Fernando L. Hernando

Proposed nomenclature for Pseudallescheria, Scedosporium and related genera

As a result of fundamental changes in the International Code of Nomenclature on the use of separate names for sexual and asexual stages of fungi, generic names of many groups should be reconsidered. Members of the ECMM/ISHAM working group on Pseudallescheria/Scedosporium infections herein advocate a novel nomenclature for genera and species in Pseudallescheria, Scedosporium and allied taxa. The generic names Parascedosporium, Lomentospora, Petriella, Petriellopsis, and Scedosporium are proposed for a lineage within Microascaceae with mostly Scedosporium anamorphs producing slimy, annellidic conidia. Considering that Scedosporium has priority over Pseudallescheria and that Scedosporium prolificans is phylogenetically distinct from the other Scedosporium species, some name changes are proposed. Pseudallescheria minutispora and Petriellidium desertorum are renamed as Scedosporium minutisporum and S. desertorum, respectively. Scedosporium prolificans is renamed as Lomentospora prolificans.

Identification of Candida albicans cell wall antigens lost during subculture in synthetic media

A high variability in reactivity was observed when Candida albicans strains freshly isolated from both patients with candidiasis and asymptomatic carriers were tested against different human sera. The highest reactivity was observed in C. albicans strains isolated from blood cultures. This high reactivity was observed when the isolates were tested against sera from patients with Candida oesophagitis, patients with vulvovaginal candidiasis, or asymptomatic carriers but not against sera from blood donors. The antigenic reactivity of the strongly reactive strains, but not that of the weakly reactive strains, decreased during subculture in synthetic media. Five major components of an apparent molecular mass of > 200, 67-70, 49-52, 33-35 and 29-31 kDa were observed in alpha-mannosidase extracts from C. albicans strains from both blood cultures (Group I) and patients with Candida oesophagitis (Group II) subcultured in synthetic media for different times. Changes in staining intensity through the different subcultures were observed for some bands. Group I strains showed a decrease in staining intensity for bands of > 200 and 67-70 kDa, an increase for bands of 33-35 and 29-31 kDa, but no changes were observed for the band of 49-52 kDa. Group II strains showed opposite changes in banding intensity. A decrease in staining intensity was observed for the proteins of 33-35 and 29-31 kDa, an increase for the protein of 49-52 kDa, and no change in intensity was observed for the band of 67-70 kDa. A component of > 200 kDa showed an irregular expression through the subcultures. The main antigen present in extracts from the first subculture of isolates from Group I and II had a molecular mass of 67-70 kDa. It could be related to the P antigens since it disappeared following subculture of the strains in synthetic media.

The aspHS gene as a new target for detecting Aspergillus fumigatus during infections by quantitative real-time PCR

Invasive aspergillosis (IA) is a serious nosocomial infection caused by Aspergillus spp. which has a high mortality rate due to the fact, among other factors, that it is difficult to diagnose. Within the Aspergillus genus, A. fumigatus is the main species causing IA. We propose a virulence factor, the aspHS gene, as a novel target for the specific detection of A. fumigatus by quantitative real-time PCR (qPCR). This target gene encodes a haemolysin, which is overexpressed in vivo during infection. We have designed specific primers and hydrolysis (Taqman) probes for the detection of this target and a chimeric internal amplification control (IC), designed to detect false negative results due to PCR inhibition. This qPCR assay was tested with DNA extracted from a wide collection of microorganisms, tissues from infected mice, and human bronchoalveolar lavage (BAL) samples. Results showed that it, together with the DNA extraction method, could detect A. fumigatus with high specificity. Furthermore, it can distinguish between germinated (first step to the development of infection) and non-germinated conidia (not detected). Our data indicate that these techniques could be sufficiently sensitive and rapid to help clinicians establish an earlier diagnosis, but the presence of PCR inhibitors in clinical samples such as BAL fluids needs to be addressed.

