Mitochondria complex I deficiency in Candida albicans arrests the cell cycle at S phase through suppressive TOR and PKA pathways.

How mutations in mitochondrial electron transport chain (ETC) proteins impact the cell cycle of Candida albicans was investigated in this study. Using genetic null mutants targeting ETC complexes I (CI), III (CIII), and IV (CIV), the cell cycle stages (G0/G1, S phase, and G2/M) were analyzed via fluorescence-activated cell sorting (FACS). Four CI null mutants exhibited distinct alterations, including extended S phase, shortened G2/M population, and a reduction in cells size exceeding 10 µM. Conversely, CIII mutants showed an increased population in G1/G0 phase. Among four CI mutants, ndh51Δ/Δ and goa1Δ/Δ displayed aberrant cell cycle patterns correlated with previously reported cAMP/PKA downregulation. Specifically, nuo1Δ/Δ and nuo2Δ/Δ mutants exhibited increased transcription of RIM15, a central hub linking cell cycle with nutrient-dependent TOR1 and cAMP/PKA pathways and Snf1 aging pathway. These findings suggest that suppression of TOR1 and cAMP/PKA pathways or enhanced Snf1 disrupts cell cycle progression, influencing cell longevity and growth among CI mutants. Overall, our study highlights the intricate interplay between mitochondrial ETC, cell cycle, and signaling pathways.

The mitochondrial complex I proteins of Candida albicans moderate phagocytosis and the production of pro-inflammatory cytokines in murine macrophages and dendritic cells.

Loss of respiratory functions impairs Candida albicans colonization of host tissues and virulence in a murine model of candidiasis. Furthermore, it is known that respiratory inhibitors decrease mannan synthesis and glucan exposure and thereby promotes phagocytosis. To understand the impact of respiratory proteins of C. albicans on host innate immunity, we characterized cell wall defects in three mitochondrial complex I (CI) null mutants (nuo1Δ, nuo2Δ and ndh51Δ) and in one CI regulator mutant (goa1Δ), and we studied the corresponding effects of these mutants on phagocytosis, neutrophil killing and cytokine production by dendritic cells (DCs). We find that reductions of phosphopeptidomannan (PPM) in goa1Δ, nuo1Δ and phospholipomannan (PLM) in nuo2Δ lead to reductions of IL-2, IL-4, and IL-10 but increase of TNF-α in infected DCs. While PPM loss is a consequence of a reduced phospho-Cek1/2 MAPK that failed to promote phagocytosis and IL-22 production in goa1Δ and nuo1Δ, a 30% glucan reduction and a defective Mek1 MAPK response in ndh51Δ lead to only minor changes in phagocytosis and cytokine production. Glucan exposure and PLM abundance seem to remain sufficient to opsonize neutrophil killing perhaps via humoral immunity. The diversity of immune phenotypes in these mutants possessing divergent cell wall defects is further supported by their transcriptional profiles in each infected murine macrophage scenario. Since metabolic processes, oxidative stress-induced senescence, and apoptosis are differently affected in these scenarios, we speculate that during the early stages of infection, host immune cells coordinate their bioactivities based upon a mixture of signals generated during host-fungi interactions.

Salivary and serum IgA and IgG responses to SARS-CoV-2-spike protein following SARS-CoV-2 infection and after immunization with COVID-19 vaccines.

Background: Secretory immunoglobulin A (sIgA) plays an important role in antiviral protective immunity. Although salivary testing has been used for many viral infections, including severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS), its use has not yet been well established with the SARS coronavirus 2 (SARS-CoV-2). Quantification of salivary IgA and IgG antibodies can elucidate mucosal and systemic immune responses after natural infection or vaccination. Here, we report the development and validation of a rapid enzyme-linked immunosorbent assay (ELISA) for anti-SARS-CoV-2 salivary IgA and serum IgG antibodies, and present quantitative results for immunized subjects both prior to or following COVID-19 infections. Objective: Total and serum SARS-CoV-2 spike-specific IgG responses were compared with salivary spike-specific IgA and IgG responses in samples obtained from patients recently infected with SARS-CoV-2 and from subjects recently immunized with COVID-19 vaccines. Methods: A total of 52 paired saliva and serum samples were collected from 26 study participants: 7 subjects after COVID-19 infection and 19 subjects who were uninfected. The ELISA results from these samples were compared with five prepandemic control serum samples. Total IgG and SARS-CoV-2 spike-specific IgG in the serum samples from the subjects who were infected and vaccinated were also measured in a commercial laboratory with an enzyme immunoassay. Results: A wide variation in antibody responses was seen in salivary and serum samples measured by both methods. Three groups of serum total and IgG spike-specific SARS-CoV-2 antibody responses were observed: (1) low, (2) intermediate, and (3) high antibody responders. A correlational analysis of salivary IgA (sIgA) responses with serum IgG concentrations showed a statistical correlation in the low and intermediate antibody responder groups but not in the high group (which we believe was a result of saturation). Conclusion: These preliminary findings suggest measuring salivary and serum IgG and IgA merit further investigation as markers of current or recent SARS-CoV-2 infections.

