Influence of growth conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata.

The effect of cultural conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata was tested. C. albicans cells grown at room temperature were more hydrophobic than cells grown at 37 degrees C. No consistent pattern was observed with C. glabrata. Relative hydrophobicity was found to vary with the growth phase and growth medium for both species. The implications for pathogenesis studies are discussed.     

New assay for measuring cell surface hydrophobicities of Candida dubliniensis and Candida albicans

Hydrophobic interactions, based on cell surface hydrophobicity (CSH), are among the many and varied mechanisms of adherence deployed by the pathogenic yeast Candida albicans. Recently it was shown that, unlike C. albicans, C. dubliniensis is a species that exhibits an outer fibrillar layer consistent with constant CSH. Previously, C. dubliniensis grown at 25 or 37 degrees C was shown to coaggregate with the oral anaerobic bacterium Fusobacterium nucleatum. C. albicans, however, demonstrated similar coaggregation only when hydrophobic or grown at 25 degrees C. This observation implied that coaggregation of Candida cells with F. nucleatum is associated with a hydrophobic yeast cell surface. To test this hypothesis, 42 C. albicans and 40 C. dubliniensis clinical isolates, including a C. albicans hydrophobic variant, were grown at 25 and 37 degrees C and tested with the established hydrophobicity microsphere assay, which determines CSH levels based on the number of microspheres attached to the yeast cells. The coaggregation assay was performed in parallel experiments. All C. dubliniensis isolates grown at either temperature, hydrophobic 25 degrees C-grown C. albicans isolates, and the C. albicans hydrophobic variant, unlike the 37 degrees C-hydrophilic C. albicans isolates, exhibited hydrophobic CSH levels with the microsphere assay and simultaneously showed maximum, 4+, coaggregation with F. nucleatum. The parallel results obtained for C. dubliniensis using both assays support the use of the CoAg assay both as a rapid assay to determine CSH and to differentiate between C. dubliniensis and C. albicans.     

Participation of yeast cell surface hydrophobicity in adherence of Candida albicans to human epithelial cells.

Recent studies have revealed that hydrophobic cells of the opportunistic pathogenic fungus Candida albicans are more virulent than hydrophilic cells. One critical step in the pathogenic process is adherence to host tissues. Adherence of C. albicans to epithelial tissues is mediated primarily by specific adhesin-receptor interactions, but whether cell surface hydrophobicity (CSH) of the yeast cells may also contribute has not been definitively demonstrated. Nineteen isolates of C. albicans were grown in Sabouraud dextrose broth at either 23 or 37 degrees C and tested for CSH by a polystyrene microsphere assay and for the ability to adhere to HeLa cells, a human cervical epithelioid carcinoma cell line. For 13 isolates, growth at 23 degrees C resulted in significantly higher levels of CSH than did growth at 37 degrees C. Three isolates were hydrophobic and two were hydrophilic regardless of growth temperature. One isolate was more hydrophobic after growth at 37 degrees C. Of the isolates that were more hydrophobic after growth at 23 degrees C, 86.5% (11 of 13) were also more adherent to HeLa cells. Growth temperature did not appear to determine adherence ability, as all isolates that did not differ in CSH after growth at either temperature also did not differ in ability to adhere. No correlation (r = 0.44) was obtained between CSH and adherence when the isolates grown at 23 degrees C were evaluated as a group. Higher correlation (r = 0.65) was obtained when the isolates were grown at 37 degrees C. Interestingly, a significantly positive correlation between CSH and adherence was obtained when individual isolates were analyzed. To accomplish this analysis, the isolates were allowed to vary in CSH over time in tissue culture medium without serum, and the corresponding adherence values determined. Only isolates that varied in CSH by greater than 10% were used. Correlation statistical analysis in which the coefficient of determination (r2) was calculated indicated that poor correlation between CSH and adherence for the isolates evaluated as a group was likely due to the fact that CSH had little effect on adherence once a moderately high level of CSH was attained. These results indicate that CSH is involved in adherence but is not the predominant mechanism and that the effect of CSH on adherence is isolate dependent.     