Qualitative and quantitative differences in recognition patterns of Candida albicans protein and polysaccharide antigens by human sera

Cytoplasmic and cell wall proteins and glycoproteins extracted from Candida albicans germ tubes were screened by Western blotting for their ability to differentiate between the serological responses of patients with candidosis and healthy individuals. Molecules of 114, 74 and 65 kDa were not recognized by any sera. Qualitative differences were observed for responses to proteins and glycoproteins from 29 to 60 kDa. Conversely, only quantitative differences were found to high molecular mass glycoproteins. Their recognition by control sera was invariably associated with reactivity against a 14-18 kDa antigen. However, despite a high level of antibodies against high molecular mass mannoproteins, some patients sera failed to react with the 14-18 kDa antigen, or lost this reactivity during the course of the disease.

Candida albicans and cancer: Can this yeast induce cancer development or progression?

Abstract There is currently increasing concern about the relation between microbial infections and cancer. More and more studies support the view that there is an association, above all, when the causal agents are bacteria or viruses. This review adds to this, summarizing evidence that the opportunistic fungus Candida albicans increases the risk of carcinogenesis and metastasis. Until recent years, Candida spp. had fundamentally been linked to cancerous processes as it is an opportunist pathogen that takes advantage of the immunosuppressed state of patients particularly due to chemotherapy. In contrast, the most recent findings demonstrate that C. albicans is capable of promoting cancer by several mechanisms, as described in the review: production of carcinogenic byproducts, triggering of inflammation, induction of Th17 response and molecular mimicry. We underline the need not only to control this type of infection during cancer treatment, especially given the major role of this yeast species in nosocomial infections, but also to find new therapeutic approaches to avoid the pro-tumor effect of this fungal species.

Phosphoglycerate kinase and fructose bisphosphate aldolase of Candida albicans as new antigens recognized by human salivary IgA.

BACKGROUND Candida albicans is an opportunistic dimorphic fungus commonly present in the human oral cavity that causes infections in immunocompromised patients. The antigen variability, influenced by growth conditions, is a pathogenicity factor. AIMS To determine the effect of nutritional and heat stress on the antigen expression of C. albicans, and to identify major antigens recognized by human salivary secretory immunoglobulin A (sIgA). METHODS Under various different nutritional conditions, heat shock was induced in C. albicans cells in stationary and exponential growth phases. The expression of protein determinants of C. albicans was assessed by Western blot analysis against human saliva. The antigens were purified and characterized by two-dimensional electrophoresis and identified by protein microsequencing. RESULTS Five antigens recognized by salivary IgA were characterized as mannoproteins due to their reactivity with concanavalin A. They did not show reactivity with anti-heat shock protein monoclonal antibodies. Two of them (42 and 36 kDa) were found to be regulated by heat shock and by nutritional stress and they were identified as phosphoglycerate kinase and fructose bisphosphate aldolase, respectively. CONCLUSIONS These glycolytic enzymes are major antigens of C. albicans, and their differential expression and recognition by the mucosal immune response system could be involved in protection against oral infection.

Scedosporium prolificans immunomes against human salivary immunoglobulin A

The filamentous fungus Scedosporium prolificans is an emerging multidrug resistant pathogen related to serious infections mainly affecting immunocompromised individuals. Considering that it is frequently isolated from anthropic environments and penetrates mainly through the airways, the human mucosal immune system may play an important protective role against S. prolificans. To advance in the search for biomarkers and targets both for diagnosis and treatment, we analysed the S. prolificans immunomes recognized by human salivary Immunoglobulin A. Using indirect immunofluorescence, it was observed that conidia were strongly recognized, while hyphae were not. By 2-D immunoblotting and peptide mass fingerprinting, 25 immunodominant antigens in conidia and 30 in hyphae were identified. These included catalase, putative glyceronetransferase, translation elongation factor-1α, serine/threonine protein kinase, putative superoxide dismutase, putative mitochondrial cyclophilin 1 and peptidyl-prolyl cis-trans isomerase in conidiospores, and putative Hsp60, ATP synthase β chain, 40S ribosomal protein S0, citrate synthase and putative ATP synthase in hyphae. The functional study showed that metabolism - and protein fate - related enzymes were the most abundant antigens in conidia, whereas metabolism - , translation - , or energy production - related enzymes were in hyphae. The immunogenic proteins identified are proposed as candidates for the development of novel diagnostic tools or therapeutic strategies.

