Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5′-CACAGCCAC-3′ and 5′-CCCTGCCCC-3′ sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -B and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The ΔpmcA and ΔpmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the ΔcalA and ΔcrzA mutant strains. However, only the A. fumigatus ΔpmcA was avirulent in the murine model of invasive pulmonary aspergillosis

N-Terminal Gly224–Gly411 Domain in Listeria Adhesion Protein Interacts with Host Receptor Hsp60

Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated.Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met(1)-Pro(223)) and N2 (Gly(224)-Gly(411)), and the ADH region contains C1 (Gly(412)-Val(648)) and C2 (Pro(649)-Val(866)). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells.The N2 subdomain exhibited the greatest affinity for Hsp60 with a K(D) of 9.50±2.6 nM. The K(D) of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P<0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells.These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies

Association of Calcineurin with the COPI Protein Sec28 and the COPII Protein Sec13 Revealed by Quantitative Proteomics

Calcineurin is a calcium-calmodulin-dependent serine/threonine specific protein phosphatase operating in key cellular processes governing responses to extracellular cues. Calcineurin is essential for growth at high temperature and virulence of the human fungal pathogen Cryptococcus neoformans but the underlying mechanism is unknown. We performed a mass spectrometry analysis to identify proteins that associate with the calcineurin A catalytic subunit (Cna1) in C. neoformans cells grown under non-stress and high temperature stress conditions. A novel prioritization strategy for mass spectrometry data from immunoprecipitation experiments identified putative substrates and proteins potentially operating with calcineurin in common pathways. Cna1 co-purified with proteins involved in membrane trafficking including the COPI component Sec28 and the COPII component Sec13. The association of Cna1 with Sec28 and Sec13 was confirmed by co-immunoprecipitation. Cna1 exhibited a dramatic change in subcellular localization during high temperature stress from diffuse cytoplasmic to ER-associated puncta and the mother-bud neck and co-localized with Sec28 and Sec13

Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

BACKGROUND:The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS), resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface. METHODOLOGY:The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen. CONCLUSIONS:The results of this study provide evidence for the cooperative role of multiple virulence determinants in C. neoformans pathogenesis and suggest new avenues for the development of anti-infective agents in the prevention of fungal tissue invasion