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1.
J Antimicrob Chemother ; 70(5): 1322-30, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25583750

RESUMO

OBJECTIVES: Here we report on a long-term outbreak from 2009 to 2012 with an XDR Pseudomonas aeruginosa on two wards at a university hospital in southern Germany. METHODS: Whole-genome sequencing was performed on the outbreak isolates and a core genome was constructed for molecular epidemiological analysis. We applied a time-place-sequence algorithm to improve estimation of transmission probabilities. RESULTS: By using conventional infection control methods we identified 49 P. aeruginosa strains, including eight environmental isolates that belonged to ST308 (by MLST) and carried the metallo-ß-lactamase IMP-8. Phylogenetic analysis on the basis of a non-recombinant core genome that contained 22 outbreak-specific SNPs revealed a pattern of four dominant clades with a strong phylogeographic structure and allowed us to determine the potential temporal origin of the outbreak to July 2008, 1 year before the index case was diagnosed. Superspreaders at the root of clades exhibited a high number of probable and predicted transmissions, indicating their exceptional position in the outbreak. CONCLUSIONS: Our results suggest that the initial expansion of dominant sublineages was driven by a few superspreaders, while environmental contamination seemed to sustain the outbreak for a long period despite regular environmental control measures.


Assuntos
Surtos de Doenças , Farmacorresistência Bacteriana Múltipla , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , Transmissão de Doença Infecciosa , Microbiologia Ambiental , Estudos Epidemiológicos , Genoma Bacteriano , Alemanha/epidemiologia , Hospitais Universitários , Humanos , Epidemiologia Molecular , Tipagem Molecular , Infecções por Pseudomonas/transmissão , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Análise de Sequência de DNA , Análise Espaço-Temporal
2.
BMC Infect Dis ; 13: 515, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24176052

RESUMO

BACKGROUND: Blood stream infections (BSI) with Pseudomonas aeruginosa lead to poor clinical outcomes. The worldwide emergence and spread of metallo-ß-lactamase (MBL) producing, often multidrug-resistant organisms may further aggravate this problem. Our study aimed to investigate the effect of MBL-producing P. aeruginosa (MBL-PA) and various other resistance phenotypes on clinical outcomes. METHODS: A retrospective cohort study was conducted in three German hospitals. Medical files from 2006 until 2012 were studied, and a number of 113 patients with P. aeruginosa BSI were included. The presence of VIM, IMP and NDM genes was detected using molecular techniques. Genetic relatedness was assessed through multilocus sequence typing (MLST). The effect of resistance patterns or MBL production on clinical outcomes was investigated by using multivariate Cox regression models. RESULTS: In-hospital mortality was significantly higher in patients with MBL-PA and multidrug-resistant P. aeruginosa. However, neither BSI with MBL-PA nor BSI with various resistance phenotypes of P. aeruginosa were independently associated with mortality or length of hospital stay. In multivariate models, the SAPS II score (HR 1.046), appropriate definitive treatment (HR range 0.25-0.26), and cardiovascular disease (HR range 0.44-0.46) were independent predictors of mortality. Concomitant infections were associated with an excess length of stay (HR < 1). CONCLUSIONS: Medication with appropriate antimicrobial agents at any time during the course of infection remains the key for improving clinical outcomes in patients with P. aeruginosa BSI and should be combined with a strict implementation of routine infection control measures.


