistance. Well-studied examples are inhibitors of ß-lactamases, clavulanic acid, sulbactam, and tazobactam, which are used in combination with ß-lactams to treat infections caused by bacterial pathogens that harbor ß-lactamase enzymes (26). The pprA-pprB system could be used to screen for chemicals that specifically increase bacterial cell membrane permeability, which can be used together with aminoglycosides to fight infections caused by multidrug-resistant P. aeruginosa isolates.
ACKNOWLEDGMENTS{@3)}, http://www.100md.com
We thank Reuben Ramphal for providing clinical isolates of P. aeruginosa.{@3)}, http://www.100md.com
Work in the authors' laboratory was supported by NIH, the American Cancer Society, and the Cystic Fibrosis Foundation.{@3)}, http://www.100md.com
REFERENCES{@3)}, http://www.100md.com
Alonso, A., E. Campanario, and J. L. Martinez. 1999. Emergence of multidrug-resistant mutants is increased under antibiotic selective pressure in Pseudomonas aeruginosa. Microbiology 145(Pt 10):2857-2862.{@3)}, http://www.100md.com
Bodey, G. P., R. Bolivar, V. Fainstein, and L. Jadeja. 1983. Infections caused by Pseudomonas aeruginosa. Rev. Infect. Dis. 5:279-313.{@3)}, http://www.100md.com
Bryan, L. E. 1988. General mechanisms of resistance to antibiotics. J. Antimicrob. Chemother. 22(Suppl. A):1-15.{@3)}, http://www.100md.com
Bryan, L. E., K. O'Hara, and S. Wong. 1984. Lipopolysaccharide changes in impermeability-type aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 26:250-255.
Burns, J. L., J. M. Van Dalfsen, R. M. Shawar, K. L. Otto, R. L. Garber, J. M. Quan, A. B. Montgomery, G. M. Albers, B. W. Ramsey, and A. L. Smith. 1999. Effect of chronic intermittent administration of inhaled tobramycin on respiratory microbial flora in patients with cystic fibrosis. J. Infect. Dis. 179:1190-1196.x\, 百拇医药
Gilligan, P. H. 1991. Microbiology of airway disease in patients with cystic fibrosis. Clin. Microbiol. Rev. 4:35-51.x\, 百拇医药
Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580.x\, 百拇医药
Holder, I. A. 1993. Pseudomonas aeruginosa virulence-associated factors and their role in burn wound infections, p. 235-245. In R. B. Fick, Jr. (ed.), Pseudomonas aeruginosa, the opportunist pathogenesis and disease. CRC Press, Inc., Boca Raton, Fla.x\, 百拇医药
Karlowsky, J. A., S. A. Zelenitsky, and G. G. Zhanel. 1997. Aminoglycoside adaptive resistance. Pharmacotherapy 17:549-555.x\, 百拇医药
LeClerc, J. E., and T. A. Cebula. 2000. Pseudomonas survival strategies in cystic fibrosis. Science 289:391-392.
Mao, W., M. S. Warren, A. Lee, A. Mistry, and O. Lomovskaya. 2001. MexXY-OprM efflux pump is required for antagonism of aminoglycosides by divalent cations in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 45:2001-2007..|np, http://www.100md.com
Martinez, M. B., M. C. Flickinger, and G. L. Nelsestuen. 1996. Accurate kinetic modeling of alkaline phosphatase in the Escherichia coli periplasm: implications for enzyme properties and substrate diffusion. Biochemistry 35:1179-1186..|np, http://www.100md.com
Martinez, M. B., F. J. Schendel, M. C. Flickinger, and G. L. Nelsestuen. 1992. Kinetic properties of enzyme populations in vivo: alkaline phosphatase of the Escherichia coli periplasm. Biochemistry 31:11500-11509..|np, http://www.100md.com
Masuda, N., E. Sakagawa, S. Ohya, N. Gotoh, H. Tsujimoto, and T. Nishino. 2000. Contribution of the MexX-MexY-OprM efflux system to intrinsic resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44:2242-2246..|np, http://www.100md.com
Masuda, N., E. Sakagawa, S. Ohya, N. Gotoh, H. Tsujimoto, and T. Nishino. 2000. Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM efflux pumps in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 44:3322-3327.
Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.^t5], http://www.100md.com
Moore, R. A., and R. E. Hancock. 1986. Involvement of outer membrane of Pseudomonas cepacia in aminoglycoside and polymyxin resistance. Antimicrob. Agents Chemother. 30:923-926.^t5], http://www.100md.com
Mouton, J. W., J. G. den Hollander, and A. M. Horrevorts. 1993. Emergence of antibiotic resistance amongst Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J. Antimicrob. Chemother. 31:919-926.^t5], http://www.100md.com
National Committee for Clinical Laboratory Standards. 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved standard M7-A4. National Committee for Clinical Laboratory Standards Wayne, Pa.^t5], http://www.100md.com
Nikaido, H. 1994. Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science 264:382-388.^t5], http://www.100md.com
Nunn, D. N., and S. Lory. 1991. Product of the Pseudomonas aeruginosa gene pilD is a prepilin leader peptidase. Proc. Natl. Acad. Sci. USA 88:3281-3285.
