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

Abstract

bCoxiella 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.T he naturally obligate intracellular pathogen Coxiella burnetii is the causative agent of Q fever in humans. The agent's high infectivity, ease of spread by aerosols, and environmental stability have led to its classification as a category B select agent by the Centers for Disease Control and Prevention (1). The primary route of infection is through inhalation of contaminated aerosols, which typical results in acute Q fever, a debilitating flu-like illness that is generally self-limiting and readily resolves without antibiotic treatment. However, under some circumstances, persistent infection can lead to chronic Q fever that presents as endocarditis or hepatitis (1). While the number of reported cases of acute Q fever in the United States has been relatively low, a marked increase occurred in 1999 when Q fever became a reportable disease. A recent outbreak of Q fever in The Netherlands resulted in over 4,000 confirmed cases, with 20% of patients requiring hospitalization (2). The dramatic increase in reported cases suggests C. burnetii is an emerging pathogen and highlights our lack of understanding of C. burnetii virulence factors.Inhalation of C. burnetii by a mammalian host results in actindependent endocytosis and internalization in an early endosome. While numerous intracellular pathogens actively subvert the default endocytic pathway to establish a unique host-derived vacuole, C. burnetii generally follows the default trafficking pathway to establish a Coxiella-containing vacuole (CCV) derived from the host lysosomal network. Generation of this unique replicative compartment requires active bacterial protein synthesis which drives homotypic and heterotypic vesicle fusions associated with generation of a spacious CCV that occupies the majority of the hos...

The SCID Mouse Model for Identifying Virulence Determinants in Coxiella burnetii

“…There are several reasons that an enhC mutant may be attenuated for growth in vivo . First, a previous report has established that a C. burnetii enhC mutant had an internalization defect, a phenotype that is also observed for enhC mutants in the closely related bacterium Legionella pneumophila (Cirillo et al, 2000 ; Weber et al, 2016 ). More recently, it was found that EnhC of L. pneumophila inhibits the function of SltL transglycosylase by interfering with peptidoglycan degradation, which benefits intracellular replication by limiting the activation of host Nod1 by cell wall components (Liu et al, 2012 ).…”

“…Here, we present a SCID mouse model of C. burnetii infection that we have used to screen the virulence potential of NMII Himar1 transposon mutants. The SCID model was used to determine that CirA is required for virulence in vivo , which was the first application of this assay (Weber et al, 2016 ). This report describes the validation of the model and its utility in comparing virulence between NMII strains.…”

The anti-apoptotic Coxiella burnetii effector protein AnkG is a strain specific virulence factor

“…These processes are dependent on the T4BSS 18 , 19 , which injects effector proteins into the host cell cytosol to manipulate the host cell for the benefit of the pathogen 20 . Roughly 150 putative C. burnetii effector proteins have been identified; however, functions have been assigned to only a few of them 21 – 29 . Anti-apoptotic activity was shown for the three effector proteins AnkG, CaeA and CaeB 23 , 26 , 30 – 33 .…”

Subversion of the Endocytic and Secretory Pathways by Bacterial Effector Proteins

“…Another essential type IV effector protein, CirA, can function as a GAP for RhoA and is hypothesized to promote cytoskeletal rearrangements to promote redirection of host vesicles to the growing CCV (Weber et al, 2016b ). CirA is predicted to encode several arginine-like finger motifs that support the observation that CirA acts as GTPase activating protein.…”