Candida albicans Shaving to Profile Human Serum Proteins on Hyphal Surface.

Candida albicans is a human opportunistic fungus and it is responsible for a wide variety of infections, either superficial or systemic. C. albicans is a polymorphic fungus and its ability to switch between yeast and hyphae is essential for its virulence. Once C. albicans obtains access to the human body, the host serum constitutes a complex environment of interaction with C. albicans cell surface in bloodstream. To draw a comprehensive picture of this relevant step in host-pathogen interaction during invasive candidiasis, we have optimized a gel-free shaving proteomic strategy to identify both, human serum proteins coating C. albicans cells and fungi surface proteins simultaneously. This approach was carried out with normal serum (NS) and heat inactivated serum (HIS). We identified 214 human and 372 C. albicans unique proteins. Proteins identified in C. albicans included 147 which were described as located at the cell surface and 52 that were described as immunogenic. Interestingly, among these C. albicans proteins, we identified 23 GPI-anchored proteins, Gpd2 and Pra1, which are involved in complement system evasion and 7 other proteins that are able to attach plasminogen to C. albicans surface (Adh1, Eno1, Fba1, Pgk1, Tdh3, Tef1, and Tsa1). Furthermore, 12 proteins identified at the C. albicans hyphae surface induced with 10% human serum were not detected in other hypha-induced conditions. The most abundant human proteins identified are involved in complement and coagulation pathways. Remarkably, with this strategy, all main proteins belonging to complement cascades were identified on the C. albicans surface. Moreover, we identified immunoglobulins, cytoskeletal proteins, metabolic proteins such as apolipoproteins and others. Additionally, we identified more inhibitors of complement and coagulation pathways, some of them serpin proteins (serine protease inhibitors), in HIS vs. NS. On the other hand, we detected a higher amount of C3 at the C. albicans surface in NS than in HIS, as validated by immunofluorescence.

Comparative proteomic study of Edwardsiella tarda strains with different degrees of virulence.

Edwardsiella tarda is an enteric opportunistic pathogen that causes a great loss in aquaculture. This species has been described as a phenotypical homogeneous group; in contrast, serological studies and molecular typing revealed a wide heterogeneity. In this work, a proteomic study of differential expression of a virulent isolate from turbot cultured in the Norwest of Spain in comparison with an avirulent collection strain was performed in order to recognize proteins involved in virulence. One hundred and three proteins that presented different abundance were successfully identified and classified into 11 functional categories according to their biological processes: amino acid, carbohydrate and lipid metabolism, tricarboxylic cycle, stress response and protein fate, protein synthesis, biogenesis of cellular components, cell rescue defence and virulence, cell membrane and transport, signal transduction and purine and pyrimidine metabolism. Twenty three protein spots detected only in turbot isolate were identified. It was shown that the same proteins appeared in different spots in the two isolates. Mass spectra obtained by MALDITOF/TOF of some of these proteins and DNA sequencing explained the changes as a result of different amino acid sequences. Several proteins related with the virulence of E. tarda (FliC, ArnA or FeSODI) were only detected in the turbot European isolate. This article is part of a Special Issue entitled: HUPO 2014.

Immunoproteomic profiling of Saccharomyces cerevisiae systemic infection in a murine model.

Saccharomyces cerevisiae is considered a safe microorganism widely used as a dietary supplement. However, in the latest decades several cases of S. cerevisiae infections have been reported. Recent studies in a murine model of systemic infection have also revealed the virulence of some S. cerevisiae dietary strains. Here we use an immunoproteomic approach based on protein separation by 2D-PAGE followed by Western-blotting to compare the serological response against a virulent dietary and a non-virulent laboratory strains leading to the identification of highly different patterns of antigenic proteins. Thirty-six proteins that elicit a serological response in mice have been identified. Most of them are involved in stress responses and metabolic pathways. Their selectivity as putative biomarkers for S. cerevisiae infections was assessed by testing sera from S. cerevisiae-infected mice against Candida albicans and C. glabrata proteins. Some chaperones and metabolic proteins showed cross-reactivity. We also compare the S. cerevisiae immunodetected proteins with previously described C. albicans antigens. The results point to the stress-related proteins Ahp1, Yhb1 and Oye2, as well as the glutamine synthetase Gln1 and the oxysosterol binding protein Kes1 as putative candidates for being evaluated as biomarkers for diagnostic assays of S. cerevisiae infections. BIOLOGICAL SIGNIFICANCE: S. cerevisiae can cause opportunistic infections, and therefore, a precise diagnosis of fungal infections is necessary. This immunoproteomic analysis of sera from a model murine infection with a virulent dietary S. cerevisiae strain has been shown to be a source of candidate proteins for being evaluated as biomarkers to develop assays for diagnosis of S. cerevisiae infections. To our knowledge, this is the first study devoted to the identification of S. cerevisiae immunogenic proteins and the results allowed the proposal of five antigens to be further investigated.

