Camphor and Eucalyptol—Anticandidal Spectrum, Antivirulence Effect, Efflux Pumps Interference and Cytotoxicity

Candidaalbicans represents one of the most common fungal pathogens. Due to its increasing incidence and the poor efficacy of available antifungals, finding novel antifungal molecules is of great importance. Camphor and eucalyptol are bioactive terpenoid plant constituents and their antifungal properties have been explored previously. In this study, we examined their ability to inhibit the growth of different Candida species in suspension and biofilm, to block hyphal transition along with their impact on genes encoding for efflux pumps (CDR1 and CDR2), ergosterol biosynthesis (ERG11), and cytotoxicity to primary liver cells. Camphor showed excellent antifungal activity with a minimal inhibitory concentration of 0.125–0.35 mg/mL while eucalyptol was active in the range of 2–23 mg/mL. The results showed camphor’s potential to reduce fungal virulence traits, that is, biofilm establishment and hyphae formation. On the other hand, camphor and eucalyptol treatments upregulated CDR1;CDR2 was positively regulated after eucalyptol application while camphor downregulated it. Neither had an impact on ERG11 expression. The beneficial antifungal activities of camphor were achieved with an amount that was non-toxic to porcine liver cells, making it a promising antifungal compound for future development. The antifungal concentration of eucalyptol caused cytotoxic effects and increased expression of efflux pump genes, which suggests that it is an unsuitable antifungal candidate.

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EFFLUX PUMP OVEREXPRESSION PROFILING IN ACINETOBACTER BAUMANNII AND STUDY OF NEW 1-(1-NAPHTHYLMETHYL)-PIPERAZINE ANALOGS AS POTENTIAL EFFLUX INHIBITORS

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00710-21 ◽

2021 ◽

Author(s):

Morgane Choquet ◽

Elodie Lohou ◽

Etienne Pair ◽

Pascal Sonnet ◽

Catherine Mullie

Keyword(s):

Antibiotic Resistance ◽

Acinetobacter Baumannii ◽

Inhibitory Activity ◽

Efflux Pump ◽

Parent Compound ◽

Efflux Pumps ◽

Quinoline Derivative ◽

Genes Encoding ◽

Efflux Pump Inhibitors ◽

Therapeutic Alternatives

Overexpression of efflux pumps extruding antibiotics currently used for the treatment of Acinetobacter baumannii infections has been described as an important mechanism causing antibiotic resistance. The first aim of this work was to phenotypically evaluate the overexpression of efflux pumps on a collection of 124 ciprofloxacin resistant A. baumannii strains. An overexpression of genes encoding one or more efflux pumps was obtained for 19 out of the 34 strains with a positive phenotypic efflux (56%). The most frequent genes overexpressed were those belonging to the RND family, with adeJ being the most prevalent (50%). Interestingly, efflux pump genes coding for MATE and MFS families were also overexpressed quite frequently: abeM (32%) and abaQ (26%). The second aim was to synthesize 1-(1-NaphthylMethyl)-Piperazine analogs as potential new efflux pump inhibitors and biologically evaluate them against strains with a positive phenotypic efflux. Quinoline and pyridine analogs were found to be more effective than their parent compound 1-(1-NaphthylMethyl)-Piperazine. Stereochemistry also played an important part in the inhibitory activity as quinoline derivative ( R )-3a was identified as being the most effective and less cytotoxic. Its inhibitory activity was also correlated to the number of efflux pumps expressed by a strain. The results obtained in this work suggest that quinoline analogs of 1-(1-NaphthylMethyl)-Piperazine are promising leads in the development of new anti- Acinetobacter baumannii therapeutic alternatives, in combination with antibiotics for which an efflux-mediated resistance is suspected.

