The mexR repressor of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: characterization of mutations compromising activity

Mutations in mexR yield a multidrug resistance phenotype in nalB mutants of Pseudomonas aeruginosa as a result of derepression of the mexAB-oprM multidrug efflux operon. MexR produced by several nalB strains carried single amino acid changes that compromised MexR stability or its ability to dimerize. Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding.     

MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719

MexAB-OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa, the latter as a result of mutational hyperexpression of the mexAB-oprM operon. While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so-called nalC mutants that demonstrate more modest mexAB-oprM expression and, thus, more modest multidrug resistance than do mexR strains. Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified. Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC. PA3721 (nalC) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two-gene operon, PA3720-PA3719, whose expression was negatively regulated by PA3721. Thus, PA3720-PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants. The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB-OprM expression to wild-type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype. Introduction of PA3719 into wild-type P. aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB-OprM expression and multidrug resistance characteristic of a nalC strain. Thus, the nalC (PA3721) mutation serves only to enhance PA3720-PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB-OprM expression. Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720-PA3719 hyperexpression somehow modulates MexR repressor activity. The deduced products of PA3720-PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur.     

Influence of mutations in the mexR repressor gene on expression of the MexA-MexB-oprM multidrug efflux system of Pseudomonas aeruginosa

Several nalB-type multidrug-resistant mutants of Pseudomonas aeruginosa overexpressed MexAB-OprM and carried mutations in the local regulatory gene, mexR. Others, dubbed nalC types, carried mutations elsewhere and overexpressed MexAB-OprM less extensively than the nalB strains. Available evidence showed that MexR acted solely as repressor. Disruption of the mexR gene at various places suggested that the 5' end of mexR may be a part of the mexAB-oprM promoter.     

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.     

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.     

Mutations in the consensus helicase domains of the Werner syndrome gene. Werner's Syndrome Collaborative Group.

Werner syndrome (WS) is an autosomal recessive disease with a complex phenotype that is suggestive of accelerated aging. WS is caused by mutations in a gene, WRN, that encodes a predicted 1,432-amino-acid protein with homology to DNA and RNA helicases. Previous work identified four WS mutations in the 3' end of the gene, which resulted in predicted truncated protein products of 1,060-1,247 amino acids but did not disrupt the helicase domain region (amino acids 569-859). Here, additional WS subjects were screened for mutations, and the intron-exon structure of the gene was determined. A total of 35 exons were defined, with the coding sequences beginning in the second exon. Five new WS mutations were identified: two nonsense mutations at codons 369 and 889; a mutation at a splice-junction site, resulting in a predicted truncated protein of 760 amino acids; a 1-bp deletion causing a frameshift; and a predicted truncated protein of 391 amino acids. Another deletion is >15 kb of genomic DNA, including exons 19-23; the predicted protein is 1,186 amino acids long. Four of these new mutations either partially disrupt the helicase domain region or result in predicted protein products completely missing the helicase region. These results confirm that mutations in the WRN gene are responsible for WS. Also, the location of the mutations indicates that the presence or absence of the helicase domain does not influence the WS phenotype and suggests that WS is the result of complete loss of function of the WRN gene product.     

Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants.

To study important epitopes on glycoprotein E2 of Sindbis virus, eight variants selected to be singly or multiply resistant to six neutralizing monoclonal antibodies reactive against E2, as well as four revertants which had regained sensitivity to neutralization, were sequenced throughout the E2 region. To study antigenic determinants in glycoprotein E1, four variants selected for resistance to a neutralizing monoclonal antibody reactive with E1 were sequenced throughout the E2 and E1 regions. All of the salient changes in E2 occurred within a relatively small region between amino acids 181 and 216, a domain that encompasses a glycosylation site at residue 196 and that is rich in charged amino acids. Almost all variants had a change in charge, suggesting that the charged nature of this domain is important for interaction with antibodies. Variants independently isolated for resistance to the same antibody were usually altered in the same amino acid, and reversion to sensitivity occurred at the sites of the original mutations, but did not always restore the parental amino acid. The characteristics of this region suggest that this domain is found on the surface of E2 and constitutes a prominent antigenic domain that interacts directly with neutralizing antibodies. Previous studies have shown that this domain is also important for penetration of cells and for virulence of the virus. Resistance to the single E1-specific neutralizing monoclonal antibody resulted from changes of Gly-132 of E1 to either Arg or Glu. Analogous to the findings with E2, these changes result in a change in charge and are found near a glycosylation site at residue 139. This domain of E1 may therefore be found near the 181 to 216 domain of E2 on the surface of the E1-E2 heterodimer; together, they could form a domain important in virus penetration and neutralization.     