Kinetics of Phagocytosis and Intracellular Killing of Candida albicans by Human Granulocytes and Monocytes

The study of the phagocytosis and intracellular killing of Candida albicans by granulocytes and monocytes has been hampered by the budding and pseudomycelium formation of this yeast during a relatively short incubation period at 37 degrees C and by the similar density of candida cells and phagocytes, which makes differential centrifugation impossible. In the present study, C. albicans was used after 5 days of preculture at 30 degrees C, after which the number of candida cells remained constant during incubation at 37 degrees C for 90 min. On this basis, phagocytosis and intracellular killing were limited to a period of 60 min. Phagocytosis of C. albicans by granulocytes and monocytes was measured with a hemocytometer, the number of extracellular candida being a measure of the ingestion of these microorganisms. After 60 min, 96% of the candida cells were ingested by normal human granulocytes and monocytes. This process was dependent on the opsonin concentration and temperature and was inhibited by mono-iodoacetic acid. Heat-inactivated serum was less active than fresh serum, reflecting the role of complement factors with respect to opsonization. Intracellular killing was measured by a microbiological assay. After 60 min of incubation of phagocytes together with C. albicans and serum, human granulocytes and monocytes killed 58 and 50% of the ingested candida, respectively. This process was inhibited by phenylbutazone. Phagocytes from patients with chronic granulomatous disease showed impaired intracellular killing.     

Variable expression of a surface determinant during proliferation of Candida albicans.

The surface expression of an antigenic determinant that is present in the cell wall of Candida albicans was investigated with monoclonal antibody 24 (MAb24), an immunoglobulin M MAb. The proportion of the cell population that expressed the epitope under different growing conditions was determined by indirect immunofluorescence microscopy. More than 90% of stationary-phase yeast cells of strain B311 grown at 28 degrees C expressed the antigen. Less than 50% of yeast cells grown exponentially at 28 degrees C or either growing or stationary-phase yeast cells cultivated at 37 degrees C expressed the epitope. Germ tubes, which were induced at 37 degrees C from stationary-phase yeast cells grown at 28 degrees C, expressed the determinant on the parent yeast but not the hyphal portion of the germ tube. The change in antigen expression by stationary-phase cells grown at 28 degrees C, when they resumed growth by bud formation, suggested that antigen expression was lost by cells in the inoculum prior to the first cell division. By using the same assay, strong positive reactions were observed in stationary-phase cultures of other isolates of C. albicans, C. guilliermondii, C. stellatoidea, and C. tropicalis, but not with isolates of C. krusei, C. parapsilosis, or Torulopsis glabrata. The identification of the antigenic determinant as a carbohydrate was based on three observations: (i) interaction with a mannan preparation from the same organism, (ii) sensitivity of the antigen to periodate but not proteases, and (iii) coincidence of the migration of antigen during electrophoresis with material which stained intensely with carbohydrate but not with protein reagents. These observations suggest that the expression of the antigenic determinant of MAb24 is dependent on the growth conditions, growth state, and morphology of the cell and that the topography of the cell surface is dynamic.     

Impairment of phagocytosis by the Klebsiella pneumoniae mannose-inhibitable adhesin-T7 receptor.

It has been previously shown that Klebsiella pneumoniae K59 carrying the mannose-inhibitable adhesin-T7 receptor (MIAT) efficiently binds to polymorphonuclear leukocytes (PMNs) incubated at 4 degrees C but is not efficiently bound and internalized by phagocytes incubated at 37 degrees C. Pretreatment of K59 with compounds that bind the MIAT ligand (D-mannose, UV-inactivated T7 phages, and pepsin-digested anti-MIAT antibodies) enables PMNs to phagocytize and kill these bacteria. In this article, we show that the incubation temperature has no direct effect on expression of either the MIAT or the PMN receptors. These receptors were always expressed at 37 degrees C when PMNs were treated with substances that impaired their ability to rearrange their surfaces (glutaraldehyde and cytochalasins B and D). Pretreatment of inert PMNs with concanavalin A or succinyl concanavalin A drastically reduced binding of K59 to phagocytes at both 4 and 37 degrees C. The same pretreatment carried out with metabolically active PMNs enabled them to efficiently phagocytize the MIAT-positive strain. When phagocytes were treated with K59 bacteria, they became unable to ingest and kill a K59 mutant not expressing the MIAT which was sensitive to phagocytosis. If this pretreatment was performed in the presence of D-mannose, UV-inactivated T7 phages, and pepsin-digested anti-MIAT antibodies, PMNs maintained their phagocytic activity against the MIAT-negative strain. In the presence of K59 bacteria, a very low chemiluminescence response was generated; in contrast, a significant response was observed when bacteria were previously absorbed with UV-inactivated T7 phages and pepsin-digested anti-MIAT antibodies. These results support our previous suggestion that the MIAT adhesin triggers changes in the cell surface, inhibiting further binding and phagocytosis.     

