Coxiella burnetii, a global zoonotic pathogen, presents a serious public health threat. Central to its pathogenesis, C. burnetii replicates within a unique phagosome-derived Coxiella-containing vacuole (CCV). Scrutinizing metabolic pathways required by C. burnetii to replicate in these normally bactericidal conditions could uncover novel therapeutic targets or preventative strategies. The shikimate pathway synthesizes aromatic compounds, notably aromatic amino acids, and is an attractive potential target, being absent in mammals yet essential in bacteria, fungi, plants, and some protozoa. Additionally, virulence reduction of bacterial strains lacking the shikimate pathway has led to successful vaccine formulation in other bacterial species.
We demonstrated that CBU0010 acts as a shikimate dehydrogenase (AroE), the fourth enzyme in the pathway, in in vitro enzyme activity assays. The function of AroE is to convert 3-dehydro-shikimate to shikimate but in the absence of AroE, 3-dehydro-shikimate is broken down to protocatechuate. A previously generated aroE (cbu0010) transposon (Tn) mutant provided an opportunity to scrutinize the importance of AroE, and the shikimate pathway overall, to C. burnetii pathogenesis. Metabolomic data verified that AroE was non-functional in the aroE::Tn mutant, with significantly increased protocatechuate amounts compared to wildtype. This aroE::Tn mutant exhibited significantly lower replication compared to wildtype in both intracellular and axenic growth assays, suggesting the shikimate pathway is necessary for efficient C. burnetii replication.
In work by others, C. burnetii required all aromatic amino acids in axenic media for substantial replication. Additionally, we found that compared to the limited replication of wildtype in medium lacking tyrosine, aroE::Tn mutant replication was significantly less, suggesting C. burnetii has some ability to synthesize certain aromatic amino acids. Metabolomic analysis detected significantly reduced abundance of phenylalanine and several other aromatic compounds in the aroE::Tn mutant compared to wildtype, providing further indication of shikimate pathway functionality. Significant changes in the abundance of metabolites in numerous metabolic pathways suggests the loss of AroE and shikimate pathway derivatives affects C. burnetii more broadly.