Figure 2.

Prediction of T4BSS secretion signal sequences, detection of T4BSS components in the ACCM during growth of C. burnetii and dependence of a functional T4BSS secretion system for the release of DotA and IcmX. (A) SignalP 4.0 was used to identify N-terminal secretion signals amongst the 23 T4BSS proteins (Petersen et al. 2011). IcmD, IcmX, DotA, DotC, DotD and DotH contained a secretion signal for type I peptidase (red text). In addition, DotC and DotD also contained a secretion signal for type II peptidase (blue text). In DotD, the type I and type II signals overlapped (purple text). Arrows designate predicted cleavage location. (B)Coxiella burnetii grown in ACCM were harvested at 96 and 168 hpi and the ACCM from these cultures was retained. T4BSS components with predicted secretion signals were independently probed for by immunoblot using antibodies against DotA, IcmX, DotH, DotC and IcmD. All T4BSS components with a predicted secretion signal are detectable in the C. burnetii lysate and only DotA and IcmX are released into the ACCM. The amount of total C. burnetii protein loaded was normalized using Com1. Equal starting volumes of an uninoculated ACCM (negative control) and the 96 and 168 hpi ACCM samples were concentrated to an equal volume and equal volumes loaded. Predicted molecular weights (kDa) are indicated on the left. (C) ACCM cultures of C. burnetii NMII and the C. burnetii NMII T4BSS ΔdotA, ΔdotB and icmD::Tn mutants were grown to 168 h post inoculation and the ACCM from the respective cultures retained. Coxiella burnetii lysates were normalized to Com1 and equal volumes of the conditioned ACCM were concentrated to an equivalent volume. Proteins were detected by immunoblot using antibodies against DotA and IcmX. Predicted molecular weights (kDa) are indicated on the left. DotA and IcmX were not detectable in the ACCM from mutant strains and are only found in ACCM from the C. burnetii NMII culture and the C. burnetii cell lysates.