Genetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species ...and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis.
We selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis.
Among 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp.
Molecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.
ABSTRACT
Background:
Malfunctioning or damaged mitochondria result in altered energy metabolism, redox equilibrium, and cellular dynamics and is a central point in the pathogenesis of neurological ...disorders such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and Amyotrophic Lateral Sclerosis. Therefore, it is of utmost importance to identify mitochondrial genetic susceptibility markers for neurodegenerative diseases. Potential markers include the respiratory chain enzymes Riboflavin kinase (
RFK
), Flavin adenine dinucleotide synthetase (
FAD
), Succinate dehydrogenase B subunit (
SDHB
), and Cytochrome C1 (
CYC1
). These enzymes are associated with neuroprotection and neurodegeneration.
Objective:
To test if variants in genes
RFK
,
FAD
,
SDHB
and
CYC1
deviate from Hardy-Weinberg Equilibrium (HWE) in different human mitochondrial haplogroups.
Methods:
Sequence variants in genes
RFK
,
FAD
,
SDHB
and
CYC1
of 2,504 non-affected individuals of the 1,000 genomes project were used for mitochondrial haplogroup assessment and HWE calculations in different mitochondrial haplogroups.
Results:
We show that
RFK
variants deviate from HWE in haplogroups G, H, L, V and W, variants of
FAD
in haplogroups B, J, L, U, and C, variants of
SDHB
in relation to the C, W, and A and
CYC1
variants in B, L, U, D, and T. HWE deviation indicates action of selective pressures and genetic drift.
Conclusions:
HWE deviation of particular variants in relation to global populational HWE, could be, at least in part, associated with the differential susceptibility of specific populations and ethnicities to neurodegenerative diseases. Our data might contribute to the epidemiology and diagnostic/prognostic methods for neurodegenerative diseases.
Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate ...identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on “DNA barcoding of human and animal pathogenic fungi.” A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.
We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis ...shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3'-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. The model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. The analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.
Summary
Enteropathogenic Escherichia coli (EPEC) forms attaching and effacing lesions in the intestinal mucosa characterized by intimate attachment to the epithelium by means of intimin (an outer ...membrane adhesin encoded by eae). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC); only tEPEC carries the EAF (EPEC adherence factor) plasmid that encodes the bundle‐forming pilus (BFP). Characteristically, after 3 h of incubation, tEPEC produces localized adherence (LA) (with compact microcolonies) in HeLa/HEp‐2 cells by means of BFP, whereas most aEPEC form looser microcolonies. We have previously identified nine aEPEC strains displaying LA in extended (6 h) assays (LA6). In this study, we analysed the kinetics of LA6 pattern development and the role of intimin in the process. Transmission electron microscopy and confocal laser microscopy showed that the invasive process of strain 1551‐2 displays a LA phenotype. An eae‐defective mutant of strain 1551‐2 prevented the invasion although preserving intense diffused adherence. Sequencing of eae revealed that strain 1551‐2 expresses the omicron subtype of intimin. We propose that the LA phenotype of aEPEC strain 1551‐2 is mediated by intimin omicron and hypothesize that this strain expresses an additional novel adhesive structure. The present study is the first to report the association of compact microcolony formation and an intense invasive ability in aEPEC.
•There is an extensive PIK polymorphism at genomic localization level in T. cruzi.•FYVE-PIK is a trypanosomatid PIK gene that might be acquired by horizontal transfer.•The localization of TOR ...proteins within Trypanosoma genus is different.•Polymorphism may be considered in designing drugs and vaccines for Chagas’ disease.
