Biology subjects

Peters, T. L.

Publications and source records attributed to Peters, T. L..

6 recordsLinked to original sources

bioRxiv2025

Relative contributions of the ERG11VF125AL and MRR1AN647T mutations to fluconazole resistance in Clade III Candidozyma (Candida) auris clinical isolates

ObjectivesCandidozyma (Candida) auris is an emerging fungal pathogen of global concern that often exhibits multi-drug resistance. Over 90% of isolates are resistant to fluconazole. Of the six described clades of C. auris, Clade III has been found to be nearly universally fluconazole resistant and almost every Clade III isolate described carries a mutation in the gene encoding the fluconazole target sterol demethylase (ERG11) leading to a VF125AL substitution and a mutation leading to a N647T substitution in the gene encoding Mrr1a, a transcriptional regulator of the Mdr1 transporter. Both mutations have been shown to contribute to fluconazole resistance in C. auris. MethodsIn the present study we introduced the Clade III MRR1A mutation into a Clade I background using CRISPR-Cas9 gene editing. In two Clade III clinical isolates we corrected the native MRR1A and ERG11 mutations to their wild-type sequences as well as disrupted MDR1. Triazole susceptibilities and MDR1 gene expression were measured in all strains. ResultsIntroduction of the N647T substitution in a Clade I background confers a modest reduction in fluconazole and voriconazole susceptibility. Similarly, correction of MRR1A or disruption of MDR1 in each Clade III background resulted in a one-dilution decrease in fluconazole and voriconazole MIC while the ERG11 correction resulted in a three-dilution decrease in fluconazole and voriconazole MIC. ConclusionsOur findings show that while the MRR1A mutation makes a modest contribution, the ERG11 mutation is responsible for most of the fluconazole resistance observed in Clade III isolates. We also show that while these mutations likewise affect voriconazole susceptibility, they have no effect on susceptibility to itraconazole, isavuconazole, or posaconazole suggesting the potential therapeutic utility of these antifungals for infections due to Clade III isolates of C. auris.

microbiology↗

bioRxiv2025

A screen to identify antifungal antagonists reveals a variety of pharmacotherapies induce echinocandin tolerance in Candida albicans.

Through screening a comprehensive collection of drugs approved for human use, we identified over 20 that oppose the antifungal activity of the echinocandins upon the infectious yeast, Candida albicans. More detailed evaluation of five such drugs, including the atypical antipsychotic aripiprazole and the tyrosine kinase inhibitor ponatinib, indicated they promote C. albicans survival following exposure to the echinocandin antifungals. The activity of the five selected antagonists was dependent upon the Mkc1p MAPK pathway, however, ponatinib was paradoxically shown to suppress phosphorylation and therefore activation of Mkc1p itself. Components of several other signaling pathways are also required, including those of calcineurin and casein kinase-2, suggesting the observed antagonism required much of the cell wall stress responses previously described for C. albicans. Transcriptome analysis revealed that the antagonists stimulated the expression of genes involved in xenobiotic and antifungal resistance, and suppressed the expression of genes associated with hyphal growth. Thus, the echinocandin antagonistic drugs modulate C. albicans physiology in ways that could impact its pathogenicity and/or response to therapeutic intervention. Finally, a mutant lacking the Efg1p transcription factor, which has a central role in the activation of C. albicans hyphal growth was found to have intrinsically high levels of echinocandin tolerance, suggesting a link between modulation of morphogenesis related signaling and echinocandin tolerance. ImportanceWe report a substantial number of previously unknown drug interactions that modulate the echinocandin sensitivity of one of the most prevalent human fungal pathogens, Candida albicans. The echinocandins are the first line therapy for treating disseminated and often lethal Candida infections, that account for >75% of invasive fungal infections in the U.S.. For largely unknown reasons, a substantial number of patients with invasive candidiasis fail to respond to treatment with these drugs. The finding of this study suggest that co-administered medications have the potential to influence the therapeutic outcomes of invasive fungal infections through modulating antifungal drug tolerance and/or fungal pathogenicity. The potential for echinocandin antagonistic medications to influence therapeutic outcomes is discussed.

microbiology↗

bioRxiv2025

Mutations in TAC1B drive CDR1 and MDR1 expression and azole resistance in C. auris