Oliver, A., R. Canton, P. Campo, F. Baquero, and J. Blazquez. 2000. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288:1251-1254.+, 百拇医药
Ouchane, S., and S. Kaplan. 1999. Topological analysis of the membrane-localized redox-responsive sensor kinase PpprB from Rhodobacter sphaeroides 2.4.1. J. Biol. Chem. 274:17290-17296.+, 百拇医药
Poole, K., and R. Srikumar. 2001. Multidrug efflux in Pseudomonas aeruginosa: components, mechanisms and clinical significance. Curr. Top. Med. Chem. 1:59-71.+, 百拇医药
Putman, M., H. W. van Veen, and W. N. Konings. 2000. Molecular properties of bacterial multidrug transporters. Microbiol. Mol. Biol. Rev. 64:672-693.+, 百拇医药
Rolinson, G. N. 1991. Evolution of beta-lactamase inhibitors. Rev. Infect. Dis. 13(Suppl. 9):S727-S732.+, 百拇医药
Saiman, L., F. Mehar, W. W. Niu, H. C. Neu, K. J. Shaw, G. Miller, and A. Prince. 1996. Antibiotic susceptibility of multiply resistant Pseudomonas aeruginosa isolated from patients with cystic fibrosis, including candidates for transplantation. Clin. Infect. Dis. 23:532-537.
Shawar, R. M., D. L. MacLeod, R. L. Garber, J. L. Burns, J. R. Stapp, C. R. Clausen, and S. K. Tanaka. 1999. Activities of tobramycin and six other antibiotics against Pseudomonas aeruginosa isolates from patients with cystic fibrosis. Antimicrob. Agents Chemother. 43:2877-2880.?;a, 百拇医药
Shearer, B. G., and N. J. Legakis. 1985. Pseudomonas aeruginosa: evidence for the involvement of lipopolysaccharide in determining outer membrane permeability to carbenicillin and gentamicin. J. Infect. Dis. 152:351-355.?;a, 百拇医药
Stover, C. K., X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. Brinkman, W. O. Hufnagle, D. J. Kowalik, M. Lagrou, R. L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L. L. Brody, S. N. Coulter, K. R. Folger, A. Kas, K. Larbig, R. Lim, K. Smith, D. Spencer, G. K. Wong, Z. Wu, and I. T. Paulsen. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406:959-964.?;a, 百拇医药
Tatterson, L. E., J. F. Poschet, A. Firoved, J. Skidmore, and V. Deretic. 2001. CFTR and Pseudomonas infections in cystic fibrosis. Front Biosci. 6:D890-D897.
Totten, P. A., J. C. Lara, and S. Lory. 1990. The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. J. Bacteriol. 172:389-396.m'vs7.i, http://www.100md.com
Totten, P. A., and S. Lory. 1990. Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK. J. Bacteriol. 172:7188-7199.m'vs7.i, http://www.100md.com
West, S. E., H. P. Schweizer, C. Dall, A. K. Sample, and L. J. Runyen-Janecky. 1994. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81-86.m'vs7.i, http://www.100md.com
Westbrock-Wadman, S., D. R. Sherman, M. J. Hickey, S. N. Coulter, Y. Q. Zhu, P. Warrener, L. Y. Nguyen, R. M. Shawar, K. R. Folger, and C. K. Stover. 1999. Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability. Antimicrob. Agents Chemother. 43:2975-2983.m'vs7.i, http://www.100md.com
Whiteley, M., M. G. Bangera, R. E. Bumgarner, M. R. Parsek, G. M. Teitzel, S. Lory, and E. P. Greenberg. 2001. Gene expression in Pseudomonas aeruginosa biofilms. Nature 413:860-864.m'vs7.i, http://www.100md.com
Xiong, Y. Q., J. Caillon, M. F. Kergueris, H. Drugeon, D. Baron, G. Potel, and A. S. Bayer. 1997. Adaptive resistance of Pseudomonas aeruginosa induced by aminoglycosides and killing kinetics in a rabbit endocarditis model. Antimicrob. Agents Chemother. 41:823-826.m'vs7.i, http://www.100md.com
Zhulin, I. B., B. L. Taylor, and R. Dixon. 1997. PAS domain S-boxes in Archaea, Bacteria and sensors for oxygen and redox. Trends Biochem. Sci. 22:331-333.(Yanping Wang Unhwan Ha Lin Zeng and Shouguang Jin)