Roy A. Khalaf

Yeast cell adhesion molecules have functional amyloid-forming sequences.

ABSTRACT The occurrence of highly conserved amyloid-forming sequences in Candida albicans Als proteins (H. N. Otoo et al., Eukaryot. Cell 7:776–782, 2008) led us to search for similar sequences in other adhesins from C. albicans and Saccharomyces cerevisiae. The β-aggregation predictor TANGO found highly β-aggregation-prone sequences in almost all yeast adhesins. These sequences had an unusual amino acid composition: 77% of their residues were β-branched aliphatic amino acids Ile, Thr, and Val, which is more than 4-fold greater than their prevalence in the S. cerevisiae proteome. High β-aggregation potential peptides from S. cerevisiae Flo1p and C. albicans Eap1p rapidly formed insoluble amyloids, as determined by Congo red absorbance, thioflavin T fluorescence, and fiber morphology. As examples of the amyloid-forming ability of the native proteins, soluble glycosylphosphatidylinositol (GPI)-less fragments of C. albicans Als5p and S. cerevisiae Muc1p also formed amyloids within a few days under native conditions at nM concentrations. There was also evidence of amyloid formation in vivo: the surfaces of cells expressing wall-bound Als1p, Als5p, Muc1p, or Flo1p were birefringent and bound the fluorescent amyloid-reporting dye thioflavin T. Both of these properties increased upon aggregation of the cells. In addition, amyloid binding dyes strongly inhibited aggregation and flocculation. The results imply that amyloid formation is an intrinsic property of yeast cell adhesion proteins from many gene families and that amyloid formation is an important component of cellular aggregation mediated by these proteins.

Characterisation of Pga1, a putative Candida albicans cell wall protein necessary for proper adhesion and biofilm formation.

The fungal pathogen Candida albicans is a leading causative agent of death in immunocompromised individuals. Many factors have been implicated in virulence including filamentation‐inducing transcription factors, adhesins, lipases and proteases. Many of these factors are glycosylphosphatidylinositol‐anchored cell surface antigenic determinant proteins. Pga1 is one such protein shown to be upregulated during cell wall regeneration. The purpose of this study was to characterise the role Pga1 plays by creating a homozygous pga1 null strain and comparing the phenotype with the parental strain. It was observed that the mutant strain showed less oxidative stress tolerance and an increased susceptibility to calcofluor white, a cell surface disrupting agent that inhibits chitin fibre assembly which translated as a 40% decrease in cell wall chitin content. Furthermore, the mutant exhibited a 50% reduction in adhesion and a 33% reduction in biofilm formation compared with the parental strain, which was reflected as a slight reduction in virulence. Our data suggest that Pga1 plays an important role in cell wall rigidity and stability. It was also observed that the pga1 null was over filamentous on both liquid and solid media and exhibited increased resistance to SDS suggesting upregulation of filamentation‐inducing genes and cell surface components to partially compensate for the deletion.

The Candida albicans Hwp2 is necessary for proper adhesion, biofilm formation and oxidative stress tolerance

Candida albicans is an important fungal pathogen of humans that is responsible for the majority of mucosal and systemic candidiasis. The host-pathogen interaction in C. albicans has been the subject of intense investigation as it is the primary step that leads to establishment of infection. Hwp2 is a cell wall GPI-anchored cell wall protein that was previously shown to be necessary for hyphal and invasive growth on solid media. The purpose of the current study is to further characterize the protein as far as its role in oxidative stress, sensitivity to cell wall disrupting agents, adhesion to human epithelial and endothelial cells, biofilm formation and chitin content. It appears that Hwp2 is necessary for proper oxidative stress tolerance, adhesion and biofilm formation as an hwp2 null is more susceptible to increasing doses of hydrogen peroxide, unable to adhere efficiently to epithelial and endothelial cell lines and unable to form wild type biofilm levels.

Characterization of Hwp2, a Candida albicans putative GPI-anchored cell wall protein necessary for invasive growth.

