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Fungi in the basidiomycete genus Malassezia are the most prevalent eukaryotic microbes resident on the skin of human and other warm-blooded animals and have been implicated in skin diseases and systemic disorders. Analysis of Malassezia genomes revealed that key adaptations to the skin microenvironment have a direct genomic basis, and the identification of mating/meiotic genes suggests a capacity to reproduce sexually, even though no sexual cycle has yet been observed. In contrast to other bipolar or tetrapolar basidiomycetes that have either two linked mating-type-determining (MAT) loci or two MAT loci on separate chromosomes, in Malassezia species studied thus far the two MAT loci are arranged in a pseudobipolar configuration (linked on the same chromosome but capable of recombining). By generating additional chromosome-level genome assemblies, and an improved Malassezia phylogeny, we infer that the pseudobipolar arrangement was the ancestral state of this group and revealed six independent transitions to tetrapolarity, seemingly driven by centromere fission or translocations in centromere-flanking regions. Additionally, in an approach to uncover a sexual cycle, Malassezia furfur strains were engineered to express different MAT alleles in the same cell. The resulting strains produce hyphae reminiscent of early steps in sexual development and display upregulation of genes associated with sexual development as well as others encoding lipases and a protease potentially relevant for pathogenesis of the fungus. Our study reveals a previously unseen genomic relocation of mating-type loci in fungi and provides insight toward the identification of a sexual cycle in Malassezia, with possible implications for pathogenicity.
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Basidiomycota , Malassezia , Humanos , Malassezia/genética , Evolución Molecular , Basidiomycota/fisiología , Hongos/genética , Filogenia , Reproducción/genética , Genes del Tipo Sexual de los Hongos/genéticaRESUMEN
Candida auris poses threats to the global medical community due to its multidrug resistance, ability to cause nosocomial outbreaks and resistance to common sterilization agents. Different variants that emerged at different geographical zones were classified as clades. Clade-typing becomes necessary to track its spread, possible emergence of new clades, and to predict the properties that exhibit a clade bias. We previously reported a colony-Polymerase Chain Reaction-based, clade-identification method employing whole genome alignments and identification of clade-specific sequences of four major geographical clades. Here, we expand the panel by identifying clade 5 which was later isolated in Iran, using specific primers designed through in silico analyses.
Candida auris, a multidrug-resistant fungal pathogen, evolves as distinct geographical clades. We describe the identification of clade 5 specific DNA sequence, which was used to design primers that distinguished clade 5 from other clades, adding to the panel of the clade-identification system.
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Candida , Candidiasis , Animales , Candida/genética , Candidiasis/epidemiología , Candidiasis/veterinaria , Candida auris , Reacción en Cadena de la Polimerasa/veterinaria , Genoma Fúngico , Antifúngicos/farmacología , Pruebas de Sensibilidad Microbiana/veterinariaAsunto(s)
Centrómero , Genómica , Centrómero/genética , ADN de Hongos/genética , Cromosomas FúngicosRESUMEN
The chaperonin complex CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring complex) participates in the folding of many crucial proteins including actin and tubulin in eukaryotes. Mutations in genes encoding its subunits can affect protein folding and in turn, the physiology of the organism. Stress response in Saccharomyces cerevisiae is important in fermentation reactions and operates through overexpression and underexpression of genes, thus altering the protein profile. Defective protein folding machinery can disturb this process. In this study, the response of cct mutants to stress conditions in general and ethanol in specific was investigated. CCT1 mutants showed decreased resistance to different conditions tested including osmotic stress, metal ions, surfactants, reducing and oxidising agents. Cct1-3 mutant with the mutation in the conserved ATP-binding region showed irreversible defects than other mutants. These mutants were found to have inherent cell wall defects and showed decreased ethanol tolerance. This study reveals that cell wall defects and ethanol sensitivity are linked. Genetic and proteomic analyses showed that the yeast genes RPS6A (ribosomal protein), SCL1 (proteasomal subunit) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase) on overexpression, improved the growth of cct1-3 mutant on ethanol. We propose the breakdown of common stress response pathways caused by mutations in CCT complex and the resulting scarcity of functional stress-responsive proteins, affecting the cell's defence against different stress agents in cct mutants. Defective cytoskeleton and perturbed cell wall integrity reduce the ethanol tolerance in the mutants which are rescued by the extragenic suppressors.
