The "Biological Weapons" of Ehrlichia chaffeensis: Novel Molecules and Mechanisms to Subjugate Host Cells

Abstract

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging, potentially fatal tick-borne infectious disease. The bacterium enters human cells via the binding of its unique outer-membrane invasin EtpE to the cognate receptor DNase X on the host-cell plasma membrane; this triggers actin polymerization and filopodia formation at the site of E. chaffeensis binding, and blocks activation of phagocyte NADPH oxidase that catalyzes the generation of microbicidal reactive oxygen species. Subsequently, the bacterium replicates by hijacking/dysregulating host-cell functions using Type IV secretion effectors. For example, the Ehrlichia translocated factor (Etf)-1 enters mitochondria and inhibits mitochondria-mediated apoptosis of host cells. Etf-1 also induces autophagy mediated by the small GTPase RAB5, the result being the liberation of catabolites for proliferation inside host cells. Moreover, Etf-2 competes with the RAB5 GTPase-activating protein, for binding to RAB5-GTP on the surface of E. chaffeensis inclusions, which blocks GTP hydrolysis and consequently prevents the fusion of inclusions with host-cell lysosomes. Etf-3 binds ferritin light chain to induce ferritinophagy to obtain intracellular iron. To enable E. chaffeensis to rapidly adapt to the host environment and proliferate, the bacterium must acquire host membrane cholesterol and glycerophospholipids for the purpose of producing large amounts of its own membrane. Future studies on the arsenal of unique Ehrlichia molecules and their interplay with host-cell components will undoubtedly advance our understanding of the molecular mechanisms of obligatory intracellular infection and may identify hitherto unrecognized signaling pathways of human hosts. Such data could be exploited for development of treatment and control measures for ehrlichiosis as well as other ailments that potentially could involve the same host-cell signaling pathways that are appropriated by E. chaffeensis.

Keywords: Ehrlichia chaffeensis; RAB5; ROS; autophagy; ferritinophagy; invasin; membrane cholesterol; type IV secretion effector.

Figure 1

Ehrlichia entry is coupled with blockade of the activation of the phagocyte NADPH oxidase (NOX2) complex. Extracellular E. chaffeensis uses the C-terminal region of its surface protein EtpE to bind DNase X on the host-cell surface. The consequent lateral redistribution of DNase X within dynamic lipid rafts brings CD147 into association with the EtpE–DNase X complex (1). CD147 relays the signal to downregulate Vav1 GEF (guanine-nucleotide exchange factor; 2A) that prevents Rac1 activation (3A) and consequently prevents activation of the NOX2 complex (4A). CD147 also recruits hnRNP-K to bind N-WASP, leading to activation of N-WASP (conformational change) (2B and 3B). Activated N-WASP binds the Arp2/3 actin-nucleation complex (4B), leading to spatiotemporal actin polymerization and filopodia formation to internalize E. chaffeensis into endosomes (5B). The drawing was modified from (Mohan Kumar et al., 2015), copyright 2015 ASM.

Figure 2

Ehrlichia chaffeensis Etf-1 localizes to mitochondria to block apoptosis of host cells. Alternatively, Etf-1 binds to the Belin 1–VPS34–RAB5-GTP complex and induces RAB5-regulated autophagy. (Right) Etf-1 is depicted in blue with the putative T4SS signal depicted in dark blue. Etf-1 has a mitochondria-targeting presequence and localizes to mitochondria. Mitochondria-localized Etf-1 blocks apoptosis of eukaryotic host cells by preventing loss of mitochondrial membrane potential. TOM: transporter outer membrane complex; TIM: transporter inner membrane complex. (Left) Etf-1 binds the Beclin 1–VPS34–RAB5-GTP complex and induces RAB5-regualted autophagy. Etf-1 autophagosomes are recruited to E. chaffeensis inclusions and deliver captured host cytoplasmic contents. If not recruited to inclusions, Etf-1 autophagosomes mature to autolysosomes, in which captured substrates are degraded and catabolites are released to the cytoplasmic to promote bacterial proliferation. The drawing was modified from (Rikihisa, 2019), copyright 2017 Taylor & Francis, and from (Rikihisa, 2017), copyright 2017 Springer.

Figure 3

Ehrlichia chaffeensis Etf-2 binds RAB5-GTP and blocks RABGAP5 from acting on RAB5. RAB5-GTP hydrolysis by the RAB5-specific GAP is required for endosome maturation and lysosomal fusion (left). Etf-2 is responsible for blocking lysosomal fusion with E. chaffeensis inclusions by localizing to E. chaffeensis inclusions via binding to RAB5-GTP and competitively blocking RABGAP5 from acting on RAB5 on the inclusion surface (right). The drawing is from (Yan et al., 2018), copyright 2018 PNAS.

Figure 4

Ehrlichia chaffeensis Etf-3 binds ferritin light chain to induce ferritinophagy to increase the labile-iron pool for acquisition of iron by E. chaffeensis. Iron homeostasis is tightly regulated in host cells to maintain the labile cellular iron pool (left). Etf-3 directly binds ferritin via ferritin light chain and induces ferritinophagy to increase the labile cellular iron pool, thereby providing Fe²⁺ for E. chaffeensis proliferation. Etf-1–induced autophagy synergizes with Etf-3 to deliver extra Fe²⁺ to Ehrlichia inclusions (right). Tf: transferrin, which binds Fe³⁺ and transports it into cells. TfR: transferrin receptor, which binds and delivers iron-saturated transferrin via endocytosis. STEAP2: Six-transmembrane epithelial antigen of prostate-2, a metalloreductase that reduces Fe³⁺ to Fe²⁺. DMT1: Divalent metal transporter 1 that transports Fe²⁺ from endosomes to the cytoplasm. NCOA4: Nuclear receptor coactivator 4, a cargo receptor that mediates ferritinophagy. The drawing is from (Yan et al., 2021), copyright 2021 PNAS.

Figure 5

Host-cell membrane lipids (cholesterol and glycerophospholipids) are trafficked to the membrane of E. chaffeensis and its inclusions. NBD-phosphatidylcholine (A), TopFluor-cholesterol (B), or DiI (C) is added to E. chaffeensis–infected RF/6A cells to monitor the intracellular distribution of the fluorescent lipids. DNAs of bacteria and host are stained with Hoechst 33342 (pseudocolored green and red, respectively). Note labeling of densely packed intraluminal E. chaffeensis (Ech, small cocci stained with Hoechst 33342) with the three fluorescent lipids (A–C) and labeling of the bacterial inclusion membrane (open arrows) and intraluminal membranes with TopFluor-cholesterol (B) and DiI (C). Bar: 3 µm. The drawing was modified from (Lin et al., 2020), copyright 2020 PNAS.