Candida albicans,plasticity and pathogenesis

Abstract

The yeast Candida albicans has emerged as a major public health problem during the past two decades. The spectrum of diseases caused by this species ranges from vaginal infections, which affect up to 75% of the women at least once in their lifetime, to deep infections in hospitalized patients which lead to high morbidity and mortality rates. Candida albicans may also play a role in the persistence or worsening of some chronic inflammatory bowel diseases. Active research is now improving our understanding of the molecular mechanisms and genetic factors in the yeast and its host which influence the development of disease. Despite these advances and the availability of a more extensive therapeutic arsenal, current progress in the control of nosocomial infections due to Candida remains limited, mainly due to the difficulties in diagnosing these infections. The biologist has a key role to play in establishing a dialogue with the clinician in order to identify the saprophyte/pathogen transition in patients as early as possible. This review provides a quick synopsis of the modern concepts of Candida pathogenesis with some representative examples illustrating the specifics traits of this yeast in terms of pathogenic adaptation.     

Ubiquitin-like epitopes associated with Candida albicans cell surface receptors.

We have recently reported the cloning of a Candida albicans polyubiquitin gene and the presence of ubiquitin in the cell wall of this fungus. The polyubiquitin cDNA clone was isolated because of its reactivity with antibodies generated against the candidal 37-kDa laminin-binding protein. In the present study, we have further investigated the relationship between ubiquitin and cell wall components displaying receptor-like activities, including the 37-kDa laminin receptor, the 58-kDa fibrinogen-binding mannoprotein, and the candidal C3d receptor. Two-dimensional electrophoretic analysis and immunoblot experiments with antibodies against ubiquitin and the individually purified receptor-like molecules confirmed that these cell surface components are ubiquitinated. In an enzyme-linked immunosorbent assay, polyclonal antisera to each receptor reacted with ubiquitin, thus demonstrating that the purified receptor preparations used as immunogens contained ubiquitin-like epitopes. It is proposed that ubiquitin may play a role in modulating the activity of these receptors and in the interaction of C. albicans cells with host structures.     

Candida albicans Als proteins mediate aggregation with bacteria and yeasts.

Candida albicans occupies a microniche on mucosal surfaces where diverse microbial populations interact within a biofilm. Because C. albicans is intimately involved with other microbes in this environment we studied the interactions of C. albicans with other fungi and bacteria that form mixed microbial aggregates. Once aggregation is initiated, aggregates form rapidly and incorporate fungal as well as bacterial cells. The fungus formed mixed microbial aggregates with homotypic cells (i.e., self to self, e.g., C. albicans or Als1p-expressing yeast cells aggregating with cells bearing Als1p); with heterotypic cells (i.e., self to non-self, e.g., C. albicans or Alsp-expressing yeast cells aggregating with other Candida species); and with xenotypic cells (e.g., C. albicans or Alsp-expressing yeast cells forming aggregates with bacteria). When either of the C. albicans adhesins Als1p or Als5p was displayed on the surface of non-adherent Saccharomyces cerevisiae cells, the S. cerevisiae also mediated these mixed microbial interactions. Thus the Als adhesins are potentially important for the co-adhesion of mixed microbial communities in biofilms and on mucus surfaces.     

Participation of C3 in Intracellular Killing of Candida albicans

Using new objective methods for measuring, independently, phagcytosis and killing, it was demonstrated that Candida albicans opsonized by C3-deficient serum was ingested by not killed in vitro by human polymorphonuclear leukocytes. Killing could be induced by adding purified C3 to the C3-deficient serum. It is concluded that C3 participates directely in the intracellular process leading to phagocytic killing of C. albicans.     

Intracellular esterase activity of Candida albicans and its correlation with pathogenicity in mice

ObjectiveTo determine the virulence of clinical Candida albicans isolates and intracellular esterase activity in differentiating the virulent strains.Materials and methodsThe virulence of 17 isolates of C. albicans was determined in BALB/c mice with Candidiasis. The esterase activity against five substrates was assayed by a colorimetric method.ResultsThe results showed that the virulence of C. albicans isolates was considerably different from each other. The esterase activity against each of the five substrates did not show significant correlation with the virulence of the isolates. Comparison of the mean esterase activity of the human and animal isolates showed significant differences against some of the substrates.ConclusionIt is concluded that this study showed a positive but not significant correlation between some intracellular esterase activities and the C. albicans virulence.     

Expression of surface hydrophobic proteins by Candida albicans in vivo.