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Pared Celular/metabolismo , Etanol/farmacología , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Pared Celular/química , Pared Celular/genética , Fermentación , Mutación , Pliegue de Proteína/efectos de los fármacos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The gut and oral microbiome is altered in people living with HIV (PLWH). While antiretroviral treatment (ART) is pivotal in restoring immune function in PLWH, several studies have identified an association between specific antiretrovirals, particularly integrase inhibitors (INSTI), and weight gain. In our study, we explored the differences in the oral and gut microbiota of PLWH under different ART regimens, and its correlation to Body Mass Index (BMI). Fecal and salivary samples were collected from PLWH (n = 69) and healthy controls (HC, n = 80). We performed taxonomy analysis to determine the microbial composition and relationship between microbial abundance and ART regimens, BMI, CD4+T-cell count, CD4/CD8 ratio, and ART duration. PLWH showed significantly lower richness compared to HC in both the oral and gut environment. The gut microbiome composition of INSTI-treated individuals was enriched with Faecalibacterium and Bifidobacterium, whereas non-nucleotide reverse transcriptase inhibitor (NNRTI)-treated individuals were enriched with Gordonibacter, Megasphaera, and Staphylococcus. In the oral microenvironment, Veillonella was significantly more abundant in INSTI-treated individuals and Fusobacterium and Alloprevotella in the NNRTI-treated individuals. Furthermore, Bifidobacterium and Dorea were enriched in gut milieu of PLWH with high BMI. Collectively, our findings identify distinct microbial profiles, which are associated with different ART regimens and BMI in PLWH on successful ART, thereby highlighting significant effects of specific antiretrovirals on the microbiome.
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Microbioma Gastrointestinal , Infecciones por VIH , Humanos , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Masculino , Femenino , Persona de Mediana Edad , Adulto , Boca/microbiología , Índice de Masa Corporal , Heces/microbiología , Antirretrovirales/uso terapéutico , Saliva/microbiologíaRESUMEN
Periodontitis is one of the world's most prevalent infectious conditions, affecting between 25 and 40% of the adult population. It is a consequence of the complex interactions between periodontal pathogens and their products, which trigger the host inflammatory response, chronic inflammation, and tissue destruction. Chronic systemic low-grade inflammation is involved in numerous diseases, and it is also known that long-lasting inflammation and chronic infections predispose one to cancer. Here, we characterized and compared the subgingival microbiota associated with periodontitis and diagnosis of malignancy in a longitudinal 10-year follow-up study. The study was conducted on 50 patients with periodontitis and 40 periodontally healthy individuals. The recorded clinical oral health parameters were periodontal attachment loss (AL), bleeding on probing (BOP), gingival index (GI), probing depth (PD), and plaque index (PI). Subgingival plaque was collected from each participant, from which DNA was extracted, and 16S rRNA gene amplicon sequencing performed. Cancer diagnoses data were collected between the years 2008-2018 from the Swedish Cancer Registry. The participants were categorized based on having cancer at the time of sample collection (CSC), having developed cancer later (DCL), and controls without any cancer. The most abundant phyla across all 90 samples were Actinobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Fusobacteria. At the genus level, Treponema, Fretibacterium, and Prevotella were significantly more abundant in samples of periodontitis patients compared to non-periodontitis individuals. With regard to samples of cancer patients, Corynebacterium and Streptococcus were more abundant in the CSC group; Prevotella were more abundant in the DCL group; and Rothia, Neisseria, and Capnocytophaga were more abundant in the control group. In the CSC group, we also found that the presence of periodontal inflammation, in terms of BOP, GI, and PLI, significantly correlated with species belonging to the genera Prevotella, Treponema, and Mycoplasma. Our results revealed that several subgingival genera were differentially enriched among the studied groups. These findings underscore the need for further research to fully understand the role that oral pathogens may play in the development of cancer.