The antifungal pipeline: the need is established. Are there new compounds?

Our review summarizes and compares the temporal development (eras) of antifungal drug discovery as well as antibacterial ventures. The innovation gap that occurred in antibacterial discovery from 1960 to 2000 was likely due to tailoring of existing compounds to have better activity than predecessors. Antifungal discovery also faced innovation gaps. The semi-synthetic antibiotic era was followed closely by the resistance era and the heightened need for new compounds and targets. With the immense contribution of comparative genomics, antifungal targets became part of the discovery focus. These targets by definition are absolutely required to be fungal- or even lineage (clade) specific. Importantly, targets need to be essential for growth and/or have important roles in disease and pathogenesis. Two types of antifungals are discussed that are mostly in the FDA phase I-III clinical trials. New antifungals are either modified to increase bioavailability and stability for instance, or are new compounds that inhibit new targets. One of the important developments in incentivizing new antifungal discovery has been the prolific number of publications of global and country-specific incidence. International efforts that champion global antimicrobial drug discovery are discussed. Still, interventions are needed. The current pipeline of antifungals and alternatives to antifungals are discussed including vaccines.

Treg-inducing capacity of genomic DNA of Bifidobacterium longum subsp. infantis.

Background: Allergic and autoimmune diseases comprise a group of inflammatory disorders caused by aberrant immune responses in which CD25+ forkhead box P3-positive regulatory T cells (Treg) cells that normally suppress inflammatory events are often poorly functioning. This has stimulated an intensive investigative effort to find ways of increasing Tregs as a method of therapy for these conditions. Commensal microbiota known to have health benefits in humans include the lactic acid-producing, probiotic bacteria B. longum subsp. infantis and Lactobacillus rhamnosus. Mechanistically, several mechanisms have been proposed to explain how probiotics may favorably affect host immunity, including the induction of Tregs. Analysis of emerging data from several laboratories, including our own, suggest that DNA methylation may be an important determinant of immune reactivity responsible for Treg induction. Although methylated CpG moieties in normal mammalian DNA are both noninflammatory and lack immunogenicity, unmethylated CpGs, found largely in microbial DNA, are immunostimulatory and display proinflammatory properties. Objective: We hypothesize that microbiota with more DNA methylation may potentiate Treg induction to a greater degree than microbiota with a lower content of methylation. The purpose of the present study was to test this hypothesis by studying the methylation status of whole genomic DNA (gDNA) and the Treg-inducing capacity of purified gDNA in each of the probiotic bacteria B. longum subsp. infantis and L. rhamnosus, and a pathogenic Escherichia coli strain B. Results: We showed that gDNA from B. longum subsp. infantis is a potent Treg inducer that displays a dose-dependent response pattern at a dose threshold of 20 µg of gDNA. No similar Treg-inducing responses were observed with the gDNA from L. rhamnosus or E. coli. We identified a unique CpG methylated motif in the gDNA sequencing of B. longum subsp. infantis which was not found in L. rhamnosus or E. coli strain B. Conclusion: Although the literature indicates that both B. longum subsp. infantis and L. rhamnosus strains contribute to health, our data suggest that they do so by different mechanisms. Further, because of its small molecular size, low cost, ease of synthesis, and unique Treg-inducing feature, this methylated CpG oligodeoxynucleotide (ODN) from B. longum would offer many attractive features for an ideal novel therapeutic vaccine candidate for the treatment of immunologic diseases, such as the allergic and autoimmune disorders, in which Treg populations are diminished.

