Coordinate control of virulence gene expression in Francisella tularensis

Francisella tularensis is a Gram-negative, intracellular pathogen and the causative agent of tularemia. Due to its low infectious dose, ability to cause potentially fatal disease, and ability to be easily aerosolized, several countries have developed F. tularensis as a potential bioweapon. Three proteins, MglA, SspA, and PigR, and the small molecule guanosine tetraphosphate (ppGpp), are transcription factors critical for the virulence of this organism. These regulators function coordinately to positively regulate the expression of genes present on the Francisella pathogenicity island, as well as many other genes that are required for the virulence of this organism. MglA and SspA form a complex that associates with RNA polymerase (RNAP); the interaction between the MglA-SspA complex and RNAP is thought to be critical for MglA and SspA to regulate gene expression. PigR, a putative DNA-binding protein, associates with the RNAP-associated MglA-SspA complex and may stabilize the binding of RNAP at regulated promoters. The interaction between the MglA-SspA complex and PigR in F. tularensis has been shown to be promoted by ppGpp. A direct interaction between the MglA-SspA complex and PigR had previously been found using a modified version of an E. coli two-hybrid assay, referred to as the bridge-hybrid assay, that permits the detection of interactions between a protein of interest and a protein complex. However, the role of this direct interaction in controlling gene expression in F. tularensis had not been investigated. Conflicting reports in the literature over the ability of PigR to interact with the MglA-SspA complex led to differing models of how PigR regulates virulence gene expression in F. tularensis. To address the importance of the interaction between the MglA-SspA complex and PigR in regulating gene expression, we used a combination of genetic approaches to identify mutants of either MglA or SspA that are specifically defective for interaction with PigR. The identified mutants of MglA and SspA were unable to functionally substitute for MglA or SspA, respectively, and were unable to promote expression of MglA- and SspA- regulated genes in F. tularensis. These results indicate that the interaction between the MglA-SspA complex and PigR is critical for expression of virulence genes in F. tularensis. Our work also identified a surface on the MglA-SspA complex that is important for the interaction with PigR and which may constitute a binding site for PigR. The small molecule ppGpp has previously been shown to promote the interaction between the MglA-SspA complex and PigR in F. tularensis. It is unknown if ppGpp directly or indirectly promotes this interaction. We determined that ppGpp is required to detect an interaction between the MglA-SspA complex and PigR in the E. coli bridge-hybrid assay, indicating that ppGpp is either directly involved in promoting this interaction or works through an indirect mechanism that is conserved between F. tularensis and E. coli. One potential conserved mechanism through which ppGpp may be influencing the interaction between the MglA-SspA complex and PigR is through regulation of the levels of the molecule polyphosphate. However, we determined that polyphosphate is not required in order for the MglA-SspA complex and PigR to detectably interact with one another in the E. coli bridge-hybrid assay. Furthermore, analysis of the role of polyphosphate in gene expression in F. tularensis revealed that polyphosphate is a negative regulator of virulence gene expression.Medical Science