ObjectiveCandida auris has emerged as a fungal pathogen of particular concern owing in part to its propensity to exhibit antifungal resistance, especially to the commonly prescribed antifungal fluconazole. In this work we aimed to determine how mutations in the transcription factor gene TAC1B, which are common among resistant isolates and confer fluconazole resistance, exert this effect. MethodsSelected TAC1B mutations from clinical isolates were introduced into a susceptible isolate and reverted to the wild-type sequence in select clinical isolates using CRISPR Cas9 gene editing. Disruption mutants were likewise generated for select genes of interest. TAC1B mutants were subjected to transcriptional profiling by RNA-seq, and relative expression of specific genes of interest was determined by qRT-PCR. Antifungal susceptibilities were determined by modified CLSI broth microdilution. ResultsTAC1B mutations leading to A640V, A657V, and F862_N866del conferred fluconazole resistance, as well as increased resistance to other triazoles, when introduced into a susceptible isolate. RNA-seq revealed that the ATP-Binding Cassette (ABC) transporter gene CDR1 as well as the Major Facilitator Superfamily (MFS) transporter gene MDR1 were both upregulated by these TAC1B mutations. Disruption of CDR1 greatly abrogated resistance in strains with TAC1B mutations whereas disruption of MDR1 had little to no effect. However, disruption of both CDR1 and MDR1 resulted in an additional reduction in resistance as compared to disruption of either gene alone. ConclusionTAC1B mutations leading to A640V, A657V, and F862_N866del all result in increased resistance to fluconazole and other triazole antifungals, and increased expression of both CDR1 and MDR1 in C. auris. CDR1 is the primary driver of resistance conferred by these TAC1B mutations.

microbiology↗

bioRxiv2024

Directed evolution of bacteriophages: impacts of prolific prophage

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods is to propagate phage on multiple bacterial hosts, pool the lysate, and repeat the propagation process until phage(s) can form plaques on the target host(s). In theory, this propagation process produces a phage lysate that contains input phages and their evolved phage progeny. However, in practice, this phage lysate can also include prophages originating from bacterial hosts. Here we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, in which we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing analysis revealed that this observed host-range expansion was due to a Casadabanvirus prophage that originated from one of the Appelmans hosts. Host-range analysis of the prophage showed that it could infect five of eight bacterial hosts initially used, allowing it to proliferate and persist through the end of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment. This work highlights the impact of prophages in directed evolution experiments and the importance of incorporating sequencing data in analyses to verify output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine presence of prophage. IMPORTANCEDirected evolution is a common strategy for evolving phages to expand host range, often targeting pathogenic strains of bacteria. In this study we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophage are active players in directed evolution and can contribute to observation of host-range expansion. Since prophage are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving experimental design of future directed evolution studies.

microbiology↗

bioRxiv2022

Phenotypic characterization and analysis of complete genomes of two distinct strains of the proposed species "L. swaminathanii"

Recently, a new Listeria species, "Listeria swaminathanii", was proposed. Here, we phenotypically and genotypically characterize two additional strains that were previously obtained from soil samples and compare the results to the type strain. Complete genomes for both strains were assembled from hybrid Illumina and Nanopore sequencing reads and annotated. Further genomic analysis including average nucleotide identity (ANI) and detection of mobile genetic elements and genes of interest (e.g., virulence-associated) were conducted. The strains showed 98.7-98.8% ANI with the type strain. The UTK C1-0015 genome contained a partial monocin locus and a plasmid while the UTK C1-0024 genome contained a full monocin locus and a prophage. Phenotypic characterization consistent with those performed on the proposed type strain was conducted to assess consistency of phenotypes across a greater diversity of the proposed species (n=3 instead of n=1). Only a few findings were notably different from those of the type strain, such as catalase activity, glycerol metabolism, starch metabolism, and growth at 41{degrees}C. This study further expands our understanding of this newly proposed sensu stricto Listeria species.

microbiology↗

bioRxiv2021

Soil collected in the Great Smoky Mountains National Park yielded a novel Listeria species, L. swaminathanii, effectively expanding the sensu stricto clade to ten species

Soil samples collected in the Great Smoky Mountains National Park yielded a Listeria isolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses confirmed this isolate to be a novel Listeria sensu stricto species with the highest similarity to L. marthii (ANI=93.9%, isDDH=55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit, an automated program for classifying bacterial genomes, further supported delineation as a novel Listeria sensu stricto species, as this tool failed to assign a species identification but identified L. marthii as the closest match. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novel Listeria species described here is phenotypically (i) non-hemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in the Listeria pathogenicity island 1 (LIPI-1), as well as the internalin genes inlA and inlB. While the type strain for the new species is phenotypically catalase-negative (an unusual characteristic for Listeria sensu stricto species), its genome contained an apparently intact catalase gene (kat); hence assessment of this phenotype with future isolates will be important. Rapid species identification systems (Listeria API, VITEK 2, VITEK MS) misidentified this novel species as either L. monocytogenes, L. innocua, or L. marthii. We propose the name L. swaminathanii, and the type strain is FSL L7-0020T (=ATCC TSD-239T). IMPORTANCEL. swaminathanii is a novel sensu stricto species that originated from a US National Park, and its place of origin is ultimately preventing this species from achieving valid status. The US National Park Service restricts strain accessibility and open access is currently a prerequisite for species validation. Essentially the only criteria that was not met for L. swaminathanii validation is accessibility of the type strain, therefore nomenclature status should not negate the significance of this discovery. As a novel sensu stricto species, L. swaminathanii expands the group of species whose presence is associated with an increased risk of an L. monocytogenes contamination, and therefore could play an important role in public health. While developers of Listeria spp. detection methods historically only included validly publish species in their validation studies, L. swaminathanii is unequivocally a sensu stricto species and should be included as well.

microbiology↗