Candida albicans modulates cell surface hydrophobicity during growth and morphogenesis in vitro. To determine if surface hydrophobicity is expressed during pathogenesis, we generated a polyclonal antiserum against yeast hydrophobic proteins. The antiserum was then used for indirect immunofluorescence analysis of tissues from mice colonized and chronically infected with C. albicans. Results demonstrated that yeast hydrophobic proteins are exposed on fungal cells present in host tissues. The polyclonal antiserum distinguished between hydrophobic and hydrophilic cell surfaces in vitro and gave similar staining patterns and intensities for C. albicans cells in vivo. Of the yeast forms present within tissue lesions, approximately half exhibited moderate to intense immunofluorescence with the antiserum. Immunoblot analysis indicated that antigens recognized by the antiserum are predominantly low-molecular-mass hydrophobic proteins that are expressed by different C. albicans isolates and are expressed regardless of growth temperature. Taken together, the immunofluorescence and immunoblot analyses of antigens indicate that C. albicans displays surface hydrophobic proteins during pathogenesis and these proteins are available for hydrophobic interactions with host tissues. The effect of hydrophobic protein exposure on the virulence of C. albicans is discussed.     

Increased phenotypic switching in strains of Candida albicans associated with invasive infections.

This study reports the rates of phenotypic switching in strains of Candida albicans isolated from superficial and invasive infections. Of 19 invasive strains, 68% showed switching activity, often at very high rates, compared with only 28% of 40 strains isolated from superficial sites (P = 0.004).     

Platelet interactions with Candida albicans.

The interaction of human platelets and Candida albicans was studied. Platelet-rich plasma was obtained from freshly drawn blood or outdated platelet concentrates. From the platelet-rich plasma, a platelet extract was derived which stimulated germ tube formation by C. albicans when incubated with yeast cells at 37 degrees C. The active component(s) was heat stable, trypsin sensitive, and ribonuclease and deoxyribonuclease insensitive, and possessed cationic properties since it readily attached to carboxymethyl-Sephadex. The active component(s) seemed to bind to heparin also, since germ tube-promoting activity was eluted from a heparin-cyanogen bromide-activated Sepharose 4B column. In addition, platelet-derived growth factor (Collaborative Research, Inc.) stimulated germination when incubated with low amounts (0.4% final concentration) of bovine calf serum. The aggregation of platelets, prepared as platelet-rich plasma by C. albicans cell wall or alkali-extracted cell wall fractions, was also studied. Aggregation of platelets was observed when cell wall or cell wall fractions were incubated with platelet-poor plasma at 37 degrees C for 20 min and then added to platelet-rich plasma. The component of platelet-poor plasma which promoted aggregation of platelets by C. albicans cell wall or alkali-extracted fractions was inactivated at 56 degrees C (30 min) and by cobra venom factor, indicating a role for the alternate complement pathway in the aggregation response.     

Influence of preformed antibody on the pathogenesis of experimental Candida albicans endocarditis.

The influence of preformed antibody on the induction of experimental Candida albicans endocarditis was investigated by both in vitro and in vivo techniques. Preincubation of C. albicans with immune serum (raised in rabbits by intravenous injection of Formalin-killed yeast cells) decreased adhesion to the constituents of nonbacterial thrombotic endocarditis, e.g., fibrin plus platelets, in vitro. Two different methods, with radiolabeled or viable yeast cells, were confirmatory and demonstrated decreased adhesion of immune serum-treated C. albicans cells to 0 to 7.8% of control values (P less than 0.001). These results correlated with protection from the development of C. albicans endocarditis in the immunized rabbits. The mean (+/- standard deviation) infectious dose for 50% of the animals was 10(5.29) +/- 10(0.07) in 48 control animals versus 10(7.11) +/- 10(0.22) in 37 immunized rabbits (P less than 0.001). These studies suggest that humoral antibody may protect against C. albicans endocarditis, perhaps through inhibition of adhesion, a crucial early step in the pathogenesis of endocarditis.     

Phosphate-containing proteins and glycoproteins of the cell wall of Candida albicans.

