Multidrug Efflux Transporter Genes Research Articles

Structures of Neisseria gonorrhoeae MtrR-operator complexes reveal molecular mechanisms of DNA recognition and antibiotic resistance-conferring clinical mutations.

Mutations within the mtrR gene are commonly found amongst multidrug resistant clinical isolates of Neisseria gonorrhoeae, which has been labelled a superbug by the Centers for Disease Control and Prevention. These mutations appear to contribute to antibiotic resistance by interfering with the ability of MtrR to bind to and repress expression of its target genes, which include the mtrCDE multidrug efflux transporter genes and the rpoH oxidative stress response sigma factor gene. However, the DNA-recognition mechanism of MtrR and the consensus sequence within these operators to which MtrR binds has remained unknown. In this work, we report the crystal structures of MtrR bound to the mtrCDE and rpoH operators, which reveal a conserved, but degenerate, DNA consensus binding site 5′-MCRTRCRN4YGYAYGK-3′. We complement our structural data with a comprehensive mutational analysis of key MtrR-DNA contacts to reveal their importance for MtrR-DNA binding both in vitro and in vivo. Furthermore, we model and generate common clinical mutations of MtrR to provide plausible biochemical explanations for the contribution of these mutations to multidrug resistance in N. gonorrhoeae. Collectively, our findings unveil key biological mechanisms underlying the global stress responses of N. gonorrhoeae.

WGS-Based Prediction and Analysis of Antimicrobial Resistance in Campylobacter jejuni Isolates From Israel.

Rapid developments in the field of whole genome sequencing (WGS) make in silico antimicrobial resistance (AMR) a target within reach. Campylobacter jejuni is a leading cause of foodborne infections in Israel with increasing rates of resistance. We applied WGS analysis to study the prevalence and genetic basis of AMR in 263 C. jejuni human and veterinary representative isolates retrieved from a national collection during 2003–2012. We evaluated the prediction of phenotypic AMR from genomic data. Genomes were screened by the NCBI AMRFinderPlus and the BioNumerics tools for acquired AMR genes and point mutations. The results were compared to phenotypic resistance determined by broth microdilution. The most prevalent resistant determinants were the multi-drug efflux transporter gene cmeABC (100%), the tetracycline resistance tet(O) gene (82.1%), the quinolone resistance gyrA T861 point mutation (75.7%), and the aadE streptomycin resistance gene. A variety of 12 known β lactam resistance genes (blaOXA variants) were detected in 241 (92%) isolates, the most prevalent being blaOXA−193, blaOXA−461, and blaOXA−580 (56, 16, and 7%, respectively). Other aminoglycoside resistance genes and the macrolide resistance point mutation were rare (<1%). The overall correlation rate between WGS-based genotypic prediction and phenotypic resistance was 98.8%, sensitivity, specificity, positive, and negative predictive values being 98.0, 99.3, 99.1, and 98.5%, respectively. wgMLST-based phylogeny indicated a high level of clonality and clustering among the studied isolates. Closely related isolates that were part of a genetic cluster (single linkage distance ≤ 15 alleles) based on wgMLST phylogeny mostly shared a homogenous AMR determinant profile. This was observed in 18 of 20 (90.0%) clusters within clonal complex-21, suggesting clonal expansion of resistant isolates. Strong association to lineage was noted for the aadE gene and the various blaOXA genes. High resistance rates to tetracycline and quinolones and a low resistance rate to macrolides were detected among the Israeli C. jejuni isolates. While a high genotypic-phenotypic correlation was found, some resistance phenotypes could not be predicted by the presence of AMR determinants, and particularly not the level of resistance. WGS-based prediction of antimicrobial resistance in C. jejuni requires further optimization in order to integrate this approach in the routine workflow of public health laboratories for foodborne surveillance.

Detection and molecular characterisation of amikacin-resistant Mycobacterium abscessus isolated from patients with pulmonary disease.

