Characterizing the Role of Cell-Wall β-1,3-Exoglucanase Xog1p in Candida albicans Adhesion by the Human Antimicrobial Peptide LL-37

Candida albicans is the major fungal pathogen of humans. Its adhesion to host-cell surfaces is the first critical step during mucosal infection. Antimicrobial peptides play important roles in the first line of mucosal immunity against C. albicans infection. LL-37 is the only member of the human cathelicidin antimicrobial peptide family and is commonly expressed in various tissues, including epithelium. We previously showed that LL-37 significantly reduced C. albicans adhesion to plastic, oral epidermoid OECM-1 cells, and urinary bladders of female BALB/c mice. The inhibitory effect of LL-37 on cell adhesion occurred via the binding of LL-37 to cell-wall carbohydrates. Here we showed that formation of LL-37–cell-wall protein complexes potentially inhibits C. albicans adhesion to polystyrene. Using phage display and ELISA, we identified 10 peptide sequences that could bind LL-37. A BLAST search revealed that four sequences in the major C. albicans cell-wall β-1,3-exoglucanase, Xog1p, were highly similar to the consensus sequence derived from the 10 biopanned peptides. One Xog1p-derived peptide, Xog1p90–115, and recombinant Xog1p associated with LL-37, thereby reversing the inhibitory effect of LL-37 on C. albicans adhesion. LL-37 reduced Xog1p activity and thus interrupted cell-wall remodeling. Moreover, deletion of XOG1 or another β-1,3-exoglucanase-encoding gene EXG2 showed that only when XOG1 was deleted did cellular exoglucanase activity, cell adhesion and LL-37 binding decrease. Antibodies against Xog1p also decreased cell adhesion. These data reveal that Xog1p, originally identified from LL-37 binding, has a role in C. albicans adhesion to polystyrene and, by inference, attach to host cells via direct or indirect manners. Compounds that target Xog1p might find use as drugs that prevent C. albicans infection. Additionally, LL-37 could potentially be used to screen for other cell-wall components involved in fungal cell adhesion

Rapid In-vitro Testing for Chemotherapy Sensitivity in Leukaemia Patients

© Springer-Verlag Berlin Heidelberg 2014. Bioluminescent bacterial biosensors can be used in a rapid in vitro assay to predict sensitivity to commonly used chemotherapy drugs in acute myeloid leukemia (AML). The nucleoside analog cytarabine (ara-C) is the key agent for treating AML; however, up to 30% of patients fail to respond to treatment. Screening of patient blood samples to determine drug response before commencement of treatment is needed. To achieve this aim, a self-bioluminescent reporter strain of Escherichia coli has been constructed and evaluated for use as an ara-C biosensor and an in vitro assay has been designed to predict ara-C response in clinical samples. Transposition mutagenesis was used to create a cytidine deaminase (cdd)-deficient mutant of E. coli MG1655 that responded to ara-C. The strain was transformed with the luxCDABE operon and used as a whole-cell biosensor for development an 8-h assay to determine ara-C uptake and phosphorylation by leukemic cells. Intracellular concentrations of 0.025 lmol/L phosphorylated ara-C were detected by significantly increased light output (P\0.05) from the bacterial biosensor. Results using AML cell lines with known response to ara-C showed close correlation between the 8-h assay and a 3-day cytotoxicity test for ara-C cell killing. In retrospective tests with 24 clinical samples of bone marrow or peripheral blood, the biosensor-based assay predicted leukemic cell response to ara-C within 8 h. The biosensor-based assay may offer a predictor for evaluating the sensitivity of leukemic cells to ara-C before patients undergo chemotherapy and allow customized treatment of drug-sensitive patients with reduced ara-C dose levels. The 8-h assay monitors intracellular ara-CTP (cytosine arabinoside triphosphate) levels and, if fully validated, may be suitable for use in clinical settings