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1.
POCUS J ; 8(2): 170-174, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38099170

RESUMO

Background: Intracerebral hemorrhage (ICH) is associated with high morbidity and mortality. ICH causes increased intracranial pressure (ICP), leading to brain herniation as the disease progresses. Neurological physical exam and monitoring of the disease progression can be challenging due to the impaired consciousness and routine clinical management in this patient population. Given the continuity of the intracranial cavity with the optic nerve subarachnoid space, an increased ICH leads to distension of the optic nerve sheath. We herein examined the correlation between the ICH volume and the optic nerve sheath diameter (ONSD) measured by point of care ultrasound (POCUS). Methods: Patients with ICH diagnosed with a head computed tomography (CT) scan were prospectively enrolled in this study. A portable ultrasound was used to measure the (ONSD); the volume of ICH hematoma, the Acute Physiology And Chronic Health Evaluation IV score, and the Intracerebral Hemorrhage score were collected. A Spearman rank correlation coefficient test was used to assess the relationship between continuous variables. A Wilcoxon rank sum test was used to assess differences in continuous variables between two groups. A p-value less than 0.05 was deemed as statistically significant. Results: A total of 28 subjects were enrolled. A moderate positive correlation was detected between hemorrhage volume and the average ONSD (correlation = 0.4214, p = 0.0255). A weak positive correlation was detected between average ONSD and APACHE IV (correlation = 0.2347, p = 0.2294). A weak moderate positive correlation was detected between average ONSD and ICH score (correlation = 0.1160, p = 0.5566). Conclusions: In this study we demonstrate that ONSD is moderately correlated with hematoma size. A potential application may include serial measurements of the ONSD with ultrasound. This may offer a quick, non-invasive technique that can be used in an intracerebral hemorrhage to monitor the stability or expansion of a hematoma indirectly, and potentially catch a catastrophic event like cerebral herniation.

2.
Genetics ; 225(3)2023 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-37811798

RESUMO

Candida albicans, a normal member of the human microbiome and an opportunistic fungal pathogen, undergoes several morphological transitions. One of these transitions is white-opaque switching, where C. albicans alternates between 2 stable cell types with distinct cellular and colony morphologies, metabolic preferences, mating abilities, and interactions with the innate immune system. White-to-opaque switching is regulated by mating type; it is repressed by the a1/α2 heterodimer in a/α cells, but this repression is lifted in a/a and α/α mating type cells (each of which are missing half of the repressor). The widely used C. albicans reference strain, SC5314, is unusual in that white-opaque switching is completely blocked when the cells are a/α; in contrast, most other C. albicans a/α strains can undergo white-opaque switching at an observable level. In this paper, we uncover the reason for this difference. We show that, in addition to repression by the a1/α2 heterodimer, SC5314 contains a second block to white-opaque switching: 4 transcription regulators of filamentous growth are upregulated in this strain and collectively suppress white-opaque switching. This second block is missing in the majority of clinical strains, and, although they still contain the a1/α2 heterodimer repressor, they exhibit a/α white-opaque switching at an observable level. When both blocks are absent, white-opaque switching occurs at very high levels. This work shows that white-opaque switching remains intact across a broad group of clinical strains, but the precise way it is regulated and therefore the frequency at which it occurs varies from strain to strain.


Assuntos
Candida albicans , Proteínas Fúngicas , Humanos , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos Tipo Acasalamento , Fenótipo , Comunicação Celular , Regulação Fúngica da Expressão Gênica
3.
mBio ; 14(4): e0137623, 2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37493629

RESUMO

The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in healthcare settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five Food and Drug Administration (FDA)-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris. To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only twofold to fivefold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1) and in the drug transporter CDR1. These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more susceptible to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent. IMPORTANCE The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated 8-hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris. Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest twofold to fivefold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500-fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated 8-hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens. Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris. Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.


