Richard Calderone

Recognition between Candida albicans and host cells

The cell surface of Candida albicans is a complex mosaic of polysaccharide and protein, of which mannoproteins constitute the major antigens and host cell recognition molecules. One group of mannoproteins is known as adhesins and has properties similar to lectins and integrins. The adhesins recognize either host cell fucosyl glycosides or peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD peptides).

Microbial Adhesion to Fibronectin in Vitro Correlates with Production of Endocarditis in Rabbits

Abstract Microbial adhesion to the constituents of nonbacterial thrombotic endocarditis (NBTE) is an important early event in the pathogenesis of infective endocarditis. Fibronectin is a ubiquitous mammalian glycoprotein with diverse functions which binds to certain bacteria but not to others. In this study, we determined that fibronectin is present on the surface of NBTE (after catheter-induced aortic valve trauma) but not on normal rabbit cardiac valvular endothelium. The adhesion of various bacteria and yeasts to human fibronectin in tissue culture wells was then measured. Microorganisms with a high isolation frequency from endocarditis cases (Staphylococcus aureus, Candida tropicalis, C albicans, Streptococcus faecalis, S. sanguis) bound significantly better (P < 0.01) to fibronectin in vitro than other organisms (Escherichia coli, C. krusei, Pseudomonas aeruginosa) rarely implicated in this disease. Microbial adhesion to fibronectin correlated closely with the propensity of each organism to produce endocarditis in rabbits (e.g., ID50) with preexistent NBTE. A similar distribution was noted after binding of soluble radiolabeled fibronectin to bacteria in suspension. The results suggest that fibronectin, expressed on the surface of NBTE, may mediate microbial adhesion of circulating organisms to initiate colonization during the early pathogenesis of infective endocarditis.

Rad52 depletion in Candida albicans triggers both the DNA‐damage checkpoint and filamentation accompanied by but independent of expression of hypha‐specific genes

We have analysed the effect of RAD52 deletion in several aspects of the cell biology of Candida albicans. Cultures of rad52Δ strains exhibited slow growth and contained abundant cells with a filamentous morphology. Filamentation with polarization of actin patches was accompanied by the induction of the hypha‐specific genes (HSG) ECE1, HWP1 and HGC1. However, filament formation occurred in the absence of the transcription factors Efg1 and Cph1, even though disruption of EFG1 prevented expression of HSG. Therefore, expression of HSG genes accompanies but is dispensable for rad52Δ filamentation. However, deletion of adenylate cyclase severely impaired filamentation, this effect being largely reverted by the addition of exogenous cAMP. Filaments resembled elongated pseudohyphae, but some of them looked like true hyphae. Following depletion of Rad52, many cells arrested at the G2/M phase of the cell cycle with a single nucleus suggesting the early induction of the DNA‐damage checkpoint. Filaments formed later, preferentially from G2/M cells. The filamentation process was accompanied by the uncoupling of several landmark events of the cell cycle and was partially dependent on the action of the cell cycle modulator Swe1. Hyphae were still induced by serum, but a large number of rad52 cells myceliated in G2/M.

Homologous recombination in Candida albicans: role of CaRad52p in DNA repair, integration of linear DNA fragments and telomere length

Chromosomal rearrangements are common in both clinical isolates and spontaneous mutants of Candida albicans. It appears that many of these rearrangements are caused by translocations around the major sequence repeat (MSR) that is present in all chromosomes except chromosome 3, suggesting that homologous recombination (HR) may play an important role in the survival of this organism. In order to gain information on these processes, we have cloned the homologue of RAD52, which in Saccharomyces cerevisiae is the only gene required for all HR events. CaRAD52 complemented poorly a rad52 mutant of S. cerevisiae. Two null Carad52Δ/Carad52Δ mutants were constructed by sequential deletion of both alleles and two reconstituted strains were obtained by reintegration of the gene. Characterization of these mutants indicated that HR plays an essential role in the repair of DNA lesions caused by both UV light and the radiomimetic compound methyl‐methane‐sulphonate (MMS), whereas the non‐homologous end‐joining pathway (NHEJ) is used only in the absence of Rad52p or after extensive DNA damage. Repair by HR is more efficient in exponentially growing than in stationary cells, probably because a larger number of cells are in late S or G2 phases of the cell cycle (and therefore, can use a sister chromatid as a substrate for recombinational repair), whereas stationary phase cells are mainly in G0 or G1, and only can be repaired using the chromosomal homologue. In addition, CaRad52pu200a isu200a absolutelyu200a requiredu200a foru200a theu200a integration of linear DNA with long flanking homologous sequences. Finally, the absence of CaRad52p results in the lengthening of telomeres, even in the presence of an active telomerase, an observation not described in any other organism. This raises the possibility that both telomerase and homologous recombination may function simultaneously at C. albicans telomeres.