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Fungi in the basidiomycete genus Malassezia are the most prevalent eukaryotic microbes resident on the skin of human and other warm-blooded animals and have been implicated in skin diseases and systemic disorders. Analysis of Malassezia genomes revealed that key adaptations to the skin microenvironment have a direct genomic basis, and the identification of mating/meiotic genes suggests a capacity to reproduce sexually, even though no sexual cycle has yet been observed. In contrast to other bipolar or tetrapolar basidiomycetes that have either two linked mating-type-determining ( MAT ) loci or two MAT loci on separate chromosomes, in Malassezia species studied thus far the two MAT loci are arranged in a pseudobipolar configuration (linked on the same chromosome but capable of recombining). By incorporating newly generated chromosome-level genome assemblies, and an improved Malassezia phylogeny, we infer that the pseudobipolar arrangement was the ancestral state of this group and revealed six independent transitions to tetrapolarity, seemingly driven by centromere fission or translocations in centromere- flanking regions. Additionally, in an approach to uncover a sexual cycle, Malassezia furfur strains were engineered to express different MAT alleles in the same cell. The resulting strains produce hyphae reminiscent of early steps in sexual development and display upregulation of genes associated with sexual development as well as others encoding lipases and a protease potentially relevant for pathogenesis of the fungus. Our study reveals a previously unseen genomic relocation of mating-type loci in fungi and provides insight towards the discovery of a sexual cycle in Malassezia , with possible implications for pathogenicity. Significance Statement: Malassezia , the dominant fungal group of the mammalian skin microbiome, is associated with numerous skin disorders. Sexual development and yeast-to-hyphae transitions, governed by genes at two mating-type ( MAT ) loci, are thought to be important for fungal pathogenicity. However, Malassezia sexual reproduction has never been observed. Here, we used chromosome-level assemblies and comparative genomics to uncover unforeseen transitions in MAT loci organization within Malassezia , possibly related with fragility of centromeric-associated regions. Additionally, by expressing different MAT alleles in the same cell, we show that Malassezia can undergo hyphal development and this phenotype is associated with increased expression of key mating genes along with other genes known to be virulence factors, providing a possible connection between hyphal development, sexual reproduction, and pathogenicity.
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BACKGROUND: Enhanced levels of a dipeptide, WG-am, have been reported among elite controllers - patients who spontaneously control their HIV-1 infection. This study aimed to evaluate anti-HIV-1 activity and mechanism of action of WG-am. METHODS: Drug sensitivity assays in TZM.bl cells, PBMCs and ACH-2 cells using WT and mutated HIV-1 strainswere performed to evaluate the antiviral mechanism of WG-am. Mass spectrometry-based proteomics and Real-time PCR analysis of reverse transcription steps were performed to unravel the second anti-HIV-1 mechanism of WG-am. RESULTS: The data suggest that WG-am binds to the CD4 binding pocket of HIV-1 gp120 and blocks its binding to the host cell receptors. Additionally, the time course assay showed that WG-am also inhibited HIV-1 at 4-6 hours post-infection, suggesting a second antiviral mechanism. Drug sensitivity assays under acidic wash conditions confirmed the ability of WG-am to internalise into the host cell in an HIV independent manner. Proteomic studies showed a clustering of all samples treated with WG-am independent of the number of doses or presence or absence of HIV-1. Differentially expressed proteins due to the WG-am treatment indicated an effect on HIV-1 reverse transcription, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). CONCLUSION: Naturally occurring in HIV-1 elite controllers, WG-am stands out as a new kind of antiviral compound with two independent inhibitory mechanisms of action on HIV-1 replication. WG-am halts HIV-1 entry to the host cell by binding to HIV-1 gp120, thereby blocking the binding of HIV-1 to the host cell. WG-am also exerts a post-entry but pre-integration antiviral effect related to RT-activity.