Assuntos
Bacteriemia/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , beta-Lactamases/biossíntese , Idoso , Antibacterianos/farmacologia , Bacteriemia/diagnóstico , Bacteriemia/epidemiologia , Bacteriemia/mortalidade , Estudos de Coortes , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla , Feminino , Alemanha/epidemiologia , Mortalidade Hospitalar , Hospitais , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/mortalidade , Pseudomonas aeruginosa/genética , Estudos Retrospectivos , Resultado do Tratamento , beta-Lactamases/genética , beta-Lactamases/metabolismo
3.
J Infect ; 67(5): 416-23, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23817209

RESUMO

OBJECTIVES: The time to positivity (TTP), measured as the time span between the start of incubation and the alert signal from the blood culture device, has been described as useful tool of prognosis in patients suffering from blood stream infection with Staphylococcus aureus, Escherichia coli and Klebsiella pneumonia. The present study investigates the relationship between TTP and in-hospital mortality in patients with monomicrobial Pseudomonas aeruginosa blood stream infection (PA-BSI). METHODS: From 2006 until 2012 a retrospective cohort study was undertaken in 3 hospitals in the region surrounding Tübingen, Germany. Seventy-four patients with monomicrobial PA-BSI were studied. TTP and clinical parameters were determined and analyzed by receiver operating characteristic (ROC) analysis and Cox regression. RESULTS: The in-hospital mortality of our clinical cohort was 33.78%. In multivariate Cox regression, a TTP ≤ 18 h proved to be independently associated with mortality (HR 3.83, P = 0.012) along with SAPS II score (HR 1.04, P = 0.006), cardiac disease (HR 0.33, P = 0.008) and appropriate definitive antimicrobial treatment (HR 0.21, P = 0.013). CONCLUSIONS: TTP is an easy-to-measure laboratory tool for prognosis in patients with monomicrobial PA-BSI, providing useful information in addition to clinical parameters.


Assuntos
Bacteriemia/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Área Sob a Curva , Bacteriemia/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Infecções por Pseudomonas/diagnóstico , Estudos Retrospectivos
4.
Antimicrob Agents Chemother ; 57(4): 1797-803, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23380719

RESUMO

The association between antimicrobial consumption and resistance in nonfermentative Gram-negative bacteria is well-known. Antimicrobial restriction, implemented in clinical routines by antibiotic stewardship programs (ASPs), is considered a means to reduce resistance rates. Whether and how antimicrobial restriction can accomplish this goal is still unknown though. This leads to an element of uncertainty when designing strategies for ASPs. From January 2002 until December 2011, an observational study was performed at the University Hospital Tübingen, Tübingen, Germany, to investigate the association between antimicrobial use and resistance rates in Pseudomonas aeruginosa. Transfer function models were used to determine such associations and to simulate antimicrobial restriction strategies. Various positive associations between antimicrobial consumption and resistance were observed in our setting. Surprisingly, impact estimations of different antimicrobial restriction strategies revealed relatively low intervention expenses to effectively attenuate the observed increase in resistance. For example, a simulated intervention of an annual 4% reduction in the use of meropenem over 3 years from 2009 until 2011 yielded a 62.5% attenuation (95% confidence interval, 15% to 110%) in the rising trend of multidrug-resistant Pseudomonas aeruginosa (three- and four-class-resistant P. aeruginosa [34MRGN-PA]). Time series analysis models derived from past data may be a tool to predict the outcome of antimicrobial restriction strategies, and could be used to design ASPs.


Assuntos
Antibacterianos/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana
5.
Antimicrob Agents Chemother ; 55(10): 4789-801, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21746957

RESUMO

Fungal infections are a serious health problem in clinics, especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses, only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapy, significant side effects, and high costs for several antifungals, there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities, we screened a compound library including more than 35,000 individual compounds derived from organic synthesis as well as combinatorial compound collections representing mixtures of compounds for antimycotic activity. In total, more than 100,000 compounds were screened using a new type of activity-selectivity assay, analyzing both the antifungal activity and the compatibility with human cells at the same time. One promising hit, an (S)-2-aminoalkyl benzimidazole derivative, was developed among a series of lead compounds showing potent antifungal activity. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole showed the highest antifungal activity and the best compatibility with human cells in several cell culture models and against a number of clinical isolates of several species of pathogenic Candida yeasts. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol pathway, in contrast to other benzimidazole derivatives, which target microtubules.


Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Animais , Antifúngicos/química , Antifúngicos/toxicidade , Benzimidazóis/química , Benzimidazóis/farmacologia , Benzimidazóis/toxicidade , Células CHO , Candida/genética , Candida/isolamento & purificação , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Linhagem Celular , Cricetinae , Farmacorresistência Fúngica , Ergosterol/antagonistas & inibidores , Genoma Fúngico , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos , Relação Estrutura-Atividade , Transcrição Genética
6.
J Med Chem ; 54(19): 6993-7, 2011 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-21711055

RESUMO

Novel nontoxic (S)-2-aminoalkylbenzimidazole derivatives were found to be effective against Candida spp. at low micromolar concentrations using high-throughput screening with infected HeLa cells. A collection of analogues defined the chemical groups relevant for activity. The most active compound was characterized by transcriptional analysis of the response of C. albicans Sc5314. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole had a strong impact on membrane biosynthesis. Testing different clinically relevant pathogenic fungi showed the selectivity of the antimycotic activity against Candida species.


Assuntos
Antimitóticos/síntese química , Benzimidazóis/síntese química , Antifúngicos/síntese química , Antifúngicos/farmacologia , Antifúngicos/toxicidade , Antimitóticos/farmacologia , Antimitóticos/toxicidade , Benzimidazóis/farmacologia , Benzimidazóis/toxicidade , Candida/efeitos dos fármacos , Candida/genética , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Humanos , Imidazóis/síntese química , Imidazóis/farmacologia , Imidazóis/toxicidade , Testes de Sensibilidade Microbiana , Micologia/métodos , Estereoisomerismo , Relação Estrutura-Atividade , Transcrição Genética/efeitos dos fármacos
7.
J Dtsch Dermatol Ges ; 9(10): 806-8, 2011 Oct.
Artigo em Inglês, Alemão | MEDLINE | ID: mdl-21501382

RESUMO

An increasing number of patients with the zoonosis tularemia have been reported in the last few years in Europe. Tularemia can be divided into different forms depending on its appearance. Tularemia must be considered in the differential diagnosis of diseases that present with an ulcer and regional lymphadenopathy. The diagnosis can be confirmed by culturing Francisella tularensis. With effective antibiotic intervention, the prognosis is favorable. Typically tularemia develops after outdoor activities; it is generally transferred by blood-sucking arthropods from infected wild animals to humans.


Assuntos
Abscesso/diagnóstico , Úlcera da Perna/diagnóstico , Linfadenite/diagnóstico , Tularemia/diagnóstico , Zoonoses , Abscesso/tratamento farmacológico , Administração Oral , Idoso , Animais , Antibacterianos/uso terapêutico , Mordeduras e Picadas/complicações , Vetores de Doenças , Doxiciclina/uso terapêutico , Humanos , Injeções Intramusculares , Úlcera da Perna/tratamento farmacológico , Linfadenite/tratamento farmacológico , Masculino , Estreptomicina/uso terapêutico , Carrapatos , Tularemia/tratamento farmacológico , Tularemia/transmissão
8.
J Clin Microbiol ; 48(2): 460-71, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20007393

RESUMO

Extended-spectrum beta-lactamases (ESBL) of the TEM, SHV, or CTX-M type confer resistance to beta-lactam antibiotics in gram-negative bacteria. The activity of these enzymes against beta-lactam antibiotics and their resistance against inhibitors can be influenced by genetic variation at the single-nucleotide level. Here, we describe the development and validation of an oligonucleotide microarray for the rapid identification of ESBLs in gram-negative bacteria by simultaneously genotyping bla(TEM), bla(SHV), and bla(CTX-M). The array consists of 618 probes that cover mutations responsible for 156 amino acid substitutions. As this comprises unprecedented genotyping coverage, the ESBL array has a high potential for epidemiological studies and infection control. With an assay time of 5 h, the ESBL microarray also could be an attractive option for the development of rapid antimicrobial resistance tests in the future. The validity of the DNA microarray was demonstrated with 60 blinded clinical isolates, which were collected during clinical routines. Fifty-eight of them were characterized phenotypically as ESBL producers. The chip was characterized with regard to its resolution, phenotype-genotype correlation, and ability to resolve mixed genotypes. ESBL phenotypes could be correctly ascribed to ESBL variants of bla(CTX-M) (76%), bla(SHV) (22%), or both (2%), whereas no ESBL variant of bla(TEM) was found. The most prevalent ESBLs identified were CTX-M-15 (57%) and SHV-12 (18%).


