Heparin-binding motifs and biofilm formation by Candida albicans.

Candida albicans is a leading pathogen in infections of central venous catheters, which are frequently infused with heparin. Binding of C. albicans to medically relevant concentrations of soluble and plate-bound heparin was demonstrable by confocal microscopy and enzyme-linked immunosorbent assay (ELISA). A sequence-based search identified 34 C. albicans surface proteins containing ≥1 match to linear heparin-binding motifs. The virulence factor Int1 contained the most putative heparin-binding motifs (n = 5); peptides encompassing 2 of 5 motifs bound to heparin-Sepharose. Alanine substitution of lysine residues K805/K806 in 804QKKHQIHK811 (motif 1 of Int1) markedly attenuated biofilm formation in central venous catheters in rats, whereas alanine substitution of K1595/R1596 in 1593FKKRFFKL1600 (motif 4 of Int1) did not impair biofilm formation. Affinity-purified immunoglobulin G (IgG) recognizing motif 1 abolished biofilm formation in central venous catheters; preimmune IgG had no effect. After heparin treatment of C. albicans, soluble peptides from multiple C. albicans surface proteins were detected, such as Eno1, Pgk1, Tdh3, and Ssa1/2 but not Int1, suggesting that heparin changes candidal surface structures and may modify some antigens critical for immune recognition. These studies define a new mechanism of biofilm formation for C. albicans and a novel strategy for inhibiting catheter-associated biofilms.

Association of two clones allows for optimal detection of human FOXP3.

FOXP3 is a key transcription factor expressed by regulatory T cells (Treg cells). However, differences in staining and analysis protocols have led to conflicting results. Moreover, the transient upregulation of FOXP3 that follows activation in non-Treg cells renders the interpretation of FOXP3 data more difficult in humans than in mice. Human peripheral blood mononuclear cells (PBMCs), isolated CD25(-) or CD25(+)CD4(+) T cells were stained with three different anti-FOXP3 clones (PCH101, 206D, and 259D) alone or in combination, and using different permeabilization methods. FOXP3 expression was evaluated following T cell activation by several pathways. Gating based on a population that did not express FOXP3 (such as CD3(-)CD4(-) T cells) allowed for the optimal characterization of Treg cells. The 206D clone detected a lower percentage of cells than PCH101 or 259D. In contrast, 259D stained a population of activated T cells that PCH101 did not. Staining with two clones together consistently increased the proportion of FOXP3(+) cells. However, it is likely that only the double positive cells are Treg cells, as they expressed the highest CD25 and lowest CD127 levels. Our results emphasize that the choice of staining protocol leads to very different results concerning the frequency of Treg cells in humans. A more consistent identification of these cells will improve the knowledge of their biology, particularly during disease processes.

Protein expression profiling of Coccidioides posadasii by two-dimensional differential in-gel electrophoresis and evaluation of a newly recognized peroxisomal matrix protein as a recombinant vaccine candidate.

Coccidioides posadasii and Coccidioides immitis are dimorphic, soil-dwelling pathogenic ascomycetes endemic to the southwestern United States. Infection can result from inhalation of a very few arthroconidia, but following natural infection, long-lived immunity is the norm. Previous work in the field has shown that spherule-derived vaccines afford more protection than those from mycelia. We have used two-dimensional differential in-gel electrophoresis coupled with nano-high-performance liquid chromatography-tandem mass spectrometry to directly assess both absolute abundance and differential expression of proteins in the spherule and the mycelial phases of C. posadasii with the intent to identify potential vaccine candidates. Peptides derived from 40 protein spots were analyzed and a probable identity was assigned to each. One spherule-abundant protein, identified as Pmp1, showed homology to allergens from Aspergillus fumigatus and other fungi, all of which exhibit similarity to yeast thiol peroxidases. Recombinant Pmp1 was reactive with serum from individuals with both acute and protracted disease, and evoked protection in two murine models of infection with C. posadasii. These results demonstrate the utility of proteomic analysis as a point of discovery for protective antigens for possible inclusion in a vaccine candidate to prevent coccidioidomycosis.

Coccidioides posadasii contains a single 1,3-beta-glucan synthase gene that appears to be essential for growth.

1,3-beta-Glucan synthase is responsible for the synthesis of beta-glucan, an essential cell wall structural component in most fungi. We sought to determine whether Coccidioides posadasii possesses genes homologous to known fungal FKS genes that encode the catalytic subunit of 1,3-beta-glucan synthase. A single gene, designated FKS1, was identified, and examination of its predicted protein product showed a high degree of conservation with Fks proteins from other filamentous fungi. FKS1 is expressed at similar levels in mycelia and early spherulating cultures, and expression decreases as the spherules mature. We used Agrobacterium-mediated transformation to create strains that harbor DeltaFKS1::hygB, a null allele of FKS1, and hypothesize that Fks1p function is essential, due to our inability to purify this allele away from a complementing wild-type FKS1 allele in a heterokaryotic strain. The heterokaryon appears normal with respect to growth rate and arthroconidium production; however, microscopic examination of strains with DeltaFKS1::hygB alleles revealed abnormal swelling of hyphal elements.

Localization within a proline-rich antigen (Ag2/PRA) of protective antigenicity against infection with Coccidioides immitis in mice.

Subunits of a proline-rich coccidioidal antigen (Ag2/PRA) of Coccidioides immitis were analyzed by comparison as vaccines in mice. The optimal dose of plasmid vaccine encoding full-length Ag2/PRA was determined to be between 10 and 100 microg. Mice vaccinated with plasmids encoding amino acids (aa) 1 to 106 were as protective as full-length Ag2/PRA (aa 1 to 194). The subunit from aa 27 to 106 was significantly but less protective. Plasmids encoding aa 90 to 151 or aa 90 to 194 were not protective. Analogous results were obtained with recombinant vaccines of the same amino acid sequences. In addition, mixtures of aa 90 to 194 with either aa 1 to 106 or aa 27 to 106 did not enhance protection compared to the active single-recombinant subunits alone. Humoral response of total immunoglobulin G (IgG) and subclasses IgG1 and IgG2a were detectable in subunit vaccinations but at significantly (100-fold) lower concentrations than after vaccination with plasmids encoding full-length Ag2/PRA. Since virtually all protection by vaccination with full-length Ag2/PRA can be accounted for in the first half of the protein (aa 1 to 106), this subunit could make a multicomponent vaccine more feasible by reducing the quantity of protein per dose and the possibility of an untoward reactions to a foreign protein.