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Comparative Genomics and the Evolution of Pathogenicity in Human Pathogenic Fungi

Eukaryotic Cell ◽

10.1128/ec.00242-10 ◽

2010 ◽

Vol 10 (1) ◽

pp. 34-42 ◽

Cited By ~ 72

Author(s):

Gary P. Moran ◽

David C. Coleman ◽

Derek J. Sullivan

Keyword(s):

Fungal Pathogens ◽

Direct Interaction ◽

Pathogenic Fungi ◽

Gene Families ◽

Fungal Pathogenesis ◽

Individual Species ◽

Fungal Virulence ◽

Content Type ◽

Sequencing Technologies ◽

Genes Encoding

ABSTRACTBecause most fungi have evolved to be free-living in the environment and because the infections they cause are usually opportunistic in nature, it is often difficult to identify specific traits that contribute to fungal pathogenesis. In recent years, there has been a surge in the number of sequenced genomes of human fungal pathogens, and comparison of these sequences has proved to be an excellent resource for exploring commonalities and differences in how these species interact with their hosts. In order to survive in the human body, fungi must be able to adapt to new nutrient sources and environmental stresses. Therefore, genes involved in carbohydrate and amino acid metabolism and transport and genes encoding secondary metabolites tend to be overrepresented in pathogenic species (e.g.,Aspergillus fumigatus). However, it is clear that human commensal yeast species such asCandida albicanshave also evolved a range of specific factors that facilitate direct interaction with host tissues. The evolution of virulence across the human pathogenic fungi has occurred largely through very similar mechanisms. One of the most important mechanisms is gene duplication and the expansion of gene families, particularly in subtelomeric regions. Unlike the case for prokaryotic pathogens, horizontal transfer of genes between species and other genera does not seem to have played a significant role in the evolution of fungal virulence. New sequencing technologies promise the prospect of even greater numbers of genome sequences, facilitating the sequencing of multiple genomes and transcriptomes within individual species, and will undoubtedly contribute to a deeper insight into fungal pathogenesis.

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The C2H2 Transcription Factor SltA Contributes to Azole Resistance by Coregulating the Expression of the Drug Target Erg11A and the Drug Efflux Pump Mdr1 in Aspergillus fumigatus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01839-20 ◽

2021 ◽

Author(s):

Wenlong Du ◽

Pengfei Zhai ◽

Tingli Wang ◽

Michael J Bromley ◽

Yuanwei Zhang ◽

...

Keyword(s):

Transcription Factor ◽

Aspergillus Fumigatus ◽

Fungal Pathogens ◽

Efflux Pump ◽

Antifungal Drugs ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Drug Efflux ◽

Mobility Shift ◽

Drug Efflux Pump

The emergence of azole-resistant fungal pathogens has posed a great threat to public health worldwide. Although the molecular mechanism of azole resistance has been extensively investigated, the potential regulators of azole resistance remain largely unexplored. Here we identified a new function of the fungal specific C2H2 zinc finger transcription factor SltA (involved in salt-tolerance pathway) in the regulation of azole resistance of the human fungal pathogen Aspergillus fumigatus. Lack of SltA results in an itraconazole hypersusceptibility phenotype. Transcriptional profiling combined with LacZ reporter analysis and electrophoretic mobility shift assays (EMSA) demonstrate that SltA is involved in its own transcriptional regulation and also regulates the expression of genes related to ergosterol biosynthesis (erg11A, erg13A and erg24A) and drug efflux pumps (mdr1, mfsC and abcE) by directly binding to the conserved 5’-AGGCA-3’ motif in their promoter regions, and this binding is dependent on the conserved cysteine and histidine within the C2H2 DNA binding domain of SltA. Moreover, overexpression of erg11A or mdr1 rescues sltA deletion defects under itraconazole conditions, suggesting that erg11A and mdr1 are related to sltA-mediated itraconazole resistance. Most importantly, deletion of SltA in laboratory-derived and clinical azole-resistant isolates significantly attenuates drug resistance. Collectively, we have identified a new function of the transcription factor SltA in regulating azole resistance by coordinately mediating the key azole target Erg11A and the drug efflux pump Mdr1, and targeting SltA may provide a potential strategy for intervention of clinical azole-resistant isolates to improve the efficiency of currently approved antifungal drugs.