Interplay of interferon-gamma and macrophage polarization during Talaromyces marneffei infection.

Talaromyces marneffei is an increasingly destructive dimorphic fungal pathogen in clinical settings that can cause lethal Talaromycosis. The activation of macrophages is known to be important for host defenses against T. marneffei, and these macrophages are known to be activated in two ways (polarization), known as M1 and M2. We investigated the plasticity of these polarizations, in order to understand if cross-conversion of macrophages may be possible even after they have been programmed. We conducted in vitro experiments using a murine macrophage cell line to investigate the ability of T. marneffei to activate these polarizations. The pre-polarized (M0) macrophage subsets were challenged with LPS as a control, and the sets of M1 markers (iNOS and CD86) and M2 markers (Arg-1 and CD206) were assessed for a possible cross-conversion among M1, M2 and M0 (unstimulated) populations. We found that either conidia or yeast forms of T. marneffei initiate the repression of Arg-1 in M2 cells with no change in the M1 subtype marker molecule iNOS. However, an additional IFN-γ stimulus caused the three macrophage groups to fully exhibit an LPS-induced M2 suppression and a shift to M1 from M0 and M2. We conclude that the conversion of macrophages is required for maintenance of sufficient iNOS production against this organism in the host. The cytokine environment is the key factor that manipulates the plasticity changes among macrophage subtypes. Furthermore, IFN-γ is a crucial host defense factor against pathogenic T. marneffei that has significant therapeutic potential to promote an M1 polarization phenotype.

Respiratory stress in mitochondrial electron transport chain complex mutants of Candida albicans activates Snf1 kinase response.

We have previously established that mitochondrial Complex I (CI) mutants of Candida albicans display reduced oxygen consumption, decreased ATP production, and increased reactive oxidant species (ROS) during cell growth. Using the Seahorse XF96 analyzer, the energetic phenotypes of Electron Transport Chain (ETC) complex mutants are further characterized in the current study. The underlying regulation of energetic changes in these mutants is determined in glucose and non-glucose conditions when compared to wild type (WT) cells. In parental cells, the rate of oxygen consumption remains constant for 2.5 h following the addition of glucose, oligomycin, and 2-DG, but glycolysis is highly active upon the addition of glucose. In comparison, over the same time period, electron transport complex mutants (CI, CIII and CIV) have heightened activities in both oxygen consumption and glycolysis upon glucose uptake. We refer to the response in these mutants as an "explosive respiration," which we believe is caused by low energy levels and increased production of reactive oxygen species (ROS). Accompanying this phenotype in mutants is a hyperphosphorylation of Snf1p which in Saccharomyces cerevisiae serves as an energetic stress response protein kinase for maintaining energy homeostasis. Compared to wild type cells, a 2.9- to 4.4-fold hyperphosphorylation of Snf1p is observed in all ETC mutants in the presence of glucose. However, the explosive respiration and hyperphosphorylation of Snf1 can be partially reduced by the replacement of glucose with either glycerol or oleic acid in a mutant-specific manner. Furthermore, Inhibitors of glutathione synthesis (BSO) or anti-oxidants (mito-TEMPO) likewise confirmed an increase of Sfn1 phosphorylation in WT or mutant due to increased levels of ROS. Our data establish the role of the C. albicans Snf1 as a surveyor of cell energy and ROS levels. We interpret the "explosive respiration" as a failed attempt by ETC mutants to restore energy and ROS homeostasis via Snf1 activation. An inherently high OCR baseline in WT C. albicans with a background level of Snf1 activation is a prerequisite for success in quickly fermenting glucose.

Hybrid histidine kinases in pathogenic fungi.

Histidine kinases (HK) sense and transduce via phosphorylation events many intra- and extracellular signals in bacteria, archaea, slime moulds and plants. HK are also widespread in the fungal kingdom, but their precise roles in the regulation of physiological processes remain largely obscure. Expanding genomic resources have recently given the opportunity to identify uncharacterised HK family members in yeasts and moulds and now allow proposing a complex classification of Basidiomycota, Ascomycota and lower fungi HK. A growing number of genetic approaches have progressively provided new insight into the role of several groups of HK in prominent fungal pathogens. In particular, a series of studies have revealed that members of group III HK, which occur in the highest number of fungal species and contain a unique N-terminus region consisting of multiple HAMP domain repeats, regulate morphogenesis and virulence in various human, plant and insect pathogenic fungi. This research field is further supported by recent shape-function studies providing clear correlation between structural properties and signalling states in group III HK. Since HK are absent in mammals, these represent interesting fungal target for the discovery of new antifungal drugs.

