Major Efflux Pump Research Articles

Functionalization of ZnO Nanoparticles by Glutamic Acid and Conjugation with Thiosemicarbazide Alters Expression of Efflux Pump Genes in Multiple Drug-Resistant Staphylococcus aureus Strains.

Efflux-mediated drug resistance in bacterial strains is regarded as a major cause of drug resistance. In this study, we aimed to evaluate the expression of some major facilitator superfamily class efflux pump genes (EPGs) in the presence of ZnO nanoparticles (NPs) conjugated to thiosemicarbazide (TSC) under amine functionalization by glutamic acid (ZnO@Glu-TSC) as well as ciprofloxacin (CIP) among multiple drug-resistant Staphylococcus aureus. Synthesized NPs were characterized by ultraviolet-visible spectroscopy, X-ray diffraction pattern, and transmission electron microscopy. Antibiogram and ethidium bromide agar cartwheel method were used to determine the efflux-mediated multidrug-resistant phenotype of clinical strains. Then, expression of EPGs, including norA, norB, norC, and tet38 among the strains, exposed to ZnO@Glu-TSC and CIP was evaluated using quantitative real-time PCR (qPCR). According to the results, the strains resistant to CIP showed minimum inhibitory concentration (MIC) values ranging from 256 to 1,024 μg/mL, while ZnO@Glu-TSC NPs showed MICs from 8 to 256 μg/mL against bacterial strains, which indicates stronger antibacterial activity of NPs (2-8-fold) compared to CIP. ZnO@Glu-TSC NPs showed a good bacterial inhibitory potential with average inhibition zones of 11, 15, and 20 mm for concentrations of 50, 100, and 150 μg/mL, respectively. Moreover, simultaneous use of ZnO@Glu-TSC NPs (1/2 MIC) in combination with CIP (1/2 MIC) significantly reduced the expression of norA, norB, norC, and tet38 by 5.4-, 3.8-, 2.1-, and 3.4-fold, respectively, compared to the CIP alone. Therefore, ZnO@Glu-TSC NPs with their potent antimicrobial effects could be used as an antimicrobial agent against S. aureus for preventive and/or therapeutic approaches.

SmvA is an important efflux pump for cationic biocides in Klebsiella pneumoniae and other Enterobacteriaceae

The multidrug resistant (MDR) opportunistic pathogen Klebsiella pneumoniae has previously been shown to adapt to chlorhexidine by increasing expression of the MFS efflux pump smvA. Here we show that loss of the regulator SmvR, through adaptation to chlorhexidine, results in increased resistance to a number of cationic biocides in K. pneumoniae and other members of the Enterobacteriaceae. Clinical Enterobacteriaceae isolates which lack smvA and smvR also have an increased susceptibility to chlorhexidine. When smvA from Salmonella and K. pneumoniae are expressed in Escherichia coli, which lacks a homologue to SmvAR, resistance to chlorhexidine increased (4-fold) but plasmid carriage of smvA alone was detrimental to the cell. Challenge of K. pneumoniae with chlorhexidine and another cationic biocide, octenidine, resulted in increased expression of smvA (approx. 70 fold). Adaptation to octenidine was achieved through mutating key residues in SmvA (A363V; Y391N) rather than abolishing the function of SmvR, as with chlorhexidine adaptation. Molecular modelling was able to predict that octenidine interacted more strongly with these mutated SmvA forms. These results show that SmvA is a major efflux pump for cationic biocides in several bacterial species and that increased efflux through SmvA can lead to increased chlorhexidine and octenidine tolerance.

IL-1β functionally attenuates ABCG2 and PDZK1 expression in HK-2 cells partially through NF-ĸB activation.