Chagas disease is caused by the protozoan Trypanosoma cruzi which affects 10 million people worldwide. Very few kinases have been characterized in this parasite, including the phosphatidylinositol kinases (PIKs) that are at the heart of one of the major pathways of intracellular signal transduction. Recently, we have classified the PIK family in T. cruzi using five different models based on the presence of PIK conserved domains. In this study, we have mapped PIK genes to the chromosomes of two different T. cruzi lineages (G and CL Brener) and determined the cellular localization of two PIK members. The kinases have crucial roles in metabolism and are assumed to be conserved throughout evolution. For this reason, they should display a conserved localization within the same eukaryotic species. In spite of this, there is an extensive polymorphism regarding PIK localization at both genomic and cellular levels, among different T. cruzi isolates and between T. cruzi and Trypanosomabrucei, respectively. We showed in this study that the cellular localization of two PIK-related proteins (TOR1 and 2) in the T. cruzi lineage is distinct from that previously observed in T. brucei. In addition, we identified a new PIK gene with peculiar feature, that is, it codes for a FYVE domain at N-terminal position. FYVE-PIK genes are phylogenetically distant from the groups containing exclusively the FYVE or PIK domain. The FYVE-PIK architecture is only present in trypanosomatids and in virus such as Acanthamoeba mimivirus, suggesting a horizontal acquisition. Our Bayesian phylogenetic inference supports this hypothesis. The exact functions of this FYVE-PIK gene are unknown, but the presence of FYVE domain suggests a role in membranous compartments, such as endosome. Taken together, the data presented here strengthen the possibility that trypanosomatids are characterized by extensive genomic plasticity that may be considered in designing drugs and vaccines for prevention of Chagas disease.
Identification of genes differentially expressed in hyphae of Candida albicans Melo, Analy Salles de Azevedo(Universidade Federal de São Paulo Escola Paulista de Medicina Departamento de Microbiologia, Imunologia e Parasitologia); Serafim, Rui Cosme(Universidade Federal de São Paulo Escola Paulista de Medicina Departamento de Microbiologia, Imunologia e Parasitologia); Briones, Marcelo Ribeiro da Silva(Universidade Federal de São Paulo Escola Paulista de Medicina Departamento de Microbiologia, Imunologia e Parasitologia)
Brazilian journal of microbiology,
11/2003, Volume:
34, Issue:
S1
Journal Article
Peer reviewed
Open access
The ability to switch from yeast to hyphal forms is essential for Candida albicans virulence. This morphological switch involves the expression of hyphal-specific genes under the control of ...transcriptional factors. To contribute to the discovery of hyphal-specific genes, we used a differential screening method where clones of a genomic DNA library were hybridized with yeast and hyphal cDNA probes. Two clones with increased expression in hyphae were selected for study. Sequencing these clones, we found that they encoded two important metabolic genes, CaHXT7 (high-affinity hexose transporter) and CaYLL34 (member of the AAA ATPase family). CaHXT7 and CaYLL34 ORFs were completely determined. Analyses of the putative proteins show that: (1) CaHxt7p has one hexose transporter domain and (2) CaYll34p has two AAA ATPase domains. These results show, for the first time, increased expression of metabolic genes in C. albicans hyphae. Also, because the proteins encoded by CaHXT7 and CaYLL34 may be necessary for the switching to hyphae, they could be new targets for antifungal drugs.
A transição morfológica de levedura para hifa é essencial para a virulência de Candida albicans. Esta transição envolve a expressão de genes hifa-específicos que estão sob o controle de fatores transcricionais. Para descobrir genes hifa-específicos utilizamos um método de triagem diferencial, onde clones de biblioteca de DNA genômico foram hibridizados com sondas de cDNA de levedura e hifa. Dois clones com aumento de expressão em hifa foram selecionados. O sequenciamento dos insertos destes clones permitiu a identificação de dois genes metabólicos importantes: CaHXT7 (high-affinity hexose transporter) e CaYLL34 (da família AAA ATPase). As ORFs completas destes genes foram caracterizadas e a análise das proteínas hipotéticas revelou que: (1) CaHxt7p tem um domínio de transportador de hexose e (2) CaYll34 tem dois domínios AAA ATPase. Este é o primeiro estudo que demonstra aumento de expressão de genes metabólicos em hifas de C. albicans. Ainda, a associação dos produtos de CaHXT7 e CaYLL34 com a formação de hifas torna estas proteínas potenciais novos alvos para drogas antifúngicas.
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