John E. Bennett, M.D.

Chief, Clinical Mycology Section

Figure 1. Scanning Electron Micrograph of Candida albicans showing buds and bud scars.

Description of Research Program

The Clinical Mycology Section focuses on pathogenesis, diagnosis, treatment, prevention, and epidemiology of mycoses, particularly cryptococcosis and candidiasis.

Candidiasis is the most common cause of deep fungal infections. Oropharyngeal candidiasis occurs in nearly all patients infected with the human immunodeficiency virus. Candidemia is the fourth or fifth most common cause of nosocomial septicemia in most hospitals. Treatment of candidiasis with azoles has provided good efficacy, low toxicity, and ease of oral administration. Increasing resistance of Candida species to all azoles has complicated management and prompted a search for newer agents.

Our work has focused on defining the molecular mechanisms by which azole resistance is occurring. The two broad areas under investigation are drug efflux pumps and sterol synthesis genes. Although azole resistance in Candida albicans has been related to both ABC (ATP binding cassette) and MFS (Major facilitator superfamily) pumps, we have concentrated on the former. Our work on genes important for conversion of lanosterol to ergosterol, the major fungal sterol, has centered around ERG3, which codes for 5,6-desaturase activity, and ERG11, which codes for the 14-demethylase. Mutations in ERG11 have clearly been identified as important for azole resistance. We have found that the Y132H and I471T change in the deduced amino acid sequence of 14 demethylase appears to increase azole resistance in the Darlington strain of C. albicans. Changes in two amino acids (A168V and T329S) in the C-5 sterol desaturase of the Darlington strain of Candida albicans account for lack of function of the expressed protein.

In C. glabrata, we have cloned, sequenced, expressed, and deleted a multidrug resistance gene, PDH1, that confers resistance to cycloheximide, oligomycin, rhodamine 6G, and cadmium. Ongoing work concerns defining other drug efflux pumps and their regulatory control in C. glabrata.

Figure 2. Cryptococcus neoformans var. neoformans: India Ink preparation of cells from a mucoid colony, note the exhuberant capsule.

Cryptococcus neoformans

The incidence of cryptococcosis has risen dramatically in the USA since the onset of the HIV-1 epidemic. Most centers now report that 95% of their patients with cryptococcosis are co-infected with HIV-1.

We are interested in an enzyme, laccase, which enhances virulence of C. neoformans. We have cloned, sequenced, and expressed this enzyme in the yeast Pichia pastoris. The heavily mannosylated expressed protein has been purified and deglycosylated for production of polyclonal and monoclonal antibody. Antibody to protein-conjugated peptides from laccase has also been prepared. We are collaborating with Peter Williamson, M.D., Ph.D., formerly of this laboratory, to use the antibodies for intracellular localization. Mutants with defective intracellular processing of laccase will be studied for clues to how laccase production may be blocked and virulence decreased.

Research Group Members

Kleper de Almeida, M.D.; Hiroshi Kakeya, M.D., Ph.D.; Brian Grimberg; Katherine Nyswaner.

Selected Recent Publications

Miyazaki T, Tsai HF, Bennett JE. Kre29p is a novel nuclear protein involved in DNA repair and mitotic fidelity in Candida glabrata. Curr Genet. 2006 Jul;50(1):11-22. Epub 2006 Apr 28.

Tsai HF, Krol AA, Sarti KE, Bennett JE. Candida glabrata PDR1, a transcriptional regulator of a pleiotropic drug resistance network, mediates azole resistance in clinical isolates and petite mutants. Antimicrob Agents Chemother. 2006 Apr;50(4):1384-92.

Miyazaki T, Miyazaki Y, Izumikawa K, Kakeya H, Miyakoshi S, Bennett JE, Kohno S. Fluconazole treatment is effective against a Candida albicans erg3/erg3 mutant in vivo despite in vitro resistance.Antimicrob Agents Chemother. 2006 Feb;50(2):580-6.

Patterson TF, Boucher HW, Herbrecht R, Denning DW, Lortholary O, Ribaud P, Rubin RH, Wingard JR, DePauw B, Schlamm HT, Troke P, Bennett JE; European Organization for Research and Treatment of Cancer (EORTC) Invasive Fungal Infections Group (IFIG); Pfizer Global Aspergillus Study Group. Strategy of following voriconazole versus amphotericin B therapy with other licensed antifungal therapy for primary treatment of invasive aspergillosis: impact of other therapies on outcome. Clin Infect Dis. 2005 Nov 15;41(10):1448-52. Epub 2005 Oct 13.

Bennett JE. Salvage therapy for aspergillosis. Clin Infect Dis. 2005 Sep 15;41 Suppl 6:S387-8.

Tsai HF, Bard M, Izumikawa K, Krol AA, Sturm AM, Culbertson NT, Pierson CA,Bennett JE. Candida glabrata erg1 mutant with increased sensitivity to azoles and to low oxygen tension. Antimicrob Agents Chemother. 2004 Jul;48(7):2483-9.

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