Assuntos
Antifúngicos , Clioquinol , Humanos , Antifúngicos/metabolismo , Candida auris , Candida , Clioquinol/farmacologia , Clioquinol/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Farmacorresistência Fúngica/genética
4.
bioRxiv ; 2023 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-36824717

RESUMO

The fungal pathogen Candida auris represents a severe threat to hospitalized patients. Its resistance to multiple classes of antifungal drugs and ability to spread and resist decontamination in health-care settings make it especially dangerous. We screened 1,990 clinically approved and late-stage investigational compounds for the potential to be repurposed as antifungal drugs targeting C. auris and narrowed our focus to five FDA-approved compounds with inhibitory concentrations under 10 µM for C. auris and significantly lower toxicity to three human cell lines. These compounds, some of which had been previously identified in independent screens, include three dihalogenated 8-hydroxyquinolines: broxyquinoline, chloroxine, and clioquinol. A subsequent structure-activity study of 32 quinoline derivatives found that 8-hydroxyquinolines, especially those dihalogenated at the C5 and C7 positions, were the most effective inhibitors of C. auris . To pursue these compounds further, we exposed C. auris to clioquinol in an extended experimental evolution study and found that C. auris developed only 2- to 5-fold resistance to the compound. DNA sequencing of resistant strains and subsequent verification by directed mutation in naive strains revealed that resistance was due to mutations in the transcriptional regulator CAP1 (causing upregulation of the drug transporter MDR1 ) and in the drug transporter CDR1 . These mutations had only modest effects on resistance to traditional antifungal agents, and the CDR1 mutation rendered C. auris more sensitive to posaconazole. This observation raises the possibility that a combination treatment involving an 8-hydroxyquinoline and posaconazole might prevent C. auris from developing resistance to this established antifungal agent. Abstract Importance: The rapidly emerging fungal pathogen Candida auris represents a growing threat to hospitalized patients, in part due to frequent resistance to multiple classes of antifungal drugs. We identify a class of compounds, the dihalogenated hydroxyquinolines, with broad fungistatic ability against a diverse collection of 13 strains of C. auris . Although this compound has been identified in previous screens, we extended the analysis by showing that C. auris developed only modest 2- to 5-fold increases in resistance to this class of compounds despite long-term exposure; a noticeable difference from the 30- to 500- fold increases in resistance reported for similar studies with commonly used antifungal drugs. We also identify the mutations underlying the resistance. These results suggest that the dihalogenated hydroxyquinolines are working inside the fungal cell and should be developed further to combat C. auris and other fungal pathogens. Tweet: Lohse and colleagues characterize a class of compounds that inhibit the fungal pathogen C. auris . Unlike many other antifungal drugs, C. auris does not readily develop resistance to this class of compounds.

5.
PLoS One ; 18(1): e0280233, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36662710

RESUMO

Candida albicans is a normal member of the human microbiome and an opportunistic fungal pathogen. This species undergoes several morphological transitions, and here we consider white-opaque switching. In this switching program, C. albicans reversibly alternates between two cell types, named "white" and "opaque," each of which is normally stable across thousands of cell divisions. Although switching under most conditions is stochastic and rare, certain environmental signals or genetic manipulations can dramatically increase the rate of switching. Here, we report the identification of two new inputs which affect white-to-opaque switching rates. The first, exposure to sub-micromolar concentrations of (E,E)-farnesol, reduces white-to-opaque switching by ten-fold or more. The second input, an inferred PKA phosphorylation of residue T208 on the transcriptional regulator Efg1, increases white-to-opaque switching ten-fold. Combining these and other environmental inputs results in a variety of different switching rates, indicating that a given rate represents the integration of multiple inputs.


