Identification of a Coxiella burnetii outer membrane porin required for intracellular replication

Coxiella burnetii is a gram-negative, obligate intracellular pathogen that causes human Q fever. Within host cells, C. burnetii proliferates in a spacious, acidic, lysosome-derived Coxiella-containing vacuole (CCV) by a process that requires the Dot/Icm type IVB secretion system to deliver effectors that manipulate host cell functions. A previous transposon mutagenesis screen identified the gene cbu0937 as being important for intracellular replication of C. burnetii. Here, the function of Cbu0937 was investigated. The cbu0937::Tn mutant had no detectable defect replicating in the axenic acidified citrate cysteine medium 2. Additionally, intracellular replication of the cbu0937::Tn mutant was not restored by co-infection of host cells with an isogenic wild-type strain of C. burnetii. Thus, the cbu0937::Tn mutant has a cell-intrinsic intracellular replication defect. Intracellular replication of the cbu0937::Tn mutant was restored by complementing the gene in trans with a plasmid encoding either untagged or an epitope-tagged version of Cbu0937. Analysis of the predicted structure of the Cbu0937 protein using AlphaFold revealed high similarity between Cbu0937 and several bacterial porins. Fractionation studies and surface labeling of C. burnetii producing a functional epitope-tagged protein indicated the localization of Cbu0937 to the bacterial outer membrane. From these data, we conclude that cbu0937 encodes a porin that plays an essential role in supporting C. burnetii intracellular replication, which likely involves the acquisition of an important metabolite in the CCV lumen.