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ABSTRACTJustice-impacted persons may inconsistently access HIV testing. This cross-sectional secondary analysis investigates lifetime HIV testing prevalence among adults with prior histories of incarceration in Southern California, United States, participating in health-focused programming (n = 3 studies). Self-reported demographic and lifetime HIV testing data were collected between 2017-2023; descriptive analyses were conducted. Across the three samples, at least 74% of participants were male; Latino and African American individuals accounted for nearly two-thirds of participants. Lifetime HIV testing ranged from 72.8% to 84.2%. Males were significantly more likely than females to report never being tested in two samples and accounted for >95% of those never tested. No statistically significant differences in testing were observed by race/ethnicity. Single young adults (ages 18-26) were less likely than their partnered peers to report testing. HIV testing is critical for ensuring that individuals access prevention and treatment. HIV testing among justice-impacted adults in this study was higher than in the general population, potentially due to opt-out testing in correctional settings. Nevertheless, these findings underscore the importance of implementing targeted interventions to reduce structural (e.g., health insurance, access to self-testing kits) and social barriers (e.g., HIV stigma) to increase HIV testing among justice-impacted males and single young adults.
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Heart failure (HF) is a major clinical and public health problem associated with significant mortality, morbidity, and health-care costs. Despite the existence of evidence-based guidelines for the optimal treatment of HF, the quality of care remains suboptimal. Our aim was to increase the use a care bundle in 50% of enrolled subjects during their hospitalization and discharge and to reduce their readmission for HF causes by 10%. We conducted an uncontrolled before-after study in eight hospitals in Argentina to evaluate the effect of a quality improvement intervention on the use of an HF care bundle in patients with HF New York Heart Association (NYHA) Class II-III. The HF bundle of care included medication, continuum of care, lifestyle habits, and predischarge examinations. Training and follow-up of multidisciplinary teams in each center were performed through learning sessions and plan-do-study-act improvement cycles. Data collectors reviewed bundle compliance in the health records of recruited patients after their hospital discharge and verified readmissions through phone calls to patients within 30-40 days after discharge. We recruited 200 patients (83 before and 127 during the intervention phase), and bundle compliance increased from 9.6% to 28.3% [odds ratio 3.71, 95% confidence interval (8.46; 1.63); P = .002]. Despite a slow improvement during the first months, bundle compliance gained momentum near the end of the intervention surpassing 80%. We observed a non-significant decreased readmission rate within 30 days of discharge due to HF in the postintervention period [8.4% vs. 5.5%, odds ratio 0.63, 95% CI (1.88; 0.21); P = .410]. Qualitative analysis showed that members of the intervention teams acknowledged the improvement of work organization and standardization of care, teamwork, shared mental model, and health record completeness as well as the utility of training fellows. Despite the challenges related to the pandemic, better care of patients with HF NYHA Class II-III was possible through simple interventions and collaborative work. Graphical abstract.
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COVID-19 , Insuficiência Cardíaca , Humanos , Pandemias , Melhoria de Qualidade , Argentina/epidemiologia , COVID-19/epidemiologia , COVID-19/terapia , Insuficiência Cardíaca/terapia , Readmissão do PacienteRESUMO
Paracoccidioidomycosis (PCM) is a life-threatening systemic mycosis widely reported in the Gran Chaco ecosystem. The disease is caused by different species from the genus Paracoccidioides, which are all endemic to South and Central America. Here, we sequenced and analyzed 31 isolates of Paracoccidioides across South America, with particular focus on isolates from Argentina and Paraguay. The de novo sequenced isolates were compared with publicly available genomes. Phylogenetics and population genomics revealed that PCM in Argentina and Paraguay is caused by three distinct Paracoccidioides genotypes, P. brasiliensis (S1a and S1b) and P. restrepiensis (PS3). P. brasiliensis S1a isolates from Argentina are frequently associated with chronic forms of the disease. Our results suggest the existence of extensive molecular polymorphism among Paracoccidioides species, and provide a framework to begin to dissect the connection between genotypic differences in the pathogen and the clinical outcomes of the disease.