Assuntos
Candida albicans , Farneseno Álcool , Proteínas Fúngicas , Humanos , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/metabolismo , Farneseno Álcool/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Técnicas Genéticas , Fenótipo , Fosforilação
7.
Am J Emerg Med ; 48: 92-95, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33866269

RESUMO

INTRODUCTION: Computed tomography (CT) is often ordered for patients in whom the diagnosis of acute pancreatitis (AP) has already been established via elevated lipase levels and typical abdominal pain. We investigated whether early CT imaging performed in the ED altered the diagnosis or management. METHODS: A retrospective chart review was performed on patients presenting to a large, academic ED between the years 2013-2015 with AP who received CT imaging. Relevant history, laboratory, imaging data, and hospital course were abstracted from the medical record and analyzed by three independent reviewers, with 100% agreement among reviewers on 30 randomly selected cases. The primary outcome was whether the CT led to a change in diagnosis or management above and beyond the ultrasound. Univariate and multivariate analyses were performed to determine association between predictor variables and outcomes. RESULTS: The electronic health record query yielded 458 patients. Of those, 174 met the American College of Gastroenterology criteria for AP and were included in the study. 145 patients (83%) had abdominal CT during their hospital course, 125 (86%) of which were performed in the ED. Of these 145 patients, 57 (39%) had imaging evidence of AP. 107 patients had abdominal ultrasound (US) during their hospital course. Of 84 patients who had both CT and US, 31 (37%) patients were diagnosed with gallstones by US versus 19 (23%) by CT. Biliary dilation/obstruction was diagnosed by US in 5 (6%) patients versus 4 (5%) by CT. CT led to the correct diagnosis or change in management in 21 (14.5%) patients. CONCLUSION: Early CT may alter the diagnosis or management in up to 15% of patients presenting to the ED with AP, especially older patients with prior episodes of pancreatitis and biliary interventions, however abdominal US may be a more sensitive screening study for biliary etiologies and thereby better direct further management.


Assuntos
Tomada de Decisão Clínica/métodos , Serviço Hospitalar de Emergência , Pancreatite/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Doença Aguda , Adulto , Idoso , Estudos Transversais , Feminino , Humanos , Modelos Lineares , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Pancreatite/terapia , Estudos Retrospectivos , Ultrassonografia
8.
J Fungi (Basel) ; 7(1)2020 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-33375490

RESUMO

The human fungal pathogen Candida albicans can form biofilms on biotic and abiotic surfaces, which are inherently resistant to antifungal drugs. We screened the Chembridge Small Molecule Diversity library containing 30,000 "drug-like" small molecules and identified 45 compounds that inhibited biofilm formation. These 45 compounds were then tested for their abilities to disrupt mature biofilms and for combinatorial interactions with fluconazole, amphotericin B, and caspofungin, the three antifungal drugs most commonly prescribed to treat Candida infections. In the end, we identified one compound that moderately disrupted biofilm formation on its own and four compounds that moderately inhibited biofilm formation and/or moderately disrupted mature biofilms only in combination with either caspofungin or fluconazole. No combinatorial interactions were observed between the compounds and amphotericin B. As members of a diversity library, the identified compounds contain "drug-like" chemical backbones, thus even seemingly "weak hits" could represent promising chemical starting points for the development and the optimization of new classes of therapeutics designed to target Candida biofilms.

9.
Genetics ; 216(2): 409-429, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32839241

RESUMO

An unusual feature of the opportunistic pathogen Candida albicans is its ability to switch stochastically between two distinct, heritable cell types called white and opaque. Here, we show that only opaque cells, in response to environmental signals, massively upregulate a specific group of secreted proteases and peptide transporters, allowing exceptionally efficient use of proteins as sources of nitrogen. We identify the specific proteases [members of the secreted aspartyl protease (SAP) family] needed for opaque cells to proliferate under these conditions, and we identify four transcriptional regulators of this specialized proteolysis and uptake program. We also show that, in mixed cultures, opaque cells enable white cells to also proliferate efficiently when proteins are the sole nitrogen source. Based on these observations, we suggest that one role of white-opaque switching is to create mixed populations where the different phenotypes derived from a single genome are shared between two distinct cell types.


