Biology subjects

Vellanki, S.

Publications and source records attributed to Vellanki, S..

6 recordsLinked to original sources

bioRxiv2026

An epigenetic mechanism of azole tolerance facilitates acquired antifungal resistance in Aspergillus fumigatus

Antibiotic tolerance paves the way for acquired resistance in bacterial pathogens. However, the mechanisms of tolerance and its evolutionary role in acquired resistance in pathogenic fungi, particularly molds, remains elusive. Here, we identified an Inhibitor of Growth domain protein (IngB) as a novel epigenetic regulator of azole tolerance in Aspergillus fumigatus. The loss of ingB promotes supra-MIC growth on agar surface despite susceptible MICs in standardized assays. Moreover, established {Delta}ingB biofilms are less susceptible to azoles in vitro and in vivo. Subsequent exposure of the tolerant strain to high azole concentrations resulted in rapid acquired resistance, most notably a frameshift mutation in a putative 20S proteasome maturation protein, UmpA, while the susceptible wildtype strain failed to acquire adaptive mutations. The data suggest that IngB-mediated tolerance provides an epistatic background for the emergence of azole resistance. Our work shows drug tolerance facilitates resistance emergence in a critical fungal pathogen. ImportanceWhile antimicrobial drug resistance causes a significant adverse effect on human health, drug tolerance can also lead to insufficient pathogen clearance, resulting in infection relapse. However, the mechanisms of antifungal drug tolerance and its evolutionary role in acquired drug resistance in pathogenic fungi, particularly the molds, remains elusive. We identified IngB as a novel regulator of azole tolerance in Aspergillus fumigatus. Importantly, loss of IngB leads to rapid azole drug resistance under azole-selective pressure. Our work identifies a novel regulator of antifungal tolerance and suggests antifungal drug tolerance can pave the way for resistance emergence in a critical fungal pathogen.

microbiology↗

bioRxiv2025

Insights into Aspergillus fumigatus morphogenesis and pathogenesis through the putative lipid transporter ArvA

Aspergillus fumigatus poses a significant threat to human well-being, in part due to the increasing emergence of strains resistant to frontline antifungal therapy. In this study, we observe that the gene, arvA, is required for A. fumigatus morphogenesis, antifungal drug susceptibility, and cell wall homeostasis. Intriguingly, our study reveals novel morphological and growth aberrations in the absence of arvA. Loss of arvA results in hyper-swollen conidia that give rise to stunted, polarity-deficient hyphae in numerous environmental conditions, indicating a pivotal role for arvA in A. fumigatus morphogenesis. Surprisingly, despite these severe in vitro morphological and cell wall defects, arvA was not required for morbidity and mortality in immunologically distinct murine models of invasive pulmonary aspergillosis (IPA). However, growth in natural calf lung surfactant was able to normalize{Delta} arvA growth with the wild-type strain suggesting lung surfactant may partially complement the severe in vitro morphological defects of arvA loss in vivo. Taken together our observations reveal arvA as a mediator of A. fumigatus antifungal drug susceptibility and highlight the complex and ill-defined pulmonary nutrient environments role in mediating A. fumigatus pathogenesis and disease progression. IMPORTANCEAspergillus fumigatus is a challenging fungal pathogen in the clinic in part due to increasing azole drug resistance. In this study, we observe that loss of the A. fumigatus gene arvA results in increased azole susceptibility and significant in vitro morphological changes highlighted by hyper-swollen conidia that yield stunted and polarity deficient hyphae. Importantly, despite these severe in vitro morphological and growth abnormalities,{Delta} arvA surprisingly retains full pathogenicity and virulence in two immunologically distinct murine models of invasive pulmonary aspergillosis. These results challenge our understanding of the in-host environment and how it mediates fungal morphogenesis and pathogenesis. These results, consequently, not only enhances our understanding of the role of arvA in A. fumigatus morphogenesis and drug susceptibility, but further emphasizes the importance of in vivo animal models in fully evaluating potential antifungal drug targets.

microbiology↗

bioRxiv2025

An antifungal with a novel mechanism of action discovered via resistance gene-guided genome mining

Invasive fungal infections claim over two million lives annually, a problem exacerbated by rising resistance to current antifungal treatments and an increasing population of immunocompromised individuals. Despite this, antifungal drug development has stagnated, with few novel agents and fewer novel targets explored in recent decades. Here, we validate acetolactate synthase (ALS), an enzyme critical for branched-chain amino acid biosynthesis and absent in humans, as a promising target for new therapeutics. Using resistance gene-guided genome mining, we discovered a biosynthetic gene cluster in Aspergillus terreus encoding HB-35018 (1), a novel spiro-cis-decalin tetramic acid that potently inhibits ALS. Biochemical and antifungal assays demonstrate that 1 surpasses existing ALS inhibitors in efficacy against Aspergillus fumigatus and other pathogenic fungi. Structural studies via cryo-electron microscopy reveal a unique covalent binding interaction between compound 1 and ALS, distinct from known inhibitors and finally, we demonstrate that ALS is essential for the virulence in a mouse model of invasive aspergillosis. These findings position ALS as a promising target for antifungal development and demonstrate the potential of resistance gene-guided genome mining for antifungal discovery.

bioengineering↗

bioRxiv2025

Transcriptional analysis of developing Aspergillus fumigatus biofilms reveals metabolic shifts required for biofilm maintenance

