Molecular pathogenesis of the obligate intracellular bacterium Coxiella burnetii

Abstract

The agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that causes acute and chronic infections. The study of C. burnetii pathogenesis has benefited from two recent fundamental advances: improved genetic tools and the ability to grow the bacterium in extracellular media. In this Review, we describe how these recent advances have improved our understanding of C. burnetii invasion and host cell modulation, including the formation of replication-permissive Coxiella-containing vacuoles. Furthermore, we describe the Dot/Icm (defect in organelle trafficking/intracellular multiplication) system, which is used by C. burnetii to secrete a range of effector proteins into the host cell, and we discuss the role of these effectors in remodelling the host cell.

Figure 1. The intracellular trafficking pathway of Coxiella burnetii

Coxiella burnetii binds to macrophages through α_Vβ₃ integrin, which triggers phagocytosis of the bacterium through an actin-dependent mechanism. The nascent Coxiella-containing vacuole (CCV) acquires RAB5 and EEA1 as early as 5 minutes after internalization and acidifies to approximately pH 5.4, which is characteristic of normal phagosomal development. By contrast to phagosomes, the CCV also acquires microtubule-associated protein light-chain 3 (LC3; an autophagosomal marker), a process that is dependent on bacterial protein synthesis. The nascent CCV develops through fusion and fission events with early endosomes and then late endosomes, leading to the disappearance of RAB5 and EEA1 and the acquisition of RAB7 and lysosome-associated membrane glycoprotein 1 (LAMP1) 40 to 60 minutes after internalization, in concurrence with a further acidification to pH 5, which is characteristic of normal phagosomal development. Lysosomal enzymes, including cathepsin D (CTSD), start accumulating in the CCV by 2 hours after internalization, at which point the vacuole is at ~pH 4.5; this is delayed significantly from normal phagolysosomal acquisition of CTSD. This pause in CCV development might allow conversion of the bacteria from small cell variants (SCVs) to the metabolically active large cell variants (LCVs). Between 8 hours and 2 days after internalization, the CCV expands to occupy an increasingly dominant portion of the cytoplasmic space of the host cell. This process is dependent on bacterial protein synthesis and involves the recruitment of both RHO GTPase and RAB1B to the CCV membrane. RHO GTPase is likely to be involved in maintenance of the large vacuole, whereas the recruitment of RAB1B from the ER might facilitate the acquisition of additional membranes to create this spacious CCV.

Figure 2. The type IVB secretion systems of Coxiella burnetii and Legionella pneumophila

Coxiella burnetii encodes 24 of the 27 Legionella pneumophila type IVB secretion system (T4BSS) components. The C. burnetii system lacks three homologues, DotJ (defect in organelle trafficking J; also known as IcmM), DotV and IcmR (intracellular multiplication R), which are depicted with a dashed outline. Studies using L. pneumophila identified a subcomplex termed the core transport complex, which links the inner and outer membranes and is composed of DotC, DotD, DotF, DotG and DotH. As this complex is also conserved in the T4ASS, it is hypothesized that C. burnetii assembles a similar complex. A second subcomplex consists of the coupling protein DotL (providing a link between the substrates and the transport complex), DotM, DotN, IcmS and IcmW. The ATPase activity of DotB is required for secretion, but the function of this activity remains to be determined. C. burnetii proteins that can substitute for the corresponding proteins in the L. pneumophila system are shown in maroon.

Figure 3. Roles of type IV secretion system effectors during Coxiella burnetii intracellular infection

A new family of type IV secretion system (T4SS) effectors has been identified that localizes to the Coxiella-containing vacuole (CCV) and might have diverse functions, such as CCV stability and fusogenicity. Located next to the Golgi apparatus, CBU0635 interferes with the host secretory pathway on ectopic expression. At least three T4SS effectors localize to the nucleus (CaeA, CBU1314 and CBU1976) when ectopically expressed in host cells, and these proteins might be involved in the transcriptional modulation that occurs during Coxiella burnetii infection. Exogenously stimulated apoptosis is modulated by C. burnetii, and an ankyrin repeat-containing protein (AnkG) has anti-apoptotic activity through an interaction with p32, a host protein that is involved in pathogen-induced apoptosis. A second T4SS effector, CaeB, alters mitochondrial membrane permeability during exogenously stimulated apoptosis. C. burnetii infection also increases the synthesis of anti-apoptotic proteins BCL2-related protein A1 (BCL2A1; also known as BFL1) and cIAP2 (also known as BIRC3) and causes activation of the pro-survival kinases AKT, ERK1 and ERK2. Other effectors might also be involved in the modulation of apoptosis. Several T4SS effectors have F-box domains and are potentially involved in promoting proteasome-mediated degradation of proteins; these effectors include CpeC, which localizes to ubiquitin-rich compartments in host cells. The effector CpeB has been localized to the autophagosome, and AnkA, AnkB and AnkF localize to the cytoplasm. Dashed arrows represent predicted effector functions that have not yet been experimentally demonstrated. All T4SS effectors are shown as green circles. BECN1, beclin 1; LCV, large cell variant; PKA, protein kinase A; SCV, small cell variant.