Biology subjects

Perlin, D. S.

Publications and source records attributed to Perlin, D. S..

15 recordsLinked to original sources

bioRxiv2026

The common evolution of fungicide persistence in cryptococcosis patients

Fungicide persistence, the ability of dormant fungal cells to survive lethal drug exposure, undermines treatment efficacy, yet its clinical evolution remains largely unexplored. We assembled a nationwide collection of clinical Cryptococcus neoformans isolates from China and revealed extensive inter strain variation in amphotericin B (AmB) persistence. This variation can arise from common evolutionary events in patients, generating previously undescribed high persistence cryptococcal variants refractory to AmB clearance. Genome wide fitness landscape analysis shows that persistence associated mutations generally impose minimal fitness costs, facilitating persistence evolution even in fungistatic resistant and hypervirulent genetic backgrounds. Machine learning identified deficient ACO2 expression as a key predictor of high persistence clinical isolates. ACO2 deficiency reproduces the high persistence phenotype by promoting POPC accumulation, which competes with AmB for its target. Moreover, the clinical stage antifungal T-2307 effectively eliminates high persistence strains across diverse genetic backgrounds. Collectively, our findings unveil common evolution of fungicide persistence in cryptococcosis patients, representing a previously overlooked clinical concern.

microbiology↗

bioRxiv2025

Candida glabrata replicating within macrophages experiences amino acid deprivation, DNA damage, and chromosome instability

Macrophages, the central players of innate immunity, control invading microbes by encapsulating them inside the phagosome, a nutrient-poor, reactive oxidant species-rich organelle. Nevertheless, some microbes, including the opportunistic yeast pathogen Candida glabrata, noted for its karyotype diversity, rapid evolution of antifungal drug resistance, and lack of meiosis, can survive and even replicate inside macrophages. However, it is not fully understood how C. glabrata responds to macrophage engulfment, and it is unknown how this presumably DNA-damaging environment influences the pathogens genome stability. In this study, we used comparative transcriptomics to identify amino acid starvation and DNA damage as conditions eliciting C. glabrata responses most similar to macrophage engulfment. Consistent with this, we found that C. glabrata intra-macrophage survival and replication require master regulator of amino acid biosynthesis GCN4 and functional DNA double-strand break repair. Furthermore, comet assays provided the first direct evidence for increased DNA breaks in intra-macrophage yeast, and pulse-field gel electrophoresis showed that chromosomal alterations occur frequently in macrophage-passaged C. glabrata. Interestingly, these alterations could not be resolved by long read DNA sequencing, suggesting that they involved highly complex repetitive regions. Finally, we identified several point mutations emerging during macrophage passaging and showed that among them, a frameshift in RME1 (repressor of meiosis in Saccharomyces cerevisiae), increased C. glabrata intra-macrophage fitness. Together, these analyses point to amino acid deprivation, reveal elevated DNA breakage and chromosome instability, and raise intriguing questions about the role of meiotic gene orthologs in C. glabrata persisting and replicating within macrophages.

microbiology↗

bioRxiv2025

Omicron Subvariants Infection Kinetics and Nirmatrelvir Efficacy in Transgenic K18-hACE2 Mice