Assuntos
Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Genes Bacterianos/genética , Análise em Microsséries/métodos , Testes de Sensibilidade Microbiana/métodos , Resistência beta-Lactâmica , beta-Lactamases/genética , Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Sensibilidade e Especificidade , Análise de Sequência de DNA
9.
Med Klin (Munich) ; 104(6): 464-73, 2009 Jun 15.
Artigo em Alemão | MEDLINE | ID: mdl-19533054

RESUMO

The increasing number of complicated soft-tissue or invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is a frequent reason for elaborate treatment regimens. Unidentified MRSA carriers may be the origin of endemic spread to other patients and medical staff. Recently, community-associated cMRSA with particular virulence factors were isolated from persons without the typical history of hospital contacts. Molecular tools for the timely detection of the mecA resistance gene for the identification of MRSA in medical test specimens have become a standard approach in MRSA-related diagnostic procedures. The actual therapy of MRSA infections requires consideration of both the appropriate spectrum of activity and the adequate pharmacological properties of a chosen antimicrobial. Preventive strategies rely on the consistent application of standard hygiene precautions, which have to be supplemented with increased barriers for the isolation of identified MRSA patients.


Assuntos
Infecções Comunitárias Adquiridas/epidemiologia , Infecção Hospitalar/epidemiologia , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas/epidemiologia , Animais , Proteínas de Bactérias/genética , Técnicas Bacteriológicas , Portador Sadio , Infecções Comunitárias Adquiridas/diagnóstico , Infecções Comunitárias Adquiridas/tratamento farmacológico , Infecções Comunitárias Adquiridas/prevenção & controle , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/prevenção & controle , Estudos Transversais , Alemanha , Humanos , Incidência , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas , Fatores de Risco , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/prevenção & controle , Precauções Universais
10.
Methods Mol Biol ; 470: 141-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19089382

RESUMO

Invasive fungal infections comprise a group of serious and life-threatening diseases affecting immunocompromised patients. Molecular analysis of fungal virulence involves the deletion of genes that are suspected for contributing to fungal pathogenesis. Phenotypic analysis of the generated mutants includes in vivo infection experiments in order to assign a function during fungal disease to a gene of interest.


Assuntos
Candida albicans/patogenicidade , Micologia/métodos , Animais , Candida albicans/genética , Candidíase/etiologia , Feminino , Fungemia/etiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Virulência/genética
12.
Infect Immun ; 74(7): 4366-9, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16790813

RESUMO

By generating a calcineurin mutant of the Candida albicans wild-type strain SC5314 with the help of a new recyclable dominant selection marker, we confirmed that calcineurin mediates tolerance to a variety of stress conditions but is not required for the ability of C. albicans to switch to filamentous growth in response to hypha-inducing environmental signals. While calcineurin was essential for virulence of C. albicans in a mouse model of disseminated candidiasis, deletion of CMP1 did not significantly affect virulence during vaginal or pulmonary infection, demonstrating that the requirement for calcineurin for a successful infection depends on the host niche.


