Urease Produced by Coccidioides posadasii Contributes to the Virulence of This Respiratory Pathogen

Abstract

Urease activity during in vitro growth in the saprobic and parasitic phases of Coccidioides spp. is partly responsible for production of intracellular ammonia released into the culture media and contributes to alkalinity of the external microenvironment. Although the amino acid sequence of the urease of Coccidioides posadasii lacks a predicted signal peptide, the protein is transported from the cytosol into vesicles and the central vacuole of parasitic cells (spherules). Enzymatically active urease is released from the contents of mature spherules during the parasitic cycle endosporulation stage. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense host inflammatory response. Ammonia production by the spherules of C. posadasii is markedly increased by the availability of exogenous urea found in relatively high concentrations at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of C. posadasii resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. BALB/c mice challenged intranasally with the mutant strain showed increased survival, a wellorganized granulomatous response to infection, and better clearance of the pathogen than animals challenged with either the parental or the reconstituted (revertant) strain. We conclude that ammonia and enzymatically active urease released from spherules during the parasitic cycle of C. posadasii contribute to host tissue damage, which exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen.

A Genetically Engineered Live Attenuated Vaccine ofCoccidioides posadasiiProtects BALB/c Mice against Coccidioidomycosis

“…This permitted comparison of expression profiles of the eight CTS genes during selected stages of spherule development. We examined levels of gene expression during spherule septation (72-h parasitic-phase cultures), digestion of the septal wall complex and initiation of endospore formation (96 h), and endospore release from ruptured spherules (120 to 132 h) (32) by quantitative real time-PCR (QRT-PCR) as reported previously (22). The generation of cDNA from parasitic cell-derived total RNA preparations was conducted as reported elsewhere (9).…”

“…Putative cts2⌬::HPH and ard1⌬/cts3⌬::HPH transformants were selected on GYE agar plates supplemented with 75 g/ml of HmB and subsequently maintained on a growth medium containing GYE plus 100 g/ml HmB. Southern hybridization was employed as reported previously (32) to confirm the targeted disruption of the CTS2 and ARD1/CTS3 genes. In brief, genomic DNA isolated from candidate transformants was digested with endonucleases selected on the basis of the restriction maps of the genes (see Fig.…”

“…The 3.6-kb fragment includes a hygromycin B phosphotransferase gene (HPH) which when expressed with the appropriate fungal promoter and terminator in C. posadasii (38) confers resistance to the growth inhibitor hygromycin B (HmB) as reported previously (40). The final 6.9-kb plasmid construct (pcts2⌬::HPH) used to disrupt the CTS2 gene was linearized by digestion of the modified Topo vector with ApaI and DraI and purified for subsequent transformation of the C. posadasii parental strain as described previously (32). The 6,891-bp ApaI/DraI plasmid construct was comprised of a 3,597-bp fragment of pAN7.1, with flanking 5Ј and 3Ј regions of the CTS2 gene (828-bp and 1,242-bp, respectively).…”

Innovation of the Parasitic Cycle of Coccidioides spp.

“…PMN are the dominant cells in this response, although macrophages are also present. However, these cells only partially disable Coccidioides growth and are unable to kill it (Vincendeau et al, 2003;Mirbod-Donovan 2006;Hung et al, 2007).…”

Virulence Regulation of Phytopathogenic Fungi by pH

“…Indeed, C. coccodes mutants with reduced ammonia production exhibited diminished pathogenicity (1, 2, 100), and tomato rboh mutants, which displayed less PCD, were less susceptible than the WT tomato to Colletotrichum necrotrophic attack (2). Effects of ammonia were observed not only in plant-fungal interactions: Coccidioides immitis, a fungal pathogen of humans that causes respiratory disease, is known to alkalinize its environment with ammonia (36, 73, 97); mutants with compromised ammonia secretion were less pathogenic (98), and ammonia was shown to compromise the rat-host immune response and cause inflammation (67). Further, in rat astrocytes and brain edema ammonia was shown to activate NADPH oxidase, which was followed by autophagy (17, 31, 68,71,74,75,105,125,133), which suggests that ammonia possesses a global role of action on NADPH oxidase and cell death.…”