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Chapter 26 : Virulence Mechanisms of Coccidioides
Category: Fungi and Fungal Pathogenesis
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This chapter examines the various mechanisms utilized by Coccidioides to survive within the hostile environment of the host. Much of the discussion which follows in this chapter is dependent on the understanding of the events that occur during conidiogenesis and stages of parasitic cell development of Coccidioides within the mammalian host. The authors have reported that extracts of Coccidioides arthroconidia can inhibit in vitro production of superoxide anion by rat alveolar macrophages. Not surprisingly, BLASTx searches of the translated genomic database of Coccidioides have identified homologs of reported fungal Cu/Zn superoxide dismutases (SODs), catalases , glutathione peroxidases, and thioredoxin peroxidases. They have cloned the ARG gene of Coccidioides and determined that its level of expression during the infection cycle within murine lung tissue is constitutive. The chapter presents evidence that Coccidioides is well equipped with mechanisms to withstand an attack by the sophisticated innate and acquired immune defense systems of the mammalian host. The final section of the chapter examines the impact of a biased Th2 pathway of the immune response to Coccidioides infection on macrophage function. This discussion of virulence mechanisms of Coccidioides is intended to stimulate research, since the majority of factors described are in need of further investigation.
The saprobic and parasitic cycles of Coccidioides.
Morphological features of arthroconidia and spherule initials. (A) Thin section of arthroconidium. The outer conidial wall (OCW) contains hydrophobins which appear as fascicles of rodlets (inset) at the cell surface. ICW, inner conidial wall; Mt, mitochondrion; N, nucleus. Bar, 1μm (inset, 20 nm). (B and C) Arthroconidia grown on plate culture containing glucose-salts medium (126) supplemented with L-DOPA (1 mM) were reacted with either a monoclonal antibody raised against C. neoformans melanin (6D2) (B) or a control monoclonal antibody raised against C. neofor-mans glucuronoxylomannan (2D10) (C), both provided by J. Nosanchuk. The cells were subsequently exposed to fluorescein isothiocyanate-conjugated goat anti-mouse Ig. Bar in panel B represents 2μm. (D) Mithramycin-stained spherule initials (germinated arthroconidia) grown at 37°C in the presence of murine tracheal explants. Bar, 4μm. (E) Thin section of spherule initial, grown as described in panel D, revealing an early stage in differentiation of the lipid-rich spherule outer wall (SOW) layer. Remnants of the outer conidial wall (OCW) are visible. Bar, 2μm. (F) Mithramycin-stained, multinucleate spherule which had nearly completed its isotropic growth phase. Note the presence of a large, central vacuole. Bar, 10μm.
Spherule segmentation and early stage of endospore differentiation. (A to C) Thin sections of spherules which show early stages of segmentation wall formation. Pm, plasma membrane; Sph. wall, spherule wall. Bar, 1μm. (D) Thin section of segmented spherule showing compartments formed by growth and fusion of invaginated segmentation wall (Seg. wall). Note the residual cytoplasm trapped between vacuolar membrane and compartments. Bar, 5μm. (E) Thin section of spherule at early stage of endosporulation. Note that the vacuolar membrane has ruptured, spheroidal endospores (End.) have been released from compartments, and the segmentation wall has partially disappeared. Arrow indicates cytoplasmic debris. Bar, 4μm.
Digestion of the segmentation wall is essential for endospore maturation. (A) Thin section of endospores contained within the maternal spherule. Note the fragments of digested segmentation wall (Seg. wall). EW, endospore wall. Bar, 1μm. (B and C) Light micrographs of endosporulating spherules (B) and sterile spherules (C) derived from cultures of the wild-type (parental) strain of C. posadasii and the ∆cts2/∆cts3 mutant strain, respectively. Bar, 20μm. (D) Survival plot of BALB/c mice challenged intranasally with arthroconidia derived from the parental strain (C735) or arthroconidia isolated from the single or double chitinase gene knockout strains. Mice were challenged separately with 50 arthroconidia of the parental, ∆cts2, and ∆cts3 strains and with 200 conidia of the ∆cts2/∆cts3 strain.
Endospore release and host response. (A) Hemotoxylin-eosin-stained paraffin section through a terminal bronchiole of a mouse lung infected with C. posadasii (C735 strain), showing an abundance of neutrophils (PMNs) which have surrounded and entered a ruptured spherule. Bar, 20μm. (B) Periodic acid-Schiff-stained paraffin section of mature, ruptured spherules in an infected murine lung. Bar, 20μm.