Functional diversity of complex I subunits in Candida albicans mitochondria.

Our interest in the mitochondria of Candida albicans has progressed to the identification of several proteins that are critical to complex I (CI) activity. We speculated that there should be major functional differences at the protein level between mammalian and fungal mitochondria CI. In our pursuit of this idea, we were helped by published data of CI subunit proteins from a broad diversity of species that included two subunit proteins that are not found in mammals. These subunit proteins have been designated as Nuo1p and Nuo2p (NADH-ubiquinone oxidoreductases). Since functional assignments of both C. albicans proteins were unknown, other than having a putative NADH-oxidoreductase activity, we constructed knock-out strains that could be compared to parental cells. The relevance of our research relates to the critical roles of both proteins in cell biology and pathogenesis and their absence in mammals. These features suggest they may be exploited in antifungal drug discovery. Initially, we characterized Goa1p that apparently regulates CI activity but is not a CI subunit protein. We have used the goa1∆ for comparisons to Nuo1p and Nuo2p. We have demonstrated the critical role of these proteins in maintaining CI activities, virulence, and prolonging life span. More recently, transcriptional profiling of the three mutants and an ndh51∆ (protein is a highly conserved CI subunit) has revealed that there are overlapping yet also different functional assignments that suggest subunit specificity. The differences and similarities of each are described below along with our hypotheses to explain these data. Our conclusion and perspective is that the C. albicans CI subunit proteins are highly conserved except for two that define non-mammalian functions.

Fungal-specific subunits of the Candida albicans mitochondrial complex I drive diverse cell functions including cell wall synthesis.

Our published research has focused on the role of Goa1p, an apparent regulator of the Candida albicans mitochondrial complex I (CI). Lack of Goa1p affects optimum cell growth, CI activity and virulence. Eukaryotic CI is composed of a core of 14 alpha-proteobacterial subunit proteins and a variable number of supernumerary subunit proteins. Of the latter group of proteins, one (NUZM) is fungal specific and the other (NUXM) is found in fungi, algae and plants, but is not a mammalian CI subunit protein. We have established that NUXM is orf19.6607 and NUZM is orf19.287 in C. albicans. Herein, we validate both subunit proteins as NADH:ubiquinone oxidoreductases (NUO) and annotate their gene functions. To accomplish these objectives, we compared null mutants of each with wild type (WT) and gene-reconstituted strains. Genetic mutants of genes NUO1 (orf19.6607) and NUO2 (orf19.287), not surprisingly, each had reduced oxygen consumption, decreased mitochondrial redox potential, decreased CI activity, increased reactive oxidant species (ROS) and decreased chronological ageing in vitro. Loss of either gene results in disassembly of CI. Transcriptional profiling of both mutants indicated significant down-regulation of genes of carbon metabolism, as well as up-regulation of mitochondrial-associated gene families that may occur to compensate for the loss of CI activity. Profiling of both mutants also demonstrated a loss of cell wall ß-mannosylation but not in a conserved CI subunit (ndh51Δ). The profiling data may indicate specific functions driven by the enzymatic activity of Nuo1p and Nuo2p. Of importance, each mutant is also avirulent in a murine blood-borne, invasive model of candidiasis associated with their reduced colonization of tissues. Based on their fungal specificity and roles in virulence, we suggest both as drug targets for antifungal drug discovery.

Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.

Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/µl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients.

System-level impact of mitochondria on fungal virulence: to metabolism and beyond.

The mitochondrion plays wide-ranging roles in eukaryotic cell physiology. In pathogenic fungi, this central metabolic organelle mediates a range of functions related to disease, from fitness of the pathogen to developmental and morphogenetic transitions to antifungal drug susceptibility. In this review, we present the latest findings in this area. We focus on likely mechanisms of mitochondrial impact on fungal virulence pathways through metabolism and stress responses, but also potentially via control over signaling pathways. We highlight fungal mitochondrial proteins that lack human homologs, and which could be inhibited as a novel approach to antifungal drug strategy.