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Multidrug-Resistant Transporter Mdr1p-Mediated Uptake of a Novel Antifungal Compound

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01504-13 ◽

2013 ◽

Vol 57 (12) ◽

pp. 5931-5939 ◽

Cited By ~ 18

Author(s):

Nuo Sun ◽

Dongmei Li ◽

William Fonzi ◽

Xin Li ◽

Lixin Zhang ◽

...

Keyword(s):

Fungal Pathogens ◽

Efflux Pump ◽

Fungal Infections ◽

Patent Application ◽

Multidrug Resistant ◽

Inhibitory Effect ◽

Major Facilitator Superfamily ◽

Antifungal Compound ◽

Specific Class ◽

Content Type

ABSTRACTThe activity of many anti-infectious drugs has been compromised by the evolution of multidrug-resistant (MDR) pathogens. For life-threatening fungal infections, such as those caused byCandida albicans, overexpression ofMDR1, which encodes an MDR efflux pump of the major facilitator superfamily (MFS), often confers resistance to chemically unrelated substances, including the most commonly used azole antifungals. As the development of new and efficacious antifungals has lagged far behind the growing emergence of resistant strains, it is imperative to develop strategies to overcome multidrug resistance. Previous advances have been mainly to deploy combinational therapy to restore azole susceptibility, which, however, requires coordination of two or more compounds. We observed a unique phenotype in which Mdr1p facilitates the uptake of a specific class of compounds. Among them, we describe a novel antifungal small molecule, bis[1,6-a:5′,6′-g]quinolizinium 8-methyl-salt (BQM) (U.S. patent application no. 61/793,090,2013), that has potent and broad antifungal activity. Notably, BQM exploits the MDR phenotype inC. albicansto promote the inhibitory effect. Rather than causing an antagonism of MDR strains, it exhibits a highly potentiated activity against a collection of clinical isolates and lab strains that overexpressMDR1. The activity of BQM againstMDR1-overexpressing isolates is due to its facilitated intracellular accumulation. Microarray comparisons showed an extensive upregulation ofMDR1as well as polyamine transporter genes in a fluconazole-resistant strain. We then demonstrated that the polyamine transporters augment the accumulation of BQM. Importantly, BQM had greater activity than fluconazole and itraconazole against various fungal pathogens, including MDRAspergillus fumigatus. Thus, our findings offer a paradigm shift to overcome MDR and the promise of improving antifungal treatment, especially in MDR pathogens.

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Pathogenesis of fungal disease

10.1093/med/9780198755388.003.0008 ◽

2017 ◽

Cited By ~ 1

Author(s):

Frank C. Odds

Keyword(s):

Virulence Factors ◽

Fungal Pathogens ◽

Hydrolytic Enzymes ◽

Fungal Disease ◽

Fungal Species ◽

Fungal Virulence ◽

Host Immune Responses ◽

Genes Encoding ◽

Different Strains

The pathogenesis of fungal disease involves an interplay between fungal virulence factors and host immune responses. Most fungal pathogens are opportunists that preferentially invade hosts with immune defects, but the fact that relative pathogenicity varies between fungal species (and even between different strains within a species) is evidence that fungi have evolved multiple, different molecular virulence factors. Experiments in which genes encoding putative virulence attributes are specifically disrupted and the resulting mutants are tested for virulence in a range of vertebrate and invertebrate hosts have identified or confirmed many gene products as significant for the pathogenesis of various types of fungal disease. These include factors determining fungal shape in vivo, biofilm formation, and a plethora of surface components, including adhesins and hydrolytic enzymes. This chapter provides an overview of fungal virulence attributes.

