Recombinant Coccidioides immitis Vacuolar ATPase assembly integral membrane protein VMA21 (VMA21)

Overview of Recombinant Coccidioides immitis Vacuolar ATPase Assembly Integral Membrane Protein VMA21 (VMA21)

Coccidioides immitis is a fungus that causes coccidioidomycosis, also known as Valley fever . An effective vaccine against coccidioidomycosis could significantly improve the health of people living in areas where the infection is common .

VMA21 as a Target for Vaccine Development

• T-cell-reactive protein (rTCRP) The identification of C. immitis antigens that stimulate T cells is important for understanding host defense and developing effective vaccines . A 48-kDa T-cell-reactive protein (TCRP) expressed by parasitic cells stimulates T cells and the production of gamma interferon in mice immunized with C. immitis spherules . Antibodies that react with recombinant TCRP (rTCRP) have been found in the serum of patients with coccidioidal infection . Immunizing mice with rTCRP provides a modest level of protection against C. immitis .

• Multivalent recombinant protein vaccine A multicomponent vaccine against coccidioidomycosis has the potential to stimulate a broader range of T-cell clones and may be more effective in evoking protection in immunologically heterogenous human populations .

VMA21 and Vacuolar ATPase

VMA21 is an assembly factor gene of the V0 domain of the V-ATPase complex, required for intracellular pH homeostasis .

Saccharomyces cerevisiae vacuolar H+-ATPase (V-ATPase) is a multi-subunit complex with a peripheral membrane sector (V1) for ATP hydrolysis and an integral membrane sector (V0) for proton translocation . Biogenesis of V0 requires an endoplasmic reticulum (ER)-localized accessory factor, Vma21p . In vma21Δ cells, the major proteolipid subunit of V0 fails to interact with the 100-kDa V0 subunit, Vph1p, indicating that Vma21p is necessary for V0 assembly .

VMA21 Structure and Function

VMA21 encodes an 8.5-kDa integral membrane protein predicted to span the membrane twice, with both the amino- and carboxy-termini facing the cytosol . The carboxy-terminus of Vma21p contains a -KKXX ER-retrieval sequence . Mutation of the -KKXX motif in Vma21p to -QQXX leads to the mislocalization of Vma21(QQ)p to the vacuolar membrane . Vma21p cycles between the ER and Golgi and may have a role in the transport of V0 subcomplex out of the ER .

VMA21 in Colorectal Cancer

VMA21 is upregulated in colorectal cancer epithelial cells . High VMA21 expression is an independent predictor of disease-specific survival (DSS) . VMA21 overexpression decreased CRC growth, whereas VMA21 knockdown increased CRC growth in vitro and in vivo . VMA21 expression suppresses CRC growth and predicts favorable DSS in patients with stage I-III disease .

Studies on Differentially Expressed Genes

Transcriptional studies have shown that the sets of genes expressed in the mycelia and spherule phases of Coccidioides are different . In C. immitis and C. posadasii, 13% of genes are preferentially expressed in hyphae, and 19% are preferentially expressed in spherules . There is substantial overlap between the differentially expressed genes in C. immitis and C. posadasii, but about 50% of the genes that are up-regulated in spherules are only up-regulated in one species .

Urease and Ureidoglycolate Hydrolase (UGH)

Deletion mutants lacking both UGH and URE make less ammonia than either mutant alone and are much less pathogenic than the wildtype organism or either mutant . The ability to produce ammonia and alkalinize the tissue around Coccidioides is a vital element of pathogenesis .

Engineered Avirulent Mutants

An engineered avirulent mutant grown in Converse medium in vitro grew as hyphae with polar swelling . The mutant was avirulent in mice, and no viable organisms could be recovered from the lung or spleen . This transcription factor influenced the expression of many of the genes coding for differentiation into spherules and pathogenicity .

Immunogenicity of Recombinant Proteins

• C57BL/6 mice immunized with rPep1 show a marked increase in percent survival after i.n. challenge with Coccidioides compared to nonvaccinated mice .

• Recombinant Plb and Amn1 are additional T-cell-reactive antigens that can be incorporated into a vaccine against coccidioidomycosis .

• Necropsied mice that had been vaccinated with either a single recombinant protein or the three combined proteins and survived to 90 days after intranasal challenge all showed that a granulomatous response had occurred in their lungs . Histopathological examinations of the lung tissue of these mice revealed well-differentiated granulomata, indicative of a protective response to coccidioidal infection .