Yeast pathogens and their interactions with host cells
Research highlights
Candida yeasts are among the major fungal pathogens in humans, causing infections that seriously affect the quality of life and health of the world population. Although C. albicans is the most frequent cause of candidemia, other species, namely C. parapsilosis and C. glabrata, have also emerged as important agents of human infections. Our general aims are the development of new tools for molecular diagnosis and typing of these pathogens and the study of the role of specific C. albicans genes in pathogenesis and virulence.
In the past three years different research projects were developed focusing mainly on: i) the epidemiology of candidemia and the identification of new microsatellite markers for differentiation of C. parapsilosis isolates; ii) evaluation of the role of C. albicans RLM1, GUP1, JEN1, and JEN2 in pathogenesis and virulence; iii) immunobiological studies of C. albicans secreted aspartic proteinases and evaluation of their role in virulence and host immune response in murine hematogenously disseminated candidiasis.
A survey of bloodstream infections in a Portuguese oncology hospital was performed showing that C. parapsilosis is the second most frequent pathogenic yeast species isolated from these patients.
We have described a microsatellite multiplex system for C. parapsilosis typing with a discriminatory power of 0.99, proving to be a valuable tool for strain differentiation.
Following our previous work we integrated a multicenter collaborative study for the standardization of C. albicans genotyping using microsatellite markers and used the same approach to group C. albicans major clades.
Studies of C. albicans sequencial bloodstream isolates showed that mice infected with the recurrent strain lived longer than those infected with the original strain, indicating that host selective pressure may result in C. albicans attenuated virulence.
Allelic variants of the CaRLM1 transcription factor were characterized and we demonstrated that an increase in the number of glutamines in the C-terminal of Rlm1p enhances resistance to stress agents.
It was shown that C. albicans Gup1p strongly interferes with the capacity of cells to develop hyphae, to adhere, to invade, and to form a biofilm, all of which are significant virulence factors, as well as in the mechanisms underlying antifungal resistance. Cagup1 null mutant was more resistant to several common antifungal, namely azoles.
The importance of carboxylate transport for the capacity of C. albicans to grow within different host niches was also studied. In C. albicans, CaJEN1 encodes a monocarboxylate permease, whereas the paralog, CaJEN2, previously assigned by homology as a lactate transporter, was demonstrated to encode a novel dicarboxylate plasma membrane transporter.
Moreover, in in vivo and ex vivo models of infection, it was found that both CaJEN1 and CaJEN2 are expressed in glucose-poor niches within the host, being important contributors to carbon metabolism during the early stages of infection.
Additionally, a collection of plasmids suitable for the in vivo cloning of C. albicans genes in S. cerevisiae, by gap repair: CIp10-2µ, CIp20-2µ and CIp30-2µ, was constructed. This is particularly useful for genes, which are difficult to clone in E. coli (like the ones encoding several membrane proteins).
We have re-assessed the importance of SAP1-SAP6 in a murine model of hematogenously disseminated candidiasis, using sap null mutant strains not affected in their URA3 gene expression.
No significant differences were observed in the number of CFUs in the kidney, limphocyte populations, co-stimulatory molecules or activation markers between the mice infected with the parental strain or with the ?sap1-3 and ?sap4-6 mutants.
Our results suggest that secreted aspartyl proteinases 1-6 do not play a significant role in C. albicans virulence in a murine model of hematogenously disseminated candidiasis and that, in this model, Sap1-3 are not necessary for successful C. albicans infection.
Ongoing and future work
Regarding the epidemiology of fungal infections, the multiplex microsatellite genotyping system described for C. parapsilosis is presently being applied in the characterization of isolates from all over the world, in a collaborative study. Future research will focus on the development of new typing systems for emergent fungal pathogens.
CaRlm1 is involved in the cell wall integrity pathway. The role of this gene in C. albicans virulence is currently under evaluation by characterization of Carlm1 mutant strains by in vivo and in vitro studies and microarray analysis.
