Abstract
BackgroundFrancisella tularensis 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 Francisella species and is required for intracellular growth. F. tularensis ripA deletion mutants escape the phagosome of infected cells, but unlike wild type organisms fail to replicate in the host cell cytoplasm.ResultsFurther analysis of ripA 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, ripA-lacZ transcriptional and translational fusions, and a RipA-tetracysteine tag fusion protein we found that both ripA 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 iglA, 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 ripA and iglA 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 ripA and iglA and that MglA and SspA are positive regulators of iglA we tested the impact of mglA and sspA deletions on ripA and iglA expression. In the deletion mutant strains iglA expression was reduced dramatically as expected, however ripA expression was increased over 2-fold.ConclusionExpression of ripA is required for growth at neutral pH, is pH sensitive, and is responsive to the intracellular environment. The intracellular expression pattern of ripA coincided with iglA, which is positively regulated by MglA and SspA. However, in contrast to their positive impact on iglA expression, MglA and SspA negatively impacted ripA expression in vitro.
Highlights
Francisella tularensis is a highly virulent, facultative intracellular pathogen and the etiologic agent of the zoonotic disease Tularemia
Intracellular growth is intricately associated with F. tularensis virulence and pathogenesis, and the intracellular lifestyle of F. tularensis is an active area of investigation
The phagosome escape mechanism employed by F. tularensis remains essentially unknown, but this property is clearly necessary for F. tularensis intracellular growth since mutants that fail to reach the cytoplasm are essentially unable to replicate within host cells [16,17]
Summary
Francisella tularensis is a highly virulent, facultative intracellular pathogen and the etiologic agent of the zoonotic disease Tularemia. F. tularensis ripA deletion mutants escape the phagosome of infected cells, but unlike wild type organisms fail to replicate in the host cell cytoplasm. Francisella tularensis is a highly virulent Gram negative bacterial pathogen and the etiologic agent of the zoonotic disease tularemia. Following uptake or invasion of a host cell wild type F. tularensis cells escape the phagosome and replicate within the cytoplasm [13,14,15] of infected cells. The phagosome escape mechanism employed by F. tularensis remains essentially unknown, but this property is clearly necessary for F. tularensis intracellular growth since mutants that fail to reach the cytoplasm are essentially unable to replicate within host cells [16,17]
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