The Rbf1, Hfl1 and Dbp4 of Candida albicans regulate common as well as transcription factor-specific mitochondrial and other cell activities.

BACKGROUND: Our interest in Candida albicans mitochondria began with the identification of GOA1. We demonstrated its role in cell energy production, cross-talk among mitochondria and peroxisomes, non-glucose energy metabolism, maintenance of stationary phase growth, and prevention of premature apoptosis. Its absence results in avirulence. However, what regulated transcription of GOA1 was unknown. RESULTS: To identify transcriptional regulators (TRs) of GOA1, we screened a C. albicans TF knockout library (TRKO) and identified Rbf1p, Hfl1p, and Dpb4p as positive TRs of GOA1. The phenotypes of each mutant (reduced respiration, inability to grow on glycerol, reduced ETC CI and CIV activities) are reasonable evidence for their required roles especially in mitochondrial functions. While the integration of mitochondria with cell metabolic activities is presumed to occur, there is minimal information on this subject at the genome level. Therefore, microarray analysis was used to provide this information for each TR mutant. Transcriptional profiles of Rbf1p and Hfl1p are more similar than that of Dpn4p. Our data demonstrate common and also gene-specific regulatory functions for each TR. We establish their roles in carbon metabolism, stress adaptation, cell wall synthesis, transporter efflux, peroxisomal metabolism, phospholipid synthesis, rRNA processing, and nuclear/mtDNA replication. CONCLUSIONS: The TRs regulate a number of common genes but each also regulates specific gene transcription. These data for the first time create a genome roadmap that can be used to integrate mitochondria with other cell processes. Of interest, the TRs are fungal-specific, warranting consideration as antifungal drug targets.

Cell surface changes in the Candida albicans mitochondrial mutant goa1Δ are associated with reduced recognition by innate immune cells.

We have previously characterized several fungal-specific proteins from the human pathogen Candida albicans that either encode subunits of mitochondria Complex I (CI) of the electron transport chain (ETC) or regulate CI activity (Goa1p). Herein, the role of energy production and cell wall gene expression is investigated in the mitochondria mutant goa1Δ. We show that downregulation of cell wall-encoding genes in the goa1Δ results in sensitivity to cell wall inhibitors such as Congo red and Calcofluor white, reduced phagocytosis by a macrophage cell line, reduced recognition by macrophage receptors, and decreased expression of cytokines such as IL-6, IL-10 and IFN-γ. In spite of the reduced recognition by macrophages, the goa1Δ is still killed to the same extent as control strains. We also demonstrate that expression of the epithelial cell receptors E-cadherin and EGFR is also reduced in the presence of goa1Δ. Together, our data demonstrate the importance of mitochondria in the expression of cell wall biomolecules and the interaction of C. albicans with innate immune and epithelial cells. Our underlying premise is thatmitochondrial proteins such as Goa1p and other fungal-specific mitochondrial proteins regulate critical functions in cell growth and in virulence. As such, they remain as valid drug targets for antifungal drug discovery.

Fluconazole Resistance Patterns in Candida Species that Colonize Women with HIV Infection.