The persistent evolution of SARS-CoV-2 has led to the emergence of antigenically distinct Omicron subvariants exhibiting increased transmissibility, immune evasion, and altered pathogenicity. Among these, recent subvariants like JN.1, KP.3.1.1, and LB.1 possess unique antigenic and virological features, underscoring the need for continued surveillance and therapeutic evaluation. As vaccines and commercial monoclonal antibodies show reduced effectiveness against these variants, the role of direct-acting antivirals, such as Nirmatrelvir, targeting conserved viral elements like the main protease inhibitor, becomes increasingly crucial. In this study, we investigated the replication kinetics, host immune responses, and therapeutic susceptibility of three recently circulating Omicron subvariants in the K18-hACE2 transgenic mouse model, using the SARS-CoV-2 parent WA1/2020 strain as a reference. Omicron subvariants exhibited a marked temporal shift in viral infection kinetics characterized by an early lung viral titer peak ([~]7-8 Log PFU) at 2 days post-infection (dpi), followed by a decline (1-3 Log PFU) by 4 dpi. Pulmonary cytokine and chemokine responses (GM-CSF, TNF-, IL-1{beta}, IL-6) showed an earlier increase in subvariant-infected mice compared to a gradual response in WA1/2020 infection. Notably, Nirmatrelvir treatment led to significant reduction in lung viral titers in subvariant-infected mice than in WA1/2020, surpassing its efficacy against the parent strain. These findings highlight that Omicron subvariants infection yields a broad dynamic range in viral burden with minimum variability, while retaining a prominent therapeutic response to Nirmatrelvir. This study provides insights to the emerging subvariants pathogenesis and therapeutic responsiveness, reinforcing the importance of continued variant monitoring and the development of effective countermeasures. ImportanceThe emergence of SARS-CoV-2 Omicron subvariants still poses a significant public health challenge due to their antigenic drift, altered pathogenesis, and immune evasion capabilities. This study comprehensively highlights the distinct replication kinetics, immune responses, and therapeutic susceptibility of the recently circulating Omicron subvariants JN.1, KP.3.1.1, and LB.1 using the K18-hACE2 mouse model. We demonstrate that while these subvariants exhibit altered virological profiles and immune activation patterns compared to the parent WA1/2020 strain, they remain susceptible to Nirmatrelvir which is a direct-acting main protease inhibitor. Notably, Nirmatrelvir demonstrated greater in vivo antiviral efficacy in Omicron subvariant infections than in the ancestral WA1/2020 strain. These findings underscore the enduring therapeutic value of protease-targeting antivirals and emphasize the critical need for ongoing variant-specific preclinical assessments.

microbiology↗

bioRxiv2025

Characterization of a Novel FKS1 Mutation in Candida lusitaniae Shows a Potential Critical Role for MKC1 in Echinocandin Resistance

Caspofungin is an echinocandin antifungal that inhibits glucan synthesis in the fungal cell wall. A Candida parapsilosis bloodstream isolate resistant to echinocandins was recovered from a patient who had undergone allogeneic hematopoietic stem cell transplantation. The FKS1 gene, encoding the target glucan synthase, contained a heterozygous mutation resulting in an I1380T amino acid change, in addition to the naturally occurring P660A polymorphism. When expressed at the equivalent position in the Fks1p protein of C. lusitaniae, P642A and I1359T, alone and in combination, led to 6-, 12-, and [≥]256-fold increases in the minimal inhibitory concentration (MIC) of caspofungin, respectively. The caspofungin concentration needed to inhibit 50% of glucan synthase activity was increased 3-, 37-, and 270-fold, respectively. At high drug concentrations, and also in drug-free medium, infrared spectroscopy revealed a decrease in {beta}-glucan content and an increase in chitin in the cell wall of the I1359T Fks1p mutants. Atomic force microscopy showed cell wall damage and cell swelling in both susceptible and resistant strains under caspofungin exposure. Analysis of susceptibility to cell-wall stressors and key factors in cell wall integrity (CWI) and high-osmolarity glycerol (HOG) pathways showed that all strains activated these pathways under caspofungin stress. In the I1359T Fks1p mutants, Mkc1p was constitutively activated even without caspofungin. Deletion of MKC1 restored caspofungin susceptibility, indicating that activation of the CWI pathway is a key molecular determinant of resistance in vitro to caspofungin in these mutants.

microbiology↗

bioRxiv2025

Broad-Spectrum Activity and Mechanisms of Action of SQ109 on a Variety of Fungi

ABSTRACTWe investigated the activity of the tuberculosis drug SQ109 against sixteen fungal pathogens: Candida albicans, C. auris, C. glabrata, C. guilliermondi, C. kefyr, C. krusei, C. lusitaniae, Candida parapsilosis, C. tropicalis, Cryptococcus neoformans, Rhizopus spp., Mucor spp., Fusarium spp., Coccidioides spp., Histoplasma capsulatum and Aspergillus fumigatus. MIC values varied widely (125 ng/mL to >64 {micro}g/mL) but in many cases we found promising (MIC[~]4 {micro}g/mL) activity as well as MFC/MIC ratios of [~]2. SQ109 metabolites were inactive. The activity of 12 analogs of SQ109 against Saccharomyces cerevisiae correlated with protonophore uncoupling activity, suggesting mitochondrial targeting, consistent with the observation that growth inhibition was rescued by agents which inhibit ROS species accumulation. SQ109 disrupted H+/Ca2+ homeostasis in S. cerevisiae vacuoles, and there was synergy (FICI[~]0.31) with pitavastatin, indicating involvement of isoprenoid biosynthesis pathway inhibition. SQ109 is, therefore, a potential antifungal agent with multi-target activity.