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Infecciones por VIH , VIH-1 , Humanos , Dipéptidos , Proteómica , Infecciones por VIH/tratamiento farmacológico , Antivirales , Controladores de Élite , Replicación ViralRESUMEN
Although mRNA SARS-CoV-2 vaccines are generally safe and effective, in certain immunocompromised individuals they can elicit poor immunogenic responses. Among these individuals, people living with HIV (PLWH) have poor immunogenicity to several oral and parenteral vaccines. As the gut microbiome is known to affect vaccine immunogenicity, we investigated whether baseline gut microbiota predicts immune responses to the BNT162b2 mRNA SARS-CoV-2 vaccine in healthy controls and PLWH after two doses of BNT162b2. Individuals with high spike IgG titers and high spike-specific CD4+ T-cell responses against SARS-CoV-2 showed low α-diversity in the gut. Here, we investigated and presented initial evidence that the gut microbial composition influences the response to BNT162b2 in PLWH. From our predictive models, Bifidobacterium and Faecalibacterium appeared to be microbial markers of individuals with higher spike IgG titers, while Cloacibacillus was associated with low spike IgG titers. We therefore propose that microbiome modulation could optimize immunogenicity of SARS-CoV-2 mRNA vaccines.
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COVID-19 , Microbioma Gastrointestinal , Infecciones por VIH , Humanos , Vacunas contra la COVID-19 , Vacuna BNT162 , COVID-19/prevención & control , SARS-CoV-2 , Vacunación , ARN Mensajero , Inmunoglobulina GRESUMEN
Candida auris, the multidrug-resistant human fungal pathogen, emerged as four major distinct geographical clades (clade 1-clade 4) in the past decade. Though isolates of the same species, C. auris clinical strains exhibit clade-specific properties associated with virulence and drug resistance. In this study, we report the identification of unique DNA sequence junctions by mapping clade-specific regions through comparative analysis of whole-genome sequences of strains belonging to different clades. These unique DNA sequence stretches are used to identify C. auris isolates at the clade level in subsequent in silico and experimental analyses. We develop a colony PCR-based clade-identification system (ClaID), which is rapid and specific. In summary, we demonstrate a proof-of-concept for using unique DNA sequence junctions conserved in a clade-specific manner for the rapid identification of each of the four major clades of C. auris. IMPORTANCE C. auris was first isolated in Japan in 2009 as an antifungal drug-susceptible pathogen causing localized infections. Within a decade, it simultaneously evolved in different parts of the world as distinct clades exhibiting resistance to antifungal drugs at varying levels. Recent studies hinted the mixing of isolates belonging to different geographical clades in a single location, suggesting that the area of isolation alone may not indicate the clade status of an isolate. In this study, we compared the genomes of representative strains of the four major clades to identify clade-specific sequences, which were then used to design clade-specific primers. We propose the utilization of whole genome sequence data to extract clade-specific sequences for clade-typing. The colony PCR-based method employed can rapidly distinguish between the four major clades of C. auris, with scope for expanding the panel by adding more primer pairs.
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Antifúngicos , Candida , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Candida/genética , Candida auris , Humanos , Japón , Pruebas de Sensibilidad Microbiana , VirulenciaRESUMEN
The upper respiratory tract (URT) microbiome can contribute to the acquisition and severity of respiratory viral infections. The described associations between URT microbiota and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are limited at microbiota genus level and by the lack of functional interpretation. Our study, therefore, characterized the URT bacterial microbiome at species level and their encoded pathways in patients with COVID-19 and correlated these to clinical outcomes. Whole metagenome sequencing was performed on nasopharyngeal samples from hospitalized patients with critical COVID-19 (n = 37) and SARS-CoV-2-negative individuals (n = 20). Decreased bacterial diversity, a reduction in commensal bacteria, and high abundance of pathogenic bacteria were observed in patients compared to negative controls. Several bacterial species and metabolic pathways were associated with better respiratory status and lower inflammation. Strong correlations were found between species biomarkers and metabolic pathways associated with better clinical outcome, especially Moraxella lincolnii and pathways of vitamin K2 biosynthesis. Our study demonstrates correlations between the URT microbiome and COVID-19 patient outcomes; further studies are warranted to validate these findings and to explore the causal roles of the identified microbiome biomarkers in COVID-19 pathogenesis.