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Roles of Efflux Pumps from Different Superfamilies in the Surface-Associated Motility and Virulence of Acinetobacter baumannii ATCC 17978

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02190-18 ◽

2019 ◽

Vol 63 (3) ◽

Cited By ~ 3

Author(s):

María Pérez-Varela ◽

Jordi Corral ◽

Jesús Aranda ◽

Jordi Barbé

Keyword(s):

Acinetobacter Baumannii ◽

Efflux Pump ◽

Efflux Pumps ◽

Major Facilitator Superfamily ◽

Content Type ◽

Small Multidrug Resistance ◽

Genes Encoding ◽

Resistance Nodulation Division ◽

Major Facilitator ◽

Reported Data

ABSTRACT Although the relationship between Acinetobacter baumannii efflux pumps and antimicrobial resistance is well documented, less is known about the involvement of these proteins in the pathogenicity of this nosocomial pathogen. In previous work, we identified the AbaQ major facilitator superfamily (MFS) efflux pump and demonstrated its participation in the motility and virulence of A. baumannii. In the present study, we examined the role in these processes of A. baumannii transporters belonging to different superfamilies of efflux pumps. Genes encoding known or putative permeases belonging to efflux pump superfamilies other than the MFS were selected, and the corresponding knockouts were constructed. The antimicrobial susceptibilities of these mutants were consistent with previously reported data. In mutants of A. baumannii strain ATCC 17978 carrying inactivated genes encoding the efflux pumps A1S_2736 (resistance nodulation division [RND]), A1S_3371 (multidrug and toxic compound extrusion [MATE]), and A1S_0710 (small multidrug resistance [SMR]), as well as the newly described ATP-binding cassette (ABC) permeases A1S_1242 and A1S_2622, both surface-associated motility and virulence were reduced compared to the parental strain. However, inactivation of the genes encoding the known ABC permeases A1S_0536 and A1S_1535, the newly identified putative ABC permeases A1S_0027 and A1S_1057, or the proteobacterial antimicrobial compound efflux (PACE) transporters A1S_1503 and A1S_2063 had no effects on bacterial motility or virulence. Our results demonstrate the involvement of antimicrobial transporters belonging at least to five of the six known efflux pump superfamilies in both surface-associated motility and virulence in A. baumannii ATCC 17978.

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Rapid, Transient Fluconazole Resistance inCandida albicans Is Associated with Increased mRNA Levels of CDR

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.10.2584 ◽

1998 ◽

Vol 42 (10) ◽

pp. 2584-2589 ◽

Cited By ~ 118

Author(s):

Kieren A. Marr ◽

Christopher N. Lyons ◽

Tiger Rustad ◽

Raleigh A. Bowden ◽

Theodore C. White

Keyword(s):

Abc Transporter ◽

Efflux Pumps ◽

Northern Blotting ◽

Ergosterol Biosynthesis ◽

Mrna Levels ◽

Genes Encoding ◽

Fluconazole Resistant ◽

Fluconazole Prophylaxis ◽

Lanosterol Demethylase ◽

Free Media

ABSTRACT Fluconazole-resistant Candida albicans, a cause of recurrent oropharyngeal candidiasis in patients with human immunodeficiency virus infection, has recently emerged as a cause of candidiasis in patients receiving cancer chemotherapy and marrow transplantation (MT). In this study, we performed detailed molecular analyses of a series of C. albicans isolates from an MT patient who developed disseminated candidiasis caused by an azole-resistant strain 2 weeks after initiation of fluconazole prophylaxis (K. A. Marr, T. C. White, J. A. H. vanBurik, and R. A. Bowden, Clin. Infect. Dis. 25:908–910, 1997). DNA sequence analysis of the gene (ERG11) for the azole target enzyme, lanosterol demethylase, revealed no difference between sensitive and resistant isolates. A sterol biosynthesis assay revealed no difference in sterol intermediates between the sensitive and resistant isolates. Northern blotting, performed to quantify mRNA levels of genes encoding enzymes in the ergosterol biosynthesis pathway (ERG7, ERG9, and ERG11) and genes encoding efflux pumps (MDR1, ABC1,YCF, and CDR), revealed that azole resistance in this series is associated with increased mRNA levels for members of the ATP binding cassette (ABC) transporter superfamily, CDRgenes. Serial growth of resistant isolates in azole-free media resulted in an increased susceptibility to azole drugs and corresponding decreased mRNA levels for the CDR genes. These results suggest that C. albicans can become transiently resistant to azole drugs rapidly after exposure to fluconazole, in association with increased expression of ABC transporter efflux pumps.