Various factors are thought to be responsible for Candida albicans virulence, such as lipases, proteases and adhesins. Many of these factors are GPI-anchored cell surface proteins responsible for pathogenicity. Hwp2 is a putative GPI-anchored protein. The purpose of this study is to characterize the role of Hwp2 regarding filamentation on various filamentation-inducing and non-inducing solid and liquid media, virulence in a mouse model of disseminated candidiasis, and drug resistance to six widely used antifungal agents, by creating a homozygous null hwp2 strain and comparing it with the parental and a revertant HWP2(+)strain. It was observed that an hwp2Delta strain was highly filamentation-deficient on solid agar media as opposed to most liquid media tested. Furthermore, the mutant strain was slightly reduced in virulence compared to the wild strain since all mice infected with the control strain died after 6 days of injection compared with 11 days for the mutant. These results indicate a possible role for Hwp2 in adhesion and invasiveness. Finally a previously unidentified 37-amino-acid-long, stretch of Hwp2, possibly involved in protein aggregation, was found to align with high sequence identity and exclusively to C. albicans cell wall proteins.

Susceptibility of Candida albicans to common and novel antifungal drugs, and relationship between the mating type locus and resistance, in Lebanese hospital isolates

The incidence of antifungal resistance is on the increase worldwide and novel drugs are constantly being developed to counter this trend. One hundred and sixteen clinical isolates of Candida albicans were collected from Lebanese hospitals in order to first determine the degree of resistance of Lebanese isolates to four common azoles: fluconazole (FL), itraconazole (IT), ketoconazole (KE), and voriconazole (VO), in addition to amphotericin B (AP) and caspofungin (CS) through the Epsilometer test method and second, determine any relationship between the allelic compositions of the mating type loci (MTLa, MTL α, MTLa/α) with drug resistance. Results showed that resistance, among C. albicans isolates, was the highest with 12% for IT, followed by 7.7% for VO, 6% for KE, 5% for FL, 1.7% for AP and 0% for CS. Three isolates (2.6%) were resistant to all azoles tested, including one that was resistant to all drugs used except CS. Eleven isolates were homozygous at the MTL locus (9.5%), five of which (45%) were resistant to at least one antifungal drug whereas 14 of the 105 heterozygous strains (13%) exhibited similar resistance (P = 0.02), indicating a strong correlation between MTL locus homozygosity and resistance.

Identification, typing, antifungal resistance profile, and biofilm formation of Candida albicans isolates from Lebanese hospital patients.

As leading opportunistic fungal pathogens identification and subtyping of Candida species are crucial in recognizing outbreaks of infection, recognizing particularly virulent strains, and detecting the emergence of drug resistant strains. In this study our objective was to compare identification of Candida albicans by the hospitals through the use of conventional versus identification based on the ITS (Internal Transcribed Spacer) and to assess biofilm forming capabilities, drug resistance patterns and correlate these with MLST typing. ITS typing revealed a 21.2% hospital misidentification rate. Multidrug resistance to three drugs out of four tested was detected within 25% of the isolates raising concerns about the followed treatment regimens. Drug resistant strains as well as biofilm formers were phylogenetically related, with some isolates with significant biofilm forming capabilities being correlated to those that were multidrug resistant. Such isolates were grouped closely together in a neighbor-joining tree generated by MLST typing indicating phylogenetic relatedness, microevolution, or recurrent infection. In conclusion, this pilot study gives much needed insight concerning C. albicans isolates circulating in Lebanese hospitals and is the first study of its kind correlating biofilm formation, antifungal resistance, and evolutionary relatedness.