Long-standing untreated hyperuricemia could lead to gout. Several recent studies have demonstrated a significant decrease of serum urate during acute gout attack, which is an aseptic inflammation process focusing on IL-1β. However, how IL-1β, by itself, alters the expression and the functional activity of urate transporters in renal tubular epithelial cells is still unclear. Herein, we revealed that IL-1β could attenuate the mRNA and protein levels of ABCG2, a major urate efflux pump, in HK-2 cells by real-time PCR and Western-blot assays. Moreover, using an ABCG2 specific inhibitor and a new sensitive and specific detection system, it was found that IL-1β also reduced the ABCG2 transporter activities. Incubation with specific inhibitors of the NF-κB pathway partly dampened the inhibitory effect of IL-1β on ABCG2, indicating that IL-1β reduced the ABCG2 expression partially through the NF-ĸB pathway. Furthermore, the decreased expression of PDZK1 induced by IL-1β, which is dependent on the NF-κB pathway, could account for the imbalance between the functions and expressions of ABCG2 on this status. These findings demonstrated a new role for IL-1β, whereby it leads to the inhibition of ABCG2 in renal tubular epithelial cells; this new role probably does not encompass its involvement in the process of renal urate excretion mediated by inflammation. Therefore, other regulation mechanisms of urate reabsorption in renal tubular epithelial cells deserve to be examined in further studies.

Identification and characterization of carbapenem binding sites within the RND-transporter AcrB

Understanding the molecular determinants for recognition, binding and transport of antibiotics by multidrug efflux systems is important for basic research and useful for the design of more effective antimicrobial compounds. Imipenem and meropenem are two carbapenems whose antibacterial activity is known to be poorly and strongly affected by MexAB-OprM, the major efflux pump transporter in Pseudomonas aeruginosa. However, not much is known regarding recognition and transport of these compounds by AcrAB-TolC, which is the MexAB-OprM homologue in Escherichia coli and by definition the paradigm model for structural studies on efflux pumps. Prompted by this motivation, we unveiled the molecular details of the interaction of imipenem and meropenem with the transporter AcrB by combining computer simulations with biophysical experiments. Regarding the interaction with the two main substrate binding regions of AcrB, the so-called access and deep binding pockets, molecular dynamics simulations revealed imipenem to be more mobile than meropenem in the former, while comparable mobilities were observed in the latter. This result is in line with isothermal titration calorimetry, differential scanning experiments, and binding free energy calculations, indicating a higher affinity for meropenem than imipenem at the deep binding pocket, while both sharing similar affinities at the access pocket. Our findings rationalize how different physico-chemical properties of compounds reflect on their interactions with AcrB. As such, they constitute precious information to be exploited for the rational design of antibiotics able to evade efflux pumps.

Acute pain alters P-glycoprotein-containing protein complexes in rat cerebral microvessels: Implications for P-glycoprotein trafficking

P-glycoprotein (PgP) is the major drug efflux pump in human cerebral microvessels. PgP prevents pathogens, toxins and therapeutic drugs from entering the CNS. Understanding the molecular regulation of PgP activity will suggest novel mechanisms to improve CNS drug delivery. Previously, we found that during peripheral inflammatory pain (PIP) (3 h after λ carrageenan injection in the rat paw), PgP traffics to the cortical microvessel endothelial cell plasma membrane concomitant with increased PgP activity. In the current study, we measured the changes in composition of PgP-containing protein complexes after PIP in rat microvessel isolates. We found that a portion of the PgP is contained in a multi-protein complex that also contains the caveolar proteins CAV1, SDPR, PTRF and PRKCDBP. With PIP, total CAV1 bound to PgP was unchanged; however, phosphorylated CAV1 (Y14P-CAV1) in the complex increased. There were few PgP/CAV1 complexes relative to total PgP and CAV1 in the microvessels suggesting CAV1 bound to PgP is unlikely to affect total PgP activity. However, both PgP and CAV1 trafficked away from the nucleus in response to PIP. These data suggest that P-CAV1 bound to PgP potentially regulates PgP trafficking and contributes to the acute PgP activity increase after a PIP stimulus.

