Influence of IgG Subclass on Human Antimannan Antibody-Mediated Resistance to Hematogenously Disseminated Candidiasis in Mice.

Candida albicans is a yeast-like pathogen and can cause life-threatening systemic candidiasis. Its cell surface is enriched with mannan that is resistant to complement activation. Previously, we developed the recombinant human IgG1 antimannan antibody M1g1. M1g1 was found to promote complement activation and phagocytosis and protect mice from systemic candidiasis. Here, we evaluate the influence of IgG subclass on antimannan antibody-mediated protection. Three IgG subclass variants of M1g1 were constructed: M1g2, M1g3, and M1g4. The IgG subclass identity for each variant was confirmed with DNA sequence and subclass-specific antibodies. These variants contain identical M1 Fabs and exhibited similar binding affinities for C. albicans yeast and purified mannan. Yeast cells and hyphae recovered from the kidney of antibody-treated mice with systemic candidiasis showed uniform binding of each variant, indicating constitutive expression of the M1 epitope and antibody opsonization in the kidney. All variants promoted deposition of both murine and human C3 onto the yeast cell surface, with M1g4 showing delayed activation, as determined by flow cytometry and immunofluorescence microscopy. M1g4-mediated complement activation was found to be associated with its M1 Fab that activates the alternative pathway in an Fc-independent manner. Treatment with each subclass variant extended the survival of mice with systemic candidiasis (P < 0.001). However, treatment with M1g1, M1g3, or M1g4, but not with M1g2, also reduced the kidney fungal burden (P < 0.001). Thus, the role of human antimannan antibody in host resistance to systemic candidiasis is influenced by its IgG subclass.

Next-generation computational genetic analysis: multiple complement alleles control survival after Candida albicans infection.

Candida albicans is a fungal pathogen that causes severe disseminated infections that can be lethal in immunocompromised patients. Genetic factors are known to alter the initial susceptibility to and severity of C. albicans infection. We developed a next-generation computational genetic mapping program with advanced features to identify genetic factors affecting survival in a murine genetic model of hematogenous C. albicans infection. This computational tool was used to analyze the median survival data after inbred mouse strains were infected with C. albicans, which provides a useful experimental model for identification of host susceptibility factors. The computational analysis indicated that genetic variation within early classical complement pathway components (C1q, C1r, and C1s) could affect survival. Consistent with the computational results, serum C1 binding to this pathogen was strongly affected by C1rs alleles, as was survival of chromosome substitution strains. These results led to a combinatorial, conditional genetic model, involving an interaction between C5 and C1r/s alleles, which accurately predicted survival after infection. Beyond applicability to infectious disease, this information could increase our understanding of the genetic factors affecting susceptibility to autoimmune and neurodegenerative diseases.

Influence of mannan and glucan on complement activation and C3 binding by Candida albicans.

The complement system is important for host resistance to hematogenously disseminated candidiasis. However, modulation of complement activation by cell wall components of Candida albicans has not been characterized. Although intact yeast display mannan on the surface, glucan, typically located in the interior, becomes exposed during C. albicans infection. We show here the distinct effects of mannan and glucan on complement activation and opsonophagocytosis. Previous studies showed that intact cells are resistant to initiation of complement activation through the alternative pathway, and antimannan antibody reverses this resistance via an Fc-independent mechanism. The present study shows that this mannan-dependent resistance can be overcome by periodate-borohydride conversion of mannose polysaccharides to polyalcohols; cells treated with periodate-borohydride initiate the alternative pathway without the need for antibody. These observations identify an inhibitory role for intact mannan in complement activation. Next, removal of the surface-displayed mannan by acid treatment of periodate-borohydride cells exposes glucan. Glucan-displaying cells or purified beta-glucan initiate the alternative pathway when incubated with the purified proteins of the alternative pathway alone, suggesting that C. albicans glucan is a natural activator of the alternative pathway. Finally, ingestion of mannan-displaying cells by human neutrophils requires anti-mannan antibody, whereas ingestion of glucan-displaying cells requires complement. These results demonstrate a contrasting requirement of natural antibody and complement for opsonophagocytosis of C. albicans cells displaying mannan or glucan. Thus, differential surface expression of mannan and glucan may influence recognition of C. albicans by the complement system.

Characteristics of Fc-independent human antimannan antibody-mediated alternative pathway initiation of C3 deposition to Candida albicans.