Aspergillus fumigatus is a filamentous fungus found in compost and soil that can cause invasive and/or chronic disease in a broad spectrum of individuals. Diagnosis and treatment of aspergillosis often occur during stages of infection when A. fumigatus has formed dense networks of hyphae within the lung. These dense hyphal networks are multicellular, encased in a layer of extracellular matrix, and have reduced susceptibility to contemporary antifungal drugs, characteristics which are defining features of a microbial biofilm. A mode of growth similar to these dense hyphal networks observed in vivo can be recapitulated in vitro using a static, submerged biofilm culture model. The mechanisms underlying filamentous fungal cell physiology at different stages of biofilm development remain to be defined. Here, we utilized an RNA sequencing approach to evaluate changes in transcript levels during A. fumigatus biofilm development. These analyses revealed an increase in transcripts associated with fermentation and a concomitant decrease in oxidative phosphorylation related transcripts. Further investigation revealed that ethanol and butanediol fermentation is important for mature biofilm biomass maintenance. Correspondingly, a gene (silG), a predicted transcription factor, was observed to also be required for mature biofilm biomass maintenance. Taken together, these data suggest temporal changes in A. fumigatus metabolism during biofilm development are required to maintain a fully mature biofilm. IMPORTANCEAspergillus fumigatus is the most common etiological agent of a collection of diseases termed aspergillosis. Invasive Pulmonary Aspergillosis (IPA), a severe form of aspergillosis, is highlighted by invasive growth of fungal hyphae into host lung tissue. Strains that are susceptible to antifungal therapies in vitro frequently fail to respond to treatment in vivo, resulting in high mortality rates even with treatment. It is now appreciated that this decreased antifungal efficacy in vivo is, in part, likely due to biofilm-like growth of the fungus. A. fumigatus biofilms have been shown to develop regions of limited oxygen availability that are hypothesized to induce cell quiescence and drug resistance. Understanding the mechanisms by which A. fumigatus induces, develops, and maintains biofilms to evade antifungal therapies is expected to illuminate biofilm-specific therapeutic targets. Here we present transcriptomics data of developing A. fumigatus biofilms and from these data define genes related to fungal fermentation and regulation of transcription important for maintenance of mature A. fumigatus biofilms.

microbiology↗

bioRxiv2019

A novel resistance pathway for calcineurin inhibitors in the human pathogenic Mucorales Mucor circinelloides

Mucormycosis is an emerging lethal fungal infection in immunocompromised patients. Mucor circinelloides is a causal agent of mucormycosis and serves as a model system to understand genetics in Mucorales. Calcineurin is a conserved virulence factor in many pathogenic fungi and calcineurin inhibition or deletion of the calcineurin regulatory subunit (CnbR) in Mucor results in a shift from hyphal to yeast growth. We analyzed thirty-six calcineurin inhibitor resistant or bypass mutants that exhibited hyphal growth in the presence of calcineurin inhibitors or in the yeast-locked cnbR{Delta} mutant background without carrying any mutations in known calcineurin components. We found that a majority of the mutants had altered sequence in a gene, named here bycA (bypass of calcineurin A). bycA encodes an amino acid permease. We verified that both bycA{Delta}, and the bycA{Delta} cnbR{Delta} double mutant are resistant to the calcineurin inhibitor FK506, thereby demonstrating a novel resistance mechanism against calcineurin inhibitors. We also found that the expression of bycA was significantly higher in the wild type strain treated with FK506 and in the cnbR{Delta} mutants, but significantly lower in the wild type without FK506. These findings suggest that bycA is a negative regulator of hyphal growth and/or a positive regulator of yeast growth in Mucor and calcineurin suppresses the bycA gene at the mRNA level to promote hyphal growth. BycA is involved in the Mucor hyphal-yeast transition as our data demonstrates a positive correlation between bycA expression, protein kinase A activity, and Mucor yeast-growth. Also calcineurin, independent of its role in morphogenesis, contributes to virulence traits including phagosome maturation blockade, host cell damages, and pro-angiogenic growth factor induction during interactions with hosts. ImportanceMucor is intrinsically resistant to most known antifungals, which makes mucormycosis treatment challenging. Calcineurin is a serine/threonine phosphatase widely conserved across eukaryotes. When calcineurin function is inhibited in Mucor, growth shifts to a less-virulent yeast growth form which makes calcineurin an attractive target for development of new antifungal drugs. Previously we identified two distinct mechanisms through which Mucor can become resistant to calcineurin inhibitors involving Mendelian mutations in the gene for FKBP12, calcineurin A or B subunits and epimutations silencing the FKBP12 gene. Here, we identified a third novel mechanism where loss of function mutations in the amino acid permease encoding the bycA gene contribute to resistance against calcineurin inhibitors. When calcineurin activity is absent, BycA can activate PKA to promote yeast growth via a cAMP-independent pathway. Our data also shows that calcineurin activity, primarily contributes to host - pathogen interactions in the pathogenesis of Mucor.

microbiology↗

bioRxiv2019

A Nanoemulsion as an Effective Treatment Against Human Pathogenic Fungi

The emergence of immunocompromising diseases such as HIV/AIDS or other immunosuppressive medical conditions have opened an opportunity for fungal infections to afflict patients globally. An increase antifungal drug resistant fungi have posed a serious threat to patients. Combining these circumstances with a limited variety of antifungal drugs available to treat patients has left us in a situation where we need to develop new therapeutic approaches that are less prone to development of resistance by pathogenic fungi. In this study we present the utilization of the nanoemulsion NB-201 to control human pathogenic fungi. We found that the NB-201 exhibited in vitro activity against C. albicans, including both planktonic growth and biofilms. Furthermore, treatments with NB-201 significantly reduced the fungal burden at the infection site and presented enhanced healing process after subcutaneous infections by multidrug resistant C. albicans in a murine host system. NB-201 also exhibited in vitro growth inhibition activity against other fungal pathogens, including Cryptococcus spp, Aspergillus fumigatus, and Mucorales. Due to the nature of the activity of this nanoemulsion, there is a minimized chance of drug resistance to develop, thus presents a novel treatment to control fungal wound or skin infections.

microbiology↗