Abstract
Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. Candida albicans is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein “High affinity glucose transporter 1” (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased in vitro phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of C. albicans.
Highlights
MATERIALS AND METHODSCandida albicans is a normal resident of the human oral cavity, the gastrointestinal tract (Strijbis et al, 2014) and vaginal mucous membranes (Peters et al, 2014)
As revealed by fluorescence activated cell sorting (FACS) analysis, a significantly higher proportion of parental SN152 yeast cells bound Factor H (FH) compared to the hgt1 / null mutant or SN152 pre-incubated with anti-Hgt1p or by the chimeric molecule FH6.7/Fc (Figure 4A)
In the present study we increased the knowledge on Hgt1p expression showing a higher expression of HGT1 in media with low glucose concentration (0.1%) – not unexpected for a glucose transporter – which resembles the physiological concentration in humans (Buu and Chen, 2014)
Summary
Candida albicans is a normal resident of the human oral cavity, the gastrointestinal tract (Strijbis et al, 2014) and vaginal mucous membranes (Peters et al, 2014). The polyclonal antibody anti-Hgt1p, used in this project, was commercially generated by ProteoGenix, Schiltigheim, France It recognizes an epitope exposed in an extracellular portion of Candida albicans cell membrane (upper arrows). The parental strain SN152 was blocked with 1 μg of anti-Hgt1p antibody, 1 μg of the chimeric molecule FH6.7/Fc or with 1 μg of an irrelevant rabbit anti-human IgG antibody (all at 10 μg/ml) for 45 min at 37◦C before opsonization. The positive population was detected by one color fluorescence density plot SSC vs FITC for anti-FH and SSC vs PE for C3b/iC3b using FACS Calibur and Cell Quest Pro software (BD Biosciences, San Diego, CA, United States). In a different set up, the parental strain SN152 was blocked with 1 μg anti-Hgt1p or with 1 μg of the chimeric FH6.7/Fc (both at 10 μg/ml) for 45 min at 37◦C before the opsonization. In all experiments a p-value of < 0.05 was considered statistically significant
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