Biology subjects

Beattie, S. R.

Publications and source records attributed to Beattie, S. R..

9 recordsLinked to original sources

bioRxiv2024

Similarities and distinctions in the activation of the Candida glabrata Pdr1 regulatory pathway by azole and non-azole drugs

Incidences of fluconazole (FLC) resistance among Candida glabrata clinical isolates is a growing issue in clinics. The pleiotropic drug response (PDR) network in C. glabrata confers azole resistance and is defined primarily by the Zn2Cys6 zinc cluster-containing transcription factor Pdr1 and target genes such as CDR1, that encodes an ATP-binding cassette transporter protein thought to act as a FLC efflux pump. Mutations in the PDR1 gene that render the transcription factor hyperactive are the most common cause of fluconazole resistance among clinical isolates. The phenothiazine class drug fluphenazine and a molecular derivative, CWHM-974, which both exhibit antifungal properties, have been shown to induce the expression of Cdr1 in Candida spp. We have used a firefly luciferase reporter gene driven by the CDR1 promoter to demonstrate two distinct patterns of CDR1 promoter activation kinetics: gradual promoter activation kinetics that occur in response to ergosterol limitations imposed by exposure to azole and polyene class antifungals and a robust and rapid CDR1 induction occurring in response to the stress imposed by fluphenazines. We can attribute these different patterns of CDR1 induction as proceeding through the promoter region of this gene since this is the only segment of the gene included in the luciferase reporter construct. Genetic analysis indicates that the signaling pathways responsible for phenothiazine and azole induction of CDR1 overlap but are not identical. The short time course of phenothiazine induction suggests that these compounds may act more directly on the Pdr1 protein to stimulate its activity. ImportanceCandida glabrata has emerged as the second-leading cause of candidiasis due in part to its ability to acquire high level resistance to azole drugs, a major class of antifungal, that acts to block the biosynthesis of the fungal sterol ergosterol. The presence of azole drugs causes the induction of a variety of genes involved in controlling susceptibility to this drug class including drug transporters and ergosterol biosynthetic genes such as ERG11. We found that the presence of azole drugs leads to an induction of genes encoding drug transporters and ERG11, while exposure of C. glabrata cells to antifungals of the phenothiazine class of drugs caused a much faster and larger induction of drug transporters but not ERG11. Coupled with further genetic analyses of the effects of azole and phenothiazine drugs, our data indicate that these compounds are sensed and responded to differentially in the yeast cell.

molecular biology↗

bioRxiv2023

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms

Multidrug resistance (MDR) transporters such as ATP Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased multidrug resistance transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of CDR transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 represents a unique example of a medicinal chemistry-based conversion of chemical scaffold from MDR-sensitive to MDR-resistant and, hence, active against fungi that have developed resistance to clinically used antifungals such as the azoles.

microbiology↗

bioRxiv2023

Novel keto-alkyl-pyridinium antifungal molecules active in models of in vivo Candida albicans vascular catheter infection and ex vivo Candida auris skin colonization

New antifungal therapies are needed for both systemic, invasive infections as well as superficial infections of mucosal and skin surfaces as well as biofilms associated with medical devices. The resistance of biofilm and biofilm-like growth phases of fungi contributes to the poor efficacy of systemic therapies to non-systemic infections. Here, we describe the identification and characterization of a novel keto-alkyl-pyridinium scaffold with broad spectrum activity (2-16 {micro}g/mL) against medically important yeasts and moulds, including clinical isolates resistant to azoles and/or echinocandins. Furthermore, these keto-alkyl-pyridinium agents retain substantial activity against biofilm phase yeast and have direct activity against hyphal A. fumigatus. Although their toxicity precludes use in systemic infections, we found that the keto-alkyl-pyridinium molecules reduce C. albicans fungal burden in a rat model of vascular catheter infection and reduce Candida auris colonization in a porcine ex vivo model. These initial pre-clinical data suggest that molecules of this class may warrant further study and development.

microbiology↗

bioRxiv2022

FKS1 is required for Cryptococcus neoformans fitness in vivo: application of copper-regulated gene expression to mouse models of cryptococcosis

