The mechanism of mAb-mediated protection against appears to be multifactorial, involving classical and nonclassical mechanisms of Ab function (1). mAb-mediated cross-linking of capsular polysaccharide molecules. These results provide new insights into Ab-mediated immunity, while suggesting a new nonclassical mechanism of Ab function, which may apply to other encapsulated pathogens. Our findings add to the growing body of evidence that Abs have direct antimicrobial functions impartial of other components of the immune system. Introduction Antibodies to microbial surfaces can promote host defense by mediating the effector functions of other components of the immune system, such as complement and phagocytic cells (1). However, binding of Abs can also mediate direct antimicrobial effects for the benefit of the host, even for encapsulated microbes, when binding occurs at a certain distance away from the cell. Examples of such direct antimicrobial effects include alterations of microbial metabolic activity, gene expression, quorum sensing, and susceptibility to drugs (2, 3). The physical mechanism(s) of such direct Ab-mediated effects upon capsule binding are poorly understood. The study of AbCcapsule conversation is important for understanding the mechanisms by which Ab-mediated immunity interacts with microbes, for identifying useful Abs for antimicrobial treatment (1), and for designing more effective vaccines. One of the best-studied microbial capsules is usually that of the fungal pathogen which is a complex polysaccharide (PS) structure that enlarges during contamination (8 the cells volume) and is essential for virulence (4, 5). The cryptococcal capsule exhibits strong antiphagocytic properties (6, 7), isolating the fungal cell from host immune factors and pattern recognition receptors on immune cells (8). Macromolecular analysis of extracted PSs suggests that the capsule is composed of various interconnected PS molecules with branch-like structural characteristics (9C11). This complex surface structure is considered the main virulence factor (4, 5) and remains a major target for the development of therapeutic strategies (12). The mAbs to glucuronoxylomannan (GXM), the main PS constituent of the capsule, can mediate protection against contamination by decreasing fungal burden and dissemination and, thus, increasing survival of lethally infected mice (13C16). One mAb, 18B7 (IgG1), was evaluated clinically as a therapeutic agent against cryptococcosis (17, 18). The mechanism of mAb-mediated protection against appears to be multifactorial, involving classical and nonclassical mechanisms of Ab function (1). Classical mechanisms of mAbs to GXM include enhancement of phagocytosis, complement activation, and recruitment of inflammatory cells (19C22). In addition, mAbs to GXM can function directly, affecting the normal function of upon binding to the PS capsule. mAbs can inhibit PS Ag release (23) and biofilm formation in vitro (24) and can increase drug susceptibility by somehow triggering changes in cryptococcal metabolism and gene expression (2, 25). The mechanism of such direct Ab-mediated effects on physiology remains poorly comprehended and requires new approaches for studying AbCcapsule conversation. The protective efficacy of mAbs to GXM against experimental cryptococcosis depends greatly on their capacity to interact with the capsule (26). For instance, the capacity of mAbs to GXM to alter the optical properties of the capsule (i.e., quellung effect or capsular swelling) and their fluorescence-binding pattern (i.e., annular or punctate) correlated with protective efficacy (22, 26C28). Other important determinants of protective efficacy are Ab isotype and epitope specificity (29), as well as the concentration and localization of these epitopes in the capsule (22). In this study, we examined the direct effect of mAbs to GXM on cellular replication and capsule mechanical properties, using light and optical tweezers microscopy analysis on intact yeast cells. Our data show that binding of protective, but not nonprotective, mAbs produces a concentration-dependent increase in the stiffness of the capsule. This binding translated into a situation whereby daughter cells are trapped in a saclike structure made from the parental capsule. The ability of mAbs to increase the capsule Rabbit Polyclonal to ABHD12 TRC051384 stiffness correlated with their capacity to cross-link PS molecules in solution. Our results show a new Ab-mediated effect on microbial function through the alteration of capsular mechanical properties. Materials and Methods Yeast culture serotype A strain H99 (ATCC 208821) was grown under constant agitation at 30C for 48 h in minimal medium (15 TRC051384 mM dextrose, 10 mM MgSO4, 29.3 mM KH2PO4, 13 mM glycine, 3 M thiamine-HCl; adjusted to pH 5.5). mAbs The GXM-specific mAbs used in this study TRC051384 were 18B7 (IgG1), 13F1 (IgM), 2D10 (IgM), and the 3E5 family of switch variants (IgG1, IgG2, IgG2b, IgG3), all previously described (14, 30, 31). The mAbs were purified from hybridoma cell supernatants recovered.