Pathogenesis of vaginal candidiasis: studies with a mutant which has reduced ability to adhere in vitro

A spontaneous, cerulenin-resistant mutant of Candida albicans (strain 4918-10) was found to adhere less readily to human vaginal mucosal cells in vitro than a wild type C. albicans (strain 4918). In a murine model of vaginal infection, strain 4918-10 was found to be less virulent than wild type C. albicans, i.e., the infection rate caused by 4918-10 was only 31% of that observed with wild type, 4918. A chitin-soluble extract (CSE) prepared from 4918 blocked attachment of yeast cells to human vaginal epithelial cells, while CSE from 4918-10 did not significantly reduce the attachment of yeasts to vaginal cells. Both 4918 and 4918-10 produced hyphae in vitro and in vivo, were negative for proteinase production and grew equally well at 28 degrees C and 37 degrees C. The data suggest that adherence to vaginal mucosa may be an important determinant in the pathogenesis of vaginal infection caused by C. albicans.

Afyap1, encoding a bZip transcriptional factor of Aspergillus fumigatus, contributes to oxidative stress response but is not essential to the virulence of this pathogen in mice immunosuppressed by cyclophosphamide and triamcinolone

Aspergillus fumigatus, an important human fungal pathogen, encounters high levels of reactive oxygen species following its ingestion by phagocytes. Reactive oxygen species are important mediators of the fungicidal activities of phagocytes. In yeasts, YAP1 encodes for transcriptional factors that contribute to their oxidative stress response and given the importance of the stress response, we hypothesized that the YAP1 homologue in A. fumigatus plays a similar role in this fungus. In this study, we found that Afyap1, the Yap1 homologue of A. fumigatus, confers protection against oxidative stress. Replacement of Afyap1 with the marker gene pyrG (DeltaAfyap1) resulted in hypersensitivity of A. fumigatus to oxidants such as H(2)O(2) and menadione. In contrast, an A. fumigatus strain harboring multiple-copy Afyap1 was resistant to these two oxidants as well as the oxidant diamide. However, DeltaAfyap1 and strain harboring multiple-copy Afyap1 were comparable in their virulence to a wild-type A. fumigatus strain in a murine model of invasive pulmonary aspergillosis. Taken together, these results demonstrate that Afyap1 is involved in oxidative stress response but is not an essential virulence factor for A. fumigatus.

Gene transcription studies of Candida albicans following infection of HEp2 epithelial cells

Previously we observed that infection of HEp2 epithelial cells with Candida albicans results in HEp2 cell actin rearrangement as well as reduced membrane ruffling and motility and that supernatants of a C. albicans culture (Candida metabolite) caused the same changes. In this study, we used microarray analysis to determine changes in gene transcription of C. albicans following infection of HEp2 cells compared to control cultures grown in the absence of HEp2 cells. We observed 201 genes whose regulation was increased at least 2-fold following a 3 h incubation with HEp2 cells as well as 87 genes that are down-regulated. Among the up-regulated genes were ALS2 and ALS5 both of which encode proteins that provide an adherence function for C. albicans. To confirm the changes in ALS transcription, we measured by RT-PCR ALS1-9 at 1 h intervals for a total of 4 h. After 1 h of infection, several of the ALS genes were up-regulated compared to C. albicans grown alone. At 2-4 h, an increase in most of the ALS genes was observed in both infected and control cultures. ALS7 transcription was observed only at 3-4 h, but transcription was similar in both infected and control cultures. By RT-PCR, ALS2 and 5, similar to the microarray data, were significantly increased in infected cells at 3 h. Our results show that gene transcription following the adherence of C. albicans to HEp2 cells includes the up-regulation of genes encoding members of a family of known host recognition adhesins that may be critical to successful colonization and invasion of the organism.

