The Type IV Secretion System Effector Protein CirA Stimulates the GTPase Activity of RhoA and Is Required for Virulence in a Mouse Model of Coxiella burnetii Infection.

Coxiella burnetii, the etiological agent of Q fever in humans, is an intracellular pathogen that replicates in an acidified parasitophorous vacuole derived from host lysosomes. Generation of this replicative compartment requires effectors delivered into the host cell by the Dot/Icm type IVb secretion system. Several effectors crucial for C. burnetii intracellular replication have been identified, but the host pathways coopted by these essential effectors are poorly defined, and very little is known about how spacious vacuoles are formed and maintained. Here we demonstrate that the essential type IVb effector, CirA, stimulates GTPase activity of RhoA. Overexpression of CirA in mammalian cells results in cell rounding and stress fiber disruption, a phenotype that is rescued by overexpression of wild-type or constitutively active RhoA. Unlike other effector proteins that subvert Rho GTPases to modulate uptake, CirA is the first effector identified that is dispensable for uptake and instead recruits Rho GTPase to promote biogenesis of the bacterial vacuole. Collectively our results highlight the importance of CirA in coopting host Rho GTPases for establishment of Coxiella burnetii infection and virulence in mammalian cell culture and mouse models of infection.

Altered host immune responses to membrane vesicles from Salmonella and Gram-negative pathogens.

Membrane vesicles (MVs), discrete nano-structures produced from the outer membrane of Gram-negative bacteria such as Salmonella enterica Typhimurium (S. Typhimurium), strongly activate dendritic cells (DCs), contain major antigens (Ags) recognized by Salmonella-specific B-cells and CD4+ T-cells, and provide protection against S. Typhimurium challenge in a mouse model. With this in mind, we hypothesized that alterations to the gene expression profile of bacteria will be reflected in the immunologic response to MVs. To test this, we assessed the ability of MVs from wild-type (WT) S. Typhimurium or a strain with a phenotype mimicking the intracellular-phase of S. Typhimurium (PhoP(c)) to activate dendritic cells and initiate a strong inflammatory response. MVs, isolated from wild-type and PhoP(c)S. Typhimurium (WTMVs and PhoPcMVs, respectively) had pro-inflammatory properties consistent with the parental bacterial strains: PhoPcMVs were less stimulatory for DC activation in vitro and were impaired for subsequent inflammatory responses compared to WTMVs. Interestingly, the reduced pro-inflammatory properties of PhoPcMVs did not completely rely on signals through TLR4, the receptor for LPS. Nonetheless, both WTMVs and PhoPcMVs contained abundant immunogenic antigens capable of being recognized by memory-immune CD4+ T-cells from mice previously infected with S. Typhimurium. Furthermore, we analyzed a suite of pathogenic Gram-negative bacteria and their purified MVs for their ability to activate DCs and stimulate inflammation in a manner consistent with the known inflammatory properties of the parental strains, as shown for S. Typhimurium. Finally, analysis of the potential vaccine utility of S. Typhimurium MVs revealed their capacity to encapsulate an exogenous model antigen and stimulate antigen-specific CD4+ and CD8+ T-cell responses. Taken together, our results demonstrate the dependence of bacterial cell gene expression for MV immunogenicity and subsequent in vitro immunologic response, as well as their potential utility as a vaccine platform.

Identification of Coxiella burnetii type IV secretion substrates required for intracellular replication and Coxiella-containing vacuole formation.

Coxiella burnetii, the etiological agent of acute and chronic Q fever in humans, is a naturally intracellular pathogen that directs the formation of an acidic Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Central to its pathogenesis is a specialized type IVB secretion system (T4SS) that delivers effectors essential for intracellular replication and CCV formation. Using a bioinformatics-guided approach, 234 T4SS candidate substrates were identified. Expression of each candidate as a TEM-1 ß-lactamase fusion protein led to the identification of 53 substrates that were translocated in a Dot/Icm-dependent manner. Ectopic expression in HeLa cells revealed that these substrates trafficked to distinct subcellular sites, including the endoplasmic reticulum, mitochondrion, and nucleus. Expression in Saccharomyces cerevisiae identified several substrates that were capable of interfering with yeast growth, suggesting that these substrates target crucial host processes. To determine if any of these T4SS substrates are necessary for intracellular replication, we isolated 20 clonal T4SS substrate mutants using the Himar1 transposon and transposase. Among these, 10 mutants exhibited defects in intracellular growth and CCV formation in HeLa and J774A.1 cells but displayed normal growth in bacteriological medium. Collectively, these results indicate that C. burnetii encodes a large repertoire of T4SS substrates that play integral roles in host cell subversion and CCV formation and suggest less redundancy in effector function than has been found in the comparative Legionella Dot/Icm model.