The impact of GUP1 role in C. albicans virulence as well as in the mechanisms underlying antifungal resistance, is even more significant when taken together with all the knowledge about GUP1 gene (from S. cerevisiae and mammals) giving consistence to the possibility that Gup1p may be part of a yeast morphogenic pathway parallel to the mammalian Hedgehog. Therefore, we are now testing the effect of the heterologous expression, in Cagup1 double mutant strain, of several GUP1 homologues, namely S. cerevisiae GUP1, Homo sapiens GUP1, Mus musculus GUP1 and Drosophila melanogaster GUP1, in order to explore that possibility.
We will continue to characterize the mechanisms of transport and regulation of carboxylic acids in C. albicans and we will extend our studies to C. glabrata. We expect to understand the mode of regulation of these permeases in response to different environmental conditions and their role in fungal virulence.
The development of antifungal resistance has created concern regarding the future ability to treat infections caused by C. albicans. Clearly, new strategies are needed to face this problem and the development of an efficient vaccine is one approach. In this theme we will continue the studies, designing and characterizing a new liposome:protein formulation to deliver antigenic molecules as an immunoprotective strategy against fungal infections.
Key References
Sabino, R., Veríssimo, C., Brandão, J., Alves, C., Sampaio, P., Rosado, L., Parada, H., Rosado, L., Paixão, E., Videira, Z., Tendeiro, T., Sampaio, P., Pais, C. 2010. Epidemiology of candidemia in oncology patients: a 6-year survey in a Portuguese central hospital. Medical Mycology 5:1-10.
Sabino, R., Sampaio, P., Rosado, L., Stevens, D.A., Clemons, K.V., Pais C. 2010. New polymorphic microsatellite markers able to distinguish among Candida parapsilosis sensu stricto isolates. Journal of Clinical Microbiology 48:1671-1682.
Garcia-Hermoso, D., Maccallum, D.M., Lott, T.J., Sampaio, P., Buitrago, M.J., Grenouillet, F., Klaassen C.H., Bretagne, S. 2010. A Multicenter Collaborative Study for the Standardization of Candida albicans Genotyping using a Polymorphic Microsatellite Marker. Journal of Clinical Microbiology 8:2578-2581.
Chavéz-Galarza, J., Pais, C., Sampaio, P. 2010. Microsatellite typing identifies the major clades of the human pathogen Candida albicans. Infection, Genetics and Evolution, 10:697-702.
Sampaio, P., Santos, M., Correia, A., Amaral, F. E., Chavéz-Galarza, J., Castro, A.G., Pedrosa, J., Pais, C. 2010. Virulence decrease of Candida albicans variants of the same strain isolated from an immunocompromised patient with recurrent blood stream infection. PLoS One. 5(4): e10155.
Ferreira, C., Silva, S., Faria-Oliveira, F., Pinho, E., Henriques, M., Lucas, C. 2010. Candida albicans virulence and drug-resistance requires the O-acyltransferase Gup1p. BMC Microbiol Sep 15;10 (1):238. [Epub ahead of print]
Vieira, N., Pereira, F., Casal, M., Brown, A.J., Paiva, S., Johansson, B. 2010. Plasmids for in vivo construction of integrative Candida albicans vectors in Saccharomyces cerevisiae Yeast. Jul 2. [Epub ahead of print].
Correia, A., Lermann, U., Teixeira, L., Cerca, F., Botelho, S., Gil da Costa, R.M., Sampaio, P., Gartner, F., Morschhaeuser, J., Vilanova, M., Pais, C. 2010. Limited role of secreted aspartyl proteinases Sap1 to Sap6 in Candida albicans virulence and host immune response in murine hematogenously disseminated candidiasis. Infection and Immunity Aug 2 [Epub ahead of print].
Sampaio, P., Nogueira, E., Sá Loureiro, A., Delgado-Silva, Y., Correia, A., Pais, C. 2009. Increased number of glutamine repeats in the C- terminal of Candida albicans Rlm1p enhances the resistance to stress agents. Antonie van Leeuwenhoek. 96:395-404.
Vieira, N., Casal, M., Johansson, B., Maccallum, D.M., Brown, A.J., Paiva, S. 2009. Functional specialization and differential regulation of short chain carboxylic acid transporters in the pathogen Candida albicans. Mol Microbiol 75: 1337-54.