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GC-MS Profile and Enhancement of Antibiotic Activity by the Essential Oil of Ocotea odorífera and Safrole: Inhibition of Staphylococcus aureus Efflux Pumps

Antibiotics ◽

10.3390/antibiotics9050247 ◽

2020 ◽

Vol 9 (5) ◽

pp. 247 ◽

Cited By ~ 1

Author(s):

Ray S. Almeida ◽

Priscilla R. Freitas ◽

Ana Carolina J. Araújo ◽

Irwin R. A. Menezes ◽

Eduardo L. Santos ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Essential Oil ◽

Ethidium Bromide ◽

Efflux Pump ◽

Efflux Pumps ◽

Phytochemical Analysis ◽

Broth Microdilution Method ◽

Genes Encoding ◽

Resistant Strains ◽

Efflux Proteins

Considering the evidence that essential oils, as well as safrole, could modulate bacterial growth in different resistant strains, this study aims to characterize the phytochemical profile and evaluate the antibacterial and antibiotic-modulating properties of the essential oil Ocotea odorífera (EOOO) and safrole against efflux pump (EP)-carrying strains. The EOOO was extracted by hydrodistillation, and the phytochemical analysis was performed by gas chromatography coupled to mass spectrometry (GC-MS). The antibacterial and antibiotic-modulating activities of the EOOO and safrole against resistant strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were analyzed through the broth microdilution method. The EP-inhibiting potential of safrole in association with ethidium bromide or antibiotics was evaluated using the S. aureus 1199B and K2068 strains, which carry genes encoding efflux proteins associated with antibiotic resistance to norfloxacin and ciprofloxacin, respectively. A reduction in the MIC of ethidium bromide or antibiotics was used as a parameter of EP inhibition. The phytochemical analysis identified 16 different compounds in the EOOO including safrole as the principal constituent. While the EOOO and safrole exerted clinically relevant antibacterial effects against S. aureus only, they potentiated the antibacterial activity of norfloxacin against all strains evaluated by our study. The ethidium bromide and antibiotic assays using the strains of S. aureus SA1119B and K2068, as well as molecular docking analysis, indicated that safrole inhibits the NorA and MepA efflux pumps in S. aureus. In conclusion, Ocotea odorifera and safrole presented promising antibacterial and antibiotic-enhancing properties, which should be explored in the development of drugs to combat antibacterial resistance, especially in strains bearing genes encoding efflux proteins.

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Progress in the Molecular Characterization of Hepatobiliary Transporters

Digestive Diseases ◽

10.1159/000450911 ◽

2017 ◽

Vol 35 (3) ◽

pp. 197-202 ◽

Cited By ~ 13

Author(s):

Dietrich Keppler

Keyword(s):