Assuntos
Ácido Aspártico Proteases/metabolismo , Candida albicans/genética , Proteínas Fúngicas/metabolismo , Interações Microbianas , Ácido Aspártico Proteases/genética , Candida albicans/metabolismo , Candida albicans/fisiologia , Proliferação de Células , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Nitrogênio/metabolismo , Peptídeos/metabolismo , Fenótipo
10.
Front Microbiol ; 11: 1027, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32523574

RESUMO

Biofilms formed by the fungal pathogen Candida albicans are resistant to many of the antifungal agents commonly used in the clinic. Previous reports suggest that protease inhibitors, specifically inhibitors of aspartyl proteases, could be effective antibiofilm agents. We screened three protease inhibitor libraries, containing a total of 80 compounds for the abilities to prevent C. albicans biofilm formation and to disrupt mature biofilms. The compounds were screened individually and in the presence of subinhibitory concentrations of the most commonly prescribed antifungal agents for Candida infections: fluconazole, amphotericin B, or caspofungin. Although few of the compounds affected biofilms on their own, seven aspartyl protease inhibitors inhibited biofilm formation when combined with amphotericin B or caspofungin. Furthermore, nine aspartyl protease inhibitors disrupted mature biofilms when combined with caspofungin. These results suggest that the combination of standard antifungal agents together with specific protease inhibitors may be useful in the prevention and treatment of C. albicans biofilm infections.

11.
G3 (Bethesda) ; 10(8): 2593-2600, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32487674

RESUMO

The fungal species Candida albicans is both a member of the human microbiome and a fungal pathogen. C. albicans undergoes several different morphological transitions, including one called white-opaque switching. Here, cells reversibly switch between two states, "white" and "opaque," and each state is heritable through many cell generations. Each cell type has a distinct cellular and colony morphology and they differ in many other properties including mating, nutritional specialization, and interactions with the innate immune system. Previous genetic screens to gain insight into white-opaque switching have focused on certain classes of genes (for example transcriptional regulators or chromatin modifying enzymes). In this paper, we examined 172 deletion mutants covering a broad range of cell functions. We identified 28 deletion mutants with at least a fivefold effect on switching frequencies; these cover a wide variety of functions ranging from membrane sensors to kinases to proteins of unknown function. In agreement with previous reports, we found that components of the pheromone signaling cascade affect white-to-opaque switching; however, our results suggest that the major effect of Cek1 on white-opaque switching occurs through the cell wall damage response pathway. Most of the genes we identified have not been previously implicated in white-opaque switching and serve as entry points to understand new aspects of this morphological transition.


Assuntos
Candida albicans , Regulação Fúngica da Expressão Gênica , Candida albicans/genética , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos Tipo Acasalamento , Humanos , Fenótipo , Feromônios , Transdução de Sinais
12.
Microorganisms ; 8(5)2020 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-32443498

RESUMO

Biofilms formed by the human fungal pathogen Candida albicans are naturally resistant to many of the antifungal agents commonly used in the clinic. We screened a library containing 1600 clinically tested drug compounds to identify compounds that inhibit C. albicans biofilm formation. The compounds that emerged from the initial screen were validated in a secondary screen and then tested for (1) their abilities to disrupt mature biofilms and (2) for synergistic interactions with representatives of the three antifungal agents most commonly prescribed to treat Candida infections, fluconazole, amphotericin B, and caspofungin. Twenty compounds had antibiofilm activity in at least one of the secondary assays and several affected biofilms but, at the same concentration, had little or no effect on planktonic (suspension) growth of C. albicans. Two calcium channel blockers, a selective serotonin reuptake inhibitor, and an azole-based proton pump inhibitor were among the hits, suggesting that members of these three classes of drugs or their derivatives may be useful for treating C. albicans biofilm infections.

13.
Proc Natl Acad Sci U S A ; 116(52): 26918-26924, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31822605

RESUMO

Differentiated cell types often retain their characteristics through many rounds of cell division. A simple example is found in Candida albicans, a member of the human microbiota and also the most prevalent fungal pathogen of humans; here, two distinct cell types (white and opaque) exist, and each one retains its specialized properties across many cell divisions. Switching between the two cell types is rare in standard laboratory medium (2% glucose) but can be increased by signals in the environment, for example, certain sugars. When these signals are removed, switching ceases and cells remain in their present state, which is faithfully passed on through many generations of daughter cells. Here, using an automated flow cytometry assay to monitor white-opaque switching over 96 different sugar concentrations, we observed a wide range of opaque-to-white switching that varied continuously across different sugar compositions of the medium. By also measuring white cell proliferation rates under each condition, we found that both opaque-to-white switching and selective white cell proliferation are required for entire populations to shift from opaque to white. Moreover, the switching frequency correlates with the preference of the resulting cell type for the growth medium; that is, the switching is adjusted to increase in environments that favor white cell proliferation. The widely adjustable, all-or-none nature of the switch, combined with the long-term heritability of each state, is distinct from conventional forms of gene regulation, and we propose that it represents a strategy used by C. albicans to efficiently colonize different niches of its human host.