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Shiga toxin-producing Escherichia coli (STEC) infection can cause mild to severe illness, such as nonbloody or bloody diarrhea, and the fatal hemolytic uremic syndrome (HUS). The molecular mechanism underlying the variable pathogenicity of STEC infection is not fully defined so far. Here, we performed a comparative genomics study on a large collection of clinical STEC strains collected from STEC-infected pediatric patients with and without HUS in Finland over a 16-year period, aiming to identify the bacterial genetic factors that can predict the risk to cause HUS and poor renal outcome. Of 240 STEC strains included in this study, 52 (21.7%) were from pediatric patients with HUS. Serotype O157:H7 was the main cause of HUS, and Shiga toxin gene subtype stx2a was significantly associated with HUS. Comparative genomics and pangenome-wide association studies identified a number of virulence and accessory genes overrepresented in HUS-associated STEC compared to non-HUS STEC strains, including genes encoding cytolethal distending toxins, type III secretion system effectors, adherence factors, etc. No virulence or accessory gene was significantly associated with risk factors for poor renal outcome among HUS patients assessed in this study, including need for and duration of dialysis, presence and duration of anuria, and leukocyte counts. Whole-genome phylogeny and multiple-correspondence analysis of pangenomes could not separate HUS STEC from non-HUS STEC strains, suggesting that STEC strains with diverse genetic backgrounds may independently acquire genetic elements that determine their varied pathogenicity. Our findings indicate that nonbacterial factors, i.e., characteristics of the host immunity, might affect STEC virulence and clinical outcomes. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) is a serious public health burden worldwide which causes outbreaks of gastrointestinal diseases and the fatal hemolytic uremic syndrome (HUS) characterized by the triad of mechanical hemolytic anemia, thrombocytopenia, and acute renal failure. Understanding the mechanism underlying the disease severity and patient outcome is of high importance. Using comparative genomics on a large collection of clinical STEC strains from STEC-infected patients with and without HUS, our study provides a reference of STEC genetic factors/variants that can be used as predictors of the development of HUS, which will aid risk assessment at the early stage of STEC infection. Additionally, our findings suggest that nonbacterial factors may play a primary role in the renal outcome in STEC-infected patients with HUS; further studies are needed to validate this.
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Infecciones por Escherichia coli , Síndrome Hemolítico-Urémico , Escherichia coli Shiga-Toxigénica , Niño , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/microbiología , Finlandia/epidemiología , Genómica , Síndrome Hemolítico-Urémico/epidemiología , Síndrome Hemolítico-Urémico/microbiología , Humanos , Toxina Shiga , Escherichia coli Shiga-Toxigénica/genéticaRESUMEN
Candida auris exhibits resistance to multiple antifungal drug classes and sterilization agents, posing threats to the immunocompromised worldwide. Among the four major geographical clades, the East Asian clade 2 isolates of C. auris are mostly drug susceptible. In this study, we experimentally evolved one such drug-susceptible isolate for multiple generations in the presence of the antifungal compound fluconazole and analyzed changes in the karyotype, DNA sequence, and gene expression profiles in three evolved drug-resistant isolates. Next-generation sequencing and electrophoretic karyotyping confirm the presence of segmental aneuploidy as supernumerary chromosomes originating from centromere-inclusive chromosomal duplication events in two such cases. A 638-kb region and a 675-kb region, both of which originated from chromosome 5 and contained its centromere region, are instances of supernumerary chromosome formation identified in two evolved fluconazole-resistant isolates. Loss of the supernumerary chromosomes from the drug-resistant isolates results in a complete reversal of fluconazole susceptibility. Transcriptome analysis of the third isolate identified overexpression of drug efflux pumps as a possible non-aneuploidy-driven mechanism of drug resistance. Together, this study reveals how both aneuploidy-driven and aneuploidy-independent mechanisms may operate in parallel in an evolving population of C. auris in the presence of an antifungal drug, in spite of starting from the same strain grown under similar conditions, to attain various levels of fluconazole resistance. IMPORTANCE Fungal pathogens develop drug resistance through multiple pathways by acquiring gene mutations, increasing the copy number of genes, or altering gene expression. In this study, we attempt to understand the mechanisms of drug resistance in the recently emerged superbug, C. auris. One approach to studying this aspect is identifying various mechanisms operating in drug-resistant clinical isolates. An alternative approach is to evolve a drug-susceptible isolate in the presence of an antifungal compound and trace the changes that result in drug resistance. Here, we evolve a drug-susceptible isolate of C. auris in the laboratory in the presence of a widely used antifungal compound, fluconazole. In addition to the already known changes like overexpression of drug efflux pumps, this study identifies a novel mechanism of azole resistance by the emergence of additional chromosomes through segmental duplication of chromosomal regions, including centromeres. The centric supernumerary chromosome helps stable amplification of a set of genes with an extra copy to confer fluconazole resistance.
