TheCoxiella burnetiiCryptic Plasmid Is Enriched in Genes Encoding Type IV Secretion System Substrates

Abstract

The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a phagolysosome-like parasitophorous vacuole (PV), in which it replicates. The organism encodes a Dot/Icm type IV secretion system (T4SS) predicted to deliver to the host cytosol effector proteins that mediate PV formation and other cellular events. All C. burnetii isolates carry a large, autonomously replicating plasmid or have chromosomally integrated plasmid-like sequences (IPS), suggesting that plasmid and IPS genes are critical for infection. Bioinformatic analyses revealed two candidate Dot/Icm substrates with eukaryotic-like motifs uniquely encoded by the QpH1 plasmid from the Nine Mile reference isolate. CpeC, containing an F-box domain, and CpeD, possessing kinesin-related and coiled-coil regions, were secreted by the closely related Legionella pneumophila Dot/Icm T4SS. An additional QpH1-specific gene, cpeE, situated in a predicted operon with cpeD, also encoded a secreted effector. Further screening revealed that three hypothetical proteins (CpeA, CpeB, and CpeF) encoded by all C. burnetii plasmids and IPS are Dot/Icm substrates. By use of new genetic tools, secretion of plasmid effectors by C. burnetii during host cell infection was confirmed using ␤-lactamase and adenylate cyclase translocation assays, and a C-terminal secretion signal was identified. When ectopically expressed in HeLa cells, plasmid effectors trafficked to different subcellular sites, including autophagosomes (CpeB), ubiquitinrich compartments (CpeC), and the endoplasmic reticulum (CpeD). Collectively, these results suggest that C. burnetii plasmid-encoded T4SS substrates play important roles in subversion of host cell functions, providing a plausible explanation for the absolute maintenance of plasmid genes by this pathogen.

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…Four of ten dot/icm genes tested complemented the corresponding mutant in L. pneumophila, suggesting functional similarity between the two systems [85,86]. Consequently, and owing to the lack of genetic tools for Coxiella, L. pneumophila was extensively used as a surrogate host to identify Coxiella T4BSS substrates using both CyaA and BlaM cytosolic translocation reporter assays [87][88][89][90][91][92][93][94][95][96]. Indeed, of the 143 currently recognized Coxiella Dot/Icm effectors, 125 were initially identified in L. pneumophila.…”

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

“…1A) (29,31). Second, the C terminus of CvpA contains a distribution of charged and hydrophobic amino acids reminiscent of a characterized Dot/Icm T4B secretion signal (19,22,35) (Fig. 1B).…”

“…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.…”

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.…”