The complement system has an important role in host resistance to systemic candidiasis but regulation of complement activation by Candida albicans remains poorly defined. Previous studies have identified a requirement for naturally occurring antimannan IgG antibody in initiation of C3 opsonization of C. albicans through either the classical or alternative pathway. This study characterized antibody-dependent initiation of the alternative pathway using the recombinant human monoclonal antimannan Fab fragment M1 and its full-length IgG1 antibody M1g1. Kinetic analysis of C3b deposition onto C. albicans with flow cytometry demonstrated the ability of M1g1 to restore the activity of either the classical or alternative pathway to the yeast-absorbed normal human serum, but the Fc-free M1 Fab restored only the activity of the alternative pathway. This Fc-independent, antimannan Fab-mediated C3 deposition through the alternative pathway was also observed in a serum-free assay containing the six alternative pathway proteins and in C1q- or C2-depleted serum but not in factor B-depleted serum. M1- or M1g1-dependent alternative pathway initiation of C3b deposition occurred in an asynchronous manner at discrete sites that expanded to cover the entire cell surface over time as revealed with immunofluorescence microscopy, in contrast to a uniform appearance of initial C3 deposition through the classical pathway. Furthermore, antimannan Fab M1 promoted the assembly of the alternative pathway convertase on the cell surface seen as colocalization of C3 and factor B with immunofluorescence microscopy. Thus, human antimannan antibody has a distinct Fc-independent effector function in regulation of C3 deposition to C. albicans.

Human recombinant antimannan immunoglobulin G1 antibody confers resistance to hematogenously disseminated candidiasis in mice.

Mannan is a major cell wall component found in Candida species. Natural antimannan antibody is present in sera from most normal adults, but its role in host resistance to hematogenously disseminated candidiasis is unknown. The purpose of this study was to develop recombinant human antimannan antibody and to study its protective function. A phage Fab display combinatorial library containing Fab genes from bone marrow lymphocytes was screened with Candida albicans yeast cells and chemically purified mannan. One antimannan Fab, termed M1, was converted to a full-length immunoglobulin G1 antibody, M1g1, and M1g1 was produced in CHO cells. The M1g1 epitope was found in C. albicans serotypes A and B, Candida tropicalis, Candida guilliermondii, Candida glabrata, and Candida parapsilosis. Its expression was active at both 23 degrees C and 37 degrees C and uniform over the cell surface. BALB/c mice passively immunized with M1g1 were more resistant than control mice to a lethal hematogenous infection by C. albicans, as evidenced by extension of survival in an M1g1 dose-dependent manner (P, 0.08 to <0.001) and by reduction in number of infection foci and their size in the kidney. In vitro studies found that M1g1 promoted phagocytosis and phagocytic killing of C. albicans yeast cells by mouse peritoneal macrophages and was required for activation of the mouse complement cascade. Thus, human antimannan antibody may have a protective role in host resistance to systemic candidiasis.

Functional and structural diversity in the Als protein family of Candida albicans.

The human fungal pathogen Candida albicans colonizes and invades a wide range of host tissues. Adherence to host constituents plays an important role in this process. Two members of the C. albicans Als protein family (Als1p and Als5p) have been found to mediate adherence; however, the functions of other members of this family are unknown. In this study, members of the ALS gene family were cloned and expressed in Saccharomyces cerevisiae to characterize their individual functions. Distinct Als proteins conferred distinct adherence profiles to diverse host substrates. Using chimeric Als5p-Als6p constructs, the regions mediating substrate-specific adherence were localized to the N-terminal domains in Als proteins. Interestingly, a subset of Als proteins also mediated endothelial cell invasion, a previously unknown function of this family. Consistent with these results, homology modeling revealed that Als members contain anti-parallel beta-sheet motifs interposed by extended regions, homologous to adhesins or invasins of the immunoglobulin superfamily. This finding was confirmed using circular dichroism and Fourier transform infrared spectrometric analysis of the N-terminal domain of Als1p. Specific regions of amino acid hypervariability were found among the N-terminal domains of Als proteins, and energy-based models predicted similarities and differences in the N-terminal domains that probably govern the diverse function of Als family members. Collectively, these results indicate that the structural and functional diversity within the Als family provides C. albicans with an array of cell wall proteins capable of recognizing and interacting with a wide range of host constituents during infection.

Endocytosis of Candida albicans by vascular endothelial cells is associated with tyrosine phosphorylation of specific host cell proteins.

Candida albicans escapes from the bloodstream by invading the endothelial cell lining of the vasculature. In vitro, C. albicans invades endothelial cells by inducing its own endocytosis. We examined whether this process is regulated by the tyrosine phosphorylation of endothelial cell proteins. We found that endocytosis of wild-type C. albicans was accompanied by the tyrosine phosphorylation of two endothelial cell proteins with molecular masses of 80 and 82 kDa. The phosphorylation of these proteins was closely associated with the endocytosis of C. albicans because these proteins were phosphorylated in response to the endocytosis of both live and killed organisms, but they were not phosphorylated in endothelial cells infected with a poorly endocytosed strain of C. albicans. The tyrosine kinase inhibitors genistein and tyrphostin 47 blocked the phosphorylation of the two endothelial cell proteins and significantly reduced endocytosis of C. albicans. Therefore, C. albicans probably induces its own endocytosis by stimulating the tyrosine phosphorylation of two endothelial cell proteins.