Effect of glyphosate on the greening process and photosynthetic metabolism in Chlorella pyrenoidosa

Summary Chlorella pyrenoidosa Pringsheim (211/8 a) was grown photoautotrophically using the herbicide, glyphosate [N-(phosphonomethyl)-gycine], in concentrations ranging from 0.1 mM to 1 mM. Chlorophyll and carotenoid content, greening process, photosynthetic and respiration rates, and photosynthetic electron transport activities were investigated. Glyphosate decreased cell density and photosynthetic pigment content; 1.0 mM did not allow growth. Bleached cells were used for the greening process. Glyphosate had two different effects upon photosynthetic pigments: inhibition of chlorophyll synthesis and a decrease in carotenoids. Oxygen uptake was not affected, but oxygen evolution was strongly inhibited. The results suggest that glyphosate acts as an electron transport inhibitor, acting on both photosystems, but its effect was greater on PS II than PS I.

Molecular and cellular responses of the pathogenic fungus Lomentospora prolificans to the antifungal drug voriconazole

The filamentous fungus Lomentospora (Scedosporium) prolificans is an emerging opportunistic pathogen associated with fatal infections in patients with disturbed immune function. Unfortunately, conventional therapies are hardly of any use against this fungus due to its intrinsic resistance. Therefore, we performed an integrated study of the L. prolificans responses to the first option to treat these mycoses, namely voriconazole, with the aim of unveiling mechanisms involved in the resistance to this compound. To do that, we used a wide range of techniques, including fluorescence and electron microscopy to study morphological alterations, ion chromatography to measure changes in cell-wall carbohydrate composition, and proteomics-based techniques to identify the proteins differentially expressed under the presence of the drug. Significantly, we showed drastic changes occurring in cell shape after voriconazole exposure, L. prolificans hyphae being shorter and wider than under control conditions. Interestingly, we proved that the architecture and carbohydrate composition of the cell wall had been modified in the presence of the drug. Specifically, L. prolificans constructed a more complex organelle with a higher presence of glucans and mannans. In addition to this, we identified several differentially expressed proteins, including Srp1 and heat shock protein 70 (Hsp70), as the most overexpressed under voriconazole-induced stress conditions. The mechanisms described in this study, which may be directly related to L. prolificans antifungal resistance or tolerance, could be used as targets to improve existing therapies or to develop new ones in order to successfully eliminate these mycoses.

Immunoproteomics-Based Analysis of the Immunocompetent Serological Response to Lomentospora prolificans.

The filamentous fungus Lomentospora prolificans is an emerging pathogen causing severe infections mainly among the immunocompromised population. These diseases course with high mortality rates due to great virulence of the fungus, its inherent resistance to available antifungals, and absence of specific diagnostic tools. Despite being widespread in humanized environments, L. prolificans rarely causes infections in immunocompetent individuals likely due to their developed protective immune response. In this study, conidial and hyphal immunomes against healthy human serum IgG were analyzed, identifying immunodominant antigens and establishing their prevalence among the immunocompetent population. Thirteen protein spots from each morph were detected as reactive against at least 70% of serum samples, and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Hence, the most seroprevalent antigens were WD40 repeat 2 protein, malate dehydrogenase, and DHN1, in conidia, and heat shock protein (Hsp) 70, Hsp90, ATP synthase β subunit, and glyceraldehyde-3-phosphate dehydrogenase, in hyphae. More interestingly, the presence of some of these seroprevalent antigens was determined on the cell surface, as Hsp70, enolase, or Hsp90. Thus, we have identified a diverse set of antigenic proteins, both in the entire proteome and cell surface subproteome, which may be used as targets to develop innovative therapeutic or diagnostic tools.