The aim of this study was to investigate the molecular mechanisms conferring amikacin (AMK) resistance in Mycobacterium abscessus clinical isolates. A total of 194M. abscessus clinical isolates were collected from patients with pulmonary disease during the period 2012-2017. AMK susceptibility was determined by the broth microdilution method. Whole-genome data were used for identification of mutations in resistance-associated genes. Quantitative reverse transcription PCR (qRT-PCR) was performed to measure the gene transcriptional level. AMK showed high in vitro killing activity against M. abscessus, with an MIC50 of 8mg/L and an MIC90 of 16mg/L. Five isolates (2.6%) were resistant to AMK (MIC>1024mg/L), of which four (80.0%) harboured a resistance-associated rrs mutation A1408G. qRT-PCR analysis showed that most of the AMK-resistant isolates (4/5; 80.0%) overexpressed the transcriptional regulator gene whiB7 and the multidrug-efflux transporter gene tap. However, overexpression of the aminoglycoside-modifying enzyme gene eis2 was only observed in one (20.0%) AMK-resistant isolate. The AMK resistance rate in M. abscessus clinical isolates in this study was low (2.6%). The A1408G mutation in rrs and overexpression of WhiB7 and Tap were the predominant mechanisms of AMK resistance in M. abscessus.

Application of next-generation sequencing to characterize novel mutations in clarithromycin-susceptible Helicobacter pylori strains with A2143G of 23S rRNA gene

BackgroundClarithromycin (CLR) resistance has become a predominant factor for treatment failure of Helicobacter pylori eradication. Although the molecular mechanism of CLR resistance has been clearly understood in H. pylori, it is lack of evidence of other genes involved in drug resistance. Furthermore, the molecular mechanism of phenotype susceptible to CLR while genotype of 23S rRNA is mutant with A2143G is unclear. Here, we characterized the mutations of CLR-resistant and -susceptible H. pylori strains to explore bacterial resistance.MethodsIn the present study, the whole genomes of twelve clinical isolated H. pylori strains were sequenced, including two CLR-susceptible strains with mutation of A2143G. Single nucleotide variants (SNVs) were extracted and analyzed from multidrug efflux transporter genes.ResultsWe did not find mutations associated with known CLR-resistant sites except for controversial T2182C outside of A2143G in the 23S rRNA gene. Although total SNVs of multidrug efflux transporter gene and the SNVs of HP0605 were significant differences (P ≤ 0.05) between phenotype resistant and susceptible strains. There is no significant difference in SNVs of RND or MFS (HP1181) family. However, the number of mutations in the RND family was significantly higher in the mutant strain (A2143G) than in the wild type. In addition, three special variations from two membrane proteins of mtrC and hefD were identified in both CLR-susceptible strains with A2143G.ConclusionsNext-generation sequencing is a practical strategy for analyzing genomic variation associated with antibiotic resistance in H. pylori. The variations of membrane proteins of the RND family may be able to participate in the regulation of clinical isolated H. pylori susceptibility profiles.

Investigating Selection of Demethylation Inhibitor Fungicide‐Insensitive Sclerotinia homoeocarpa Isolates by Boscalid, Flurprimidol, and Paclobutrazol

Dollar spot, caused by Sclerotinia homoeocarpa F.T. Bennett, is the most economically important disease of golf course turfgrass in the northern United States. Fungicide resistance, especially to demethylation inhibitor (DMI) fungicides, is common for S. homoeocarpa. Plant growth regulators (PGRs) are commonly applied on golf courses and have a similar chemistry to DMIs. Previous research has suggested multiple resistance among the PGRs flurprimidol and paclobutrazol, the succinase dehydrogenase inhibitor fungicide boscalid, and the DMI propiconazole, with evidence for the involvement of the multidrug efflux transporter gene, ShPDR1. The objective of this research was to investigate if flurprimidol, paclobutrazol, and boscalid select for DMI insensitivity in the field. We tested eight treatments: flurprimidol alone, tank‐mixed with propiconazole, and rotated with propiconazole; paclobutrazol alone, tank‐mixed with propiconazole, and rotated with propiconazole; propiconazole alone; and boscalid alone. S. homoeocarpa was isolated from infected turfgrass three times per year in 2010 and 2011 following treatment applications. A significant shift in DMI insensitivity was observed from the initial sampling in 2010 to the final sampling in 2011 for all treatments, except the PGRs alone and the untreated control. Both PGRs induced overexpression of ShPDR1, and we confirmed the functional involvement of ShPDR1 in reducing S. homoeocarpa sensitivity to PGRs using the ShPDR1 overexpressing yeast mutant. These results have important implications for managing DMI resistance, further investigating the fungistatic activity of PGRs, and developing new fungicides for S. homoeocarpa.