Acinetobacter nosocomialis: Defining the Role of Efflux Pumps in Resistance to Antimicrobial Therapy, Surface Motility, and Biofilm Formation

Acinetobacter nosocomialis is a member of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex. Increasingly, reports are emerging of the pathogenic profile and multidrug resistance (MDR) phenotype of this species. To define novel therapies to overcome resistance, we queried the role of the major efflux pumps in A. nosocomialis strain M2 on antimicrobial susceptibility profiles. A. nosocomialis strains with the following mutations were engineered by allelic replacement; ΔadeB, ΔadeJ, and ΔadeB/adeJ. In these isogenic strains, we show that the ΔadeJ mutation increased susceptibility to beta-lactams, beta-lactam/beta-lactamase inhibitors, chloramphenicol, monobactam, tigecycline, and trimethoprim. The ΔadeB mutation had a minor effect on resistance to certain beta-lactams, rifampicin and tigecycline. In addition, the ΔadeJ mutation resulted in a significant decrease in surface motility and a minor decrease in biofilm formation. Our results indicate that the efflux pump, AdeIJK, has additional roles outside of antibiotic resistance in A. nosocomialis.

Analysis of resistance genes of clinical Pannonibacter phragmitetus strain 31801 by complete genome sequencing.

To clarify the resistance mechanisms of Pannonibacter phragmitetus 31801, isolated from the blood of a liver abscess patient, at the genomic level, we performed whole genomic sequencing using a PacBio RS II single-molecule real-time long-read sequencer. Bioinformatic analysis of the resulting sequence was then carried out to identify any possible resistance genes. Analyses included Basic Local Alignment Search Tool searches against the Antibiotic Resistance Genes Database, ResFinder analysis of the genome sequence, and Resistance Gene Identifier analysis within the Comprehensive Antibiotic Resistance Database. Prophages, clustered regularly interspaced short palindromic repeats (CRISPR), and other putative virulence factors were also identified using PHAST, CRISPRfinder, and the Virulence Factors Database, respectively. The circular chromosome and single plasmid of P. phragmitetus 31801 contained multiple antibiotic resistance genes, including those coding for three different types of β-lactamase [NPS β-lactamase (EC 3.5.2.6), β-lactamase class C, and a metal-dependent hydrolase of β-lactamase superfamily I]. In addition, genes coding for subunits of several multidrug-resistance efflux pumps were identified, including those targeting macrolides (adeJ, cmeB), tetracycline (acrB, adeAB), fluoroquinolones (acrF, ceoB), and aminoglycosides (acrD, amrB, ceoB, mexY, smeB). However, apart from the tripartite macrolide efflux pump macAB-tolC, the genome did not appear to contain the complete complement of subunit genes required for production of most of the major multidrug-resistance efflux pumps.

Robust, pleiotropic drug resistance 5 (Pdr5)-mediated multidrug resistance is vigorously maintained in Saccharomyces cerevisiae cells during glucose and nitrogen limitation.

Saccharomyces cerevisiae has sophisticated nutrient-sensing programs for responding to harsh environments containing limited nutrients. As a result, yeast cells can live in diverse environments, including animals, as a commensal or a pathogen. Because they live in mixed populations with other organisms that excrete toxic chemicals, it is of interest to know whether yeast cells maintain functional multidrug resistance mechanisms during nutrient stress. We measured the activity of Pdr5, the major Saccharomyces drug efflux pump under conditions of limiting nutrients. We demonstrate that the steady-state level of this transporter remains unchanged during growth in low concentrations of glucose and nitrogen even though two-dimensional gel electrophoresis revealed a decrease in the level of many proteins. We also evaluated rhodame 6G transport and resistance to three xenobiotic agents in rich (synthetic dextrose) and starvation medium. We demonstrate that Pdr5 function is vigorously maintained under both sets of conditions.

Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment.