The distribution of phosphate, carbohydrate, and protein in the cell wall components extracted from intact yeast cells of Candida albicans by beta-mercaptoethanol (beta ME) at pH 8.6 was examined by analysis of the material separated by DEAE-cellulose chromatography. All protein peaks did not coincide with peaks of both carbohydrate and phosphate. Subsequent analysis was performed on material obtained from yeast cells and germ tubes which were grown in medium containing [32P]phosphate. Two extracts were obtained by treating cells with beta ME or with zymolyase following beta ME. The extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. beta ME-extracted material contained high-molecular-mass (HMM), greater than or equal to 200 kDa, polydisperse material and a major and minor band of 19 to 20 kDa, Zymolyase extracts contained (i) three components of less than or equal to 40 kDa, one of which may correspond to the major beta ME band; (ii) four bands within the HMM region which may correspond to previously reported bands; and (iii) one band of 100 to 120 kDa. After longer exposures, additional midrange bands were detected in the zymolyase extract. In extracts treated with endo-beta-N-acetylglucosaminidase H, the HMM polydisperse material increased in mobility although retaining sufficient radiolabel for detection. Western immunoblot analysis of extracts with germ tube-specific antiserum and a germ tube-specific monoclonal antibody and concanavalin A showed that not all components contained detectable phosphate, not all glycoproteins contained detectable phosphate, and at least one 19- to 20-kDa protein may be phosphorylated in the absence of carbohydrate.     

High-frequency switching in Candida albicans.

Most strains of Candida albicans are capable of switching frequently and reversibly between a number of phenotypes distinguishable by colony morphology. A number of different switching systems have been defined according to the limited set of phenotypes in each switching repertoire, and each strain appears to possess a single system. Switching can affect many aspects of cellular physiology and morphology and appears to be a second level of phenotypic variability superimposed upon the bud-hypha transition. The most dramatic switching system so far identified is the "white-opaque transition." This system dramatizes the extraordinary effects switching can have on the budding cell phenotype, including the synthesis of opaque-specific antigens, the expression of white-specific and opaque-specific genes, and the genesis of unique cell wall structures. Switching has been demonstrated to occur at sites of infection and between episodes of recurrent vaginitis, and it may function to generate variability in commensal and infecting populations for adaptive reasons. Although the molecular mechanisms involved in the switch event are not understood, recent approaches to its elucidation are discussed and an epigenetic mechanism is proposed.     

Candida albicans outbreak associated with total parenteral nutrition in the neonatal unit

Background: The most frequently isolated fungi in patients using TPN belongs to the Candida genus. Various infections including venous catheter infections, fungemia, endocarditis and ophthalmitis may be encountered. Objective: Upon growth of Candida in the blood cultures from the pediatric (neonatal) unit of our hospital, a surveillance was performed in this unit and involving the health care workers. Clonal relationships of the isolates were investigated with molecular tests. Methods: Blood samples obtained from the patients in pediatric neonatal unit were studied with automatized blood culture [BacT/Alert (Bio Mérioux, France)]. Yeast isolates from environmental surveillance cultures (TPN solutions, hands of healthcare personnel, étagère, etc) and patients were identified as C. albicans with conventional methods and ID 32 C and ATB^TM Fungus 3 (Biomerieux, France) kits. Clonal similarity was determined by using AP-PCR as initial method and we have also typified all strains by the method of REP-PCR (diversilab system,bioMérieux). Finally; Pulsed Field Gel Electrophoresis (PFGE) was used for confirmation. Results: C. albicans was isolated in blood cultures of seven patients. Similar antifungal susceptibility patterns were observed in all isolates. AP-PCR and REP-PCR showed that the C. albicans isolates grown in the TPN solution and from the patients’ blood cultures were clonally same strains. PFGE analysis further confirmed this clonality. Conclusion: According to results of the molecular methods, we thought that a C. albicans outbreak had occurred in the neonatal pediatric unit, due to contamination of TPN solution.     

A single strain of Candida albicans associated with separate episodes of fungemia and meningitis.

Four isolates of Candida albicans recovered from the blood and cerebral spinal fluid of a 66-year-old man during episodes of systemic infection separated by 3 months and antifungal therapy were analyzed by a variety of molecular typing methods. All four isolates were shown to represent the same strain, indicating a relapse of infection rather than reinfection.     

Evidence for the presence of collagenous domains in Candida albicans cell surface proteins.