Campylobacter jejuni type VI secretion system: roles in adaptation to deoxycholic acid, host cell adherence, invasion, and in vivo colonization.

The recently identified type VI secretion system (T6SS) of proteobacteria has been shown to promote pathogenicity, competitive advantage over competing microorganisms, and adaptation to environmental perturbation. By detailed phenotypic characterization of loss-of-function mutants, in silico, in vitro and in vivo analyses, we provide evidence that the enteric pathogen, Campylobacter jejuni, possesses a functional T6SS and that the secretion system exerts pleiotropic effects on two crucial processes – survival in a bile salt, deoxycholic acid (DCA), and host cell adherence and invasion. The expression of T6SS during initial exposure to the upper range of physiological levels of DCA (0.075%–0.2%) was detrimental to C. jejuni proliferation, whereas down-regulation or inactivation of T6SS enabled C. jejuni to resist this effect. The C. jejuni multidrug efflux transporter gene, cmeA, was significantly up-regulated during the initial exposure to DCA in the wild type C. jejuni relative to the T6SS-deficient strains, suggesting that inhibition of proliferation is the consequence of T6SS-mediated DCA influx. A sequential modulation of the efflux transporter activity and the T6SS represents, in part, an adaptive mechanism for C. jejuni to overcome this inhibitory effect, thereby ensuring its survival. C. jejuni T6SS plays important roles in host cell adhesion and invasion as T6SS inactivation resulted in a reduction of adherence to and invasion of in vitro cell lines, while over-expression of a hemolysin co-regulated protein, which encodes a secreted T6SS component, greatly enhanced these processes. When inoculated into B6.129P2-IL-10tm1Cgn mice, the T6SS-deficient C. jejuni strains did not effectively establish persistent colonization, indicating that T6SS contributes to colonization in vivo. Taken together, our data demonstrate the importance of bacterial T6SS in host cell adhesion, invasion, colonization and, for the first time to our knowledge, adaptation to DCA, providing new insights into the role of T6SS in C. jejuni pathogenesis.

Effects of acute or chronic exposure to dietary organic anions on secretion of methotrexate and salicylate by Malpighian tubules ofDrosophila melanogasterlarvae

The effects of dietary exposure to organic anions on the physiology of isolated Malpighian tubules and on tubule gene expression were examined using larvae of Drosophila melanogaster. Acute (24 h) or chronic (7 d) exposure to type I organic anions (fluorescein or salicylate) was associated with increased fluid secretion rates and increased fluxes of both salicylate and the type II organic anion methotrexate. By contrast, chronic exposure to dietary methotrexate was associated with increased fluid secretion rate and increased flux of methotrexate, but not salicylate. Exposure to methotrexate in the diet resulted in increases in the expression of a multidrug efflux transporter gene (MET; CG30344) in the Malpighian tubules. There were also increases in expression of genes for either a Drosophila multidrug resistance-associated protein (dMRP; CG6214) or an organic anion transporting polypeptide (OATP; CG3380), depending on the concentration of methotrexate in the diet. Exposure to salicylate in the diet was associated with an increase in expression of dMRP and with decreases of MET and OATP. Exposure to dietary salicylate or methotrexate was also associated with different patterns of expression of heat shock protein genes. The results suggest that exposure to specific type I or type II organic anions has multiple effects and results not only in increased organic anion transport but also in increased rates of inorganic ion transport, which drives osmotically-obliged fluid secretion. Increased fluid secretion may enhance secretion of organic anions by eliminating diffusive backflux from the tubule lumen to the hemolymph.