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Antifúngicos , Fluconazol , Antifúngicos/farmacología , Azoles , Candida auris , Candida/genética , Farmacorresistencia Fúngica , Pruebas de Sensibilidad Microbiana , CromosomasRESUMEN
The HIV epidemic in Eastern Europe and Russia is large and not well-controlled. To describe the more recent molecular epidemiology of HIV-1, transmitted drug resistance, and the relationship between the epidemics in this region, we sequenced the protease and reverse transcriptase genes of HIV-1 from 812 people living with HIV from Ukraine (n = 191), Georgia (n = 201), and Russia (n = 420) before the initiation of antiretroviral therapy. In 190 Ukrainian patients, the integrase gene sequence was also determined. The most reported route of transmission was heterosexual contact, followed by intravenous drug use, and men having sex with men (MSM). Several pre-existing drug resistance mutations were found against non-nucleoside reverse transcriptase inhibitors (RTIs) (n = 103), protease inhibitors (n = 11), and nucleoside analogue RTIs (n = 12), mostly polymorphic mutations or revertants. In the integrase gene, four strains with accessory integrase strand transfer inhibitor mutations were identified. Sub-subtype A6 caused most of the infections (713/812; 87.8%) in all three countries, including in MSM. In contrast to earlier studies, no clear clusters related to the route of transmission were identified, indicating that, within the region, the exchange of viruses among the different risk groups may occur more often than earlier reported.
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Infecciones por VIH , Seropositividad para VIH , VIH-1 , Minorías Sexuales y de Género , Masculino , Humanos , VIH-1/genética , Farmacorresistencia Viral/genética , Epidemiología Molecular , Homosexualidad Masculina , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Nucleósidos/uso terapéutico , Filogenia , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , Mutación , Europa Oriental/epidemiología , Inhibidores de Proteasas/uso terapéutico , ADN Polimerasa Dirigida por ARN/genética , Integrasas/genética , Péptido Hidrolasas/genéticaRESUMEN
CCT (Chaperonin containing TCP-1) is a constitutively expressed eukaryotic chaperonin complex involved in the proper folding of proteins like actin and tubulin. Temperature sensitive mutants of CCT complex have been employed in various genetic screens, acting as models to study human CCT, the defects of which are implicated in disease conditions like neurodegeneration. Mutants of CCT complex are sensitive to cell wall stress agents. In this study, we have tested the effects of sorbitol and protein kinase C overexpression on the temperature sensitivity of cct mutants. We report that both the factors alleviated temperature sensitivity of cct mutants, indicating the possible role of CCT in maintaining cell wall integrity in S. cerevisiae.
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BACKGROUND AND OBJECTIVES: Monosomy of chromosome 5 associated with utilization of non-canonical sugar L-sorbose is one of the well-studied aneuploidies in Candida albicans. Stress-induced ploidy changes are crucial determinants for pathogenicity and genetic diversity in C. albicans. The five scattered regulatory regions (A, B, C, 135, and 139) comprising of two functionally redundant pathways (SUR1 and SUR2) were found to be responsible for the growth on L-sorbose. So far, three genes such as CSU51, CSU53 and CSU57 have been identified in region A, region 135 and region C, respectively. In this study we have verified the role of region B in this regulatory pathway. MATERIALS AND METHODS: We employed a combinatorial gene deletion approach to verify the role of region B followed by co-over expression studies and qRT-PCR to identify the regulatory role of this region. RESULTS: We confirmed the role of region B in the regulation of SOU1 gene expression. The qRT-PCR results showed that regulation occurs at transcriptional level along with other two regions in SUR1 pathway. A previously uncharacterized open reading frame in region B has been implicated in this regulation and designated as CSU52. Integrating multiple copies of CSU52 in the genome at tandem, suppresses the growth of recipient strain on L-sorbose, establishing it as a repressor of SOU1 gene. CONCLUSION: This finding completes the identification of regulators in SUR1 pathway. This result paves the way to study the underlying molecular mechanisms of SOU1 gene regulation that in-turn helps to understand stress induced aneuploidy.