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Variação Genética/genética , Genômica , Paracoccidioides/genética , Paracoccidioidomicose/genética , Argentina/epidemiologia , Ecossistema , Genética Populacional , Genoma Fúngico/genética , Genótipo , Humanos , Paracoccidioides/classificação , Paracoccidioides/patogenicidade , Paracoccidioidomicose/classificação , Paracoccidioidomicose/epidemiologia , Paracoccidioidomicose/microbiologia , Paraguai/epidemiologia , FilogeniaRESUMO
The description of cryptic species with different pathogenic potentials has changed the perspectives on sporotrichosis. Sporothrix schenckii causes a benign chronic subcutaneous mycosis, Sporothrix brasiliensis is highly virulent, and Sporothrix globosa mainly causes fixed cutaneous lesions. Furthermore, S. brasiliensis is the prevalent species related to cat-transmitted sporotrichosis. Sources of infection, transmission, and distribution patterns also differ between species, and variability differs between species because of different degrees of clonality. The present review article will cover several aspects of the biology of clinically relevant agents of sporotrichosis, including epidemiological aspects of emerging species. Genomic information of Sporothrix spp. is also discussed. The cell wall is an essential structure for cell viability, interaction with the environment, and the host immune cells and contains several macromolecules involved in virulence. Due to its importance, aspects of glycosylation and cell wall polysaccharides are reviewed. Recent genome data and bioinformatics analyses helped to identify specific enzymes of the biosynthetic glycosylation routes, with no homologs in mammalian cells, which can be putative targets for development of antifungal drugs. A diversity of molecular techniques is available for the recognition of the clinically relevant species of Sporothrix. Furthermore, antigens identified as diagnostic markers and putative vaccine candidates are described. Cell-mediated immunity plays a key role in controlling infection, but Sporothrix species differ in their interaction with the host. The adaptive branch of the immune response is essential for appropriate control of infection.
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Sporothrix/fisiologia , Esporotricose/diagnóstico , Esporotricose/imunologia , Animais , Antígenos de Fungos/imunologia , Parede Celular/química , Parede Celular/metabolismo , Genoma Fúngico , Especificidade de Hospedeiro/imunologia , Humanos , Técnicas de Diagnóstico Molecular , Sporothrix/classificação , Sporothrix/imunologia , Esporotricose/microbiologia , Esporotricose/transmissão , VirulênciaRESUMO
Paracoccidioides spp. are dimorphic fungal pathogens responsible for one of the most relevant systemic mycoses in Latin America, paracoccidioidomycosis (PCM). Their exact ecological niche remains unknown; however, they have been isolated from soil samples and armadillos (Dasypus novemcinctus), which have been proposed as animal reservoir for these fungi. Human infection occurs by inhalation of conidia or mycelia fragments and is mostly associated with immunocompetent hosts inhabiting and/or working in endemic rural areas. In this review focusing on the pathogen perspective, we will discuss some of the microbial attributes and molecular mechanisms that enable Paracoccidioides spp. to tolerate, adapt, and ultimately avoid the host immune response, establishing infection.
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Tatus/microbiologia , Evasão da Resposta Imune , Paracoccidioides/patogenicidade , Esporos Fúngicos/patogenicidade , Fatores de Virulência , Animais , Tatus/imunologia , Sistemas CRISPR-Cas , Parede Celular/metabolismo , Meio Ambiente , Estrogênios/metabolismo , Inativação Gênica , Humanos , Melaninas/química , Paracoccidioides/genética , Paracoccidioidomicose/genética , Pigmentação , Polissacarídeos/química , RNA Antissenso/genética , Microbiologia do Solo , Esporos Fúngicos/genéticaRESUMO
Sporotrichosis and cutaneous leishmaniasis are skin infections with similar clinical manifestations but different treatment methods. The present study aimed to evaluate qPCR and conventional PCR for differential detection of the etiological agents of both infections in multiplex format. Assays were designed using two sets of reported primers: SS1/SS2, designed on the 18S ribosomal RNA gene from Sporothrix spp., and JW11/JW12, designed on the kinetoplast DNA (kDNA) minicircles of Leishmania spp. qPCR detected 200 fg of DNA per reaction for both Sporothrix and Leishmania. Melting curve analysis revealed two distinctive Tm peaks for Sporothrix spp. (85.5°C), and Leishmania spp. (82.6°C). A detection limit of 20 pg was determined for the diagnosis of both with conventional PCR. No other clinically important organisms were detected by either PCR or qPCR. However, a Blast analysis on GenBank databases, using as query the sequence of the PCR fragment obtained with primers SS1/SS2, showed 100% identity to environmental fungi of the Ophiostomales order. Lower percentages of identity (≤80%), with mismatches at primers' sequence regions were obtained for other environmental or clinically important fungi. Proper handling of clinical samples is required to avoid false negatives due to contamination with environmental fungi of the Ophiostomales order.
