Sec-mediated secretion by Coxiella burnetii

Abstract

Background: Coxiella burnetii is a Gram-negative intracellular bacterial pathogen that replicates within a phagolysosome-like parasitophorous vacuole (PV) of macrophages. PV formation requires delivery of effector proteins directly into the host cell cytoplasm by a type IVB secretion system. However, additional secretion systems are likely responsible for modification of the PV lumen microenvironment that promote pathogen replication.

Results: To assess the potential of C. burnetii to secrete proteins into the PV, we analyzed the protein content of modified acidified citrate cysteine medium for the presence of C. burnetii proteins following axenic (host cell-free) growth. Mass spectrometry generated a list of 105 C. burnetii proteins that could be secreted. Based on bioinformatic analysis, 55 proteins were selected for further study by expressing them in C. burnetii with a C-terminal 3xFLAG-tag. Secretion of 27 proteins by C. burnetii transformants was confirmed by immunoblotting culture supernatants. Tagged proteins expressed by C. burnetii transformants were also found in the soluble fraction of infected Vero cells, indicating secretion occurs ex vivo. All secreted proteins contained a signal sequence, and deletion of this sequence from selected proteins abolished secretion. These data indicate protein secretion initially requires translocation across the inner-membrane into the periplasm via the activity of the Sec translocase.

Conclusions: C. burnetii secretes multiple proteins, in vitro and ex vivo, in a Sec-dependent manner. Possible roles for secreted proteins and secretion mechanisms are discussed.

Figure 1

Multiple Coxiella burnetii proteins are present in growth medium supernatant.C. burnetii was cultured in modified ACCM-2 for 7 days, then supernatant was collected and analyzed by SDS-PAGE and silver staining. Lane 1, uninoculated media; lane 2, C. burnetii growth media.

Figure 2

Expression of FLAG-tagged secretion candidates by C. burnetii transformants confirms secretion and not cell lysis.C. burnetii transformed with plasmids encoding FLAG-tagged secretion candidates were cultured for 48 h, then expression of tagged protein induced by addition of aTc for 24 h. Supernatants were harvested, TCA precipitated and analyzed by immunoblotting using antibody directed against the FLAG-tag. Immunoblots were also probed with antibody directed against the cytosolic protein EF-Ts to control for bacterial lysis. Whole cell lysate of C. burnetii expressing FLAG-tagged CBU1764a was used as a positive control (+ve).

Figure 3

C. burnetii secretes proteins during growth in mammalian host cells. Vero cells were infected for 5 days with C. burnetii transformants expressing the FLAG-tagged proteins CBU0110, CBU1135 or CBU1984, then protein expression was induced for 18 h. Host cells were lysed and lysates centrifuged to pellet intact bacteria and cell debris. Proteins present in the pellet and supernatant were separated by SDS-PAGE, transferred to nitrocellulose and analyzed by immunoblotting with antibodies directed against the FLAG-tag and EF-Ts. Uninfected Vero cells were employed as a negative control.

Figure 4

Secretion requires an intact signal sequence. Five secreted proteins (CBU0110, CBU0915, CBU1135, CBU1173 and CBU1984) without their respective signal sequence were expressed as described in Figure 2. Pellets and TCA precipitated supernatants were analyzed by immunoblotting using antibody directed against the FLAG-tag.

Figure 5

Possible Sec-mediated secretion mechanisms of C. burnetii. Genome analysis predicts a TolC homolog that could mediate type I-like secretion from the periplasm after proteins are transported across the inner membrane by the Sec translocase. C. burnetii also encodes a set of core T4P proteins. T4P are evolutionarily related to type II secretion machinery and have been shown to mediate secretion of several proteins by F. novicida. Sequestration of periplasmic or surface proteins by OMVs is a third option for release of proteins into media supernatants.

Figure 6

C. burnetii produces OMVs. (A) High and low magnification scanning electron micrographs of C. burnetii within the PV of infected Vero cells. Bacteria show membrane blebbing and OMVs (arrowheads). (B) Transmission electron micrographs of C. burnetii cultured in ACCM-2 for 2 days (upper panel) and 6 days (lower panel) showing membrane blebbing and OMVs (arrowheads). Scale bars = 0.2 μm.