Environmental and intracellular regulation of <it>Francisella tularensis ripA</it>

• Main Authors: Taft-Benz Sharon, Kijek Todd M, Fuller James R, Kawula Thomas H
• Format: Article
• Language: English
• Published: BMC 2009-10-01
• Series: BMC Microbiology
• Online Access: http://www.biomedcentral.com/1471-2180/9/216

<p>Abstract</p> <p>Background</p> <p><it>Francisella tularensis </it>is a highly virulent, facultative intracellular pathogen and the etiologic agent of the zoonotic disease Tularemia. RipA is a cytoplasmic membrane protein that is conserved among <it>Francisella </it>species and is required for intracellular growth. <it>F. tularensis ripA </it>deletion mutants escape the phagosome of infected cells, but unlike wild type organisms fail to replicate in the host cell cytoplasm.</p> <p>Results</p> <p>Further analysis of <it>ripA </it>with respect to environmental effects on the growth of mutant strains and expression levels revealed that RipA is required for optimal growth at pH 7.5 but not pH 6.5. Using a combination of RT-PCR, <it>ripA-lacZ </it>transcriptional and translational fusions, and a RipA-tetracysteine tag fusion protein we found that both <it>ripA </it>transcription and RipA protein levels were elevated in organisms grown at pH 7.5 as compared to organisms grown at pH 5.5. A number of genes, including <it>iglA</it>, that are required for intracellular growth are regulated by the transcriptional regulators MglA and SspA, and are induced upon infection of host cells. We quantified <it>ripA </it>and <it>iglA </it>expression at different stages of intracellular growth and found that the expression of each increased between 1 and 6 hours post infection. Given the similar intracellular expression patterns of <it>ripA </it>and <it>iglA </it>and that MglA and SspA are positive regulators of <it>iglA </it>we tested the impact of <it>mglA </it>and <it>sspA </it>deletions on <it>ripA </it>and <it>iglA </it>expression. In the deletion mutant strains <it>iglA </it>expression was reduced dramatically as expected, however <it>ripA </it>expression was increased over 2-fold.</p> <p>Conclusion</p> <p>Expression of <it>ripA </it>is required for growth at neutral pH, is pH sensitive, and is responsive to the intracellular environment. The intracellular expression pattern of <it>ripA </it>coincided with <it>iglA</it>, which is positively regulated by MglA and SspA. However, in contrast to their positive impact on <it>iglA </it>expression, MglA and SspA negatively impacted <it>ripA </it>expression <it>in vitro</it>.</p>