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Leishmania/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sporothrix/isolamento & purificação , Esporotricose/diagnóstico , DNA Fúngico/genética , DNA de Cinetoplasto/genética , DNA de Protozoário/química , Diagnóstico Diferencial , Humanos , Leishmania/classificação , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Sporothrix/classificação , Sporothrix/genética , Esporotricose/microbiologia , Esporotricose/patologiaRESUMO
BACKGROUND: Sporotrichosis is a cutaneous and subcutaneous fungal disease of humans and other mammals, known to be caused by the Sporothrix schenckii species complex, which comprises four species of clinical importance: S. brasiliensis, S. globosa, S. luriei, and S. schenckii sensu stricto. Of them, S. globosa and S. schenckii s. str. show global distribution and differences in global frequency as causal agents of the disease. In the Americas, only three species are present: S. schenckii s. str., S. brasiliensis (so far, only reported in Brazil), and S. globosa. In Venezuela, since the first case of sporotrichosis reported in 1935, S. schenckii have been considered its unique etiological agent. In the present work, the presence of more than one species in the country was evaluated. METHODS: By phenotypic key features and molecular phylogeny analyses, we re-examined 30 isolates from diverse Venezuelan regions belonging to the fungi collection of Instituto de Biomedicina, Caracas, Venezuela, and national reference center for skin diseases. All isolates were collected between 1973 and 2013, and maintained in distilled water. RESULTS: Sporotrichosis in Venezuela is mainly caused by S. schenckii s. str. (70%). However, a significant proportion (30%) of sporotrichosis cases in the country can be attributable to S. globosa. A correlation between intraspecific genotypes and clinical presentation is proposed. CONCLUSIONS: Our data suggest that sporotrichosis various clinical forms might be related to genetic diversity of isolates, and possibly, to diverse virulence profiles previously reported in the S. schenckii species complex. Sporothrix globosa was found to be the causative agent of 30% of sporotrichosis for the Venezuelan cases re-examined, the highest frequency of this species so far reported in the Americas. The high genetic variability presented by S. schenckii s. str. indicates that species distinction based on phenotypic key features could be a challenging and uncertain task; molecular identification should be always employed.
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DNA Fúngico/análise , Sporothrix/genética , Esporotricose/epidemiologia , Variação Genética , Genótipo , Humanos , Epidemiologia Molecular , Filogenia , Sporothrix/isolamento & purificação , Esporotricose/microbiologia , Venezuela/epidemiologiaRESUMO
BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.
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Doenças do Gato/microbiologia , Proteínas Fúngicas/genética , Sporothrix/genética , Esporotricose/transmissão , Fatores de Virulência/genética , Adaptação Biológica , Animais , Doenças do Gato/transmissão , Gatos , Evolução Molecular , Especiação Genética , Genoma Mitocondrial , Humanos , Filogenia , Sporothrix/classificação , Sporothrix/patogenicidade , Esporotricose/microbiologia , Esporotricose/veterináriaRESUMO
Paracoccidioides is a fungal pathogen and the cause of paracoccidioidomycosis, a health-threatening human systemic mycosis endemic to Latin America. Infection by Paracoccidioides, a dimorphic fungus in the order Onygenales, is coupled with a thermally regulated transition from a soil-dwelling filamentous form to a yeast-like pathogenic form. To better understand the genetic basis of growth and pathogenicity in Paracoccidioides, we sequenced the genomes of two strains of Paracoccidioides brasiliensis (Pb03 and Pb18) and one strain of Paracoccidioides lutzii (Pb01). These genomes range in size from 29.1 Mb to 32.9 Mb and encode 7,610 to 8,130 genes. To enable genetic studies, we mapped 94% of the P. brasiliensis Pb18 assembly onto five chromosomes. We characterized gene family content across Onygenales and related fungi, and within Paracoccidioides we found expansions of the fungal-specific kinase family FunK1. Additionally, the Onygenales have lost many genes involved in carbohydrate metabolism and fewer genes involved in protein metabolism, resulting in a higher ratio of proteases to carbohydrate active enzymes in the Onygenales than their relatives. To determine if gene content correlated with growth on different substrates, we screened the non-pathogenic onygenale Uncinocarpus reesii, which has orthologs for 91% of Paracoccidioides metabolic genes, for growth on 190 carbon sources. U. reesii showed growth on a limited range of carbohydrates, primarily basic plant sugars and cell wall components; this suggests that Onygenales, including dimorphic fungi, can degrade cellulosic plant material in the soil. In addition, U. reesii grew on gelatin and a wide range of dipeptides and amino acids, indicating a preference for proteinaceous growth substrates over carbohydrates, which may enable these fungi to also degrade animal biomass. These capabilities for degrading plant and animal substrates suggest a duality in lifestyle that could enable pathogenic species of Onygenales to transfer from soil to animal hosts.