BACKGROUND: The Women's Interagency HIV Study was established in 1993 to study the natural history of HIV disease among women in the United States. It currently has enrolled 2,895 women testing positive for HIV infection and 972 women without HIV infection recruited from 6 national metropolitan locations. The clinical database information collected for each HIV-positive individual included CD4 cell counts, viral load, and antiviral treatment to evaluate HIV prognosis and related conditions in women. OBJECTIVE: To provide a baseline for fluconazole treatment prospects in women who test positive for HIV infection. As part of the ongoing Women's Interagency HIV Study project, we investigated the fluconazole susceptibility of Candida spp. isolated from women with HIV in comparison to volunteer women without HIV. The implication of antifungal treatment on fluconazole susceptibility was evaluated by reviewing antifungal medication use for the past 2 years in each participant. In addition, genotyping of Candida spp. at oral and vaginal sites was monitored for 4 months in 9 patients. METHODS: In a cohort of 59 women with HIV and 24 women without HIV, colonization by Candida albicans and non-albicans species of the oral and vaginal sites was first determined. Fluconazole susceptibility was surveyed in vitro according to Clinical and Laboratory Standards Institute protocol. Antifungal drug treatment history was investigated for each patient to correspond with fluconazole susceptibility. Finally, series of isolates from several patients were followed for resistance and susceptibility. Their lineage was verified by genotyping multilocus sequence typing (MLST). RESULTS: A total of 280 Candida strains were recovered from oral and vaginal swabs of women with and without HIV infection. We found that patients with HIV were colonized with Candida spp. more frequently than women without HIV. The percent of isolates that were susceptibility dose dependent or resistant to fluconazole was higher in Candida glabrata compared with C. albicans isolates, but higher for C. albicans than other published data. Resistance was noted to be more common in vaginal sites. Fluconazole resistance in either species was not associated with relative CD4 cell counts or viral load. However an association with systemic application of fluconazole and resistance was noted. CONCLUSIONS: Systemic antifungal therapy, including a vaginal topical regimen in women with HIV infection correlated with reduced fluconazole susceptibility of oral and vaginal isolates. Genotype profiling has disclosed that a majority of isolates from the same individual are clustered together, suggesting the likelihood of an original strain with some microevolution. We observed a change from a susceptibility dose dependent to a resistant phenotype of isolates in 2 women with HIV infection, even though no treatments were received during the 4-month study and the prior 2 years.

Histone-like transcription factor Hfl1p in Candida albicans harmonizes nuclear and mitochondrial genomic network in regulation of energy metabolism and filamentation development.

Candida albicans is an opportunistic fungal pathogen known for surviving in various nutrient-limited conditions within the host and causing infections. Our prior research revealed that Hfl1p, an archaeal histone-like or Hap5-like protein, is linked to mitochondrial ATP generation and yeast-hyphae morphogenesis. However, the specific roles of Hfl1p in these virulence behaviours, through its function in the CBF/NF-Y complex or as a DNA polymerase II subunit, remain unclear. This study explores Hfl1p's diverse functions in energy metabolism and morphogenesis. By combining proteomic analysis and phenotypic evaluations of the hfl1Δ/hfl1Δ mutant with ChIP data, we found that Hfl1p significantly impacts mitochondrial DNA-encoded CI subunits, the tricarboxylic acid (TCA) cycle, and morphogenetic pathways. This influence occurs either independently or alongside other transcription factors recognizing a conserved DNA motif (TAXXTAATTA). These findings emphasize Hfl1p's critical role in linking carbon metabolism and mitochondrial respiration to the yeast-to-filamentous form transition, enhancing our understanding of C. albicans' metabolic adaptability during morphological transition, an important pathogenic trait of this fungus. This could help identify therapeutic targets by disrupting the relationship between energy metabolism and cell morphology in C. albicans.

Multidrug-resistant transporter mdr1p-mediated uptake of a novel antifungal compound.

The 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 by Candida albicans, overexpression of MDR1, 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 in C. albicans to 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 overexpress MDR1. The activity of BQM against MDR1-overexpressing isolates is due to its facilitated intracellular accumulation. Microarray comparisons showed an extensive upregulation of MDR1 as 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 MDR Aspergillus fumigatus. Thus, our findings offer a paradigm shift to overcome MDR and the promise of improving antifungal treatment, especially in MDR pathogens.

Azole susceptibility and transcriptome profiling in Candida albicans mitochondrial electron transport chain complex I mutants.