Rabbit polyclonal antibodies (PAbs) directed towards the amino-terminal cysteine-rich 7S domain (PAb anti-7S), the major internal collagenous domain (PAb anti-type IV), and the C-terminal noncollagenous region (PAb anti-NC1) of the type IV collagen molecule were probed by indirect immunofluorescence against Candida albicans blastoconidia and germinated blastoconidia. Most nongerminating cells and mother blastoconidia from which germ tubes originated showed strong fluorescence when PAb anti-7S was used, whereas with PAb anti-type IV, fluorescence was found almost exclusively on the surface of filamentous forms. A patched fluorescent pattern rather than a homogenous confluent fluorescence was observed in all cases. No fluorescent cells were observed with PAb anti-NC1. By Western immunoblotting, PAb anti-type IV cross-reacted primarily with a polypeptide of 37 kDa present in wall extracts obtained from intact cells of both growth forms by treatment with beta-mercaptoethanol, whereas PAb anti-7S recognized a major 58-kDa antigen also present in both extracts, along with some other high-molecular-mass (> 106-kDa) polydisperse species present only in the material released from blastoconidia with beta-mercaptoethanol. No reactive bands were observed when PAb anti-NC1 was used as a probe in Western immunoblotting experiments. The sensitivities or resistances to collagenase digestion of the different polypeptides that cross-reacted with PAbs anti-type IV and anti-7S suggest the existence of cell wall components in C. albicans that contain epitopes that mimic the collagenous domains of the type IV collagen molecule.     

Candida albicans strain delineation.

Candida albicans is a major opportunistic pathogen causing a wide spectrum of disease in human beings. Methods for strain delineation of this species to assess or predict virulence or to conduct epidemiologic or pathogenetic investigations have been developed. Although factors associated with virulence have been identified, there is no rapid system to quantitate them in a clinical laboratory. Therefore, many typing methods are based on variable phenotypic characteristics within this species including morphotyping, serotyping, antibiogram, resistogram typing, biotyping, biotyping based on commercial carbon assimilation patterns, enzyme profiles, sensitivity to yeast killer toxins, and typing based on protein variability. Phenotypically defined strains generally do not correlate with the pathogenic potential of a strain with the exception of morphotyping. However, these methods can be useful in epidemiologic investigations; for example, they have revealed that most individuals harbor one strain and that infections are frequently due to an endogenous strain. Problems with these methods usually relate to their discriminatory power. When this is maximized, reproducibility (especially between laboratories) suffers. Recently, methods based on differences in DNA structure (genotyping) for strain delineation have been developed, including electrophoretic karyotyping and restriction enzyme fragment length polymorphisms. The development of a computer-assisted data bank and analysis for these genotypic strain delineators will open investigations into the pathogenesis of this infection and permit epidemiologic studies previously not possible with this important human pathogen.     

Physiological traits associated with success of Candida albicans strains as commensal colonizers and pathogens.

DNA fingerprinting with the moderately repetitive sequence Ca3 has repeatedly identified groups of genetically similar strains of Candida albicans that are more frequently isolated than other groups of strains from human hosts in a geographical locale. Members of these groups are found in approximately 30% of healthy individuals and in up to 70% of patients suffering from candidiasis. The high prevalence of these strains implies that they are more successful in colonizing human hosts and in causing disease than other strains (J. Schmid, Clin. Adv. Treatment Fungal Infect. 4(6):12-16, 1993). In the present study, we have compared one such group of highly prevalent strains with other strains from the same locale to identify physiological traits a larger number of chemicals than other strains in a resistogram assay. When resistance to individual chemicals used in the resistogram assay was analyzed, strains from the group of highly prevalent strains were significantly more often resistant to boric acid, cetrimide, chlorhexidine, 5-fluorocytosine, and high sodium chloride concentrations than other strains. Strains from the group of highly prevalent strains also adhered significantly (1.5 times) better to saliva-coated surfaces than did other strains. Because members of highly prevalent groups of strains are the most common infectious agents in candidiasis, these physiological traits may be involved in determining not only the success of C. albicans in colonizing human hosts in general but also its ability to cause disease. Sodium chloride resistance and increased adherence were also associated with infectious isolates outside the group of highly prevalent strains, indicating that they may be of particular importance in pathogenesis.     

Assessment of Candida albicans genes expressed during infections as a tool to understand pathogenesis.

Candida albicans is the most common fungal opportunistic pathogen of humans and causes mucocutaneous, bloodstream and deep organ infections. Screening for C. albicans genes that are preferentially expressed within infected hosts represents a strategy to identify novel virulence factors and define global expression patterns relevant to pathogenesis. Until recently, C. albicans has not been amenable to screening using existing technologies. This has begun to change with the development of new molecular genetic tools and the sequencing of the C. albicans genome. In this paper, we review studies using recently developed techniques to identify genes expressed by C. albicans during infections, as well as work from our laboratory using a human antibody-based strategy. Along with others, we have shown that selected in vivo expressed genes encode known and previously unrecognized candidal virulence factors. Future studies in this area will identify additional novel virulence factors, as well as advance our understanding of pathogenesis.