The histidine kinases of Candida albicans: regulation of cell wall mannan biosynthesis

Previously, we have used both biochemical and immunological approaches to determine that the two-component, histidine kinase Chk1p regulates cell wall biosynthesis in Candida albicans. These data were obtained by comparing wild-type cells to a strain of C. albicans deleted in CHK1. The dysregulation of cell wall biosynthesis in the mutant reduces its adherence to human esophageal tissue and results in avirulence. In the current study, we used transmission immune electron microscopy (IEM) to visualize the cell surface of both wild-type (CAF2) and the chk1 mutant (CHK21). IEM was performed using two IgM monoclonal antibodies to either an acid-stable mannan epitope (Mab B6) or to an acid-labile mannan epitope (Mab B6.1). We observed that the cell surface of the CHK21 mutant was more reactive than wild-type cells with Mab B6, while the reactivity of Mab B6.1 was similar for both CAF2 and CHK21. These observations correlate with previous data on the Western blotting of mutant and wild-type cells using the same monoclonal antibodies, i.e., greater activity with Mab B6 than with Mab B6.1. In addition to CHK1, two other histidine kinases (SLN1 and NIK1) have been described in C. albicans. Mutants in both sln1Delta and nik1Delta were compared by Western blotting using Mab B6 and Mab B6.1. Reactivity of each mutant to Mab B6 was similar to that observed with the chk1 mutant; on the other hand, the mannoprotein profiles obtained with Mab B6.1 in all mutants were similar to wild-type cells. We also compared the expression of 29 genes involved in mannan synthesis by reverse transcription-polymerase chain reaction (RT-PCR) and found that expression of a subset of six genes (ALG2, ALG6, ALG8, MNT3, PMT6, KRT2) was upregulated in all histidine kinase mutants, while increased expression of ALG7 was only observed in the sln1 and nik1 mutants, MNN1 was upregulated in the chk1 and nik1 mutants, and MNN4 was upregulated in the nik1Delta. Our data indicate that each of the C. albicans HK proteins may regulate similar functions in cell wall biosynthesis. This activity could be achieved in either a common or parallel, redundant signal transduction pathway(s).

Rad52 function prevents chromosome loss and truncation in Candida albicans

RAD52 is required for almost all recombination events in Saccharomyces cerevisiae. We took advantage of the heterozygosity of HIS4 in the Candida albicans SC5314 lineage to study the role of Rad52 in the genomic stability of this important fungal pathogen. The rate of loss of heterozygosity (LOH) at HIS4 in rad52‐ΔΔ strains was ∼10−3, at least 100‐fold higher than in Rad52+ strains. LOH of whole chromosome 4 or truncation of the homologue that carries the functional HIS4 allele was detected in all 80 rad52‐ΔΔ His auxotrophs (GLH –GL lab His‐) obtained from six independent experiments. Isolates that had undergone whole chromosome LOH, presumably due to loss of chromosome, carried two copies of the remaining homologue. Isolates with truncations carried centric fragments of broken chromosomes healed by de novo telomere addition. GLH strains exhibited variable degrees of LOH across the genome, including two strains that became homozygous for all the heterozygous markers tested. In addition, GLH strains exhibited increased chromosomal instability (CIN), which was abolished by reintroduction of RAD52. CIN of GLH isolates is reminiscent of genomic alterations leading to cancer in human cells, and support the mutator hypothesis in which a mutator mutation or CIN phenotype facilitate more mutations/aneuploidies.

Role of the homologous recombination genes RAD51 and RAD59 in the resistance of Candida albicans to UV light, radiomimetic and anti-tumor compounds and oxidizing agents.

We have cloned and characterized the RAD51 and RAD59 orthologs of the pathogenic fungus Candida albicans. CaRad51 exhibited more than 50% identity with several other eukaryotes and the conserved the catalytic domain of a bacterial RecA. As compared to the parental strain, null strains of rad51 exhibited a filamentous morphology, had a decreased grow rate and exhibited a moderate sensitivity to UV light, oxidizing agents, and compounds that cause double-strand breaks (DSB), indicating a role in DNA repair. By comparison, the rad52 null had a higher percentage of filaments, a more severe growth defect and a greater sensitivity to DNA-damaging compounds. Null strains of rad59 showed a UV-sensitive phenotype but behaved similarly to the parental strain in the rest of the assays. As compared to Saccharomyces cerevisiae, C. albicans was much more resistant to bleomycin and the same was true for their respective homologous recombination (HR) mutants. These results indicate that, as described in S. cerevisiae, RAD52 plays a more prominent role than RAD51 in the repair of DSBs in C. albicans and suggest the existence of at least two Rad52-dependent HR pathways, one dependent and one independent of Rad51.