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Onygenales/genética , Paracoccidioides/genética , Paracoccidioidomicose/microbiologia , Proteínas Quinases/genética , Metabolismo dos Carboidratos/genética , Sistemas de Liberação de Medicamentos , Evolução Molecular , Genoma Fúngico , Genoma Mitocondrial/genética , Humanos , Família Multigênica/genética , Onygenales/enzimologia , Paracoccidioides/enzimologia , Filogenia , Proteólise , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNARESUMO
Background: Postsurgical spinal infections are a severe complication and a challenge to the neurosurgeon due to their complex management. Revision surgeries and the removal of hardware are usually necessary. Recently, advances in nuclear medicine have made it possible to employ radiotracers to identify infections. The radiolabeled antimicrobial peptide technetium-99m-ubiquicidin (99mTc-UBI) (29-41) has been demonstrated to detect bacterial infections. UBI 29-41 is a peptide sequence with selective binding to the anionic cell membrane of bacteria, which has recently been used to differentiate between infection and inflammation. Here, we describe the clinical utility of 99mTc-UBI 29-41 single-photon emission computed tomography-computed tomography (SPECT-CT) in a patient suspected of a postoperative infection. Case description: A 54-year-old male who presented with conus medullaris syndrome secondary to T12 spondylodiscitis and multiple abscess collections was initially managed with debridement, corpectomy, and minimally invasive lateral instrumentation. The patient developed postsurgical empyema near the surgical site. The image study avoided the need for a second surgery and hardware removal. Conclusion: The use of 99mTc-UBI 29-41 SPECT-CT served as a tool to avoid a second invasive procedure; instead, conservative management with antibiotics was performed with an effective outcome after two weeks. This radiotracer has utility in cases in which infection is suspected, but the location is not entirely clear, and information is needed to guide the therapeutic approach.
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The geographical distribution and ecological niche of the two circulating species of the Sporothrix genus in Venezuela was established. For this, 68 isolates of Sporothrix spp. from patients of different regions of the country were analyzed. A molecular taxonomy analysis was conducted using a fragment of the calmodulin gene (CAL), and ITS regions, confirming the presence of S. schenckii (62%) and S. globosa (38%). Computational models of ecological niche for each species were obtained by the maximum entropy method using the MaxEnt software, which predicted the best environmental conditions for the presence of the two species. These models predict that the main variables influencing the presence of S. schenckii were altitude and annual mean temperature, while for S. globosa, the more influent variable was the land use, with 82% of S. globosa located at urban areas vs 56% for S. schenckii. The results here presented could contribute to understand the specific environmental factors that might modulate the occurrence of Sporothrix spp. as well as its transmission. To our knowledge, our analyses show for the first time Sporothrix spp.-specific ecological niche data, a valuable tool to promote evidence-based public health policymaking within endemic areas of sporotrichosis.
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Sporothrix/isolamento & purificação , Esporotricose/microbiologia , Ecossistema , Humanos , Modelos Biológicos , Filogenia , Sporothrix/classificação , Sporothrix/genética , Esporotricose/epidemiologia , População Urbana/estatística & dados numéricos , Venezuela/epidemiologiaRESUMO
Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main causative agents of sporotrichosis, a human subcutaneous mycosis. Differences in virulence patterns are associated with each species but remain largely uncharacterized. The S. schenckii and S. brasiliensis cell wall composition and virulence are influenced by the culturing media, with little or no influence on S. globosa. By keeping constant the culturing media, we compared the cell wall composition of three S. schenckii and two S. brasiliensis strains, previously described as presenting different virulence levels on a murine model of infection. The cell wall composition of the five Sporothrix spp. strains correlated with the biochemical composition of the cell wall previously reported for the species. However, the rhamnose-to-ß-glucan ratio exhibits differences among strains, with an increase in cell wall rhamnose-to-ß-glucan ratio as their virulence increased. This relationship can be expressed mathematically, which could be an important tool for the determination of virulence in Sporothrix spp. Also, structural differences in rhamnomannan were found, with longer side chains present in strains with lower virulence reported for both species here studied, adding insight to the importance of this polysaccharide in the pathogenic process of these fungi.