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Millions of individuals currently living with HIV globally are receiving antiretroviral therapy (ART) that suppresses viral replication and improves host immune responses. The involvement of gut microbiome during HIV infection has been studied, exposing correlation with immune status and inflammation. However, the direct effect of ART on gut commensals of HIV-infected individuals has been mostly overlooked in microbiome studies. We used 16S rRNA sequencing (Illumina MiSeq) for determining the microbiota composition of stool samples from 16 viremic patients before and one year after ART. We also tested the direct effect of 15 antiretrovirals against four gut microbes, namely, Escherichia coli, Enterococcus faecalis, Bacteroides, and Prevotella to assess their in vitro antibacterial effect. 16S rRNA analysis of fecal samples showed that effective ART for one year does not restore the microbiome diversity in HIV-infected patients. A significant reduction in α-diversity was observed in patients under non-nucleoside reverse transcriptase inhibitors; (NNRTI; 2 NRTI+NNRTI; NRTIs are nucleoside reverse transcriptase inhibitors) as compared to ritonavir-boosted protease inhibitors (PI/r; 2 NRTI+PI/r). Prevotella (P = 0.00001) showed a significantly decreased abundance in patients after ART (n = 16). We also found the direct effect of antivirals on gut microbes, where zidovudine (ZDV) and efavirenz (EFV) showed in vitro antimicrobial activity against Bacteroides fragilis and Prevotella. EFV also inhibited the growth of E. faecalis. Therefore, we observed that ART does not reverse the HIV-induced gut microbiome dysbiosis and might aggravate those microbiota alterations due to the antibacterial effect of certain antiretrovirals (like EFV, ZDV). Our results imply that restructuring the microbiota could be a potential therapeutic target in HIV-1 patients under ART.
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Fármacos Anti-VIH , Microbioma Gastrointestinal , Infecciones por VIH , Alquinos , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Benzoxazinas , Ciclopropanos , Infecciones por VIH/tratamiento farmacológico , Humanos , ARN Ribosómico 16S/genética , Zidovudina/uso terapéuticoRESUMEN
Gut microbiome plays a significant role in HIV-1 immunopathogenesis and HIV-1-associated complications. Previous studies have mostly been based on 16S rRNA gene sequencing, which is limited in taxonomic resolution at the genus level and inferred functionality. Herein, we performed a deep shotgun metagenomics study with the aim to obtain a more precise landscape of gut microbiome dysbiosis in HIV-1 infection. A reduced tendency of alpha diversity and significantly higher beta diversity were found in HIV-1-infected individuals on antiretroviral therapy (ART) compared to HIV-1-negative controls. Several species, such as Streptococcus anginosus, Actinomyces odontolyticus, and Rothia mucilaginosa, were significantly enriched in the HIV-1-ART group. Correlations were observed between the degree of immunodeficiency and gut microbiome in terms of microbiota composition and metabolic pathways. Furthermore, microbial shift in HIV-1-infected individuals was found to be associated with changes in microbial virulome and resistome. From the perspective of methodological evaluations, our study showed that different DNA extraction protocols significantly affect the genomic DNA quantity and quality. Moreover, whole metagenome sequencing depth affects critically the recovery of microbial genes, including virulome and resistome, while less than 5 million reads per sample is sufficient for taxonomy profiling in human fecal metagenomic samples. These findings advance our understanding of human gut microbiome and their potential associations with HIV-1 infection. The methodological assessment assists in future study design to accurately assess human gut microbiome.