Rapid killing of monocytes in vitro by Candida albicans yeast cells.

To study the interaction between Candida albicans blastoconidia and human phagocytes, we incubated peripheral leukocytes with fungi for 1 h at 37 degrees C and stained the cells with fluorescent vital stains ethidium bromide (EB) and fluorescein diacetate. Fungi that had been phagocytosed showed little staining; however, some leukocytes containing blastoconidia exhibited nuclear staining with EB, even though their cell membranes showed no signs of penetration by fungi. The number of EB-positive leukocytes was related to viability of the yeast cells and the temperature at which they were maintained before use. Because efforts to quantitate EB-positive leukocytes microscopically were frustrated by cell aggregation, we labeled the leukocytes with 51Cr and measured isotope release. We determined that leukocytes incubated with viable fungi released significantly more isotope than cells incubated alone or with killed blastoconidia. Furthermore, 51Cr release correlated directly with concentration of fungi in the assay, time of incubation, and temperature at which fungi were maintained before use. Using a number of isolates of C. albicans and several other species of Candida, we found that all exhibited cytotoxic activity against leukocytes, but the level of activity varied among organisms. Finally, we depleted or enriched peripheral leukocytes for specific cell populations and determined that only monocytes released more 51Cr after incubation with viable blastoconidia. Blastoconidia can lyse phagocytic cells through germination and penetration of cell membranes within 1 to 2 h, but the cytotoxic phenomenon we describe occurs within 15 to 30 min after yeast cells have been phagocytosed. Therefore, this capacity may represent a more immediate response by blastoconidia against phagocytosis and killing by monocytes.     

Factors influencing the interaction of Candida albicans with fibroblast cell cultures.

The interaction of Candida albicans clinical isolates with primary and established fibroblast cultures was studied. The intent was to determine whether yeast adherence and invasion of nonendothelial cell monolayer cultures could be quantitated reproducibly and whether this system could be used for future studies on yeast pathogenesis. Our results demonstrated that specific interactions between the yeast cells and fibroblasts only occurred at 37 degrees C and correlated with the germination process. Fluorescent-antibody staining indicated that invasion or tight associations between the germinating yeast cells and mammalian cells occurred after less than 3 h of incubation. Yeast adherence was estimated radiometrically and trypsin-resistant interaction with individual mammalian cells (infection) was measured microscopically after inoculated monolayer cells were detached with trypsin. We demonstrated that both types of association were time dependent at 37 degrees C; neither was affected by the concentration of glucose used to grow the yeast cells. Primary and established fibroblast cell lines were equally susceptible to infection, but primary cells appeared to have more yeast-binding sites. Fibroblasts maintained in confluent culture for an extended period of time also appeared to have more binding sites, and while not quantitatively more susceptible to infection, the older cells were more susceptible to infection-related cell death. An established kidney epithelial cell line (MDCK) was not susceptible to either type of yeast interaction, indicating that the yeast-fibroblast associations were specific.     

Differential adherence of hydrophobic and hydrophilic Candida albicans yeast cells to mouse tissues.