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Sporothrixschenckii is one of the etiological agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. Its cell wall contains a glycoconjugate composed of rhamnose, mannose, glucuronic acid, and proteins, named peptidorhamnomannan, which harbors important Sporothrix-specific immunogenic epitopes. Although the peptidorhamnomannan carbohydrate moiety has been extensively studied, thus far, little is known about the protein core. Here, using LC-MS/MS, we analyzed the S.schenckii peptidorhamnomannan peptide fraction and generated mass signals of 325 proteins, most of them likely to be moonlighting proteins. Among the identified proteins, chaperonin GroEL/Hsp60 and the uncharacterized protein Pap1 were selected for further analysis. Both proteins were heterologously expressed in bacteria, and they showed adhesive properties to the extracellular matrix proteins laminin, elastin, fibrinogen, and fibronectin, although Pap1 also was bound to type-I and type-II collagen. The inoculation of concentrations higher than 40 µg of these proteins, separately, increased immune effectors in the hemolymph of Galleriamellonella larvae and protected animals from an S.schenckii lethal challenge. These observations were confirmed when yeast-like cells, pre-incubated with anti-rHsp60 or anti-rPap1 antibodies were used to inoculate larvae. The animals inoculated with pretreated cells showed increased survival rates when compared to the control groups. In conclusion, we report that Hsp60 and Pap1 are part of the cell wall peptidorhamnomannan, can bind extracellular matrix components, and contribute to the S.schenckii virulence. To our knowledge, this is the first report about moonlighting protein in the S.schenckii cell wall with an important role during the pathogen-host interaction.
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On January 2020, the WHO Director General declared that the outbreak constitutes a Public Health Emergency of International Concern. The world has faced a worldwide spread crisis and is still dealing with it. The present paper represents a white paper concerning the tough lessons we have learned from the COVID-19 pandemic. Thus, an international and heterogenous multidisciplinary panel of very differentiated people would like to share global experiences and lessons with all interested and especially those responsible for future healthcare decision making. With the present paper, international and heterogenous multidisciplinary panel of very differentiated people would like to share global experiences and lessons with all interested and especially those responsible for future healthcare decision making.
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COVID-19/epidemiologia , Saúde Global , Pandemias , Pesquisa Biomédica , COVID-19/diagnóstico , COVID-19/terapia , Vacinas contra COVID-19 , Atenção à Saúde/organização & administração , Política de Saúde , Acessibilidade aos Serviços de Saúde , Disparidades nos Níveis de Saúde , Disparidades em Assistência à Saúde , Humanos , Cooperação Internacional , Vacinação em Massa/organização & administração , Pandemias/prevenção & controle , Política , Atenção Primária à Saúde/organização & administração , Telemedicina/organização & administraçãoRESUMO
Five Paracoccidioides brasiliensis isolates were grown in the presence of caspofungin (0 to 1 µg/ml). Inhibition of the yeast phase ranged from 20 to 65%, while in the mycelial form it ranged from 75% to 82%. Such variability was loosely related to the amount of cell wall ß-1,3-glucan. No association with point mutations in the ß-1,3-glucan synthase was detected. Caspofungin induced physical changes and cytoplasmic deterioration in both fungal phases.
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Antifúngicos/farmacologia , Equinocandinas/farmacologia , Paracoccidioides/efeitos dos fármacos , Proteínas de Bactérias/genética , Caspofungina , Glucosiltransferases/genética , Lipopeptídeos , Micélio/citologia , Micélio/efeitos dos fármacos , Micélio/genética , Paracoccidioides/citologia , Paracoccidioides/genética , Mutação Puntual/genética , Leveduras/citologia , Leveduras/efeitos dos fármacos , Leveduras/genéticaRESUMO
We report the isolation and sequencing of CHS3, a gene that encodes one of several chitin synthases in Paracoccidioides brasiliensis, a medically important fungus restricted geographically to Latin America. The gene contains a single open reading frame of 3817 bp with two introns (71 and 86 bp) and encodes a 1220 amino acid polypeptide with high similarity to other fungal chitin synthases. Northern analysis reveals a high expression of CHS3 in the pathogenic yeast-like phase of the fungus and at the end of the mycelium-yeast transition. Expression of P. brasiliensis CHS3 in a Saccharomyces cerevisiae chs3 null mutant enhanced calcofluor white staining in parallel to an increase in total chitin synthase activity and chitin content in its cell wall.