14.
PLoS One ; 14(6): e0218037, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31170229

RESUMO

Candida albicans, a species of fungi, can thrive in diverse niches of its mammalian hosts; it is a normal resident of the GI tract and mucosal surfaces but it can also enter the bloodstream and colonize internal organs causing serious disease. The ability of C. albicans to thrive in these different host environments has been attributed, at least in part, to its ability to assume different morphological forms. In this work, we examine one such morphological change known as white-opaque switching. White cells are the default state of C. albicans, and most animal studies have been carried out exclusively with white cells. Here, we compared the proliferation of white and opaque cells in two murine models of infection and also monitored, using specially constructed strains, switching between the two states in the host. We found that white cells outcompeted opaque cells in many niches; however, we show for the first time that in some organs (specifically, the heart and spleen), opaque cells competed favorably with white cells and, when injected on their own, could colonize these organs. In environments where the introduced white cells outcompeted the introduced opaque cells, we observed high rates of opaque-to-white switching. We did not observe white-to-opaque switching in any of the niches we examined.


Assuntos
Candida albicans/citologia , Candida albicans/patogenicidade , Candidíase/microbiologia , Especificidade de Órgãos , Pigmentação , Animais , Candidíase/patologia , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Corantes Fluorescentes/metabolismo , Intestinos/microbiologia , Intestinos/patologia , Rim/microbiologia , Rim/patologia , Camundongos
15.
Curr Protoc Microbiol ; 50(1): e60, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29995344

RESUMO

Candida albicans is a normal member of the human microbiota that asymptomatically colonizes healthy individuals, however it is also an opportunistic pathogen that can cause severe infections, especially in immunocompromised individuals. The medical impact of C. albicans depends, in part, on its ability to form biofilms, communities of adhered cells encased in an extracellular matrix. Biofilms can form on both biotic and abiotic surfaces, such as tissues and implanted medical devices. Once formed, biofilms are highly resistant to antifungal agents and the host immune system, and can act as a protected reservoir to seed disseminated infections. Here, we present several in vitro biofilm protocols, including protocols that are optimized for high-throughput screening of mutant libraries and antifungal compounds. We also present protocols to examine specific stages of biofilm development and protocols to evaluate interspecies biofilms that C. albicans forms with interacting microbial partners. © 2018 by John Wiley & Sons, Inc.


Assuntos
Biofilmes , Candida albicans/fisiologia , Técnicas de Cultura de Células/métodos , Colorimetria/métodos , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Fluorescência/métodos , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candidíase/microbiologia , Humanos
16.
Nat Rev Microbiol ; 16(1): 19-31, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29062072

RESUMO

Candida albicans is among the most prevalent fungal species of the human microbiota and asymptomatically colonizes healthy individuals. However, it is also an opportunistic pathogen that can cause severe, and often fatal, bloodstream infections. The medical impact of C. albicans typically depends on its ability to form biofilms, which are closely packed communities of cells that attach to surfaces, such as tissues and implanted medical devices. In this Review, we provide an overview of the processes involved in the formation of C. albicans biofilms and discuss the core transcriptional network that regulates biofilm development. We also consider some of the advantages that biofilms provide to C. albicans in comparison with planktonic growth and explore polymicrobial biofilms that are formed by C. albicans and certain bacterial species.