Assuntos
Calcineurina/fisiologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Estresse Oxidativo/fisiologia , Animais , Calcineurina/genética , Candida albicans/genética , Candida albicans/fisiologia , Candidíase/imunologia , Candidíase/microbiologia , Modelos Animais de Doenças , Feminino , Inativação Gênica , Imunidade Inata/genética , Camundongos , Estresse Oxidativo/genética , Virulência/genética
13.
Curr Biol ; 15(22): 2021-6, 2005 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-16303561

RESUMO

The ascomycete Candida albicans is the most common fungal pathogen in immunocompromised patients . Its ability to change morphology, from yeast to filamentous forms, in response to host environmental cues is important for virulence . Filamentation is mediated by second messengers such as cyclic adenosine 3',5'-monophosphate (cAMP) synthesized by adenylyl cyclase . The distantly related basidiomycete Cryptococcus neoformans is an encapsulated yeast that predominantly infects the central nervous system in immunocompromised patients . Similar to the morphological change in C. albicans, capsule biosynthesis in C. neoformans, a major virulence attribute, is also dependent upon adenylyl cyclase activity . Here we demonstrate that physiological concentrations of CO2/HCO3- induce filamentation in C. albicans by direct stimulation of cyclase activity. Furthermore, we show that CO2/HCO3- equilibration by carbonic anhydrase is essential for pathogenesis of C. albicans in niches where the available CO2 is limited. We also demonstrate that adenylyl cyclase from C. neoformans is sensitive to physiological concentrations of CO2/HCO3-. These data demonstrate that the link between cAMP signaling and CO2/HCO3- sensing is conserved in fungi and reveal CO2 sensing to be an important mediator of fungal pathogenesis. Novel therapeutic agents could target this pathway at several levels to control fungal infections.


Assuntos
Adenilil Ciclases/metabolismo , Candida albicans/enzimologia , Candida albicans/patogenicidade , Dióxido de Carbono/metabolismo , Cryptococcus neoformans/enzimologia , AMP Cíclico/metabolismo , Transdução de Sinais/fisiologia , Animais , Bicarbonatos/metabolismo , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candidíase/mortalidade , Anidrases Carbônicas/metabolismo , Cryptococcus neoformans/metabolismo , Indução Enzimática/fisiologia , Feminino , Teste de Complementação Genética , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sobrevida , Virulência
14.
Infect Immun ; 73(10): 7061-3, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16177393

RESUMO

SAP7 of Candida albicans is induced after vaginal infection of mice. Conversely, virulence during vaginal infection was not affected in a Deltasap7/Deltasap7 mutant strain. Only a partial virulence phenotype was detectable after intravenous injection. In conclusion, SAP7 expression does not correlate with C. albicans virulence in mice.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Candida/enzimologia , Candida/patogenicidade , Candidíase/microbiologia , Proteínas Fúngicas/metabolismo , Doenças Vaginais/microbiologia , Animais , Ácido Aspártico Endopeptidases/genética , Candida/genética , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/genética , Deleção de Genes , Camundongos , Mutação , Vagina/microbiologia , Virulência
15.
Mol Microbiol ; 57(5): 1318-41, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16102003

RESUMO

Candida albicans is one of the most common fungal pathogens in humans. The cell wall is the first contact site between host and pathogen and thus is critical for colonization and infection of the host. We have identified Tsa1p, a protein that is differentially localized to the cell wall of C. albicans in hyphal cells but remains in the cytosol and nucleus in yeast-form cells. This is different from Saccharomyces cerevisiae, where the homologous protein solely has been found in the cytoplasm. We report here that TSA1 confers resistance towards oxidative stress as well as is involved in the correct composition of hyphal cell walls. However, no significant change of the cell wall composition was observed in a TSA1 deletion strain in yeast-form cells, which is in good agreement with the observation that Tsa1p is absent from the yeast-form cell wall. This indicates that Tsa1p of C. albicans might represent a moonlighting protein with specific functions correlating to its respective localization. Furthermore, the translocation of Tsa1p to the hyphal cell wall of C. albicans depends on Efg1p, suggesting a contribution of the cAMP/PKA pathway to the localization of this protein. In a strain deleted for TUP1 that filaments constitutively Tsa1p can be found in the cell wall under all conditions tested, confirming the result that Tsa1p localization to the cell wall is correlated to the morphology of C. albicans.


Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/fisiologia , Estresse Oxidativo , Peroxidases/fisiologia , Candida albicans/patogenicidade , Candida albicans/ultraestrutura , Núcleo Celular/química , Parede Celular/química , Parede Celular/genética , Parede Celular/metabolismo , Citoplasma/química , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Proteínas Nucleares/metabolismo , Estresse Oxidativo/genética , Peroxidases/análise , Peroxidases/genética , Peroxirredoxinas , Transporte Proteico , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Fatores de Transcrição/genética , Transcrição Genética , Virulência , Vitamina K 3/farmacologia , Água/farmacologia
16.
Infect Immun ; 73(3): 1828-35, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15731084

RESUMO

Vaginal infections caused by the opportunistic yeast Candida albicans are a significant problem in women of child-bearing age. Several factors are recognized as playing a crucial role in the pathogenesis of superficial candidiasis; these factors include hyphal formation, phenotypic switching, and the expression of virulence factors, including a 10-member family of secreted aspartic proteinases. In the present investigation, we analyzed the secreted aspartic proteinase gene (SAP) expression profile of C. albicans that is elicited in the course of vaginal infection in mice and how this in vivo expression profile is associated with hyphal formation. We utilized two different genetic reporter systems that allowed us to observe SAP expression on a single-cell basis, a recombination-based in vivo expression technology and green fluorescent protein-expressing Candida reporter strains. Of the six SAP genes that were analyzed (SAP1 to SAP6), only SAP4 and SAP5 were detectably induced during infection in this model. Expression of both of these genes was associated with hyphal growth, although not all hyphal cells detectably expressed SAP4 and SAP5. SAP5 expression was induced soon after infection, whereas SAP4 was expressed at later times and in fewer cells compared with SAP5. These findings point to a link between morphogenetic development and expression of virulence genes during Candida vaginitis in mice, where host signals induce both hyphal formation and expression of SAP4 and SAP5, but temporal gene expression patterns are ultimately controlled by other factors.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Candida albicans/patogenicidade , Candidíase Vulvovaginal/fisiopatologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Animais , Ácido Aspártico Endopeptidases/genética , Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Candidíase Vulvovaginal/microbiologia , Feminino , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Hifas/enzimologia , Hifas/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C
17.
Mol Microbiol ; 55(2): 546-60, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15659169

RESUMO

Protein O-mannosyltransferases (Pmt proteins) initiate O-mannosylation of secretory proteins. The PMT gene family of the human fungal pathogen Candida albicans consists of PMT1 and PMT6, as well as three additional PMT genes encoding Pmt2, Pmt4 and Pmt5 isoforms described here. Both PMT2 alleles could not be deleted and growth of conditional strains, containing PMT2 controlled by the MET3- or tetOScHOP1-promoters, was blocked in non-permissive conditions, indicating that PMT2 is essential for growth. A homozygous pmt4 mutant was viable, but synthetic lethality of pmt4 was observed in combination with pmt1 mutations. Hyphal morphogenesis of a pmt4 mutant was defective under aerobic induction conditions, yet increased in embedded or hypoxic conditions, suggesting a role of Pmt4p-mediated O-glycosylation for environment-specific morphogenetic signalling. Although a PMT5 transcript was detected, a homozygous pmt5 mutant was phenotypically silent. All other pmt mutants showed variable degrees of supersensitivity to antifungals and to cell wall-destabilizing agents. Cell wall composition was markedly affected in pmt1 and pmt4 mutants, showing a significant decrease in wall mannoproteins. In a mouse model of haematogenously disseminated infection, PMT4 was required for full virulence of C. albicans. Functional analysis of the first complete PMT gene family in a fungal pathogen indicates that Pmt isoforms have variable and specific roles for in vitro and in vivo growth, morphogenesis and antifungal resistance.