Cryptococcus neoformans and Cryptococcus gattii species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, C. neoformans H99 and C. gattii R265. Deletion of AFR1 in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when AFR2 or MDR1 was deleted in both strains. However, when these genes were deleted in combination with AFR1, a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in afr1Δ mutants of both strains, but rhodamine 6G accumulation increased only in the mdr1Δ mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. AFR1 and AFR2 expression was upregulated in H99 and R265 when treated with fluconazole. However, MDR1 expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

RND transporters in the living world

Transporters of the RND superfamily are well-known as the major drug efflux pumps of Gram-negative bacteria. However, they are widespread in organisms ranging from Archaea to Eukaryotes, and perform diverse functions. This review gives a brief overview of these diverse members of the superfamily with emphasis on their structure and functions.

Abnormal Ergosterol Biosynthesis Activates Transcriptional Responses to Antifungal Azoles.

Fungi transcriptionally upregulate expression of azole efflux pumps and ergosterol biosynthesis pathway genes when exposed to antifungal agents that target ergosterol biosynthesis. To date, these transcriptional responses have been shown to be dependent on the presence of the azoles and/or depletion of ergosterol. Using an inducible promoter to regulate Neurospora crassa erg11, which encodes the major azole target, sterol 14α-demethylase, we were able to demonstrate that the CDR4 azole efflux pump can be transcriptionally activated by ergosterol biosynthesis inhibition even in the absence of azoles. By analyzing ergosterol deficient mutants, we demonstrate that the transcriptional responses by cdr4 and, unexpectedly, genes encoding ergosterol biosynthesis enzymes (erg genes) that are responsive to azoles, are not dependent on ergosterol depletion. Nonetheless, deletion of erg2, which encodes C-8 sterol isomerase, also induced expression of cdr4. Deletion of erg2 also induced the expression of erg24, the gene encoding C-14 sterol reductase, but not other tested erg genes which were responsive to erg11 inactivation. This indicates that inhibition of specific steps of ergosterol biosynthesis can result in different transcriptional responses, which is further supported by our results obtained using different ergosterol biosynthesis inhibitors. Together with the sterol profiles, these results suggest that the transcriptional responses by cdr4 and erg genes are associated with accumulation of specific sterol intermediate(s). This was further supported by the fact that when the erg2 mutant was treated with ketoconazole, upstream inhibition overrode the effects by downstream inhibition on ergosterol biosynthesis pathway. Even though cdr4 expression is associated with the accumulation of sterol intermediates, intra- and extracellular sterol analysis by HPLC-MS indicated that the transcriptional induction of cdr4 did not result in efflux of the accumulated intermediate(s). This study demonstrates, by detailed genetic and chemical analysis, that transcriptional responses by a major efflux pump and genes of the ergosterol biosynthesis pathway to ergosterol biosynthesis inhibitors can be independent of the presence of the drugs and are linked with the accumulation of ergosterol intermediate(s).

Molecular Modeling of Multidrug Properties of Resistance Nodulation Division (RND) Transporters.

Efflux pumps of the resistance nodulation division (RND) superfamily are among the major contributors to intrinsic and acquired multidrug resistance in Gram-negative bacteria. Structural information on AcrAB-TolC and MexAB-OprM, major efflux pumps of Escherichia coli and Pseudomonas aeruginosa respectively, boosted intensive research aimed at understanding the molecular mechanisms ruling the active extrusion processes. In particular, several studies were devoted to the understanding of the determinants behind the extraordinary broad specificity of the RND transporters AcrB and MexB. In this chapter, we discuss the ever-growing role computational methods have been playing in deciphering key structural and dynamical features of these transporters and of their interaction with substrates and inhibitors. We further discuss and illustrate examples from our lab of how molecular docking, homology modeling, all-atom molecular dynamics simulations and in silico free energy estimations can all together give precious insights into the processes of recognition and extrusion of substrates, as well as on the possible inhibition strategies.