Using an ex vivo binding assay, we previously demonstrated that yeast cells grown at 37 degrees C display binding specificity in mouse spleen, lymph node, and kidney tissues. In spleen and lymph node tissues, binding was predominantly in regions rich in macrophages. Here, we tested the possibility that hydrophobic and hydrophilic cells bind differentially to host tissues. Hydrophobic and hydrophilic yeast cells of four Candida albicans strains were incubated for 15 min at 4 degrees C with cryostat sections of organs that had been rapidly frozen after removal from BALB/cByJ mice. Unattached cells were removed by washing, and the sections were examined. Hydrophobic cells bound diffusely and abundantly to all tissues, while hydrophilic cell attachment was restricted to specific sites. For example, hydrophobic cells bound to the white and red pulp and the marginal zones in spleens, whereas hydrophilic cells attached primarily to the marginal zones. Hydrophobic yeast cells attached throughout lymph node tissue including paracortical areas, but hydrophilic cell attachment occurred primarily at the subcapsular and trabecular sinuses, EDTA inhibited the adherence of hydrophilic cells but not hydrophobic cells to mouse tissues. Hydrophobic C. albicans strains displaying similar levels of hydrophobicity differed quantitatively in their levels of attachment to kidney and spleen tissues, confirming our earlier observation that surface hydrophobicity is not the sole determinant in adherence to host cells. Other studies have shown that hydrophobic and hydrophilic cells display different virulence characteristics related to their surface properties and that hydrophobic cells are more virulent than hydrophilic cells. Taken together, the present results suggest that the enhanced virulence of hydrophobic cells over hydrophilic cells may be due, in part, to the potential of hydrophobic cells to bind throughout various organs following clearance from the bloodstream.     

Quantitative analysis of opsonophagocytosis and of killing of Candida albicans by human peripheral blood leukocytes by using flow cytometry.

We describe a simple, rapid, automated procedure for measuring opsonophagocytosis and killing of Candida albicans by human peripheral blood leukocytes. Yeast cells are labelled by allowing uptake and cleavage of membrane-permeable bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester to its membrane-impermeable fluorescent derivative bis-carboxyethyl-carboxyfluorescein. The yeast cells are added to cell-rich plasma obtained after dextran sedimentation of erythrocytes. Opsonophagocytosis and killing are quantified by using automated fluorescent cell analysis, and the following parameters can be obtained: (i) relative percentage of phagocytes that participate in opsonophagocytosis, (ii) relative percentage of yeast cells that become associated with phagocytes, and (iii) percentage of killing of C. albicans. The first two parameters are obtained through the additional use of a phycoerythrin-conjugated monoclonal antibody that selectively labels monocytes and polymorphonuclear granulocytes in peripheral blood. Killing is assessed by solubilizing blood cells with deoxycholate to liberate yeast cells from the phagocytes. Viable yeast cells retain carboxyfluorescein, but nonviable cells lose the fluorescent marker; thus, the reduction in number of fluorescent particles directly reflects phagocytic killing. Results obtained by the present method correlated excellently with parallel enumerations by colony counting. Test results with seven healthy individuals revealed a marked dissociation between the process of opsonophagocytosis, which was essentially complete after 20 min at 37 degrees C, and killing rates, which were 48% +/- 11% and 63% +/- 9% (standard deviation) after 1 and 2 h, respectively, when yeast cell-to-phagocyte ratios were in the range of 0.5:1 to 2:1. The described assay is unrivaled in simplicity, rapidity, and reproducibility and generates results for a large number of samples within hours.     

Phagocytosis measured as inhibition of uridine uptake by Candida albicans.

Inhibition of 3H-uridine incorporation into Candida albicans can be used as a sensitive index of phagocytic function because: 1) there is a linear correlation between uridine incorporation and yeast number; 2) phagocytic cells do not incorporate significant amounts of uridine in short term cultures; and 3) C. albicans replicating inside phagocytic cells does not take up uridine from culture medium. Appropriate conditions for measuring phagocytic capacity of human polymorphonuclear cells (PMN's) were 5 x 10(5) C. albicans and 5 x 10(4) PMNs in 0.5 ml of medium containing 2.5% AB serum. This mixture was incubated for 30 min at 37 degrees C. Aliquots were then transferred into microtiter wells and incubated for a further 60 min in the presence of 3H-uridine. Under these conditions PMN leucocytes from 25 healthy individuals caused suppression of uridine incorporation ranging from 33 to 75% (50 +/- 12).     