Assuntos
Antifúngicos/farmacologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Regulação Fúngica da Expressão Gênica , Manosiltransferases/metabolismo , Família Multigênica , Animais , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candidíase/microbiologia , Candidíase/fisiopatologia , Feminino , Humanos , Isoenzimas , Manosiltransferases/genética , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Morfogênese , Mutação , Virulência
18.
Infect Immun ; 72(4): 2386-9, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15039365

RESUMO

The Tec1p transcription factor is involved in the expression of hypha-specific genes in Candida albicans. Although the induction of the hypha-associated SAP5 gene by serum in vitro depends on Tec1p, deletion of all Tec1p binding site consensus sequences from the SAP5 promoter did not affect its activation. In two different animal models of candidiasis, the SAP5 promoter was induced even in a Deltatec1 deletion mutant, demonstrating that the requirement for Tec1p in gene expression in C. albicans depends on the environmental conditions within the host.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Candida albicans/patogenicidade , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas , Regulação Fúngica da Expressão Gênica , Hifas/crescimento & desenvolvimento , Peritonite/fisiopatologia , Fatores de Transcrição/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candidíase/microbiologia , Candidíase/fisiopatologia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Deleção de Genes , Humanos , Camundongos , Peritonite/microbiologia , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Virulência
19.
J Clin Microbiol ; 41(11): 5033-40, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14605135

RESUMO

In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were >/=99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.


Assuntos
Anaplasma phagocytophilum/genética , Proteínas de Bactérias/genética , Variação Genética/genética , Ixodes/microbiologia , Anaplasma phagocytophilum/classificação , Anaplasma phagocytophilum/isolamento & purificação , Animais , Sequência de Bases , Primers do DNA , Alemanha , Filogenia , Reação em Cadeia da Polimerase
20.
Infect Immun ; 71(9): 5344-54, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12933882

RESUMO

Calcineurin is a conserved Ca(2+)-calmodulin-activated, serine/threonine-specific protein phosphatase that regulates a variety of physiological processes, e.g., cell cycle progression, polarized growth, and adaptation to salt and alkaline pH stresses. In the pathogenic yeast Cryptococcus neoformans, calcineurin is also essential for growth at 37 degrees C and virulence. To investigate whether calcineurin plays a role in the virulence of Candida albicans, the major fungal pathogen of humans, we constructed C. albicans mutants in which both alleles of the CMP1 gene, encoding the calcineurin catalytic subunit, were deleted. The C. albicans Delta cmp1 mutants displayed hypersensitivity to elevated Na(+), Li(+), and Mn(2+) concentrations and to alkaline pH, phenotypes that have been described after calcineurin inactivation in the related yeast Saccharomyces cerevisiae. Unlike S. cerevisiae calcineurin mutants, which exhibit reduced susceptibility to high Ca(2+) concentrations, growth of C. albicans was inhibited in the presence of 300 mM CaCl(2) after the deletion of CMP1, demonstrating that there are also differences in calcineurin-mediated cellular responses between these two yeast species. In contrast to C. neoformans, inactivation of calcineurin did not cause temperature sensitivity in C. albicans. In addition, hyphal growth, an important virulence attribute of C. albicans, was not impaired in the Delta cmp1 mutants under a variety of inducing conditions. Nevertheless, the virulence of the mutants was strongly attenuated in a mouse model of systemic candidiasis, demonstrating that calcineurin signaling is essential for virulence in C. albicans.


Assuntos
Calcineurina/fisiologia , Candida albicans/patogenicidade , Sequência de Aminoácidos , Animais , Sequência de Bases , Calcineurina/química , Calcineurina/genética , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/fisiologia , Candidíase/etiologia , DNA Fúngico/genética , Feminino , Deleção de Genes , Genes Fúngicos , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Subunidades Proteicas , Homologia de Sequência de Aminoácidos , Virulência/genética
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