肺炎克雷伯菌gyrA基因与parC基因的突变研究

目的探讨肺炎克雷伯菌对环丙沙星和左氧氟沙星的药物敏感性,及对喹诺酮敏感和耐药菌株中gyrA与parC基因的突变情况。方法收集肺炎克雷伯菌临床分离株231株,采用K-B纸片法测定肺炎克雷伯菌对环丙沙星和左氧氟沙星的敏感性,随机选取对环丙沙星和左氧氟沙星均耐药菌株4株和均敏感的菌株3株,分别PCR扩增gyrA基因和parC基因的耐药决定区,扩增片段长度分别为625、319bp,PCR扩增产物经纯化后测序并做序列分析。结果肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药率分别为51.1%（118/231）和45.9%（106/231）;gyrA和parC基因经序列分析显示,耐药株均有gyrA基因的突变,其中1株出现第83、87和27位氨基酸的改变,2株出现第83位氨基酸的改变,1株出现第47位点的改变;环丙沙星敏感株中未出现gyrA基因的突变。4株耐药株均有parC基因的突变,引起相应氨基酸Ser80→Arg的改变,2株环丙沙星敏感株也发生了同样的改变。结论哈尔滨地区肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药性显著,在喹诺酮耐药株中有gyrA和parC基因的同时突变,在敏感株中也发现了parC基因的突变。     

Mutations affecting DNA-binding activity of the MexR repressor of mexR-mexA-mexB-oprM operon expression

We have isolated 25 MexR mutants that retained their dimerizing ability but were unable to bind mexOP DNA. Surprisingly, 20 mutations were located in the hydrophobic core region at alpha4, W1, alpha2, alpha3, and beta2, and only 3 were in positively charged residues. These results verified that DNA binding is mediated by distinct regions of MexR and showed the importance of the hydrophobic core region of the DNA-binding domain.     

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor

The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.     

Molecular heterogeneity of acute intermittent porphyria: identification of four additional mutations resulting in the CRIM-negative subtype of the disease.

Four mutations of the porphobilinogen (PBG) deaminase gene that result in cross-reacting immunological material (CRIM)-negative forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA from patients and by cloning of the amplified products in a bacterial expression vector. One mutation is a single base deletion which causes a frameshift and which is expected to result in the synthesis of a truncated protein. Two other mutations consist of single base substitutions and lead to amino acid changes. The fourth mutation is a single base substitution producing an aberrant splicing and resulting in an mRNA which would encode a protein missing three amino acids. DNAs from 16 unrelated CRIM-negative AIP patients were screened for the presence of these four mutations, by hybridization with oligonucleotides specific for each of the mutations, but none of the four mutations was identified in additional patients. The results indicate that mutations responsible for CRIM-negative AIP are highly heterogenous.     

An osmosensing histidine kinase mediates dicarboximide fungicide resistance in Botryotinia fuckeliana (Botrytis cinerea)

A two-component histidine protein kinase gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from a single ascospore isolate of Botryotinia fuckeliana. A series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. Genetic crosses of the mutants with an authentic Daf1 strain showed that the phenotypes mapped to this locus. Single point mutations (seven transitions, one transversion, and one short deletion) were detected in the alleles of the nine mutants sequenced. The mutational changes were shown to cosegregate with the dicarboximide resistance and osmotic sensitivity phenotypes in progeny obtained from crossing selected resistant strains with a sensitive strain. All mutations detected are predicted to result in amino acid changes in the coiled-coil region of the putative Daf1 histidine kinase, and it is proposed that dicarboximide fungicides target this domain.     

