Identification of ElpA, a Coxiella burnetii Pathotype-Specific Dot/Icm Type IV Secretion System Substrate

Abstract

Coxiella burnetii causes human Q fever, a zoonotic disease that presents with acute flu-like symptoms and can result in chronic life-threatening endocarditis. In human alveolar macrophages, C. burnetii uses a Dot/Icm type IV secretion system (T4SS) to generate a phagolysosome-like parasitophorous vacuole (PV) in which to replicate. The T4SS translocates effector proteins, or substrates, into the host cytosol, where they mediate critical cellular events, including interaction with autophagosomes, PV formation, and prevention of apoptosis. Over 100 C. burnetii Dot/Icm substrates have been identified, but the function of most remains undefined. Here, we identified a novel Dot/Icm substrate-encoding open reading frame (CbuD1884) present in all C. burnetii isolates except the Nine Mile reference isolate, where the gene is disrupted by a frameshift mutation, resulting in a pseudogene. The CbuD1884 protein contains two transmembrane helices (TMHs) and a coiled-coil domain predicted to mediate protein-protein interactions. The C-terminal region of the protein contains a predicted Dot/Icm translocation signal and was secreted by the T4SS, while the N-terminal portion of the protein was not secreted. When ectopically expressed in eukaryotic cells, the TMH-containing N-terminal region of the CbuD1884 protein trafficked to the endoplasmic reticulum (ER), with the C terminus dispersed nonspecifically in the host cytoplasm. This new Dot/Icm substrate is now termed ElpA (ER-localizing protein A). Full-length ElpA triggered substantial disruption of ER structure and host cell secretory transport. These results suggest that ElpA is a pathotype-specific T4SS effector that influences ER function during C. burnetii infection.

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

“…Effectors have consequently evolved to contain ELFs in the form of domains or motifs that functionally mimic eukaryotic host cell proteins [113]. The higher frequency of ELFs in bacterial effectors versus noneffectors provides a powerful predictive tool for assembling candidate effector lists [73,88,[90][91][92][94][95][96]107,113]. Indeed, recognition of a cohort of Coxiella genes encoding 33 amino acid ankyrin repeat domains (ARDs) resulted in identification of the first class of Coxiella Dot/Icm effectors [90,92].…”

“…Eukaryotic-like features (ELF), a C-terminal Dot/Icm translocation signal and similarity to known Coxiella effectors are strong predictors of effector proteins. Indicators of effector genes are upstream PmrA regulatory motifs and genomic location [73,[87][88][89][90][91][92][94][95][96]107]. When assembling candidate effector gene lists, the predictive power of these features is amplified when multiple criteria are combined in machine learning [95] and manual approaches [88,94].…”

“…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.…”

“…However, detection of native effectors is exceedingly difficult; thus, ectopic expression of effector-fluorescent protein chimeras has been extensively used for trafficking studies, despite known artifacts of this procedure [120]. Effectors traffic to mitochondria [87,92,94,121], endoplasmic reticulum [91,94,96,122], Golgi apparatus [87], lysosomes [87,92,107,122], autophagosomes [91,93], endocytic vesicles [73,107], nucleus [87,94,[121][122][123], microtubules [92,122] and ubiquitinated proteins [91,93]. An interesting subset of five effectors designated Cvp (Coxiella vacuolar protein) also traffic to the CCV [73,107].…”

“…Also relying on ectopic expression is the SEAP assay used to identify effectors that potentially disturb mammalian secretory trafficking. Several effectors inhibit release of SEAP in a HEK293T reporter cell system including CBU0635 [87], CBU1566 (CvpC), CBU1825, CBUA0019 [94] and CBUD1884 (ElpA) [96].…”