Species-Specific Differences in Rhodamine 6G Accumulation of Candida Isolates Detected by Flow Cytometric Analysis

Fluorescent dye Rhodamine 6G (R6G) is a substrate of multidrug resistance pumps and its accumulation is reduced in some azole-resistant Candida isolates with the upregulation of multidrug efflux transporter genes. Despite reports on species-specific differences in azole susceptibility in various Candida species, only a few studies have been reported on the R6G accumulation among clinical isolates of Candida species. In this study, we compared R6G accumulation between six different Candida species. The intracellular accumulation of R6G and minimal inhibitory concentrations (MICs) of three triazole agents were investigated in 48 strains of six Candida species (14 C. albicans, 9 C. tropicalis, 8 C. glabrata, 8 C. krusei, 7 C. parapsilosis, and 2 C. haemulonii). R6G accumulation was measured by using flow cytometry and the geometric mean of the fluorescence intensity (GMF) was used to compare the accumulation between the Candida isolates. The GMF values for the C. tropicalis, C. albicans, C. krusei, C. parapsilosis, and C. glabrata isolates were 167.3+/-18.5, 126.9+/-6.6, 88.5+/-18.5, 50.8+/-7.0, and 38.1+/-3.9, respectively. C. glabrata had a significantly lower mean GMF than all the other Candida species (P<0.05). While some Candida strains with trailing growth phenomenon and increased fluconazole MIC did not have a reduced GMF, three Candida strains with increased MICs to all three triazole agents had a reduced GMF. This study found species-specific differences in R6G accumulation in Candida. In addition, the intracellular R6G accumulation can be used to investigate the drug efflux mechanism in azole-resistant Candida strains.

Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket and Transcription Activation through Tyrosine Expulsion

BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets, that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.

Novel macrolide-specific ABC-type efflux transporter in Escherichia coli.

In the Escherichia coli genome, five putative open reading frame (ORF) clusters, mdlAB, ybjYZ, yddA, yojHI, and yhiH, have been assumed to be possible genes for ABC drug efflux transporters (I. T. Paulsen, M. K. Sliwinski, and M. H. Saier, Jr., J. Mol. Biol. 277:573-592, 1998). We cloned all of these ORFs in multicopy plasmids and investigated the drug resistance of drug-supersensitive host cells lacking constitutive multidrug efflux transporter genes acrAB. Among them, only ybjYZ gave significant erythromycin resistance and significantly decreased the accumulation of [(14)C]erythromycin. Therefore, ybjYZ was renamed macAB (macrolide-specific ABC-type efflux carrier). Plasmids carrying both the macA and -B genes conferred resistance against macrolides composed of 14- and 15-membered lactones but no or weak resistance against 16-membered ones. Neither of the two genes produced resistance alone. The DNA sequence suggests that MacB is an integral membrane protein with four transmembrane segments and one nucleotide-binding domain, while MacA belongs to a membrane fusion protein (MFP) family with a signal-like sequence at its N terminus. The expression of the histidine-tagged proteins confirmed that MacB is an integral membrane protein and MacA is a peripheral membrane protein. In addition, MacAB required TolC for its function in a way similar to that of most of the MFP-dependent transporters in E. coli. MacB is thus a novel ABC-type macrolide efflux transporter which functions by cooperating with the MFP MacA and the multifunctional outer membrane channel TolC. This is the first case of an experimentally identified ABC antibiotic efflux transporter in gram-negative organisms.

Membrane transporters and antifungal drug resistance.

Over the last 30 years or so, the incidence of invasive fungal infections in man has risen dramatically. Patients that become severely immunocompromised because of underlying diseases such as leukemia or recently, acquired immunodeficiency syndrome or patients who undergo cancer chemotherapy or organ transplantation, are particularly susceptible to opportunistic fungal infections. Although Candida species continue to be the major pathogenic fungi in these patients, cryptococcosis, aspergillosis, and coccidioidomycosis, among others, have become increasingly important mycoses. Antifungal drugs currently being used in clinic include polyene antibiotics, azole derivatives and 5-fluorocytosine. With the exception of the latter, all other drugs possess mechanisms of action aimed at disrupting the integrity of the fungal cell membrane by either interfering with the biosynthesis of membrane sterols or by inhibiting sterol functions. However, one significant obstacle preventing successful antifungal therapy is the dramatic increase in drug resistance, especially against azole antimycotics. Among the major mechanisms by which fungi invoke drug resistance is the overexpession of extrusion pumps able to facilitate the efflux of cytotoxic drugs from the cell thus leading to decreased drug accumulation and diminished concentrations. Since the initial observations that azole resistance by fungi may be caused by overexpression of multidrug efflux transporter genes, significant advances have been achieved primarily with Saccharomyces cerevisiae and Candida albicans. The purpose of this review is to discuss various aspects of multidrug resistance in fungi such as antifungal drug mechanisms of action and fungal molecular genetics in the context of targeted drug discovery. The role that membrane transporter proteins play in drug resistance in various species of Candida, Aspergillus and Cryptococcus will be address in more detail, as will be their importance as selective drug targets in the design of novel antifungal agents.

