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Macrophage reporter cell assay for screening immunopharmacological activity of cell wall-active antifungals.

ABSTRACT: Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-?B and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus fumigatus that conditionally express genes involved in cell wall synthesis were also tested with the reporter macrophages. We found that growth of fungi in subinhibitory concentrations of glucan synthesis inhibitors (caspofungin and enfumafungin A) or repression of the ?-glucan catalytic subunit of glucan synthase, FKS1, increased macrophage NF-?B/AP-1 activation in a dectin-1-dependent manner. This pattern of activation was also transiently observed with repression of chitin synthesis in C. albicans or when yeast cells were incubated in low concentrations of the chitin synthesis inhibitor nikkomycin Z.

SUBMITTER: Lewis RE

PROVIDER: S-EPMC3957890 | biostudies-other | 2014

REPOSITORIES: biostudies-other

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Macrophage reporter cell assay for screening immunopharmacological activity of cell wall-active antifungals.

Lewis Russell E RE Liao Guangling G Young Katherine K Douglas Cameron C Kontoyiannis Dimitrios P DP

Antimicrobial agents and chemotherapy 20140106 3

Antifungal exposure can elicit immunological effects that contribute to activity in vivo, but this activity is rarely screened in vitro in a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-κB and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates of Candida albicans and Aspergillus fumi ...[more]

PMID: 24395226

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Project description:UnlabelledA high-throughput phenotypic screen based on a Citrobacter freundii AmpC reporter expressed in Escherichia coli was executed to discover novel inhibitors of bacterial cell wall synthesis, an attractive, well-validated target for antibiotic intervention. Here we describe the discovery and characterization of sulfonyl piperazine and pyrazole compounds, each with novel mechanisms of action. E. coli mutants resistant to these compounds display no cross-resistance to antibiotics of other classes. Resistance to the sulfonyl piperazine maps to LpxH, which catalyzes the fourth step in the synthesis of lipid A, the outer membrane anchor of lipopolysaccharide (LPS). To our knowledge, this compound is the first reported inhibitor of LpxH. Resistance to the pyrazole compound mapped to mutations in either LolC or LolE, components of the essential LolCDE transporter complex, which is required for trafficking of lipoproteins to the outer membrane. Biochemical experiments with E. coli spheroplasts showed that the pyrazole compound is capable of inhibiting the release of lipoproteins from the inner membrane. Both of these compounds have significant promise as chemical probes to further interrogate the potential of these novel cell wall components for antimicrobial therapy.ImportanceThe prevalence of antibacterial resistance, particularly among Gram-negative organisms, signals a need for novel antibacterial agents. A phenotypic screen using AmpC as a sensor for compounds that inhibit processes involved in Gram-negative envelope biogenesis led to the identification of two novel inhibitors with unique mechanisms of action targeting Escherichia coli outer membrane biogenesis. One compound inhibits the transport system for lipoprotein transport to the outer membrane, while the other compound inhibits synthesis of lipopolysaccharide. These results indicate that it is still possible to uncover new compounds with intrinsic antibacterial activity that inhibit novel targets related to the cell envelope, suggesting that the Gram-negative cell envelope still has untapped potential for therapeutic intervention.

Dataset Information

Macrophage reporter cell assay for screening immunopharmacological activity of cell wall-active antifungals.

Publications

Macrophage reporter cell assay for screening immunopharmacological activity of cell wall-active antifungals.

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