Identification of Type 4B Secretion System Substrates That Are Conserved among Coxiella burnetii Genomes and Promote Intracellular Growth

Coxiella burnetii is a Gram-negative pathogen that infects a variety of mammalian hosts. Infection of domesticated ewes can cause fetal abortion, whereas acute human infection normally manifests as the flu-like illness Q fever. Successful host infection requires replication of the pathogen within the lysosomal Coxiella-containing vacuole (CCV). The bacterium encodes a type 4B secretion system (T4BSS) that delivers effector proteins into the host cell. Disruption of C. burnetii T4BSS effector export abrogates CCV biogenesis and bacterial replication. Over 150 C. burnetii T4BSS substrates have been designated often based on heterologous protein translocation by the Legionella pneumophila T4BSS. Cross-genome comparisons predict that many of these T4BSS substrates are truncated or absent in the acute-disease reference strain C. burnetii Nine Mile. This study investigated the function of 32 proteins conserved among diverse C. burnetii genomes that are reported to be T4BSS substrates. Despite being previously designated T4BSS substrates, many of the proteins were not translocated by C. burnetii when expressed fused to the CyaA or BlaM reporter tags. CRISPR interference (CRISPRi) indicated that of the validated C. burnetii T4BSS substrates, CBU0122, CBU1752, CBU1825, and CBU2007 promote C. burnetii replication in THP-1 cells and CCV biogenesis in Vero cells. When expressed in HeLa cells tagged at its C or N terminus with mCherry, CBU0122 localized to the CCV membrane and the mitochondria, respectively. Collectively, these data further define the repertoire of bona fide C. burnetii T4BSS substrates. IMPORTANCE Coxiella burnetii secretes effector proteins via a T4BSS that are required for successful infection. Over 150 C. burnetii proteins are reported to be T4BSS substrates and often by default considered putative effectors, but few have assigned functions. Many C. burnetii proteins were designated T4BSS substrates using heterologous secretion assays in L. pneumophila and/or have coding sequences that are absent or pseudogenized in clinically relevant C. burnetii strains. This study examined 32 previously reported T4BSS substrates that are conserved among C. burnetii genomes. Of the proteins tested that were previously designated T4BSS substrates using L. pneumophila, most were not exported by C. burnetii. Several T4BSS substrates that were validated in C. burnetii also promoted pathogen intracellular replication and one trafficked to late endosomes and the mitochondria in a manner suggestive of effector activity. This study identified several bona fide C. burnetii T4BSS substrates and further refined the methodological criteria for their designation.