The relationship to pathogenesis of the spontaneous phenotypic switching of Candida albicans is uncertain. Since neutrophils are critical in containment of disseminated candidiasis, we used these cells and some of their potentially microbicidal oxidative products to define effects on a C. albicans strain (WO-1) that exhibits characteristic, easily recognized switching between the white and opaque phenotypes. Blastoconidia of the opaque phenotypes were more susceptible than those of the white to killing by either intact neutrophils or cell-free oxidants, including reagent hydrogen peroxide or the myeloperoxidase-H202-ClV system. Paralleling these findings, opaque blastoconidia were 2.8to 3.6-fold more potent stimuli of neutrophil superoxide generation than were the white cells. In addition, both neutrophils and oxidants (reagent H202 or hypochlorous acid as well as the myeloperoxidase-H202-Clsystem) induced unidirectional increases in spontaneous rates of switching from white to opaque phenotypes. Differences in expression of C. albicans phenotypes therefore may determine relative susceptibility to neutrophil fungicidal mechanisms, and neutrophils themselves appear to be capable of selectively augmenting the switching process.

We previously showed that unopsonized Candida albicans hyphae stimulated a delayed rise in the putative neutrophil second messengers Ca2+ and inositol 1,4,5-trisphosphate and subsequent O2- release, as compared with opsonized hyphae or zymosan. Therefore, cytoskeletal and degranulation temporal responses to these stimuli were examined. Unopsonized zymosan elicited no neutrophil responses under the experimental condition used. Neutrophil actin polymerization (quantitated by fluorescent measurements of NBD phallacidin) was rapid after stimulation by opsonized hyphae or zymosan (peaking at 1 and 2 min, respectively). This corresponded to observed changes in microscopic actin polymerization, measured with rhodamine phalloidin, which progressed from initially diffuse to collarlike to cylinderlike staining patterns surrounding the hyphae. Compared with opsonized hyphae, unopsonized hyphae resulted in a delayed appearance of the last two visible patterns (P less than 0.05) and in quantitative actin polymerization despite similarly rapid initial contact and spreading over the hyphae by neutrophils. Unlike other neutrophil responses, degranulation did not follow the delayed patterns of responses to stimulation with unopsonized hyphae. In the absence of the release of the cytoplasmic marker lactate dehydrogenase, the release of beta-glucuronidase, an azurophil granule marker, gradually and progressively rose in response to all of the stimuli but unopsonized zymosan. The low but significant levels observed were within a range consistent with published results for degranulation responses to particulate stimuli without cytochalasin B. A quantitative immunoassay of lactoferrin, a specific granule marker, detected no release into supernatants, and immunofluorescent staining indicated concomitant depletion of lactoferrin from neutrophil granules and binding to hyphal and neutrophil surfaces after stimulation by unopsonized hyphae. Thus, the delayed actin polymerization response to unopsonized hyphae occurred subsequent to neutrophil attachment and spreading and resembled the temporal sequence of other neutrophil responses linked to the respiratory burst. In contrast, the degranulation responses to all stimuli appeared to begin and progress gradually after observed attachment and spreading of the neutrophil over hyphal surfaces without a clear temporal relationship to rises in cytoplasmic Ca2+ or F-actin. In addition, the avid binding of released lactoferrin to cell surfaces eliminates its value as a quantitative marker of enzyme release but raises the possibility that it might participate in fungicidal activity.(ABSTRACT TRUNCATED AT 400 WORDS)