In vitroselection ofPhytomonas serpenscells resistant to the calpain inhibitor MDL28170: alterations in fitness and expression of the major peptidases and efflux pumps

The species Phytomonas serpens is known to express some molecules displaying similarity to those described in trypanosomatids pathogenic to humans, such as peptidases from Trypanosoma cruzi (cruzipain) and Leishmania spp. (gp63). In this work, a population of P. serpens resistant to the calpain inhibitor MDL28170 at 70 µ m (MDLR population) was selected by culturing promastigotes in increasing concentrations of the drug. The only relevant ultrastructural difference between wild-type (WT) and MDLR promastigotes was the presence of microvesicles within the flagellar pocket of the latter. MDLR population also showed an increased reactivity to anti-cruzipain antibody as well as a higher papain-like proteolytic activity, while the expression of calpain-like molecules cross-reactive to anti-Dm-calpain (from Drosophila melanogaster) antibody and calcium-dependent cysteine peptidase activity were decreased. Gp63-like molecules also presented a diminished expression in MDLR population, which is probably correlated to the reduction in the parasite adhesion to the salivary glands of the insect vector Oncopeltus fasciatus. A lower accumulation of Rhodamine 123 was detected in MDLR cells when compared with the WT population, a phenotype that was reversed when MDLR cells were treated with cyclosporin A and verapamil. Collectively, our results may help in the understanding of the roles of calpain inhibitors in trypanosomatids.

Dyslipidemia alters sperm maturation and capacitation in LXR-null mice.

Lipid metabolism disorders (dyslipidemia) are causes of male infertility, but little is known about their impact on male gametes when considering post-testicular maturation events, given that studies concentrate most often on endocrine dysfunctions and testicular consequences. In this study, three-month-old wild-type (wt) and Liver-X-Receptors knock out (Lxrα;β-/- ) males were fed four weeks with a control or a lipid-enriched diet containing 1.25% cholesterol (high cholesterol diet (HCD)). The HCD triggered a dyslipidemia leading to sperm post-testicular alterations and infertility. Sperm lipids were analyzed by LC-MS and those from Lxrα;β-/- males fed the HCD showed higher chol/PL and PC/PE ratios compared to wt-HCD (P < 0.05) and lower oxysterol contents compared to wt (P < 0.05) or Lxrα;β-/- (P < 0.05). These modifications impaired membrane-associated events triggering the tyrosine phosphorylation normally occurring during the capacitation process, as shown by phosphotyrosine Western blots. Using flow cytometry, we showed that a smaller subpopulation of spermatozoa from Lxrα;β-/- -HCD males could raise their membrane fluidity during capacitation (P < 0.05 vs wt or wt-HCD) as well as their intracellular calcium concentration (P < 0.05 vs Lxrα;β-/- and P < 0.001 vs wt). The accumulation of the major sperm calcium efflux pump (PMCA4) was decreased in Lxrα;β-/- males fed the HCD (P < 0.05 vs Lxrα;β-/- and P < 0.001 vs wt). This study is the first showing an impact of dyslipidemia on post-testicular sperm maturation with consequences on the capacitation signaling cascade. It may lead to the identification of fertility prognostic markers in this pathophysiological situation, which could help clinicians to better understand male infertilities which are thus far classified as idiopathic.

Expression of TolC and Organic Solvent Tolerance of Escherichia Coli Ciprofloxacin Resistant Mutants.

AcrAB-TolC is a major efflux pump in Escherichia coli. It was reported that tolC is overexpressed and involves in improving the organic solvent tolerance level in Escherichia colimarR mutants that are resistant to several antibiotics, such as ciprofloxacin. Low and intermediate levels resistance did not improve organic solvent tolerance. Thus, it was decided to measure tolC expression and organic solvent tolerance in high level ciprofloxacin resistant mutants. tolC expression was measured by real time PCR and organic solvent tolerance assay was conducted by counting bacterial colonies on LBGMg agar. Results showed that tolC expression was increased significantly (P<0.05) and organic solvent tolerance was slightly improved in high resistant mutants. It was concluded that high organic solvent tolerance may need higher expression of tolC.

