Andoni Ramirez-Garcia

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.

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.

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.

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.

Cyclophilin and enolase are the most prevalent conidial antigens of Lomentospora prolificans recognized by healthy human salivary IgA and cross-react with Aspergillus fumigatus

The study of the immunocompetent airways immune response may provide important information to improve the therapeutic efficacy against Lomentospora (Scedosporium) prolificans. So, this study aimed to identify the most prevalent conidial antigens of this multiresistant fungus recognized by healthy human salivary immunoglobulin A, and to study their expression and cross‐reactivity with other fungal species.

Scedosporium and Lomentospora: an updated overview of underrated opportunists

Species of Scedosporium and Lomentospora are considered as emerging opportunists, affecting immunosuppressed and otherwise debilitated patients, although classically they are known from causing trauma-associated infections in healthy individuals. Clinical manifestations range from local infection to pulmonary colonization and severe invasive disease, in which mortality rates may be over 80%. These unacceptably high rates are due to the clinical status of patients, diagnostic difficulties, and to intrinsic antifungal resistance of these fungi. In consequence, several consortia have been founded to increase research efforts on these orphan fungi. The current review presents recent findings and summarizes the most relevant points, including the Scedosporium/Lomentospora taxonomy, environmental distribution, epidemiology, pathology, virulence factors, immunology, diagnostic methods, and therapeutic strategies.

Aspergillus fumigatus transcriptome response to a higher temperature during the earliest steps of germination monitored using a new customized expression microarray.

Aspergillus fumigatus is considered to be the most prevalent airborne pathogenic fungus and can cause invasive diseases in immunocompromised patients. It is known that its virulence is multifactorial, although the mechanisms of pathogenicity remain unclear. With the aim of improving our understanding of these mechanisms, we designed a new expression microarray covering the entire genome of A. fumigatus. In this first study, we analysed the transcriptomes of this fungus at the first steps of germination after being grown at 24 and 37 °C. The microarray data revealed that 1249 genes were differentially expressed during growth at these two temperatures. According to our results, A. fumigatus modified significantly the expression of genes related to metabolism to adapt to new conditions. The high percentages of genes that encoded hypothetical or unclassified proteins differentially expressed implied that many as yet unknown genes were involved in the establishment of A. fumigatus infection. Furthermore, amongst the genes implicated in virulence upregulated at 37 °C on the microarray, we found those that encoded proteins mainly related to allergens (Asp F1, Asp F2 and MnSOD), gliotoxin biosynthesis (GliP and GliZ), nitrogen (NiiA and NiaD) or iron (HapX, SreA, SidD and SidC) metabolism. However, gene expression in iron and nitrogen metabolism might be influenced not only by heat shock, but also by the availability of nutrients in the medium, as shown by the addition of fresh medium.