microbiology↗

bioRxiv2024

Structural and Biophysical Dynamics of Fungal Plasma Membrane Proteins and Implications for Echinocandin Action in Candida glabrata

Fungal plasma membrane proteins represent key therapeutic targets for antifungal agents, yet their structure and spatial distribution in the native context remain poorly characterized. Herein, we employ an integrative multimodal approach to elucidate the structural and functional organization of plasma membrane protein complexes in Candida glabrata, focusing on prominent and essential membrane proteins, the polysaccharide synthase {beta}-(1,3)-glucan synthase (GS) and the proton pump Pma1. Cryo-electron tomography (cryo-ET) and live cell imaging reveal that GS and Pma1 are heterogeneously distributed into distinct plasma membrane microdomains. Treatment with caspofungin, an echinocandin antifungal that targets GS, alters the plasma membrane and disrupts the native distribution of GS and Pma1. Based on these findings, we propose a model for echinocandin action that considers how drug interactions with the plasma membrane environment lead to inhibition of GS. Our work underscores the importance of interrogating the structural and dynamic characteristics of fungal plasma membrane proteins in situ to understand function and facilitate precisely targeted development of novel antifungal therapies.

cell biology↗

bioRxiv2023

In Vivo Antiviral Efficacy of LCTG-002, a Pooled, Purified Human Milk Secretory IgA product, Against SARS-CoV-2 in a Murine Model of COVID-19

Immunoglobulin A (IgA) is the most abundant antibody (Ab) in human mucosal compartments including the respiratory tract, with the secretory form of IgA (sIgA) being dominant and uniquely stable in these environments. sIgA is naturally found in human milk, which could be considered a global resource for this biologic, justifying the development of human milk sIgA as a dedicated airway therapeutic for respiratory infections such as SARS-CoV-2. In the present study, methods were therefore developed to efficiently extract human milk sIgA from donors who were either immunologically naive to SARS-CoV-2 (pooled as a control IgA) or had recovered from a PCR-confirmed SARS-CoV-2 infection that elicited high-titer anti-SARS-CoV-2 Spike sIgA Abs in their milk (pooled together to make LCTG-002). Mass spectrometry determined that proteins with a relative abundance of 1.0% or greater were all associated with sIgA. None of the proteins exhibited statistically significant differences between batches. Western blot demonstrated all batches consisted predominantly of sIgA. Compared to control IgA, LCTG-002 demonstrated significantly higher binding to Spike, and was also capable of blocking the Spike - ACE2 interaction in vitro with 6.3x greater potency compared to control IgA (58% inhibition at [~]240ug/mL). LCTG-002 was then tested in vivo for its capacity to reduce viral burden in the lungs of K18+hACE2 transgenic mice inoculated with SARS-CoV-2. LCTG-002 was demonstrated to significantly reduce SARS-CoV-2 titers in the lungs compared to control IgA when administered at either 250ug/day or 1 mg/day, as measured by TCID50, plaque forming units (PFU), and qRT-PCR, with a maximum reduction of 4.9 logs. This innovative study demonstrates that LCTG-002 is highly pure, efficacious, and well tolerated in vivo, supporting further development of milk-derived, polyclonal sIgA therapeutics against SARS-CoV-2 and other mucosal infections.

immunology↗

bioRxiv2023

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness

Small colony variants (SCVs) are relatively common among some bacterial species and are associated with poor prognosis and recalcitrant infections. Similarly, Candida glabrata - a major intracellular fungal pathogen - produces small and slow-growing respiratory-deficient colonies, termed "petite." Despite reports of clinical petite C. glabrata strains, our understanding of petite behavior in the host remains obscure. Moreover, controversies exist regarding in-host petite fitness and its clinical relevance. Herein, we employed whole-genome sequencing (WGS), dual-RNAseq, and extensive ex vivo and in vivo studies to fill this knowledge gap. WGS identified multiple petite-specific mutations in nuclear and mitochondrially-encoded genes. Consistent with dual-RNAseq data, petite C. glabrata cells did not replicate inside host macrophages and were outcompeted by their non-petite parents in macrophages and in gut colonization and systemic infection mouse models. The intracellular petites showed hallmarks of drug tolerance and were relatively insensitive to the fungicidal activity of echinocandin drugs. Petite-infected macrophages exhibited a pro-inflammatory and type I IFN-skewed transcriptional program. Interrogation of international C. glabrata blood isolates (n=1000) showed that petite prevalence varies by country, albeit at an overall low prevalence (0-3.5%). Collectively, our study sheds new light on the genetic basis, drug susceptibility, clinical prevalence, and host-pathogen responses of a clinically overlooked phenotype in a major fungal pathogen. ImportanceCandida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite". This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omicstechnologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and therefore are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex-vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

microbiology↗

bioRxiv2023

Novel non hot spot modification in Fks1 of Candida auris confers echinocandin resistance

