Because Candida dubliniensis is closely related to Candida albicans, we tested whether it underwent whiteopaque switching and mating and whether white-opaque switching depended on MTL homozygosity and mating depended on switching, as they do in C. albicans. We also tested whether C. dubliniensis could mate with C. albicans. Sequencing revealed that the MTL␣ locus of C. dubliniensis was highly similar to that of C. albicans. Hybridization with the MTLa1, MTLa2, MTL␣1, and MTL␣2 open reading frames of C. albicans further revealed that, as in C. albicans, natural strains of C. dubliniensis exist as a/␣, a/a, and ␣/␣, but the proportion of MTL homozygotes is 33%, 10 times the frequency of natural C. albicans strains. C. dubliniensis underwent white-opaque switching, and, as in C. albicans, the switching was dependent on MTL homozygosis. C. dubliniensis a/a and ␣/␣ cells also mated, and, as in C. albicans, mating was dependent on a switch from white to opaque. However, white-opaque switching occurred at unusually high frequencies, opaque cell growth was frequently aberrant, and white-opaque switching in many strains was camouflaged by an additional switching system. Mating of C. dubliniensis was far less frequent in suspension cultures, due to the absence of matingdependent clumping. Mating did occur, however, at higher frequencies on agar or on the skin of newborn mice. The increases in MTL homozygosity, the increase in switching frequencies, the decrease in the quality of switching, and the decrease in mating efficiency all reflected a general deterioration in the regulation of developmental processes, very probably due to the very high frequency of recombination and genomic reorganization characteristic of C. dubliniensis. Finally, interspecies mating readily occurred between opaque C. dubliniensis and C. albicans strains of opposite mating type in suspension, on agar, and on mouse skin. Remarkably, the efficiency of interspecies mating was higher than intraspecies C. dubliniensis mating, and interspecies karyogamy occurred readily with apparently the same sequence of nuclear migration, fusion, and division steps observed during intraspecies C. albicans and C. dubliniensis mating and Saccharomyces cerevisiae mating.Beginning in 1990, a number of studies of the genetic relatedness of Candida albicans isolates derived from human immunodeficiency virus (HIV)-positive individuals identified atypical strains (9,29,31,33,49). These strains were subsequently grouped into the separate species C. dubliniensis in 1995 by Coleman, Sullivan, and coworkers (52). This new species shared a number of phenotypic characteristics with C. albicans, including two that in the past had been used to distinguish C. albicans from other species, namely, chlamydospore and true hypha formation (52). Like C. albicans, C. dubliniensis was diploid, contained a homolog to the dispersed complex repeat sequence RPS, and underwent the bud-hypha transition and colony-based 3153A-like phenotypic switching (10,17,19,37,50,51). The basic phenotypic char...

Previous studies employing transmembrane assays suggested that Candida albicans and related species, as well as Saccharomyces cerevisiae, release chemoattractants for human polymorphonuclear leukocytes (PMNs). Because transmembrane assays do not definitively distinguish between chemokinesis and chemotaxis, singlecell chemotaxis assays were used to confirm these findings and test whether mating-type or white-opaque switching affects the release of attractant. Our results demonstrate that C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. glabrata release bona fide chemoattractants for PMNs. S. cerevisiae, however, releases a chemokinetic factor but not a chemoattractant. Characterization of the C. albicans chemoattractant revealed that it is a peptide of approximately 1 kDa. Whereas the mating type of C. albicans did not affect the release of chemoattractant, switching did. White-phase cells released chemoattractant, but opaque-phase cells did not. Since the opaque phase of C. albicans represents the mating-competent phenotype, it may be that opaque-phase cells selectively suppress the release of chemoattractant to facilitate mating.Polymorphonuclear leukocytes (PMNs) represent the first line of defense in combating microbial infections. By sensing chemoattractants, PMNs migrate toward sites of infection. In vitro, PMNs orient and chemotax up spatial gradients of formyl methyl peptides (11,(53)(54)(55), which are released by bacteria. These peptides bind to a receptor on the surface of PMNs (26, 31). The receptor-mediated signal is transduced through regulatory cascades that control the actin cytoskeleton in order to polarize and direct the movement of the PMNs toward the site of infection (6, 18, 52). The spatial, and possibly temporal, patterns of receptor occupancy dictate orientation and subsequent chemotaxis (11,12).The process of PMN chemotaxis may play a role in controlling the levels of Candida spp. carriage in the commensal state, as well as in clearing yeast from sites of infection (10, 34). The attraction of PMNs to sites of infection may also contribute to the inflammatory response associated with vaginitis and other forms of mucosal candidiasis. Immunocompromised individuals are far more prone to mucosal candidiasis and candidemias (3), supporting the suggestion that the cellular immune response plays a role in suppressing such infections. The attraction of PMNs to sites of fungal infection may be mediated by an attractant either released by the colonizing yeast cells or released by human cells activated by the yeast. Interestingly, however, in animal models of vaginitis and in human cases of vaginitis the frequent absence of PMNs has been noted (10). The absence of PMNs at the site of a fungal infection may be due either to unresponsive PMNs or to the failure by the colonizing yeast to release a chemoattractant. The release of PMN chemoattractants by colonizing yeast and the responsiveness of PMNs to such chemoattractants, therefore, warrant careful scrutiny as they relate to yeast...