Assuntos
Quitina Sintase/genética , Proteínas Fúngicas/genética , Paracoccidioides/enzimologia , Paracoccidioides/genética , Saccharomyces cerevisiae/genética , Parede Celular/química , Quitina/análise , Quitina/biossíntese , Quitina Sintase/química , Quitina Sintase/metabolismo , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , Paracoccidioides/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Transformação GenéticaRESUMO
In this report we identified orthologues of fungal AGS1, RHO1, RHO2, RAC1 and CDC42 genes in the dimorphic fungus Paracoccidioides brasiliensis. Based on its homology to known fungal sequences, P. brasiliensis Ags1 was identified as an alpha-1,3-glucan synthase, while Rho1, Rho2, Rac1 and Cdc42 proteins were classified into the Rho1, Rho2, Rac1 and Cdc42 subgroups of fungal Rho GTPases, respectively. Of them, Rho1 is one of two subunits of a putative beta-1,3-glucan synthase complex, the other being the synthase itself (Fks1), while Rho2 has been associated to the alpha-1,3-glucan synthase (Ags1). Expression studies showed that mRNAs levels of RHO2 and AGS1 kept a direct relationship but the levels of RHO1 and FKS1 did not. P. brasiliensis RHO1 successfully restored growth of Saccharomyces cerevisiae rho1 mutant under restrictive temperature conditions. Chemical analyses of P. brasiliensis alpha-1,3-glucan, synthesized by Ags1p, indicated that it is essentially a linear polysaccharide, with <3% of alpha-1,4-linked glucose branches, occasionally attached as single units to the alpha-1,3-backbone.
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Parede Celular/enzimologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Paracoccidioides/enzimologia , Parede Celular/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Teste de Complementação Genética , Glucanos/análise , Glucanos/química , Glucanos/metabolismo , Espectroscopia de Ressonância Magnética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrofotometria InfravermelhoRESUMO
The secretory pathway in Candida albicans involves the protein translocation into the lumen of the endoplasmic reticulum and transport to the Golgi complex, where proteins undergo posttranslational modifications, including glycosylation and proteolysis. The Golgi-resident Kex2 protease is involved in such processing and disruption of its encoding gene affected virulence and dimorphism. These previous studies were performed using cells without URA3 or with URA3 ectopically placed into the KEX2 locus. Since these conditions are known to affect the cellular fitness and the host-fungus interaction, here we generated a kex2Δ null mutant strain with URA3 placed into the neutral locus RPS1. The characterization of this strain showed defects in the cell wall composition, with a reduction in the N-linked mannan content, and the increment in the levels of O-linked mannans, chitin, and ß-glucans. The defects in the mannan content are likely linked to changes in Golgi-resident enzymes, as the α-1,2-mannosyltransferase and α-1,6-mannosyltransferase activities were incremented and reduced, respectively. The mutant cells also showed reduced ability to stimulate cytokine production and phagocytosis by human mononuclear cells and macrophages, respectively. Collectively, these data showed that loss of Kex2 affected the cell wall composition, the protein glycosylation pathways, and interaction with innate immune cells.
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Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are etiological agents of sporotrichosis, a human subcutaneous mycosis. Although the protocols to evaluate Sporothrix virulence in animal models are well described, the cell preparation before inoculation is not standardized, and several culturing media are used to grow yeast-like cells. Here, we found that carbon or nitrogen limitation during fungal cell preparation negatively impacted the ability of S. schenckii and S. brasiliensis to kill Galleria mellonella larvae, but not S. globosa. The fungal growth conditions associated with the short median survival of animals were accompanied by increased hemocyte countings, phenoloxidase activity, and cytotoxicity. The fungal growth under carbon or nitrogen limitation also affected the cell wall composition of both S. schenckii and S. brasiliensis and showed increased exposure of ß-1,3-glucan at the cell surface, while those growing conditions had a minimal impact on the S.globosa wall, which had higher levels of this polysaccharide exposed on the wall regardless of the culture condition. This polysaccharide exposure was linked to the increased ability of insect hemocytes to uptake fungal cells, suggesting that this is one of the mechanisms behind the lower virulence of S.globosa or cells from the other species grown in carbon or nitrogen limitation.