Molecular Insights on the Recognition of Substrates by the Promiscuous Efflux Pump AcrB

Efflux pumps of the Resistance Nodulation Division (RND) superfamily play a major role in multi-drug resistance (MDR) to antibiotics in Gram-negatives. Understanding the molecular mechanism by which these pumps expel so many unrelated drugs is essential to develop antibiotics able to escape efflux machineries, as well as efflux pumps inhibitors. The drug/proton antiporter AcrB, part of the major efflux pump AcrAB-TolC in Escherichia coli, is the paradigm RND transporter. Unfortunately, only few compounds have been co-crystallized with AcrB, and thus computational approaches are essential in deciphering determinants of the interactions between diverse ligands and this promiscous protein.Previously we used a truncated model of AcrB to characterize the binding of several substrates and inhibitors. Here we performed docking calculations, molecular dynamics (MD) simulations and free energy calculations to investigate the interaction of 10 congeneric molecules with AcrB. Importantly, we use a full model of the protein in the membrane in order to exploit interactions with all the possible binding sites.Flexible ensemble-docking was performed using almost 40 non-redundant conformations of extracted from experiments and extensive MD simulations of AcrB and different protonation states for each compound. The top poses for each ligand (for a total of 200 AcrB-ligand structures) were then subjected to MD μs- long simulations and analyzed with respect to descriptors of their structure, dynamics, and thermodynamics of binding. The outcome of this study, unprecedented for its extensiveness, will provide key information to medicinal chemists for the design of new compounds with improved efficiency.

다양한 암세포주에서 Jaspine B의 함암활성 비교

Jaspine B는 석회해면류에서 추출된 sphingosine유도체로 인간 암세포에서의 항암활성이 보고되었다. 그러므로 본 연구는 다양한 인간 암세포주에서 항암활성을 비교하고, 암세포주에서의 Jaspine B의 농도를 측정하여 항암 활성과의 연관성을 확인하고자 하였다. 항암활성은 MTT 방법을 이용하여 측정하였고, <TEX>$EC_{50}$</TEX> 값으로 표현하였다. 암세포주내 Jaspine B의 농도는 LC-MS/MS를 이용하여 분석하였다. 항암활성은 세포주마다 다양하게 나타났는데, 유방암과 흑색종 세포주에서 항암활성이 높게 나타났으며(<TEX>$EC_{50}$</TEX> 각각 <TEX>$2.3{\mu}M$</TEX>과 <TEX>$2.6{\mu}M$</TEX>), 신장암세포주에서는 <TEX>$EC_{50}$</TEX> 값이 <TEX>$29.4{\mu}M$</TEX>이었다. 암세포주에서의 <TEX>$EC_{50}$</TEX> 값은 동일한 세포에서의 Jaspine B 농도와 높은 상관성을 나타내었으며(r=0.838), 암세포내 약물농도를 조절하는 것으로 잘 알려진 P-glycoprotein과 breast cancer resistance protein 등의 배출수송계와는 관련이 없음을 확인하였다. 이상의 결과는 세포내 약물농도를 높게 유지하는 것이 항암활성에 매우 중요하며, 세포내 약물농도가 암세포주에 따라 다른 약효를 보이는 원인으로 사료된다. Jaspine B is an anhydrophytosphingosine that is isolated from a marine sponge. Because of its structural similarity to sphingosine, it shows anti-cancer effects in human carcinomas. Therefore, this study aims to investigate its anti-proliferative effect on various cancer cells and to correlate its association with the intracellular accumulation of Jaspine B in relevant cancer cells. The anti-proliferative effect of Jaspine B in various cancer cells was determined by a cell viability test, and the intracellular concentration of Jaspine B in relevant cancer cells was determined using mass spectrometry coupled with liquid chromatography. The correlation coefficient and p value between the cytotoxicity and the cell accumulation of Jaspine B were determined using SPSS 16.1. The cytotoxicity of Jaspine B varied depending on the type of cancer cell when compared the <TEX>$EC_{50}$</TEX> values of Jaspine B. Breast and melanoma cancer cells were susceptible to Jaspine B, whereas renal carcinoma cells were resistant. The intracellular concentrations of Jaspine B had a reciprocal correlation with the <TEX>$EC_{50}$</TEX> values in the same cells (r = 0.838). The results suggested that the anti-proliferative effect of Jaspine B was associated with the cellular accumulation of this compound. However, Jaspine B was not a substrate for P-glycoprotein and breast cancer resistance protein, as major efflux pumps caused multidrug resistance. The maintenance of a high intracellular concentration is crucial for the cytotoxic effect of Jaspine B; however, efflux pumps may not be a controlling factor for Jaspine B-related resistance in cancer cells.

Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii.

ABSTRACTMultidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii. A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii. Furthermore, the method identified a new transcriptional regulator that controls expression of amvA. In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii.

The Crystal Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens.

Bacillus possesses the peptide toxin Sporulation-Delaying Protein (SDP), which can kill cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. The four-component transporter YknWXYZ acts as a major SDP efflux pump to protect cells against the endogenous SDP toxin, for which YknYZ is a non-canonical ATP-binding cassette (ABC)-type transporter. YknYZ consists of the following two components: (1) an individual protein (YknY) and (2) a respective permease (YknZ). To date, the crystal structure, molecular function, and mechanism of action of the integral membrane protein YknZ remain to be elucidated. In this study, to characterize the structural and biochemical roles of YknZ in the functional assembly of YknWXYZ, we predicted and overexpressed the YknZ extracellular domain. We determined the crystal structure of B. amyloliquefaciens YknZ at a resolution of 2.0 Å. The structure revealed that the YknZ extracellular region exhibits significant structural similarity with the MacB periplasmic domain, which is a non-canonical ABC-type transporter in the tripartite macrolide-specific efflux pump in Gram-negative bacteria. We also found that the YknZ extracellular domain can directly bind to an extracellular component of YknX. This structural and biochemical study provides insights into the assembly of YknWXYZ, which may be relevant to understanding cannibalistic peptide toxin resistance in Bacillus and controlling bacterial growth.

Antifungal Action of Methylene Blue Involves Mitochondrial Dysfunction and Disruption of Redox and Membrane Homeostasis in C. albicans.

Candida albicans is known to cause infections ranging from superficial and systemic in immunocompromised person. In this study, we explored that the antifungal action of Methylene blue (MB) is mediated through mitochondrial dysfunction and disruption of redox and membrane homeostasis against C. albicans. We demonstrated that MB displayed its antifungal potential against C. albicans and two clinical isolates tested. We also showed that MB is effective against two non- albicans species as well. Notably, the antifungal effect of MB seems to be independent of the major drug efflux pumps transporter activity. We explored that MB treated Candida cells were sensitive on non-fermentable carbon source leading us to propose that MB inhibits mitochondria. This sensitive phenotype was reinforced with the fact that sensitivity of Candida cells to MB could be rescued upon the supplementation of ascorbic acid, an antioxidant. This clearly suggests that disturbances in redox status are linked with MB action. We further demonstrated that Candida cells were susceptible to membrane perturbing agent viz. SDS which was additionally confirmed by transmission electron micrographs showing disruption of membrane integrity. Moreover, the ergosterol levels were significantly decreased by 66% suggesting lipid compositional changes due to MB. Furthermore, we could demonstrate that MB inhibits the yeast to hyphal transition in C. albicans which is one of the major virulence attribute in most of the hyphal inducing conditions. Taken together, the data generated from present study clearly establishes MB as promising antifungal agent that could be efficiently employed in strategies to treat Candida infections.