Pathogenic potential of Saccharomyces strains isolated from dietary supplements.

Saccharomyces cerevisiae plays a beneficial role in health because of its intrinsic nutritional value and bio-functional properties, which is why it is also used as a dietary supplement. However, the perception that S. cerevisiae is harmless has changed due to an increasing number of infections caused by this yeast. Given this scenario, we have tested whether viable strains contained in dietary supplements displayed virulence-associated phenotypic traits that could contribute to virulence in humans. We have also performed an in vivo study of the pathogenic potential of these strains using a murine model of systemic infection by intravenous inoculation. A total of 5 strains were isolated from 22 commercial products and tested. Results highlight one strain (D14) in terms of burden levels in brains and kidneys and ability to cause death, whereas the other two strains (D2 and D4) were considered of low virulence. Our results suggest a strong relationship between some of the virulence-associated phenotypic traits (ability to grow at 39°C and pseudohyphal growth) and the in vivo virulence in a mouse model of intravenous inoculation for isolates under study. The isolate displaying greatest virulence (D14) was evaluated in an experimental murine model of gastrointestinal infection with immunosuppression and disruption of mucosal integrity, which are common risk factors for developing infection in humans, and results were compared with an avirulent strain (D23). We showed that D14 was able to spread to mesenteric nodes and distant organs under these conditions. Given the widespread consumption of dietary supplements, we recommend only safe strains be used.

Immunoproteomic analysis of the protective response obtained with subunit and commercial vaccines against Glässer's disease in pigs.

An immunoproteomic analysis of the protective response of subunit and commercial vaccines in colostrum-deprived pigs against Glässer's disease was carried out. A mixture of proteins with affinity to porcine transferrin (PAPT) from Haemophilus parasuis Nagasaki strain (serovar 5) was inoculated intramuscularly (PAPT(M)) and intratracheally (PAPT(Cp)), along with a commercial bacterin. PAPT were separated using 2 dimensional electrophoresis (2DE) gels and with them, 2DE Western blots were carried out. A total of 17 spots were identified as positive with sera of pigs from any of the three vaccinated groups, the highest number of immunoreactive proteins being detected in those having received PAPT(Cp). Among them, six proteins (FKBP-type peptidyl-prolyl cis-trans isomerase, neuraminidase exo-α-sialidase, xanthine-guanine phosphoribosyl transferase, CMP-N-acetylneuraminic acid synthetase, phenylalanyl-tRNA synthetase and glyceraldehyde 3-phosphate dehydrogenase) were found to be novel immunogens in H. parasuis. These proteins showed a high potential as candidates in future subunit vaccines against Glässer's disease. The three experimental groups developed specific systemic total IgG (IgGt), IgG1, IgG2 and IgM antibodies after immunizations. In addition, those receiving PAPT(Cp) yielded a serum IgA response.

Differences in activation of MAP kinases and variability in the polyglutamine tract of Slt2 in clinical and non-clinical isolates of Saccharomyces cerevisiae.

The concept of Saccharomyces cerevisiae as an emerging opportunistic pathogen is relatively new and it is due to an increasing number of human infections during the past 20 years. There are still few studies addressing the mechanisms of infection of this yeast species. Moreover, little is known about how S. cerevisiae cells sense and respond to the harsh conditions imposed by the host, and whether this response is different between clinical isolates and non-pathogenic strains. In this regard, mitogen-activated protein kinase (MAPK) pathways constitute one of the major mechanisms for controlling transcriptional responses and, in some cases, virulence in fungi. Here we show differences among clinical and non-clinical isolates of S. cerevisiae in the level of activation of the MAPKs Kss1, which controls pseudohyphal and invasive growth, and Slt2, which is required for maintaining the integrity of the cell wall under stress conditions and in the absence of stimulating conditions. Moreover, we report for the first time the existence of length variability in SLT2 alleles of strains with a clinical origin. This is due to the expansion in the number of glutamine-encoding triplets in the microsatellite region coding for the polyglutamine (poly-Q) tract of this gene, which range from 12 to more than 38 repetitions. We suggest that this variability may influence biological features of the Slt2 protein, allowing it to adapt swiftly in order to survive in unusual environments.