Inner membrane efflux components are responsible for beta-lactam specificity of multidrug efflux pumps in Pseudomonas aeruginosa.

A major feature of the MexAB-OprM multidrug efflux pump which distinguishes it from the MexCD-OprJ and MexEF-OprN multidrug efflux systems in Pseudomonas aeruginosa is its ability to export a wide variety of beta-lactam antibiotics. Given the periplasmic location of their targets it is feasible that beta-lactams exit the cell via the outer membrane OprM without interaction with MexA and MexB, though the latter appear to be necessary for OprM function. To test this, chimeric MexAB-OprJ and MexCD-OprM efflux pumps were reconstituted in delta mexCD delta oprM and delta mexAB delta oprJ strains, respectively, and the influence of the exchange of outer membrane components on substrate (i.e., beta-lactam) specificity was assessed. Both chimeric pumps were active in antibiotic efflux, as evidenced by their contributions to resistance to a variety of antimicrobial agents, although there was no change in resistance profiles relative to the native pumps, indicating that OprM is not the determining factor for the beta-lactam specificity of MexAB-OprM. Thus, one or both of inner membrane-associated proteins MexA and MexB are responsible for drug recognition, including recognition of beta-lactams.     

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial

Background

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes.

Results

The PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans.

Conclusion

Genes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.     

The MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa is growth-phase regulated

Intrinsic antibiotic resistance in Pseudomonas aeruginosa is attributed to low outer membrane permeability and drug efflux mediated by the products of mexAmexBoprM efflux operon. Using a mexA-phoA fusion, expression of the efflux genes was assessed as a function of growth in a variety of strains. The efflux operon was growth-phase regulated in both wild-type and nalB strains, being minimally expressed in lag phase and increasing in log to late log phase. MexR, the only known regulator of MexAMexBOprM and target of mutation in nalB strains, was not involved in the growth-phase regulation. The las cascade regulates genes based on increased cell-density, but a deletion in lasR had no effect on mexAmexBoprM expression. Putative recognition sequences for AlgT/U and RpoN were identified upstream of mexA, but algT/U and rpoN null mutants also had no effect on mexAmexBoprM expression.     

Evidence of MexT-independent overexpression of MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa in presence of metabolic stress

Background

The Pseudomonas aeruginosa MexEF-OprN efflux pump confers resistance to clinically significant antibiotics. Regulation of mexEF-oprN operon expression is multifaceted with the MexT activator being one of the most prominent regulatory proteins.

Methodology

We have exploited the impaired metabolic fitness of a P. aeruginosa mutant strain lacking several efflux pump of the resistance nodulation cell division superfamily and the TolC homolog OpmH, and isolated derivatives (large colony variants) that regained fitness by incubation on nutrient-rich medium in the absence of antibiotics. Although the mexEF-oprN operon is uninducible in this mutant due to a 8-bp mexT insertion present in some P. aeruginosa PAO1 strains, the large colony variants expressed high levels of MexEF-OprN. Unlike large colony variants obtained after plating on antibiotic containing medium which expressed mexEF-oprN in a MexT-dependent fashion as evidenced by clean excision of the 8-bp insertion from mexT, mexEF-oprN expression was MexT-independent in the large colony variants obtained by plating on LB alone since the mexT gene remained inactivated. A search for possible regulators of mexEF-oprN expression using transposon mutagenesis and genomic library expression approaches yielded several candidates but proved inconclusive.

Significance

Our results show that antibiotic and metabolic stress lead to up-regulation of MexEF-OprN expression via different mechanisms and that MexEF-OprN does not only extrude antimicrobials but rather serves other important metabolic functions.     

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

The self‐assembling MexA‐MexB‐OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR‐wt as well as a selected set of MDR single mutants distant from the proposed DNA‐binding helix. Although DNA affinity and MexA‐MexB‐OprM repression were both drastically impaired in the selected MexR‐MDR mutants, MexR‐wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR‐MDR mutants, secondary structure content and oligomerization properties were very similar to MexR‐wt despite their lack of DNA binding. Despite this, the MexR‐MDR mutants showed highly varying stabilities compared with MexR‐wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA‐binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR‐wt in both free and DNA‐bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations—stability, domain interactions, and internal hydrophobic surfaces—are also critical for the regulation of MexR DNA binding.     