There is an urgent need for new antifungals to treat cryptococcal meningoencephalitis, a leading cause of mortality in people living with HIV/AIDS. An important aspect of antifungal drug development is the validation of targets to determine whether they are required for the survival of the organism in animal models of disease. In Cryptococcus neoformans, a copper-regulated promoter (pCTR4-2) has been used to modulate gene expression in vivo previously. The premise for these experiments is that copper concentrations vary depending on the host niche. Here, we directly test this premise and confirm that the expression of CTR4, the promoter used to regulate gene expression, is much lower in the mouse lung compared to the brain. To further explore this approach, we applied to the gene encoding 1,3-{beta}-glucan synthase, FKS1. In vitro, reduced expression of FKS1 has little effect on growth but does activate the cell wall integrity stress response and increase susceptibility to caspofungin, a direct inhibitor of Fks1. These data suggest that compensatory pathways that reduce C. neoformans resistance do so through post-transcriptional effects. In vivo, however, a less pronounced reduction in FKS1 expression leads to a much more significant reduction in lung fungal burden (~1 log10 CFU), indicating that the compensatory responses to a reduction in FKS1 expression are not as effective in vivo as they are in vitro. In summary, use of copper-regulated expression of putative drug targets in vitro and in vivo can provide insights into the biological consequences of reduced activity of the target during infection. ImportanceConditional expression systems are widely used to genetically validate antifungal drug targets in mouse models of infection. Copper-regulated expression using the promoter of the CTR4 gene has been sporadically used for this purpose in C. neoformans. Here, we show that CTR4 expression is low in the lung and high in the brain, establishing the basic premise behind this approach. We applied it to the study of FKS1, the gene encoding for the target of the echinocandin class of 1,3-{beta}-glucan synthase inhibitors. Our in vitro and in vivo studies indicate that C. neoformans tolerates extremely low levels of FKS1 expression. This observation provides a potential explanation for the poor activity of 1,3-{beta}-glucan synthase inhibitors toward C. neoformans.

microbiology↗

bioRxiv2022

Genetic interaction analysis reveals that Cryptococcus neoformans utilizes multiple acetyl-CoA-generating pathways during infection

Cryptococcus neoformans is an important human fungal pathogen for which the external environment is its primary niche. To cause infection and disease, C. neoformans must adapt to a plethora of conditions and stresses inherent to the host environment. Of these stresses, the role of central carbon metabolism has been of interest not only at the level of fundamental pathobiology but also as a potential target for new antifungal drug therapy. Previous work has shown that two non-essential acetyl-CoA metabolism enzymes, ATP-citrate lyase (ACL1) and acetyl-CoA synthetase (ACS1), play important roles in C. neoformans infection. Here, we took a genetic interaction approach to studying the interplay between these two enzymes along with an enzyme initially called ACS2 but which we have found is an acetoacetyl-CoA synthetase; we have renamed the gene 2-ketobutyryl CoA synthetase 1 (KBC1) based on its biochemical activity and the systematic name of its substrate. ACL1 and ACS1 represent a synthetic lethal pair of genes based on our genetic interaction studies. Double mutants of KBC1 with either ACS1 or ACL1 do not have significant synthetic phenotypes in vitro, although we find that deletion of any one of these enzymes reduces fitness within macrophages. Importantly, the acs1{Delta} kbc1{Delta} double mutant has significantly reduced fitness in the CNS relative to either single mutant as well as WT (~2 log10 CFU reduction in fungal burden), indicating the important role these enzymes play during infection. The expression of both ACS1 and KBC1 is increased in vivo relative to in vitro conditions. The acs1{Delta} mutant is hypersusceptible to fluconazole in vivo despite its minimal in vitro phenotypes. These data not only provide insights into the in vivo mechanism of action for a new class of antifungal Acs inhibitors but also into metabolic adaptations of C. neoformans to the host environment. Author SummaryThe adaptation of environmental fungal pathogens to the mammalian host is critical to pathogenesis. Of these adaptations, the remodeling of carbon metabolism is particularly important. Here, we generated a focused set of double mutants of non-essential genes (ACL1, ACS1, KBC1) involved in acetyl-CoA metabolism and examined their fitness in vitro and during CNS infection. From these studies, we found that all three enzymes play important roles during infection but that the role of ACS1/KBC1 was minimal in vitro. Consistent with these observations, the expression of ACS1 and KBC1 was increased in vivo relative to standard in vitro conditions. Furthermore, strains lacking both ACL1 and ACS1 were not viable. These data clearly show that C. neoformans employs multiple carbon metabolism pathways to adapt to the host environment. They also provide insights into the potential mechanism of action for anti-cryptococcal Acs inhibitors.

