Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils.

Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever) in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis) as well as upon contact (by serum-dependent adhesion and phagocytosis). This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.

Identification, molecular characterization, and expression analysis of a DOMON-like type 9 carbohydrate-binding module domain-containing protein of Coccidioides posadasii.

Previously, we investigated the effect of N-acetylglucosamine (GlcNAc) on Coccidioides posadasii chitinolytic enzymes during in vitro spherule-endospore (S/E) phase culture. During those studies, sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of supernatants from S/E phase cultures grown in Converse medium with or without added GlcNAc revealed a ∼ 28-kDa band (CFP28), whose abundance was increased by GlcNAc in parallel with the chitinolytic enzymes. Mass spectrometry (MS) of the CFP28 band revealed peptides that matched an open reading frame found in the tentative consensus sequence, TC20325, retrieved from the Dana Farber Cancer Institute C. posadasii Gene Index Database. The TC20325 cDNA sequence was used to design internal primers based on MS peptides and a full-length cDNA was isolated using a combination of rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. The deduced amino acid sequence of the full-length cDNA consists of 231 amino acid residues with a 19 aa signal peptide. The mature protein has a calculated molecular mass of ∼ 24.5 kDa, a theoretical pI of 6.09, and consists of a single DOMON-like type 9 carbohydrate-binding module (CBM9-like-3) conserved domain. The protein shares the highest sequence similarity (≥57%) to hypothetical proteins from fungi within the Pezizomycotina subphylum of Ascomycota. Antiserum against a recombinant version of CFP28 recognized native CFP28 in S/E phase cells and culture supernatants. CFP28 mRNA and protein expression were detectable in S/E phase in Converse medium, but were increased in the presence of added GlcNAc. Purified native CFP28 reacted with pooled sera from patients with coccidioidomycosis.

Early treatment with fluconazole may abrogate the development of IgG antibodies in coccidioidomycosis.

BACKGROUND: We have observed a number of patients who fail to develop coccidioidal complement fixing (CF) antibody (immunoglobulin [IgG]) after the initiation of early antifungal therapy. Although this is the first description of this phenomenon in mycology, a precedent for the abrogation of the immune response has been observed in other conditions, including primary syphilis and primary Lyme disease. METHODS: We conducted a retrospective case-control study to determine any patient-specific risk factors associated with this observation. Additionally, in vitro analysis of the coccidioidal CF (IgG) antigen (Cts1) was performed after Coccidioides was grown under escalating fluconazole concentrations. RESULTS: Seventeen patients persistently positive for coccidioidal IgM antibodies without developing an IgG response (cases) were compared with 64 consecutive patients who did develop coccidioidal CF (IgG) antibodies (controls). Early treatment with antifungals (within 2 weeks of symptom onset) was associated with an abrogation of IgG antibody production (P < .001). With immunodiffusion testing, control serum demonstrated a lack of IgG seroreactivity when Coccidioides posadasii grown in the presence of escalating fluconazole doses (0.5-128 µg/mL) was used as the antigen; however, control serum remained seroreactive for the presence of IgM. The coccidioidal IgG antigen (Cts1) was shown to be diminished when cultures were grown in the presence of fluconazole, lending further in vitro plausibility to our findings. CONCLUSIONS: The abrogation of an IgG response in patients treated early in the course of coccidioidal infection may complicate serodiagnosis and epidemiologic studies, and further study to determine the potential clinical implications should be performed.

Molecular cloning, characterization and expression analysis of two beta-N-acetylhexosaminidase homologs of Coccidioides posadasii.

Two full-length cDNAs were isolated from Coccidioides posadasii that encode two deduced proteins (CpHEX1 and CpHEX2) with homology to the glycosyl hydrolase 20 family of beta-N-acetylhexosaminidases. CpHEX1 consists of 595 amino acids, has a predicted molecular mass of 68 kDa and shares the highest identity with the N-acetylhexosaminidase (NAGA) of Aspergillus nidulans, while CpHEX2 consists of 603 amino acids, has a predicted molecular mass of 68.5 kDa and shares the highest identity with NAG1 from Paracoccidioides brasiliensis. CpHEX1 and CpHEX2 share only 23% identity and have dissimilar homologies showing more identity with other fungal beta-N-acetylhexosaminidases than with each other. Phylogenetic analysis of selected beta-N-acetylhexosaminidases placed CpHEX1 in a cluster with the orthologs from A. nidulans, Aspergillus oryzae, Penicillium chrysogenum and Candida albicans, while CpHEX2 grouped with the orthologs from P. brasiliensis and the Trichoderma spp. beta-N-acetylhexosaminidase activity and transcripts encoding CpHEX1 and CpHEX2 were detected in vitro during the spherule-endospore (SE) phase. Expression of the Cphex1 transcript exhibited a temporal increase that correlated with beta-N-acetylhexosaminidase activity, while the Cphex2 transcript remained relatively constant. The addition of N-acetylglucosamine to the cultures increased beta-N-acetylhexosaminidase activity and the expression of the Cphex1 transcript. A native beta-N-acetylhexosaminidase enzyme was purified from in vitro SE phase and identified as CpHEX1 by mass spectrometric analysis. Both the CpHEX1 and CpHEX2 cDNAs were expressed as recombinant fusion proteins and purified under denaturing conditions to apparent homogeneity but they lacked enzymatic activity.

Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii Cu,Zn superoxide dismutase identified by proteomic analysis of the coccidioidal T27K vaccine.

Previous studies have demonstrated that the coccidioidal T27K vaccine preparation is protective in mice against respiratory challenge using Coccidioides posadasii (C. posadasii) arthroconidia. Proteomic methods have been employed to define the molecular components within the vaccine. This method has led to the identification of novel and previously uncharacterized coccidioidal proteins including a Cu,Zn superoxide dismutase. A two-dimensional gel of the T27K vaccine was run and spots were excised for mass spectrometric analysis. One peptide was obtained from the T27K gel that matched a TIGR C. posadasii 2.0 gene index tentative consensus sequence, TC1072, which is similar to fungal Cu,Zn superoxide dismutase. Activity assays performed with native PAGE gels of the T27K vaccine showed that the vaccine contains superoxide dismutase. The cDNA encoding the enzyme has been cloned and sequenced and expressed as a recombinant protein.

Characteristics of the protective subcellular coccidioidal T27K vaccine.

While the whole killed spherule vaccine, protective in mice and monkeys, did not prevent coccidioidal disease in humans, the 27K vaccine, a soluble derivative, retains protective activity in mice with little irritant action. Gel filtration and anion exchange fractions of thimerosal-inactivated spherules (T27K), when administered with alum adjuvant, also protect mice against lethal respiratory coccidioidal challenge. However, the superb protection afforded by T27K antigens is maintained for some 3 months, but may then diminish. This appears unrelated to the aging of the mice. Prolongation of the protective action may require addition of a different adjuvant or administration of booster doses of vaccine.

Molecular cloning and expression of a cDNA encoding a Coccidioides posadasii 1,2-alpha-mannosidase identified in the coccidioidal T27K vaccine by immunoproteomic methods.

The coccidioidal T27K vaccine is protective in mice against respiratory challenge with Coccidioides posadasii (C. posadasii) arthroconidia. The vaccine is a subcellular multicomponent preparation that has not been fully characterized. To identify potential protective antigens in the heterogeneous mixture, the vaccine has been separated by two-dimensional gel electrophoresis and then analyzed for seroreactive proteins using immunoblot analysis with pooled sera from patients with coccidioidomycosis. Two seroreactive spots of identical apparent molecular weight were identified and sequenced using tandem mass spectrometry. Three peptides were generated, two of which matched a tentative consensus sequence in the TIGR C. posadasii 2.0 gene index database that is similar to fungal 1,2-alpha-mannosidases. The 5' and 3' ends of the mannosidase cDNA were mapped using rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR), and a full-length cDNA was then obtained using reverse-transcription (RT) PCR. The cDNA was cloned and sequenced and expressed as a recombinant protein. The predicted protein consists of 519 amino acids, has a theoretical molecular weight and pI of 56,918 Da and 4.84, respectively, and is very similar (>60%) to other fungal 1,2-alpha-mannosidases. Class I 1,2-alpha-mannosidase enzyme activity was also detected in the T27K vaccine using the substrate, Man-alpha-1,2-Man-alpha-OCH(3) in a spectrophotometric assay.

Method for the simultaneous determination of retinol and beta-carotene concentrations in human tissues and plasma.

To understand differential tissue distribution of retinoids and carotenoids, as it might influence biological processes in humans, we developed and demonstrated a method for measuring them in selected human tissues. The method includes internal standards and a secondary reference standard to eliminate the need for external standard calibration and to minimize sample-handling errors. Tissues were digested (saponified) in ethanolic KOH. Retinol and beta-carotene were extracted with organic solvent containing internal standards. Analytes were separated using isocratic liquid chromatography and quantified at 325 nm for retinol and 450 nm for beta-carotene. Plasma was analyzed in a similar way but without saponification. Retinal-O-ethyloxime and beta-apo-12'-carotenal-O-t-butyloxime served as internal standards. Plasma, breast, and fat from breast surgery patients and colon, liver, muscle, and fat from colon surgery patients were analyzed. Within-day relative standard deviations (RSDs) for plasma were <0.04 for beta-carotene and <0.03 for retinol, between-day RSDs were <0.05 for beta-carotene and <0.04 for retinol. Saponification ensured complete extraction of retinol and beta-carotene and removal of triglycerides that "foul" chromatographic columns. It seems retinol and beta-carotene concentrations in tissues and blood of cancer patients are the same or higher than those in corresponding tissues of patients without these cancers.