Characterization of egress mechanisms of Coxiella burnetii

Intracellular bacteria evolved mechanisms to invade host cells, establish an intracellular niche allowing survival and replication and exit the host cell after completion of the replication cycle to infect new target cells. Coxiella burnetii (C.b.) is a Gram-negative, obligate intracellular pathogen and the causative agent of the zoonotic disease Q fever. Resident alveolar macrophages are the first target cells, but C.b. spread to other organs and cell types. While we have information about the initial step of C.b. uptake and the maturation process of the C. burnetii-containing vacuole (CCV), it is not well studied how C.b. spreads, i.e. how it exits its host cell to enter new target cells. Understanding this essential step will be crucial to develop novel strategies to prevent C.b. from spreading and, thus, disease.

Here, we used human endothelial EA.hy926 cells and epithelial HeLa cells, as well as mouse fibroblasts lacking the lysosomal associated membrane proteins 1 and 2 (LAMP-1/ -2) to study the escape of intracellular C. burnetii with live cell imaging, confocal laser scanning microscopy, electron microscopy, a trans-well system, and by establishing a FACS-based spreading assay.

We could demonstrate that C.b. inhibits host cell apoptosis early during infection, but induces and/or increases apoptosis at later stages of infection. Egress depends on previously established large bacteria-filled vacuoles and a functional intrinsic apoptotic cascade. The released bacteria are not enclosed by a host cell membrane and can infect and replicate in new target cells. However, in addition to cell death, lysosomal exocytosis might be involved, as egress is inhibited in LAMP-1/-2 deficient cells.

In summary, our data argue that C. burnetii egress in a non-synchronous way at late stages of infection. Apoptosis-induction is important for C.b. egress, but other pathways such as lysosomal exocytosis most likely contribute.