It has been assumed that the natural chemotactic signal that attracts human polymorphonuclear leukocytes (PMNs) over long distances to sites of infection is in the form of a standing spatial gradient of chemoattractant. We have questioned this assumption on the grounds, first, that standing spatial gradients may not be stable over long distances for long periods of time and, second, that in the one animal cell chemotaxis system in which the natural chemotactic signal has been described in space and time, aggregation of Dicytostelium discoideum, the signal is in the form of an outwardly relayed, nondissipating wave of attractant. Here, it is demonstrated that PMNs alter their behavior in each of the four phases of a wave of PMN chemoattractant, fashioned after the Dictyostelium wave, in a manner similar to Dictyostelium. These results demonstrate that PMNs have all of the machinery to respond to a natural wave of attractant, providing support to the hypothesis that the natural signal that attracts PMNs over large distances to sites of infection in the human body may also be in the form of a wave.

Shwachman-Diamond Syndrome (SDS) is a rare autosomal recessive, multisystem disorder presenting in childhood with intermittent neutropenia and pancreatic insufficiency. It is characterized by recurrent infections independent of neutropenia, suggesting a functional neutrophil defect. While mutations at a single gene locus (SBDS) appear to be responsible for SDS in a majority of patients, the function of that gene and a specific defect in SDS neutrophil behavior have not been elucidated. Therefore, employing 2D and 3D computer-assisted motion analysis systems, we have analyzed the basic motile behavior and chemotactic responsiveness of individual polymorphonuclear leukocytes (PMNs) of 14 clinically diagnosed SDS patients. It is demonstrated that the basic motile behavior of SDS PMNs is normal in the absence of chemoattractant, that SDS PMNs respond normally to increasing and decreasing temporal gradients of the chemoattractant fMLP, and that SDS PMNs exhibit a normal chemokinetic response to a spatial gradient of fMLP. fMLP receptors were also distributed uniformly through the plasma membrane of SDS PMNs as in control PMNs. SDS PMNs, however, were incapable of orienting in and chemotaxing up a spatial gradient of fMLP. This unique defect in orientation was manifested by the PMNs of every SDS patient tested. The PMNs of an SDS patient who had received an allogenic hematopoietic stem cell transplant, as well as PMNs from a cystic fibrosis patient, oriented normally. These results suggest that the defect in SDS PMNs is in a specific pathway emanating from the fMLP receptor that is involved exclusively in regulating orientation in response to a spatial gradient of fMLP. This pathway must function in parallel with additional pathways, intact in SDS patients, that emanate from the fMLP receptor and regulate responses to temporal rather than spatial changes in receptor occupancy.

To investigate the role played by filopodia in the motility and chemotaxis of amoeboid cells, a computer-assisted 3D reconstruction and motion analysis system, DIAS 4.0, has been developed. Reconstruction at short time intervals of Dictyostelium amoebae migrating in buffer or in response to chemotactic signals, revealed that the great majority of filopodia form on pseudopodia, not on the cell body; that filopodia on the cell body originate primarily on pseudopodia and relocate; and that filopodia on the uropod are longer and more stable than those located on other portions of the cell. When adjusting direction through lateral pseudopod formation in a spatial gradient of chemoattractant, the temporal and spatial dynamics of lateral pseudopodia suggest that filopodia may be involved in stabilizing pseudopodia on the substratum while the decision is being made by a cell either to turn into a pseudopodium formed in the correct direction (up the gradient) or to retract a pseudopodium formed in the wrong direction (down the gradient). Experiments in which amoebae were treated with high concentrations of chemoattractant further revealed that receptor occupancy plays a role both in filopod formation and retraction. As phosphorylation-dephosphorylation of myosin II heavy chain (MHC) plays a role in lateral pseudopod formation, turning and chemotaxis, the temporal and spatial dynamics of filopod formation were analyzed in MHC phosphorylation mutants. These studies revealed that MHC phosphorylation-dephosphorylation plays a role in the regulation of filopod formation during cell migration in buffer and during chemotaxis. The computer-assisted technology described here for reconstructing filopodia at short time intervals in living cells, therefore provides a new tool for investigating the role filopodia play in the motility and chemotaxis of amoeboid cells.