Effects of storage and radiation on human neutrophil function in vitro

To better understand the process of time-related functional deterioration which occurs in human polymorphonuclear leukocytes (PMNs), we examined the effects of in vitro storage on multiple functional parameters of human PMNs. Single-donor, phlebotomy-collected PMNs were stored at both room temperature and 37°C for 24 and 48 h, then compared to fresh cells from the same donor. Similar numbers of cells were recovered from each storage condition. Cell viability decreased after 37°C storage for 48 h. Cells stored at room temperature for 24 h showed significant depression of multiple functions (bactericidal activity, chemotaxis, aggregation, superoxide production, and oxygen consumption) compared to fresh cells. They contained less vitamin B₁₂ binding protein activity than fresh cells, and by fluorescenceactivated cell-sorter analysis, their forward light scatter and membrane depolarization responses were abnormal. For all parameters examined, cells stored at 37°C were more abnormal than cells stored at room temperature. Stored cells from a patient with myeloperoxidase deficiency lost bactericidal and chemotactic activity after storage at 37°C for 24 h, but cells from a patient with chronic granulomatous disease retained their original bactericidal and chemotactic activity after 37°C storage for 24 h. Radiation, in doses used to prevent graft vs. host disease in leukocyte-transfusion recipients (2500–5000 rads) caused a significant decrease in the mean percentage of continuous flow centrifugation leukapheresis (CFCL) collected PMNs capable of reducing nitroblue tetrazolium. Human PMNs show deterioration of multiple in vitro functions when they are stored and are susceptible to damage by radiation when they are collected by CFCL.     

Non-serum-dependent chemotactic factors produced by Candida albicans stimulate chemotaxis by binding to the formyl peptide receptor on neutrophils and to an unknown receptor on macrophages

Serum-free culture filtrates of six Candida species and Saccharomyces cerevisiae were found to contain chemoattractants for human polymorphonuclear leukocytes (PMNs) and a mouse macrophage-like cell line, J774. The chemotactic factors differed for the PMN and J774 cells, however, in terms of heat stability, kinetics of liberation by the yeast cells, and divalent cation requirements for production. The chemoattractant in Candida albicans culture filtrates appeared to act through the formyl peptide receptor (FPR) of PMNs, since it was found to induce chemotaxis of Chinese hamster ovary (CHO) cells that were expressing the human FPR but did not induce chemotaxis of wild-type CHO cells. The C. albicans culture filtrates also induced migration of PMNs across confluent monolayers of a human gastrointestinal epithelial cell line, T84; migration occurred in the basolateral-to-apical direction but not the reverse direction, unless the epithelial tight junctions were disrupted. J774 cells did not migrate toward the formylated peptide (fMet-Leu-Phe; fMLF), and chemotaxis toward the C. albicans culture filtrate was not inhibited by an FPR antagonist (t-butoxycarbonyl-Met-Leu-Phe), suggesting that a different receptor mediated J774 cell chemotaxis. In conclusion, we have identified a receptor by which a non-serum-dependent chemotactic factor (NSCF) produced by C. albicans induced chemotaxis of PMNs. Additionally, we have shown that NSCF was active across epithelial monolayers. These findings suggest that NSCFs produced by C. albicans and other yeast species may influence host-pathogen interactions at the gastrointestinal tract mucosal surface by inducing phagocytic-cell infiltration.     

Killing of Cryptococcus neoformans strains by human neutrophils and monocytes.

The susceptibility of various strains of Cryptococcus neoformans to killing by human polymorphonuclear leukocytes (PMNs) and monocytes was investigated. Five previously characterized strains of C. neoformans serotype A, a capsule-free mutant, and six recent clinical isolates were compared. PMNs and monocytes were isolated from normal peripheral blood and allowed to adhere to the flat-bottom wells of microtiter plates. Yeast cells of C. neoformans were added in the presence of normal human serum, and the plates were incubated at 37 degrees C. After 4 h, killing was determined by comparing the quantitative plate counts of viable yeast cells in experimental wells with counts in control wells containing yeast cells in the absence of leukocytes. No appreciable growth of yeast cells occurred in the wells during the incubation period. Both PMNs and monocytes effectively killed yeast cells at effector-to-target ratios as low as 1:1, although monocytes failed to kill the capsule-free strain 602 at a 1:1 ratio. With 9 of 12 strains, PMNs killed C. neoformans more effectively than did monocytes. Significant interstrain variation in killing occurred for both monocytes and PMNs, and the recent, clinical isolates were more resistant to killing by monocytes and PMNs than were the previously characterized strains. The extent to which different strains were killed by monocytes and PMNs was not consistently related to the size of the capsule or the entire cell. Normal PMNs and monocytes are remarkably effective in killing strains of C. neoformans in the absence of specific antibody and appear to constitute a significant defense mechanism in the peripheral circulation.     