Genetic basis of quinolone resistance and epidemiology of resistant and susceptible isolates of porcine Campylobacter coli strains

AIMS: The aims of this study were to investigate the epidemiology of quinolone-resistant and -susceptible porcine isolates of Campylobacter coli and to characterize the genetic basis of quinolone resistance. METHODS AND RESULTS: Penner serotyping and flagellin gene sequence polymorphisms were used to investigate the epidemiology of the C. coli isolates. A total of 55 isolates were included, of which 30 were paired resistant and susceptible isolates from 15 pigs. Amplification of gyrA, gyrB and parC, followed by direct sequencing of amplicons was used to identify mutations in the targets of quinolones. Overall, 31 of the isolates were resistant to ciprofloxacin (minimum inhibitory concentrations (MIC), 2- >or = 32 microg x ml(-1)). Thirteen DdeI-flaA profiles were observed and resistant and susceptible strains were identified for nine profiles. The majority of resistant strains exhibited either profile 1 or 6. While profile 1 comprised susceptible and resistant strains, all of the strains with profile 6 were resistant to ciprofloxacin. The serogroup (O:24) of the profile 6 strains was identical. The only other serogroup to be uniformly associated with quinolone resistance was O:5. Strains with this phenotype comprised a number of genotypes, including profile 1. Only four of the paired isolates from individual pigs had the same profile. The genetic basis of quinolone resistance was investigated in two strains with ciprofloxacin MICs of 2 and > or = 32 miccrog x ml(-1), respectively. The amino acid substitution of isoleucine for threonine at position 86 was identified in the GyrA proteins from both strains. No mutations were identified in the GyrB proteins. CONCLUSIONS: There was an association between two of the genotypes, serotypes 5 and 24, and quinolone resistance. The association between genotype, serotype and resistance in C. coli isolates has not been reported previously. Only the mutation in GyrA associated with quinolone resistance was identified. No mutations in GyrB were identified. Amplification products of parC were not obtained and it may be that this gene is not present in some Campylobacter spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on the distribution of ciprofloxacin resistance between subtypes of C. coli.     

Analysis of the Membrane Proteome of Ciprofloxacin-Resistant Macrophages by Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

Overexpression of multidrug transporters is a well-established mechanism of resistance to chemotherapy, but other changes may be co-selected upon exposure to drugs that contribute to resistance. Using a model of J774 macrophages made resistant to the fluoroquinolone antibiotic ciprofloxacin and comparing it with the wild-type parent cell line, we performed a quantitative proteomic analysis using the stable isotope labeling with amino acids in cell culture technology coupled with liquid chromatography electrospray ionization Fourier transform tandem mass spectrometry (LC-ESI-FT-MS/MS) on 2 samples enriched in membrane proteins (fractions F1 and F2 collected from discontinuous sucrose gradient). Nine hundred proteins were identified with at least 3 unique peptides in these 2 pooled fractions among which 61 (F1) and 69 (F2) showed a significantly modified abundance among the 2 cell lines. The multidrug resistance associated protein Abcc4, known as the ciprofloxacin efflux transporter in these cells, was the most upregulated, together with Dnajc3, a protein encoded by a gene located downstream of Abcc4. The other modulated proteins are involved in transport functions, cell adhesion and cytoskeleton organization, immune response, signal transduction, and metabolism. This indicates that the antibiotic ciprofloxacin is able to trigger a pleiotropic adaptative response in macrophages that includes the overexpression of its efflux transporter.     

Amino acid substitutions in mitochondrial ATPase subunit 9 of Saccharomyces cerevisiae leading to oligomycin or venturicidin resistance

A series of isonuclear oligomycin-resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial oli1 gene has been studied. DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex. A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane. Certain amino acid substitutions also confer cross-resistance to venturicidin: these residues define an inner domain for venturicidin resistance. The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.