Mitochondrial dysfunction in pathogenic fungi or model yeast causes altered susceptibilities to antifungal drugs. Here we have characterized the role of mitochondrial complex I (CI) of Candida albicans in antifungal susceptibility. Inhibitors of CI to CV, except for CII, increased the susceptibility of both patient and lab isolates, even those with a resistance phenotype. In addition, in a C. albicans library of 12 CI null mutants, 10 displayed hypersusceptibility to fluconazole and were severely growth inhibited on glycerol, implying a role for each gene in cell respiration. We chose two other hypersusceptible null mutants of C. albicans, the goa1Δ and ndh51Δ mutants, for transcriptional profiling by RNA-Seq. Goa1p is required for CI activity, while Ndh51p is a CI subunit. RNA-Seq revealed that both the ndh51Δ mutant and especially the goa1Δ mutant had significant downregulation of transporter genes, including CDR1 and CDR2, which encode efflux proteins. In the goa1Δ mutant, we noted the downregulation of genes required for the biogenesis and replication of peroxisomes, as well as metabolic pathways assigned to peroxisomes such as ß-oxidation of fatty acids, glyoxylate bypass, and acetyl coenzyme A (acetyl-CoA) transferases that are known to shuttle acetyl-CoA between peroxisomes and mitochondria. The transcriptome profile of the ndh51Δ mutant did not include downregulation of peroxisome genes but had, instead, extensive downregulation of the ergosterol synthesis gene family. Our data establish that cell energy is required for azole susceptibility and that downregulation of efflux genes may be an outcome of that dysfunction. However, there are mutant-specific changes that may also increase the susceptibility of both of these C. albicans mutants to azoles.

Impact of eating probiotic yogurt on colonization by Candida species of the oral and vaginal mucosa in HIV-infected and HIV-uninfected women.

BACKGROUND: Candidiasis in HIV/AIDS patients continues to be a public health problem. Antifungal therapies are not always effective and may result in complications, such as the development of drug-resistant strains of Candida species. OBJECTIVES: This study evaluated the impact of probiotic consumption on Candida colonization of the oral and vaginal mucosa. PATIENTS/METHODS: A pilot study was conducted in 24 women (17 HIV-infected, 7 HIV-uninfected) from the Women's Interagency HIV Study. The women underwent a 60-day initiation period with no probiotic consumption, followed by two 15-day consumption periods, with a different probiotic yogurt (DanActive™ or YoPlus™ yogurt) during each interval. There was a 30-day washout period between the two yogurt consumption periods. Oral and vaginal culture swabs were collected on days 0, 60, 74, and 120. Candida was detected by inoculating each swab in both Sabouraud's dextrose agar with or without chloramphenicol and CHROMagar. RESULTS: Less fungal colonization among women was observed when the women consumed probiotic yogurts (54 % of the women had vaginal fungal colonization during the non-probiotic yogurt consumption period, 29 % during the DanActive™ period, and 38 % during YoPlus™ yogurt consumption period), and HIV-infected women had significantly lower vaginal fungal colonization after they consumed DanActive™ yogurt compared to the non-intervention periods (54 vs 29 %, p = 0.03). CONCLUSIONS: These data are promising, but as expected in a small pilot study, there were some significant changes but also some areas where colonization was not changed. This type of conflicting data is supportive of the need for a larger trial to further elucidate the role of probiotic yogurts in fungal growth in HIV-infected women.

Amino acid-derived 1,2-benzisothiazolinone derivatives as novel small-molecule antifungal inhibitors: identification of potential genetic targets.

We have identified four synthetic compounds (DFD-VI-15, BD-I-186, DFD-V-49, and DFD-V-66) from an amino acid-derived 1,2-benzisothiazolinone (BZT) scaffold that have reasonable MIC(50) values against a panel of fungal pathogens. These compounds have no structural similarity to existing antifungal drugs. Three of the four compounds have fungicidal activity against Candida spp., Cryptococcus neoformans, and several dermatophytes, while one is fungicidal to Aspergillus fumigatus. The kill rates of our compounds are equal to those in clinical usage. The BZT compounds remain active against azole-, polyene-, and micafungin-resistant strains of Candida spp. A genetics-based approach, along with phenotype analysis, was used to begin mode of action (MOA) studies of one of these compounds, DFD-VI-15. The genetics-based screen utilized a homozygous deletion collection of approximately 4,700 Saccharomyces cerevisiae mutants. We identified mutants that are both hypersensitive and resistant. Using FunSpec, the hypersensitive mutants and a resistant ace2 mutant clustered within a category of genes related directly or indirectly to mitochondrial functions. In Candida albicans, the functions of the Ace2p transcription factor include the regulation of glycolysis. Our model is that DFD-VI-15 targets a respiratory pathway that limits energy production. Supporting this hypothesis are phenotypic data indicating that DFD-VI-15 causes increased cell-reactive oxidants (ROS) and a decrease in mitochondrial membrane potential. Also, the same compound has activity when cells are grown in a medium containing glycerol (mitochondrial substrate) but is much less active when cells are grown anaerobically.