Chitin synthase activity and the rate of chitin formation in cell-division cycle mutant Saccharomyces cerevisiae cdc 24

At the nonpermissive temperature (37 degrees C) the cells of the temperature-sensitive mutant Saccharomyces cerevisiae cdc 24 accumulated chitin 10 times faster than at 22 degrees C. In situ determinations of the activity of chitin synthase revealed that in the cells grown at 37 degrees C more than 37% of the total chitin synthase were in the active state whereas in cells grown at 22 degrees C only 7% of the potential enzyme activity were expressed. When the enzyme activity was calculated per cell number unit, there was 10 times more of the active chitin synthase per cell in the cells grown at 37 degrees C than in the cells grown at 22 degrees C, a value which correlated well with the observed difference in the rates of chitin accumulation at different temperatures.     

Coaggregation of Streptococcus sanguis and other streptococci with Candida albicans.

Thirteen strains of viridans group streptococci and two strains of other streptococci were tested for coaggregation with Candida albicans. Streptococcus sanguis strains generally exhibited low levels of adherence to 28 degrees C-grown exponential-phase yeast cells, but starvation of yeast cells for glucose at 37 degrees C (or at 28 degrees C) increased their coaggregating activity with these streptococci by at least tenfold. This was a property common to four C. albicans strains tested, two of which were able to form mycelia (6406 and MEN) and two of which were not (MM2002 and CA2). The expression of the coaggregation adhesin during yeast cell starvation was inhibited by addition of trichodermin or amphotericin B. The strains of S. sanguis, Streptococcus gordonii, and Streptococcus oralis tested for coaggregating activity encompassed a diverse range of physiological and morphological types, yet all exhibited saturable coaggregation with starved C. albicans cells. There was no correlation of cell surface hydrophobicity, of either yeast or streptococcal cells, with their abilities to coaggregate. Strains of Streptococcus anginosus also coaggregated with starved yeast cells; Streptococcus salivarius and Streptococcus pyogenes coaggregated to a lesser degree with C. albicans, and the coaggregation with S. pyogenes was not promoted by yeast cell starvation; Streptococcus mutans and Enterococcus faecalis did not coaggregate with yeast. The coaggregation reactions of S. sanguis and S. gordonii with C. albicans were inhibited by EDTA and by heat or protease treatment of the yeast cells and were not reversible by the addition of lactose or other simple sugars. These observations extend the range of intergeneric coaggregations that are known to occur between oral microbes and suggest that coaggregations of C. albicans with viridans group streptococci may be important for colonization of oral surfaces by the yeast.     

Coaggregation of Candida dubliniensis with Fusobacterium nucleatum

The binding of microorganisms to each other and oral surfaces contributes to the progression of microbial infections in the oral cavity. Candida dubliniensis, a newly characterized species, has been identified in human immunodeficiency virus-seropositive patients and other immunocompromised individuals. C. dubliniensis phenotypically resembles Candida albicans in many respects yet can be identified and differentiated as a unique Candida species by phenotypic and genetic profiles. The purpose of this study was to determine oral coaggregation (CoAg) partners of C. dubliniensis and to compare these findings with CoAg of C. albicans under the same environmental conditions. Fifteen isolates of C. dubliniensis and 40 isolates of C. albicans were tested for their ability to coaggregate with strains of Fusobacterium nucleatum, Peptostreptococcus micros, Peptostreptococcus magnus, Peptostreptococcus anaerobius, Porphyromonas gingivalis, and Prevotella intermedia. When C. dubliniensis and C. albicans strains were grown at 37 degrees C on Sabouraud dextrose agar, only C. dubliniensis strains coaggregated with F. nucleatum ATCC 49256 and no C. albicans strains showed CoAg. However, when the C. dubliniensis and C. albicans strains were grown at 25 or 45 degrees C, both C. dubliniensis and C. albicans strains demonstrated CoAg with F. nucleatum. Heating the C. albicans strains (grown at 37 degrees C) at 85 degrees C for 30 min or treating them with dithiothreitol allowed the C. albicans strains grown at 37 degrees C to coaggregate with F. nucleatum. CoAg at all growth temperatures was inhibited by mannose and alpha-methyl mannoside but not by EDTA or arginine. The CoAg reaction between F. nucleatum and the Candida species involved a heat-labile component on F. nucleatum and a mannan-containing heat-stable receptor on the Candida species. The CoAg reactions between F. nucleatum and the Candida species may be important in the colonization of the yeast in the oral cavity, and the CoAg of C. dubliniensis by F. nucleatum when grown at 37 degrees C provides a rapid, specific, and inexpensive means to differentiate C. dubliniensis from C. albicans isolates in the clinical laboratory.     

