Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii

Abstract

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for acute and chronic Q fever. This bacterium harbors a type IV secretion system (T4SS) highly similar to the Dot/Icm of Legionella pneumophila that is believed to be essential for its infectivity. Protein substrates of the Coxiella T4SS are predicted to facilitate the biogenesis of a phagosome permissive for its intracellular growth. However, due to the lack of genetic systems, protein transfer by the C. burnetii Dot/Icm has not been demonstrated. In this study, we report the identification of 32 substrates of the C. burnetii Dot/Icm system using a fluorescence-based β-lactamase (TEM1) translocation assay as well as the calmodulin-dependent adenylate cyclase (CyaA) assay in the surrogate host L. pneumophila. Notably, 26 identified T4SS substrates are hypothetical proteins without predicted function. Candidate secretion substrates were obtained by using (i) a genetic screen to identify C. burnetii proteins interacting with DotF, a component of the T4SS, and (ii) bioinformatic approaches to retrieve candidate genes that harbor characteristics associated with previously reported substrates of the Dot/Icm system from both C. burnetii and L. pneumophila. Moreover, we have developed a shuttle plasmid that allows the expression of recombinant proteins in C. burnetii as TEM fusion products. Using this system, we demonstrated that a Dot/Icm substrate identified with L. pneumophila was also translocated by C. burnetii in a process that requires its C terminus, providing direct genetic evidence of a functional T4SS in C. burnetii.effectors | protein translocation | transporter

Identification of Novel Coxiella burnetii Icm/Dot Effectors and Genetic Analysis of Their Involvement in Modulating a Mitogen-Activated Protein Kinase Pathway

“…We also excluded ORFs considered pseudogenes according to other C. burnetii isolates, since we wanted to investigate effectors with similar functions across the different C. burnetii isolates (25). To examine effector translocation, we used the wellestablished L. pneumophila model of Icm/Dot translocation that was shown to correlate entirely with effector translocation by C. burnetii (21,24,25). We conducted two learning and validation phases and a third final learning phase to predict additional putative effectors.…”

“…In order to determine whether the 13 newly validated effectors (listed in Table 2) are expressed in C. burnetii, the levels of their mRNA were measured using RT-qPCR with C. burnetii Nine Mile phase II grown in ACCM-2 axenic medium as well as in HEK293T cells (see Materials and Methods). The levels of expression of these effector-encoding genes were compared to those of two known C. burnetii effector-encoding genes (CBU1751-coxDFB5 and CBU0635) (24,25) (Fig. 2).…”

“…The C. burnetii effector proteins were identified thus far based on different criteria, including (i) existence of eukaryotic motifs such as ankyrin domains (18,19), (ii) presence of a PmrA binding site in their upstream regulatory region (20,21), (iii) identification of their C-terminal translocation signal (17), and (iv) location on plasmids (22,23). Most intriguingly, most of the effectors identified are unique proteins not present in any other sequenced organism (24,25).…”

Computational modeling and experimental validation of the Legionella and Coxiella virulence-related type-IVB secretion signal

“…C. burnetii has been shown to contain a functional Icm/Dot secretion system that translocates effector proteins into host cells (10,32). Most of the C. burnetii effectors were validated using L. pneumophila as a translocation platform, suggesting that effectors from both bacteria share a similar secretion signal (13,14,33,34). For the experimental examination of C. burnetii predicted effectors, a cutoff score of 6 (instead of 5) was used to improve the chances for accurate prediction.…”

Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

“…To date, over 80 C. burnetii genes that encode T4BSS substrates have been identified (17)(18)(19)(20)(21)(22)(23). These substrates have largely been identified using L. pneumophila as a surrogate host and adenylate cyclase or β-lactamase-based translocation assays.…”