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The thermotolerant multidrug-resistant ascomycete Candida auris rapidly emerged since 2009 causing systemic infections worldwide and simultaneously evolved in different geographical zones. The molecular events that orchestrated this sudden emergence of the killer fungus remain mostly elusive. Here, we identify centromeres in C. auris and related species, using a combined approach of chromatin immunoprecipitation and comparative genomic analyses. We find that C. auris and multiple other species in the Clavispora/Candida clade shared a conserved small regional GC-poor centromere landscape lacking pericentromeres or repeats. Further, a centromere inactivation event led to karyotypic alterations in this species complex. Interspecies genome analysis identified several structural chromosomal changes around centromeres. In addition, centromeres are found to be rapidly evolving loci among the different geographical clades of the same species of C. auris Finally, we reveal an evolutionary trajectory of the unique karyotype associated with clade 2 that consists of the drug-susceptible isolates of C. aurisIMPORTANCECandida auris, the killer fungus, emerged as different geographical clades, exhibiting multidrug resistance and high karyotype plasticity. Chromosomal rearrangements are known to play key roles in the emergence of new species, virulence, and drug resistance in pathogenic fungi. Centromeres, the genomic loci where microtubules attach to separate the sister chromatids during cell division, are known to be hot spots of breaks and downstream rearrangements. We identified the centromeres in C. auris and related species to study their involvement in the evolution and karyotype diversity reported in C. auris We report conserved centromere features in 10 related species and trace the events that occurred at the centromeres during evolution. We reveal a centromere inactivation-mediated chromosome number change in these closely related species. We also observe that one of the geographical clades, the East Asian clade, evolved along a unique trajectory, compared to the other clades and related species.
Asunto(s)
Candida/genética , Centrómero/genética , Centrómero/metabolismo , Cromosomas/genética , Evolución Molecular , Genoma Fúngico , Antifúngicos/farmacología , Candida/clasificación , Candida/efectos de los fármacos , Candidiasis/microbiología , Centrómero/clasificación , Cromosomas/clasificación , Genómica , VirulenciaRESUMEN
The female genital tract (FGT) is an important site of human immunodeficiency virus (HIV) infection. Discerning the nature of HIV-specific local immune responses is crucial for identifying correlates of protection in HIV-exposed seronegative (HESN) individuals. The present study involved a comprehensive analysis of soluble immune mediators, secretory immunoglobulins (sIg), natural killer (NK) cells, CXCR5+ CD8+ T cells, T follicular helper (Tfh) cells, and T regulatory cells (Tregs) in the vaginal mucosa as well as the nature and composition of the cervicovaginal microbiome in HESN women. We found significantly elevated antiviral cytokines, soluble immunoglobulins, and increased frequencies of activated NK cells, CXCR5+ CD8+ T cells, and Tfh cells in HESN females compared to HIV-unexposed healthy (UH) women. Analysis of the genital microbiome of HESN women revealed a greater bacterial diversity and increased abundance of Gardnerella spp. in the mucosa. The findings suggest that the female genital tract of HESN females represents a microenvironment equipped with innate immune factors, antiviral mediators, and critical T cell subsets that protect against HIV infection. IMPORTANCE The vast majority of human immunodeficiency virus (HIV) infections across the world occur via the sexual route. The genital tract mucosa is thus the primary site of HIV replication, and discerning the nature of HIV-specific immune responses in this compartment is crucial. The role of the innate immune system at the mucosal level in exposed seronegative individuals and other HIV controllers remains largely unexplored. This understanding can provide valuable insights to improve vaccine design. We investigated mucosal T follicular helper (Tfh) cells, CXCR5+ CD8+ T cells, natural killer (NK) cells subsets, soluble immune markers, and microbiome diversity in HIV-exposed seronegative (HESN) women. We found a significantly higher level of mucosal CXCR5+ CD8+ T cells, CD4+ Tfh cells, activated NK cell subsets, and antiviral immune cell mediators in HESN women. We also found a higher abundance of Gardnerella spp., microbiome dysbiosis, and decreased levels of inflammatory markers to be associated with reduced susceptibility to HIV infection. Our findings indicate that increased distribution of mucosal NK cells, CXCR5+ CD8+ T cells, Tfh cells, and soluble markers in HIV controllers with a highly diverse cervicovaginal microbiome could contribute effectively to protection against HIV infection. Overall, our findings imply that future vaccine design should emphasize inducing these highly functional cell types at the mucosal sites.