Interaction of monoclonal antibodies with pertussis toxin and its subunits

The pathogenicity of Shigella spp. involves the ability of the bacteria to penetrate and replicate within the epithelial cells of the large intestine. Model systems for examining the virulence of shigellae employ Henle intestinal epithelial cells in tissue culture and an in vivo assay for virulence in guinea pig eyes (Sereny test). Using these systems, we studied the genetic and physiological bases for the ability of shigellae to invade epithelial cells. We found that expression of virulence in Shigella spp. is dependent on the temperature at which the bacteria are grown. When grown at 37 degrees C, strains of Shigella flexneri 2a, Shigella sonnei, and Shigella dysenteriae 1 were fully virulent and invaded Henle cells. They also produced keratoconjunctivitis in guinea pigs. When grown at 30 degrees C, the bacteria neither penetrated Henle cells nor produced conjunctivitis in the Sereny test and were phenotypically avirulent. Strains grown at 33 degrees C were only partially invasive in the Henle assay, whereas strains grown at 35 degrees C were as invasive as strains grown at 37 degrees C. Using the Henle cell assay, we determined that the loss of ability to penetrate epithelial cells was completely reversed by shifting the growth temperature from 30 to 37 degrees C. The percentage of Henle cells invaded by bacteria increased with increasing time of growth at 37 degrees C. Restoration of invasiveness after growth at 30 degrees C required protein synthesis. When shigellae were grown at 30 degrees C and shifted to 37 degrees C for 2 h in the presence of chloramphenicol, the bacteria remained noninvasive. Similarly treated bacteria grown at 37 degrees C were still invasive. These results suggested that expression of one or more genes required for virulence of Shigella spp. are subject to regulation by growth temperature.     

The presumptive identification of Candida albicans with germ tube induced by high temperature

For direct identification of Candida albicans from other Candida species, the chlamydospore formation and the mycelial transition induced by high temperature and by sera were examined in 198 Candida isolates. The germ tubes of C. albicans developed early at 30 min in high temperature-induction, but at 60 min in serum-induction. C. albicans generated germ tubes well at concentrations lower than 2 x 10(7) cells/ml, but the germ tube formation was markedly restrained at concentrations higher than 4 x 10(7) cells/ml. In a serum-free, yeast extract-peptone-dextrose (YEPD) medium, C. albicans grew as a yeast form at 30 degrees C and as a mycelial form at 35-42 degrees C. Mycelial development was maximal at 37 degrees C in serum and at 39 degrees C in YEPD. Germ tubes were formed within 30 min in YEPD at 39 degrees C, but after 60 min in serum at 37 degrees C. Our examination showed that the 39 degrees C-induced germ tube formation tests were very reliable (sensitivity 100%, specificity 100%) at discerning C. albicans from other Candida species. These results suggest that the high temperature-induced germ tube formation testing could be a useful identification method of C. albicans in clinical laboratories.     

Characterization of Candida albicans adherence to human vaginal epithelial cells in vitro

Certain environmental, physical, and biochemical aspects of Candida albicans adherence to human vaginal epithelial cells were characterized by using an in vitro radiometric adherence assay. Blastospores harvested from cultures grown at 25 degrees C adhered to vaginal epithelial cells in significantly greater numbers than did blastospores isolated from cultures grown at 37 degrees C. C. albicans viability was not essential for adherence, but severe methods used to kill the blastospores did reduce their attachment. The addition of sodium chloride, divalent cations, sugars, mannan, or mannoprotein to the assay had no effect on attachment. Pretreatment of the blastospores with detergents, salts, urea, glycosidases, lipase, or pepsin did not affect adherence, but treatment with reducing agents or five proteolytic enzymes did render C. albicans nonadherent. Cell wall fragments prepared from C. albicans, but not from Candida krusei, adhered to vaginal epithelial cells. Loss of adherence after the cell walls were treated with alpha-mannosidase or papain suggests that cell wall mannoprotein is an essential component of the C. albicans adhesin.