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Infect. Immun. doi:10.1128/IAI.00610-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Adaptation of Francisella tularensis to the mammalian environment is governed by cues which can be mimicked in vitro

Center for Immunology and Microbial Disease, and Center for Cardiovascular Sciences, Albany Medical College, 47 New Scotland Avenue, Albany NY 12208, USA. Department of Bacteriology, Umea University, SE-901 85 Umea, Sweden. Department of Material Sciences and Engineering & Bioinformatics and Computational Biology, Iowa State University, Ames, IA 50011, USA

^* To whom correspondence should be addressed. Email: Hazlett{at}mail.amc.edu.

	Abstract

The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and fresh water amoeba. It has previously been established that the conventional media used for in vitro propagation of this microbe does not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacterium's infection-derived phenotype. We compared Francisella LVS grown in brain-heart infusion broth (BHI, a standard microbiological medium rarely used in Francisella research) to those grown in Muellar Hinton broth (MHB, the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supra-physiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that of extracellular bacteria; BHI-grown bacteria mirror the latter, MHB-grown bacteria resemble neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of pro-inflammatory mediators than those exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella that have emerged from macrophages.