microbiology↗

bioRxiv2021

An Alanine Aminotransferase is Required for Polysaccharide Regulation and Resistance of Aspergillus fumigatus Biofilms to Echinocandin Treatment

Alanine metabolism has been suggested as an adaptation strategy to oxygen limitation in organisms ranging from plants to mammals. Within the pulmonary infection microenvironment A. fumigatus forms biofilms with steep oxygen gradients defined by regions of oxygen limitation. A significant increase in alanine levels was observed in A. fumigatus cultured under oxygen limiting conditions. An alanine aminotransferase, AlaA, was observed to function in alanine catabolism and is required for several aspects of A. fumigatus biofilm physiology. Loss of alaA, or its catalytic activity, results in decreased adherence of biofilms through a defect in the maturation of the extracellular matrix polysaccharide galactosaminogalactan (GAG). Additionally, exposure of cell wall polysaccharides is also impacted by loss of alaA and loss of AlaA catalytic activity confers increased biofilm susceptibility to echinocandin treatment which is correlated with enhanced fungicidal activity. The increase in echinocandin susceptibility is specific to biofilms and chemical inhibition of alaA by the alanine aminotransferase inhibitor {beta}-chloro-L-alanine is sufficient to sensitize A. fumigatus biofilms to echinocandin treatment. Finally, loss of alaA increases susceptibility of A. fumigatus to in vivo echinocandin treatment in a murine model of invasive pulmonary aspergillosis. Our results provide insight into the interplay of metabolism, biofilm formation, and antifungal drug resistance in A. fumigatus and describes a mechanism of increasing susceptibility of A. fumigatus biofilms to the echinocandin class of antifungal drugs. eLife DigestAspergillus fumigatus is a ubiquitous filamentous fungus that causes an array of diseases depending on the immune status of an individual, collectively termed aspergillosis. Antifungal therapy for invasive pulmonary aspergillosis (IPA) or chronic pulmonary aspergillosis (CPA) is limited and too often ineffective. This is in part due to A. fumigatus biofilm formation within the infection environment and the resulting emergent properties, particularly increased antifungal resistance. Thus, insights into biofilm formation and mechanisms driving increased antifungal drug resistance are critical for improving existing therapeutic strategies and development of novel antifungals. In this work, we describe an unexpected observation where alanine metabolism, via the alanine aminotransferase AlaA, is required for several aspects of A. fumigatus biofilm physiology including resistance of A. fumigatus biofilms to the echinocandin class of antifungal drugs. Importantly, we observed that chemical inhibition of alanine aminotransferases is sufficient to increase echinocandin susceptibility and that loss of alaA increases susceptibility to echinocandin treatment in a murine model of IPA.

microbiology↗

bioRxiv2019

Derivatives of the antimalarial drug mefloquine are broad spectrum antifungal molecules with activity against drug-resistant clinical isolates

The antifungal pharmacopeia is critically small, particularly in light of the recent emergence of multi-drug-resistant pathogens such as Candida auris. Herein, we report that derivatives of the anti-malarial drug mefloquine have broad spectrum antifungal activity against pathogenic yeasts and molds. In addition, the mefloquine derivatives have activity against clinical isolates that are resistant to one or more of the three classes of drugs currently used to treat invasive fungal infections, indicating that they have a novel mechanism of action. Importantly, the in vitro toxicity profiles using human cell lines indicate that the mefloquine derivatives are very similar to the parent mefloquine despite being up to 64-fold more active against fungal cells. In addition to direct antifungal activity, sub-inhibitory concentrations of the mefloquine derivatives inhibit the expression of virulence traits including filamentation in C. albicans and capsule formation/melanization in C. neoformans. Mode/mechanism of action experiments indicate that the mefloquine derivatives interfere with both mitochondrial and vacuolar function as part of a multi-target mechanism of action. The broad-spectrum scope of activity, blood-brain-barrier penetration, and large number of previously synthesized analogs available combine to support the further optimization and development of the antifungal activity of this general class of drug-like molecules.

microbiology↗

bioRxiv2019

Host carbon dioxide concentration is an independent stress for Cryptococcus neoformans that affects virulence and antifungal susceptibility