Sensitivity of Coccidioides cell types to nitric oxide exposure. Arthroconidia, spherule initials (germinated arthroconidia after 48 h of incubation in glucose-salts medium), and segmented/endosporulating spherules (from 144-h parasitic-phase cultures) were isolated, washed in PBS, and transferred to 96-well plates (105 cells in 100 μl of PBS per well), to which different amounts of SIN-1 were added as indicated. The 96-well plates were sealed and incubated in the absence of light at 37°C for 3 or 6 h. Control cells were incubated in PBS alone. Serial dilutions of the cell suspensions were plated onto GYE medium to determine number of viable fungal cells, which were recorded as CFU. The data are presented as percentage of growth inhibition, which was calculated on the basis of the formula [1 — (CFU of SIN-1-exposed cells ÷ CFU of control cell suspension)] × 100%.
Coccidioides down-regulates TNF-α production by macrophages. (A) Thin-layer chromatographic separation of SOW polar lipids using chloroform-acetic acid-methanol-water (150:50:10:4.4) as the solvent. (B) Arthroconidia (Arth.) and/or spherule initials (Sph.In) (as described in the legend to Fig. 6 ) were harvested, washed in PBS, and coincubated with a macrophage cell line (RAW 2647, American Type Culture Collection, Manassas, Va.) (1:1 ratio) for 4 h in Dulbecco modified Eagle medium as reported ( 109 ). The supernatants were isolated, and the TNF-α was quantified by ELISA using an OptEIA mouse TNF-α detection kit (PharMingen, San Diego, Calif.). In a separate experiment, macrophages were exposed to cytochalasin D (2.5μg/ml of 1% dimethyl sulfoxide [final concentration]; Biosource, Camarillo, Calif.) for 2 h prior to incubation with fungal cells in order to inhibit phagocytosis. Also, in a separate experiment, spherule initials were extracted with chloroform-methanol (2:1; high-performance liquid chomatography grade) (chlor:meth extract) for 30 min at 4°C as reported previously ( 79 ), the extract supernatant was collected by centrifugation, and the lipid layer was aspirated, dried in an N2 stream, and resuspended in petroleum ether (PE). No protein could be detected in this extract by SDS-PAGE. Aliquots of the resolubilized extract were tested for their effect on TNF-α production by macrophages as described in the text. The extracted, intact spherule initials were also tested for their influence on TNF-α production. (C) Quantitative real-time PCR analysis of C. posadasii PLB expression in vitro during stages of parasitic cell development. Relative amounts of PLB transcript produced by spherule initials (Sph. In.) and endosporulating spherules (End. Sph.) were compared to that of segmented spherules (Seg. Sph.), which was assigned an arbitrary value of 1.
SOWgp is an immunodominant, parasitic cell surface antigen. (A) Structure of the SOWgp antigen (Silveira isolate). (B) Bacterialy expressed, full-length recombinant SOWgp protein (mature protein [MP]) and fragments of SOWgp (83-residue N-terminal fragment [N-t], 179-residue repeat fragment [Rpt.], and 67-residue C-terminal fragment [C-t]) were separated by SDS-PAGE and tested in an immunoblot assay for reactivity with antiserum from a patient with confirmed coccidioidomycosis. The lower-molecular-mass band in the lane containing the C-t fragment is an Escherichia coli peptide contaminant. (C) Results of ELISA showing reactivity of the recombinant repeat fragment (Rpt.) of SOWgp with control human sera and sera from patients with confirmed coccidioidal infection. The concentration of the recombinant repeat protein bound to wells of the microtiter plate was 10 ng/well. Goat anti-human IgG (H + L) conjugated to peroxiclase was used for detection of adsorbed antibody. Antibody titers were determined as reported previously ( 128 ).
Mep1 digestion of SOWgp permits endospores to evade host detection. A phase contrast micrograph (A) and a matching immunofluorescence light micrograph (B) of a spherule which has released its endospores are shown. The cells in panel B were incubated with antibody raised against purified, native SOWgp. Note the absence of fluorescent antibody reactivity with the surface of endospores, contrasting with the high avidity of antibody for the walls of the ruptured and nonruptured spherules. Bar, 40μm.