We determined echinocandin susceptibility and FKS1 genotypes of thirteen clinical isolates of Candida auris recovered from four patients at a tertiary care center in Salvador, Brazil. Three isolates were categorized as echinocandin-resistant and harbored a novel FKS1 mutation leading to an amino acid change W691L located downstream from hot-spot 1. When introduced to echinocandin-susceptible C. auris strains by CRISPR/Cas9, Fks1 W691L induced elevated MIC values to all echinocandins (ANF 16-32x; CAS >64x; MCF >64x).

microbiology↗

bioRxiv2022

A novel oral GyrB/ParE dual binding inhibitor effective against multidrug resistant Neisseria gonorrhoeae and other high-threat pathogens

Drug resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance, as well as limit development of new resistance. We describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6- fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2- oxa-8-azaspiro[4.5]decan-3-yl)methanol], which simultaneously binds to ATP binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae including drug-resistant strains. A phosphate prodrug JSF-2659 was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram positive organisms, and JSF-2659 was shown in a deep tissue model of VRSA and a model of C. difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.

microbiology↗

bioRxiv2021

Rare modification in the ergosterol biosynthesis pathway leads to amphotericin B resistance in Candida auris clinical isolates

We determined amphotericin B (AmB) susceptibility and sequenced key genes of the ergosterol biosynthesis pathway implicated in AmB resistance (ERG2, ERG3, ERG6, ERG11) of 321 clinical isolates of Candida auris. In antifungal susceptibility testing, 19 (5.9%) isolates were categorized as AmB-resistant (MIC [≥]2 mg/l). Only one AmB-resistant isolate presented a unique non-wild-type ERG6 genotype that was confirmed to confer amphotericin B resistance (MIC >32 mg/l) when introduced into a susceptible strain (MIC = 0.5 mg/l).

microbiology↗

bioRxiv2021

Sex differences in cardio-pulmonary pathology of SARS-CoV2 infected and Trypanosoma cruzi co-infected mice

BackgroundCoronavirus disease-2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; CoV2) is a deadly contagious infectious disease. For those who survived COVID-19, post-COVID cardiac damage poses a major threat for the progression of cardiomyopathy and heart failure. Currently, the number of COVID-related cases and deaths are increasing in Latin America, where a major COVID comorbidity is Chagas heart disease (caused by the parasite Trypanosoma cruzi). Here, we investigated the effect of T. cruzi infection on the pathogenesis and severity of CoV2 infection and, conversely, the effect of CoV2 infection on heart pathology during coinfection. Methodology/findingsWe used transgenic human angiotensin-converting enzyme 2 (huACE2) mice infected with CoV2, T. cruzi, or coinfected with both in this study. Our study shows for the first time that white adipose tissue (WAT) serves as a reservoir for CoV2 and the persistence of CoV2 in WAT alters adipose tissue morphology and adipocyte physiology. Our data demonstrate a correlation between the loss of fat cells and the pulmonary adipogenic signaling (via adiponectin isomers) and pathology in CoV2 infection. The viral load in the lungs is inversely proportional to the viral load in WAT, which differs between male and female mice. Our findings also suggest that adiponectin-PPAR signaling may differently regulate Chagas cardiomyopathy in coinfected males and females. ConclusionWe conclude that adipogenic signaling may play important roles in cardio-pulmonary pathogenesis during CoV2 infection and T. cruzi coinfection. The levels of adiponectin isomers differ between male and female mice during CoV2 infection and coinfection with T. cruzi, which may differently regulate inflammation, viral load, and pathology in the lungs of both the sexes. Our findings are in line with other clinical observations that reported that males are more susceptible to COVID-19 than females and suffer greater pulmonary damage.

pathology↗

bioRxiv2021

Impact of Erg11 amino acid substitutions identified in Candida auris clade III isolates on triazole drug susceptibility