Mutant analysis shows that alanine racemases from Pseudomonas aeruginosa and Escherichia coli are dimeric

Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor D-alanine. We present evidence that the enzymes from Pseudomonas aeruginosa and Escherichia coli function exclusively as homodimers. Moreover, we demonstrate that expression of a K35A Y235A double mutation of dadX in E. coli suppresses bacterial growth in a dominant negative fashion.     

Gene cloning and properties of the RND-type multidrug efflux pumps MexPQ-OpmE and MexMN-OprM from Pseudomonas aeruginosa

We cloned two operons for putative RND-type multidrug efflux pumps from Pseudomonas aeruginosa by a PCR method. We designated the genes in one operon mexPQ(-opmE) and in another operon mexMN. Introduction of the mexPQ-opmE into drug hypersensitive cells resulted in elevated MICs of macrolides, fluoroquinolones and some other drugs. Introduction of the mexMN into the hypersensitive cells possessing oprM, but not into cells not possessing oprM, resulted in elevated MICs of chloramphenicol and thiamphenicol. Thus, we conclude that MexPQ-OpmE and MexMN-OprM are functional multidrug efflux pumps when expressed in P. aeruginosa.     

mvaT mutation modifies the expression of the Pseudomonas aeruginosa multidrug efflux operon mexEF-oprN

Pseudomonas aeruginosa carries several multidrug efflux operons, including mexEF-oprN, that contribute to its resistance to multiple antibiotics. mvaT affects the expression of several P. aeruginosa genes. In this study, we show that the mvaT mutant PAODeltamvaT is more resistant than its parent PAO1 strain to chloramphenicol and norfloxacin but more sensitive to imipenem; yet both were less resistant to chloramphenicol, norfloxacin, and imipenem than 'typical'nfxC-type mutants. Neither strain carries the deletion described for nfxC-type mutants in mexT, the mexEF-oprN regulatory gene. Expression of mexEF-oprN is increased by five- to sixfold in PAODeltamvaT, while the expression of oprD is reduced by approximately twofold. mvaT mutation had no effect on the expression of other multidrug resistance operons, although it increased the expression of several ATP-binding cassette transporter genes. We show that mvaT mutation does not affect mexEF-oprN expression through mexT or mexS. We also explored several other potential mechanisms.     

Measurement of Pseudomonas aeruginosa multidrug efflux pumps by quantitative real-time polymerase chain reaction

Multidrug efflux pumps contribute to multiple antibiotic resistance in Pseudomonas aeruginosa. Pump expression usually has been quantified by Western blotting. Quantitative real-time polymerase chain reaction has been developed to measure mRNA expression for genes of interest. Whether this method correlates with pump protein quantities is unclear. We devised a real-time PCR for mRNA expression of MexAB-OprM and MexXY-OprM multidrug efflux pumps. In laboratory strains differing in MexB and MexY expression and in several clinical isolates, protein and mRNA expression correlated well. Quantitative real-time PCR should be a useful alternative in quantitating expression of multidrug efflux pumps by P. aeruginosa isolates in clinical laboratories.     

Mutational analysis of the OprM outer membrane component of the MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa

OprM is the outer membrane component of the MexA-MexB-OprM efflux system of Pseudomonas aeruginosa. Multiple-sequence alignment of this protein and its homologues identified several regions of high sequence conservation that were targeted for site-directed mutagenesis. Of several deletions which were stably expressed, two, spanning residues G199 to A209 and A278 to N286 of the mature protein, were unable to restore antibiotic resistance in OprM-deficient strains of P. aeruginosa. Still, mutation of several conserved residues within these regions did not adversely affect OprM function. Mutation of the highly conserved N-terminal cysteine residue, site of acylation of this presumed lipoprotein, also did not affect expression or activity of OprM. Similarly, substitution of the OprM lipoprotein signal, including consensus lipoprotein box, with the signal peptide of OprF, the major porin of this organism, failed to impact on expression or activity. Apparently, acylation is not essential for OprM function. A large deletion at the N terminus, from A12 to R98, compromised OprM expression to some extent, although the deletion derivative did retain some activity. Several deletions failed to yield an OprM protein, including one lacking an absolutely conserved LGGGW sequence near the C terminus of the protein. The pattern of permissive and nonpermissive deletions was used to test a topology model for OprM based on the recently published crystal structure of the OprM homologue, TolC (V. Koronakis, A. Sharff, E. Koronakis, B. Luisi, and C. Hughes, Nature 405:914-919, 2000). The data are consistent with OprM monomer existing as a substantially periplasmic protein with four outer membrane-spanning regions.     