The ability of Cryptococcus neoformans to cause disease in humans varies significantly among strains with highly related genotypes. In general, environmental isolates of pathogenic species such as C. neoformans var. grubii have reduced virulence relative to clinical isolates, despite having no differences in the expression of the canonical virulence traits (high temperature growth, melanization and capsule formation). In this observation, we report that environmental isolates of C. neoformans tolerate host CO2 concentrations poorly compared to clinical isolates and that CO2 tolerance correlates well with the ability of the isolates to cause disease in mammals. Initial experiments also suggest that CO2 tolerance is particularly important for dissemination of C. neoformans from the lung to the brain. Furthermore, CO2 concentrations affect the susceptibility of both clinical and environmental C. neoformans isolates to the azole class of antifungal drugs, suggesting that antifungal testing in the presence of CO2 may improve the correlation between in vitro azole activity and patient outcome.\n\nImportanceA number of studies comparing either patient outcomes or model system virulence across large collections of Cryptococcus isolates have found significant heterogeneity in virulence even among strains with highly related genotypes. Because this heterogeneity cannot be explained by variations in the three well-characterized virulence traits (growth at host body temperature; melanization; and polysaccharide capsule formation), it has been widely proposed that additional C. neoformans virulence traits must exist. C. neoformans natural niche is in the environment where the carbon dioxide concentration is very low ([~]0.04%); in contrast, mammalian host tissue carbon dioxide concentrations are 125-fold higher (5%). We have found that the ability to grow in the presence of 5% carbon dioxide distinguishes low virulence strains from high virulence strains, even those with a similar genotype. Our findings suggest that carbon dioxide tolerance is a previously un-recognized virulence trait for C. neoformans.

microbiology↗

bioRxiv2018

Characterizing the pathogenic, genomic, and chemical traits of Aspergillus fischeri, the closest sequenced relative of the major human fungal pathogen Aspergillus fumigatus

Aspergillus fischeri is closely related to Aspergillus fumigatus, the major cause of invasive mold infections. Even though A. fischeri is commonly found in diverse environments, including hospitals, it rarely causes invasive disease; why that is so is unclear. Comparison of A. fischeri and A. fumigatus for diverse pathogenic, genomic, and secondary metabolic traits revealed multiple differences for pathogenesis-related phenotypes, including that A. fischeri is less virulent than A. fumigatus in multiple animal models of disease, grows slower in low oxygen environments, and is more sensitive to oxidative stress. In contrast, the two species exhibit high genomic similarity; ~90% of the A. fumigatus proteome is conserved in A. fischeri, including 48/49 genes known to be involved in A. fumigatus virulence. However, only 10/33 A. fumigatus biosynthetic gene clusters (BGCs) likely involved in secondary metabolite production are conserved in A. fischeri and only 13/48 A. fischeri BGCs are conserved in A. fumigatus. Detailed chemical characterization of A. fischeri cultures grown on multiple substrates identified multiple secondary metabolites, including two new compounds and one never before isolated as a natural product. Interestingly, an A. fischeri deletion mutant of laeA, a master regulator of secondary metabolism, produced fewer secondary metabolites and in lower quantities, suggesting that regulation of secondary metabolism is at least partially conserved. These results suggest that the non-pathogenic A. fischeri possesses many of the genes important for A. fumigatus pathogenicity but is divergent with respect to its ability to thrive under host-relevant conditions and its secondary metabolism.\n\nImportanceAspergillus fumigatus is the primary cause of aspergillosis, a devastating ensemble of diseases associated with severe morbidity and mortality worldwide. A. fischeri is a close relative of A. fumigatus, but is not generally observed to cause human disease. To gain insights into the underlying causes of this remarkable difference in pathogenicity, we compared two representative strains (one from each species) for a range of host-relevant biological and chemical characteristics. We found that disease progression in multiple A. fischeri mouse models was slower and caused less mortality than A. fumigatus. The two species also exhibited different growth profiles when placed in a range of stress-inducing conditions encountered during infection, such as low levels of oxygen and the presence of reactive oxygen species-inducing agents. Interestingly, we also found that the vast majority of A. fumigatus genes known to be involved in virulence are conserved in A. fischeri, whereas the two species differ significantly in their secondary metabolic pathways. These similarities and differences that we identified are the first step toward understanding the evolutionary origin of a major fungal pathogen.

microbiology↗