Bile Acids ◽

Driving Forces ◽

Efflux Pump ◽

Functional Characterization ◽

Efflux Pumps ◽

Renal Elimination ◽

Genes Encoding ◽

Uptake Transporters ◽

Hepatobiliary Elimination

Over the last 25 years, our understanding of the driving forces for hepatobiliary elimination and knowledge of the molecular basis of uptake and efflux transport in hepatocytes have undergone fundamental changes. This refers to bile acids and many other endogenous substances as well as to drugs that are eliminated on the hepatobiliary route. In this development, not only molecular cloning, functional characterization, and localization of transporters were decisive, but also the discovery of hereditary mutations in genes encoding sinusoidal uptake transporters and canalicular export pumps in humans and rodents. Uptake by passive diffusion and elimination into bile driven by the electrochemical gradient are no longer considered relevant for hepatobiliary elimination in the intact organism. Furthermore, insights into the relative roles of uptake transporters and unidirectional ATP-driven efflux pumps were obtained when we established double-transfected polarized cell lines stably expressing, as an example, the hepatocellular uptake transporter OATP1B3 and the apical (canalicular) efflux pump multidrug resistance protein 2 (MRP2; ABCC2). ATP-dependent efflux transporters localized to the basolateral (sinusoidal) hepatocyte membrane, particularly MRP3 (ABCC3) and MRP4 (ABCC4), pump substances from hepatocytes into sinusoidal blood. Bile acids are substrates for human MRP4 in the presence of physiological concentrations of reduced glutathione, which undergoes co-transport. These efflux pumps have been recognized in recent years to play an important compensatory role in cholestasis and to contribute to the balance between uptake and efflux of bile acids and other organic anions during the vectorial transport from blood into bile. This sinusoidal efflux not only enables subsequent renal elimination but also facilitates the re-uptake of substances into neighboring hepatocytes located more centrally and downstream in the sinusoid.

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Genomewide Location Analysis of Candida albicans Upc2p, a Regulator of Sterol Metabolism and Azole Drug Resistance

Eukaryotic Cell ◽

10.1128/ec.00070-08 ◽

2008 ◽

Vol 7 (5) ◽

pp. 836-847 ◽

Cited By ~ 76

Author(s):

Sadri Znaidi ◽

Sandra Weber ◽

Osman Zin Al-Abdin ◽

Perrine Bomme ◽

Saloua Saidane ◽

...

Keyword(s):

Drug Resistance ◽

Candida Albicans ◽

Efflux Pump ◽

Ergosterol Biosynthesis ◽

Zinc Cluster ◽

Dna Motif ◽

C Terminus ◽

Genes Encoding ◽

Important Regulator

ABSTRACT Upc2p, a transcription factor of the zinc cluster family, is an important regulator of sterol biosynthesis and azole drug resistance in Candida albicans. To better understand Upc2p function in C. albicans, we used genomewide location profiling to identify the transcriptional targets of Upc2p in vivo. A triple hemagglutinin epitope, introduced at the C terminus of Upc2p, conferred a gain-of-function effect on the fusion protein. Location profiling identified 202 bound promoters (P < 0.05). Overrepresented functional groups of genes whose promoters were bound by Upc2p included 12 genes involved in ergosterol biosynthesis (NCP1, ERG11, ERG2, and others), 18 genes encoding ribosomal subunits (RPS30, RPL32, RPL12, and others), 3 genes encoding drug transporters (CDR1, MDR1, and YOR1), 4 genes encoding transcription factors (INO2, ACE2, SUT1, and UPC2), and 6 genes involved in sulfur amino acid metabolism (MET6, SAM2, SAH1, and others). Bioinformatic analyses suggested that Upc2p binds to the DNA motif 5′-VNCGBDTR that includes the previously characterized Upc2p binding site 5′-TCGTATA. Northern blot analysis showed that increased binding correlates with increased expression for the analyzed Upc2p targets (ERG11, MDR1, CDR1, YOR1, SUT1, SMF12, and CBP1). The analysis of ERG11, MDR1, and CDR1 transcripts in wild-type and upc2Δ/upc2Δ strains grown under Upc2p-activating conditions (lovastatin treatment and hypoxia) showed that Upc2p regulates its targets in a complex manner, acting as an activator or as a repressor depending upon the target and the activating condition. Taken together, our results indicate that Upc2p is a key regulator of ergosterol metabolism. They also suggest that Upc2p may contribute to azole resistance by regulating the expression of drug efflux pump-encoding genes in addition to ergosterol biosynthesis genes.

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