ERG11 sequencing of 28 Candida auris clade III isolates revealed the presence of concomitant V125A and F126L substitutions. Heterologous expression of Erg11-V125A/F126L in Saccharomyces cerevisiae led to reduced fluconazole and voriconazole susceptibilities. Generation of single substitution gene variants through site-directed mutagenesis uncovered that F126L primarily contributes to the elevated triazole MICs. A similar, yet diminished pattern of reduced susceptibility was observed with long-tailed triazoles posaconazole and itraconazole for V125A/F126L, F126L, Y132F, and K143R alleles.

microbiology↗

bioRxiv2021

Multifactorial role of mitochondria in echinocandin tolerance revealed by transcriptome analysis of drug-tolerant cells

Fungal infections cause significant mortality and morbidity worldwide, and the limited existing antifungal reservoir is further weakened by the emergence of strains resistant to echinocandins, a first line of antifungal therapy. Candida glabrata is an opportunistic fungal pathogen that rapidly develops mutations in the echinocandin drug target, {beta}-1,3-glucan synthase (GS), which are associated with drug resistance and clinical failure. Although echinocandins are considered fungicidal in Candida, a subset of C. glabrata cells survive echinocandin exposure, forming a drug-tolerant cell reservoir, from which resistant mutations are thought to emerge. Despite their importance, the physiology of rare drug-tolerant cells is poorly understood. We used fluorescence-activated cell sorting to enrich for echinocandin-tolerant cells, followed by modified single cell RNA sequencing to examine their transcriptional landscape. This analysis identified a transcriptional signature distinct from the stereotypical yeast environmental stress response and characterized by upregulation of pathways involved in chromosome structure and DNA topology and downregulation of oxidative stress responses, the latter of which was observed despite increased levels of reactive oxygen species. Further analyses implicated mitochondria in echinocandin tolerance, wherein inhibitors of mitochondrial complexes I and IV reduced echinocandin-mediated cell killing, but mutants lacking various mitochondrial components all showed an echinocandin hypo-tolerant phenotype. Finally, GS enzyme complexes purified from mitochondrial mutants exhibited normal in vitro inhibition kinetics, indicating that mitochondrial defects influence cell survival downstream of the drug-target interaction. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal a multifactorial role of mitochondria in echinocandin tolerance. ImportanceEchinocandin drugs are a first line therapy to treat invasive candidiasis, which is a major source of morbidity and mortality worldwide. The opportunistic fungal pathogen Candida glabrata is a prominent bloodstream fungal pathogen and it is notable for rapidly developing echinocandin-resistant strains associated with clinical failure. Echinocandin resistance is thought to emerge within a small echinocandin-tolerant subset of C. glabrata cells that are not killed by drug exposure, but mechanisms underlying echinocandin tolerance are still unknown. Here we describe the unique transcriptional signature of echinocandin-tolerant cells and the results of follow-up analyses, which reveal a multifactorial role of mitochondria in C. glabrata echinocandin tolerance. In particular, although chemical inhibition of respiratory chain enzymes increased echinocandin tolerance, deletion of multiple mitochondrial components made C. glabrata cells hypo-tolerant to echinocandins. Together, these results provide new insights into the C. glabrata response to echinocandins and reveal the involvement of mitochondria in echinocandin tolerance.

microbiology↗

bioRxiv2020

A non-canonical DNA damage checkpoint response in a major fungal pathogen

To protect genome integrity, eukaryotic cells respond to DNA damage by triggering highly conserved checkpoint mechanisms involving the phosphorylation of Rad53/CHK2 kinase. Budding yeast Candida glabrata, closely related to model eukaryote Saccharomyces cerevisiae, is an opportunistic pathogen characterized by high genetic diversity and rapid emergence of drug resistant mutants. However, the mechanisms enabling this genetic variability are unclear. Here we show that C. glabrata cells exposed to DNA damage neither induce CgRad53 phosphorylation nor accumulate in S phase, and exhibit higher lethality than S. cerevisiae. Furthermore, time-lapse microscopy showed C. glabrata cells continuing to divide in the presence of DNA damage, resulting in mitotic errors and cell death. Finally, RNAseq analysis revealed transcriptional rewiring of the DNA damage response in C. glabrata and identified several key protectors of genome stability upregulated by DNA damage in S. cerevisiae but downregulated in C. glabrata, including PCNA. Together, our results reveal a non-canonical fungal DNA damage response, which may contribute to rapidly generating genetic change and drug resistance.

microbiology↗