Contribution of multidrug efflux pumps to multiple antibiotic resistance in veterinary clinical isolates of Pseudomonas aeruginosa

The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB OprM efflux system whereas two isolates simultaneously expressed the MexEF OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB-oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA-mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence.     

Diversity in the oligomeric channel structure of the multidrug efflux pumps in Pseudomonas aeruginosa

MexAB-OprM, the multidrug efflux pump of Pseudomonas aeruginosa, contributes to the high resistance of this organism to a wide variety of antibiotics. To investigate the structure and function of OprM, the outer membrane channel of MexAB-OprM, we examined the oligomeric states of OprM and its homologues OprJ and OprN. These proteins were treated with crosslinking reagent after their reconstitution into liposome membranes. The crosslinked products indicated that OprM and OprN formed trimers, while OprJ unexpectedly appeared to form a tetramer. In order to test whether differences in oligomeric structure might be intimately related to channel function, we examined the channel-forming activity of these proteins by liposome swelling assay. However, no significant differences in channel characteristics were detected among OprM, OprJ, and OprN. We proposed the probable explanation for the diversity in the oligomeric structure of the channel proteins.     

Functional interaction sites of OprM with MexAB in the Pseudomonas aeruginosa multidrug efflux pump

Subunit-swapping between Pseudomonas aeruginosa MexAB-OprM and MexEF-OprN efflux pumps has shown that OprM can interact with MexEF to produce a functional efflux pump, but that OprN cannot functionally interact with MexAB. Taking advantage of this subunit selectivity, we carried out experiments using chimeric proteins composed of OprM and OprN to determine which regions of OprM are necessary for functional interaction with MexAB. We constructed two types of chimeric proteins: one with the N-terminal half of OprM and the C-terminal half of OprN (OprMN), and the second with these halves reversed (OprNM). Introduction of either of the chimeric protein genes into a mutant expressing MexEF alone restored the functionality of the efflux pump. However, expression of OprMN or OprNM in the presence of MexAB did not restore the pump functionality, indicating that the both the N- and C-terminal halves of OprM are necessary for a functional interaction with MexAB.     

nalD encodes a second repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa

The Pseudomonas aeruginosa nalD gene encodes a TetR family repressor with homology to the SmeT and TtgR repressors of the smeDEF and ttgABC multidrug efflux systems of Stenotrophomonas maltophilia and Pseudomonas putida, respectively. A sequence upstream of mexAB-oprM and overlapping a second promoter for this efflux system was very similar to the SmeT and TtgR operator sequences, and NalD binding to this region was, in fact, demonstrated. Moreover, increased expression from this promoter was seen in a nalD mutant, consistent with NalD directly controlling mexAB-oprM expression from a second promoter.     

The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel.

OprM, the outer membrane component of the MexAB-OprM multidrug efflux pump of Pseudomonas aeruginosa, has been assumed to facilitate the export of antibiotics across the outer membrane of this organism. Here we purified to homogeneity the OprM protein, reconstituted it into liposome membranes, and tested its channel activity by using the liposome swelling assay. It was demonstrated that OprM is a channel-forming protein and exhibits the channel property that amino acids diffuse more efficiently than saccharides. However, antibiotics showed no significant diffusion through the OprM channel in the liposome membrane, suggesting that OprM functions as a gated channel. We reasoned that the protease treatment may cause the disturbance of the gate structure of OprM. Hence, we treated OprM reconstituted in the membranes with alpha-chymotrypsin and examined its solute permeability. The results demonstrated that the protease treatment caused the opening of an OprM channel through which antibiotics were able to diffuse. To elucidate which cleavage is intimately related to the opening, we constructed mutant OprM proteins where the amino acid at the cleavage site was replaced with another amino acid. By examining the channel activity of these mutant proteins, it was shown that the proteolysis at tyrosine 185 and tyrosine 196 of OprM caused the channel opening. Furthermore, these residues were shown to face into the periplasmic space and interact with other component(s). We considered the possible opening mechanism of the OprM channel based on the structure of TolC, a homologue of OprM.