Deletion of the Candida albicans PIR32 Results in Increased Virulence, Stress Response, and Upregulation of Cell Wall Chitin Deposition

Candida albicans is a common opportunistic pathogen that causes a wide variety of diseases in a human immunocompromised host leading to death. In a pathogen, cell wall proteins are important for stability as well as for acting as antigenic determinants and virulence factors. Pir32 is a cell wall protein and member of the Pir protein family previously shown to be upregulated in response to macrophage contact and whose other member, Pir1, was found to be necessary for cell wall rigidity. The purpose of this study is to characterize Pir32 by generating a homozygous null strain and comparing the phenotype of the null with that of the wild-type parental strain as far as filamentation, virulence in a mouse model of disseminated candidiasis, resistance to oxidative stress and cell wall disrupting agents, in addition to adhesion, biofilm capacities, and cell wall chitin content. Our mutant was shown to be hyperfilamentous, resistant to sodium dodecyl sulfate, hydrogen peroxide, sodium chloride, and more virulent in a mouse model when compared to the wild type. These results were unexpected, considering that most cell wall mutations weaken the wall and render it more susceptible to external stress factors and suggests the possibility of a cell surface compensatory mechanism. As such, we measured cell wall chitin deposition and found a twofold increase in the mutant, possibly explaining the above-observed phenotypes.

Microsatellite DNA Identification and Genotyping of Candida albicans from Lebanese Clinical Isolates

The present study involves collecting 125 isolates labeled as C. albicans from five different Lebanese hospitals and utilizing the microsatellite genotyping test to determine the following: first, the accuracy of hospital identification by comparing microsatellite results to hospital results. Second, the frequency and genotypes of infectious strains present relative to tissue and hospital location- a possible indicator of nosocomial infection, and third, a possible relationship between lack of microsatellite heterozygosity to azole resistance. Our results showed that the error in hospital identification varied from 2 to 33%, averaging at 7%, with the highest identification error in stool. Misidentified isolates were mainly Candida tropicalis followed by C. glabrata and C. parapsilosis. Strains with similar genotypes were also found to occur within certain hospitals suggesting the possibility of nosocomial infection. Finally, a relationship between lack of heterozygosity and azole resistance was observed since nine out of 10 homozygous isolates sharing a common allele with a heterozygote strain were sensitive to all drugs tested, whereas the homozygous genotype was resistant to at least one drug.

Natural Killer Cell Immunoglobulin-like Receptor (KIR) genotypes in Follicular Lymphoma patients: Results of a pilot study

AIMS The Natural Killer Cell Immunoglobulin-like Receptor (KIR) genotype profiling in Follicular Lymphoma has not been reported before in the literature. MATERIALS AND METHODS DNA extracted from 20 Follicular Lymphoma patients and 62 healthy controls was analyzed for KIR genotyping using a polymerase chain reaction/sequence specific primers technique (PCR/SSP) for the presence of 16 KIR gene and pseudogene loci. RESULTS The AA, AB, and BB genotype frequencies were, respectively, 20%, 60% and 20% with an A:B ratio of 1:1. KIR 2DL4, KIR 3DL2, KIR 3DL3, and KIR 3DP1*003 were presented in all individuals. No significant difference between patients and controls was detected. CONCLUSION KIR genotyping profile does not seem to be associated with Follicular Lymphoma. The results presented in this pilot research represent the first international report about this important clinical entity.

The Candida albicans Dse1 Protein Is Essential and Plays a Role in Cell Wall Rigidity, Biofilm Formation, and Virulence

The fungal pathogen Candida albicans is one of the leading causative agents of death in immunocompromised individuals. It harbors an arsenal of cell wall anchored factors that are implicated in virulence such as filamentation inducing factors, adhesins, lipases, proteases, and superoxide dismutases. Dse1 is a cell wall protein involved in cell wall metabolism. The purpose of this study is to characterize the role Dse1 plays in virulence. Dse1 appears to be an essential gene as no homozygous null mutant was possible. The heterozygote mutant exhibited increased susceptibility to calcofluor white, a cell wall disrupting agent, with a subsequent reduction in cell wall chitin content, decreased oxidative stress tolerance, a 30% reduction in biofilm formation, and a delay in adhesion that was mirrored by a reduction in virulence in a mouse model of infection. Dse1 thus appears to be an important protein involved in cell wall integrity and rigidity.