Host exposure to SOWgp contributes to Th2-biased immune response to Coccidioides infection. (A) C57BL/6 mice were challenged by the intranasal route with 35 viable arthroconidia of C. posadasii (C735 strain) and euthanized 30 days later. Total splenocytes were pooled from three infected animals, and T cells were isolated as reported previously ( 128 ). The T cells were transferred to RPMI plus 10% FBS, stimulated with two different concentrations of purified native SOWgp, and incubated for 48 h. Control cells were incubated in medium alone. The super-natants were tested for the concentration of selected cytokines as reported previously ( 220 ). (B) Normal C57BL/6 mice (Igh-6 +/+) or B-cell knockout mice (Igh-6+/+) were challenged as in panel A, their T cells were isolated and stimulated with the recombinant repeat fragment of SOWgp (rSOWgp rpt.) (10μg/ml), and the concentrations of selected cytokines were determined as above. (C) Results of ELISA with mouse sera obtained from Coccidioides-infected animals immunized with the rSOWgp repeat fragment (1μg/dose; twice) versus nonimmunized controls. Mice were challenged intranasally 4 weeks after immunization, and their antibody titers against the recombinant repeat protein were determined as reported previously ( 101 ).
Macrophage engulfment of the SOW/SOWgp complex stimulates production of Th2-type cytokines. (A) Thin section of presegmented spherule showing the surface layer of SOW plus SOWgp (stained with lipophilic osmium tetroxide). Fragments of this stained layer have been shed and engulfed by phagocytes. Bar, 4μm. (B) Groups of BALB/c mice were challenged separately by the intranasal route with an equal number of viable arthroconidia (approximately 80) derived from either the parental strain (C735) or the ∆sowgp mutant strain. BAL fluid samples were collected at various times post-challenge, and the concentration of selected cytokines at each time point was determined by ELISA as reported previously ( 220 ). BAL fluid samples from three mice per time point were examined.
Summary of metabolic events which occur in murine macrophages in response to Th2-type cytokine stimulation. A shift in balance between arginase I and iNOS activity in the direction of the former results in depletion of arginine and reduction in NO production but increased synthesis of ornithine and urea. Fungal uptake of urea and the concomitant increase in intracellular and extracellular urease activity results in high concentrations of NH3/NH4 + secreted by the pathogen at sites of infection.
An elevated level of host arginase I production compromises the host response to coccidioidal infection. (A) Quantitative real-time PCR of murine arginase I (ARGI) expression in the lungs of C57BL/6 mice at 7 days after intranasal infection with 80 arthroconidia isolated from either the parental (P) or mutant (∆sowgp) strain of C. posadasii, compared to expression of the constitutive murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Control mice were challenged with PBS alone. The data are presented as the mean values from analyses of three mice for each group. The P value indicates a significant difference between the two groups of infected C57BL/6 mice. (B) BAL fluid samples collected from C57BL/6 mice at 7 days after intranasal infection with 80 arthroconidia from either the parental (P) or mutant (∆sowgp) strain were separated by SDS-PAGE and immunoblotted using anti-arginase I (α-Arg I) monoclonal antibody (1:500; BD Biosciences PharMingen, Franklin Lakes, N.J.). (C) Quantitative real-time PCR of murine iNOS gene expression in the lungs of mice infected with either the parental (P) or mutant (∆sowgp) strain of C. posadasii. Infection conditions were the same as in the experiment in panel A. (D) BALB/c mice treated with an inhibitor of arginase I activity (nor-LOHA; Alexis, San Diego, Calif.) prior to and after intranasal challenge with a lethal inoculum of C. posadasii (80 arthroconidia) showed a significant increase in percent survival compared to untreated mice. Noninfected control mice were treated with nor-LOHA alone. Administration of nor-LOHA was via the intraperitoneal route, beginning 1 h prior to infection (6 mg/kg of body weight in 100μl of PBS; lipopolysaccharide free), followed by intraperitoneal administration of the same amount of nor-LOHA once per day for 45 days post-challenge.
(A) Summary of putative virulence factors expressed by arthroconidia, spherule initials, and endospores of Coccidioides during their interaction with host phagocytes. (B) Summary of putative virulence factors expressed by mature spherules and endosporulating spherules of Coccidioides during their extracellular association with the host. ArgI, host arginase I; FA, fatty acids; Mϕ, macrophage; PGs, prostaglandins; Plb, phospholipase B; Mep1, Coccidioides secreted metalloproteinase; PMN, polymorphonuclear neutrophil.
Affymetrix mouse array 6 days post-challenge (U74A chip)