Experimental induction of fluconazole resistance in Candida tropicalis ATCC 750.

Candida tropicalis is less commonly isolated from clinical specimens than Candida albicans. Unlike C. albicans, which can be occasionally found as a commensal, C. tropicalis is almost always associated with the development of fungal infections. In addition, C. tropicalis has been reported to be resistant to fluconazole (FLC). To analyze the development of FLC resistance in C. tropicalis, an FLC-susceptible strain (ATCC 750) (MIC = 1.0 microg/ml) was cultured in liquid medium containing increasing FLC concentrations from 8.0 to 128 microg/ml. The strain developed variable degrees of FLC resistance which paralleled the concentrations of FLC used in the medium. The highest MICs of FLC were 16, 256, and 512 microg/ml for strains grown in medium with 8.0, 32, and 128 microg of FLC per ml, respectively. Development of resistance was rapid and could be observed already after a single subculture in azole-containing medium. The resistant strains were cross-resistant to itraconazole (MIC > 1.0 microg/ml) and terbinafine (MIC > 512 microg/ml) but not to amphotericin B. Isolates grown in FLC at concentrations of 8.0 and 32 microg/ml reverted to low MICs (1.0 microg/ml) after 12 and 11 passages in FLC-free medium, respectively. The MIC for one isolate grown in FLC (128 microg/ml) (128 R) reverted to 16 microg/ml but remained stable over 60 passages in FLC-free medium. Azole-resistant isolates revealed upregulation of two different multidrug efflux transporter genes: the major facilitators gene MDR1 and the ATP-binding cassette transporter CDR1. The development of FLC resistance in vitro correlated well with the results obtained in an experimental model of disseminated candidiasis. While FLC given at 10 mg/kg of body weight/day was effective in reducing the fungal burden of mice infected with the parent strain, the same dosing regimen was ineffective in mice infected with strain 128 R. Finally, the acquisition of in vitro FLC resistance in strain 128 R was related to a loss of virulence. The results of our study elucidate important characteristics and potential mechanisms of FLC resistance in C. tropicalis.

The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents.

The resistance mechanisms to azole antifungal agents were investigated in this study with two pairs of Candida glabrata clinical isolates recovered from two separate AIDS patients. The two pairs each contained a fluconazole-susceptible isolate and a fluconazole-resistant isolate, the latter with cross-resistance to itraconazole and ketoconazole. Since the accumulation of fluconazole and of another unrelated substance, rhodamine 6G, was reduced in the azole-resistant isolates, enhanced drug efflux was considered as a possible resistance mechanism. The expression of multidrug efflux transporter genes was therefore examined in the azole-susceptible and azole-resistant yeast isolates. For this purpose, C. glabrata genes conferring resistance to azole antifungals were cloned in a Saccharomyces cerevisiae strain in which the ATP binding cassette (ABC) transporter gene PDR5 was deleted. Three different genes were recovered, and among them, only C. glabrata CDR1 (CgCDR1), a gene similar to the Candida albicans ABC transporter CDR genes, was upregulated by a factor of 5 to 8 in the azole-resistant isolates. A correlation between upregulation of this gene and azole resistance was thus established. The deletion of CgCDR1 in an azole-resistant C. glabrata clinical isolate rendered the resulting mutant (DSY1041) susceptible to azole derivatives as the azole-susceptible clinical parent, thus providing genetic evidence that a specific mechanism was involved in the azole resistance of a clinical isolate. When CgCDR1 obtained from an azole-susceptible isolate was reintroduced with the help of a centromeric vector in DSY1041, azole resistance was restored and thus suggested that a trans-acting mutation(s) could be made responsible for the increased expression of this ABC transporter gene in the azole-resistant strain. This study demonstrates for the first time the determinant role of an ABC transporter gene in the acquisition of resistance to azole antifungals by C. glabrata clinical isolates.