Assuntos
Biofilmes , Candida albicans/fisiologia , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/classificação , Candida albicans/efeitos dos fármacos , Farmacorresistência Fúngica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos
17.
Artigo em Inglês | MEDLINE | ID: mdl-28507115

RESUMO

White and opaque cells of Candida albicans have the same genome but differ in gene expression patterns, metabolic profiles, and host niche preferences. We tested whether these differences, which include the differential expression of drug transporters, resulted in different sensitivities to 27 antifungal agents. The analysis was performed in two different strain backgrounds; although there was strain-to-strain variation, only terbinafine hydrochloride and caspofungin showed consistent, 2-fold differences between white and opaque cells across both strains.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/microbiologia , Regulação Fúngica da Expressão Gênica , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Caspofungina , Equinocandinas/farmacologia , Proteínas Fúngicas/genética , Lipopeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Naftalenos/farmacologia , Fenótipo , Terbinafina
18.
Artigo em Inglês | MEDLINE | ID: mdl-28289028

RESUMO

Candida albicans biofilms have a significant medical impact due to their rapid growth on implanted medical devices, their resistance to antifungal drugs, and their ability to seed disseminated infections. Biofilm assays performed in vitro allow for rapid, high-throughput screening of gene deletion libraries or antifungal compounds and typically serve as precursors to in vivo studies. Here, we compile and discuss the protocols for several recently published C. albicansin vitro biofilm assays. We also describe improved versions of these protocols as well as novel in vitro assays. Finally, we consider some of the advantages and disadvantages of these different types of assays.


Assuntos
Antifúngicos/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Adesão Celular/fisiologia , Microfluídica/métodos , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Caspofungina , Equinocandinas/farmacologia , Humanos , Lipopeptídeos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Infecções Relacionadas à Prótese/microbiologia
19.
mBio ; 7(6)2016 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-27879329

RESUMO

The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state. IMPORTANCE: Epigenetic transitions are an important mechanism by which microbes adapt to external stimuli. For Candida albicans, such transitions are crucial for adaptation to complex, fluctuating environments, and therefore contribute to its success as a human pathogen. The white-opaque switch modulates multiple C. albicans attributes, from sexual competency to niche specificity. Here, we demonstrate that metabolic circuits are extensively rewired between white and opaque states, so that the two cell types exhibit optimal fitness under different nutritional conditions and at different temperatures. We thereby establish that epigenetic events can profoundly alter the metabolism of fungal cells. We also demonstrate that epigenetic switching regulates filamentation and biofilm formation, two phenotypes closely associated with pathogenesis. These experiments reveal that white cells, considered the most clinically relevant form of C. albicans, are a "general-purpose" state suited to many environments, whereas opaque cells appear to represent a more metabolically specialized form of the species.


Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Epigênese Genética , Regulação Fúngica da Expressão Gênica , Fenótipo , Estresse Fisiológico , Animais , Antifúngicos/metabolismo , Candida albicans/efeitos dos fármacos , Candida albicans/efeitos da radiação , Candidíase/microbiologia , Metabolismo dos Carboidratos , Modelos Animais de Doenças , Concentração de Íons de Hidrogênio , Transdução de Sinais , Temperatura
20.
mBio ; 7(5)2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27624133

RESUMO

UNLABELLED: Candida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation. IMPORTANCE: Biofilm formation by the opportunistic fungal pathogen C. albicans is a major cause of life-threatening infections. This work provides a global characterization of secreted proteolytic activity produced specifically by C. albicans biofilms. We identify activity from the proteases Sap5 and Sap6 as highly upregulated during C. albicans biofilm formation and develop Sap-cleavable fluorogenic substrates that enable the detection of biofilms from C. albicans and also from additional pathogenic Candida species. Furthermore, SAP5 and SAP6 deletions confirm that both proteases are required for proper biofilm development in vitro and in vivo We propose that secreted proteolysis is a promising marker for the diagnosis and potential therapeutic targeting of Candida biofilm-associated infections.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Biofilmes/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida albicans/fisiologia , Proteínas Fúngicas/metabolismo , Proteólise , Animais , Candidíase/microbiologia , Infecções Relacionadas a Cateter/microbiologia , Modelos Animais de Doenças , Proteoma/análise , Ratos
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