Role and Function of the Type IV Secretion System in Anaplasma and Ehrlichia Species

Abstract

The obligatory intracellular pathogens Anaplasma phagocytophilum and Ehrlichia chaffeensis proliferate within membrane-bound vacuoles of human leukocytes and cause potentially fatal emerging infectious diseases. Despite the reductive genome evolution in this group of bacteria, genes encoding the type IV secretion system (T4SS), which is homologous to the VirB/VirD4 system of the plant pathogen Agrobacterium tumefaciens, have been expanded and are highly expressed in A. phagocytophilum and E. chaffeensis in human cells. Of six T4SS effector proteins identified in them, roles and functions have been described so far only for ankyrin repeat domain-containing protein A (AnkA), Anaplasma translocated substrate 1 (Ats-1), and Ehrlichia translocated factor 1 (Etf-1, ECH0825). These effectors are abundantly produced and secreted into the host cytoplasm during infection, but not toxic to host cells. They contain eukaryotic protein motifs or organelle localization signals and have distinct subcellular localization, target to specific host cell molecules to promote infection. Ats-1 and Etf-1 are orthologous proteins, subvert two important innate immune mechanisms against intracellular infection, cellular apoptosis and autophagy, and manipulate autophagy to gain nutrients from host cells. Although Ats-1 and Etf-1 have similar functions and roles in obligatory intracellular infection, they are specifically adapted to the distinct membrane-bound intracellular niche of A. phagocytophilum and E. chaffeensis, respectively. Ectopic expression of these effectors enhances respective bacterial infection, whereas intracellular delivery of antibodies against these effectors or targeted knockdown of the effector with antisense peptide nucleic acid significantly impairs bacterial infection. Thus, both T4SSs have evolved as important survival and nutritional virulence mechanism in these obligatory intracellular bacteria. Future studies on the functions of Anaplasma and Ehrlichia T4SS effector molecules and signaling pathways will undoubtedly advance our understanding of the complex interplay between obligatory intracellular pathogens and their hosts. Such data can be applied toward the treatment and control of anaplasmosis and ehrlichiosis.

Binding of Host Cell Surface Protein Disulfide Isomerase by Anaplasma phagocytophilum Asp14 Enables Pathogen Infection

“…This discrepancy is likely due to an inherent limitation of the in vitro model. A. phagocytophilum inhibits apoptosis (55)(56)(57). Due to the inhibitory effect of BD34 on the bacterium's ability to invade cells, we rationalize that most BD34-treated neutrophils did not get infected and consequently apoptosed, leaving behind a surviving population consisting mainly of infected cells.…”

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

“…eauclairensis, studies on HF strain will help illuminate this bacterium and its pathogenesis. Despite reductive genome evolution resulting in a budget genome, P44/Msp2/OMP-1/P28/P30 genes were expanded by duplication in Ehrlichia and Anaplasma species (Rikihisa, 2010), and genes encoding VirB/D type IV secretion apparatus are expanded among the order Rickettsiales (Rikihisa, 2017), suggesting these duplications are important for the bacterial life cycle. But not all of these genes are required for in vitro growth in mammalian cell cultures in the absence of in vivo immune pressure.…”

“…Of remaining ORFs, ∼60 ORFs of the HF strain are likely required for macrophage infection, as orthologs of E. chaffeensis are essential for infection of macrophages including type IV secretion system apparatus (23 ORFs) and effector proteins (3), two-component regulatory system (6), and outer membrane proteins (porin, lipoproteins, invasin; ∼30 ORFs) etc. (Kumagai et al, 2006(Kumagai et al, , 2008Huang et al, 2008;Mohan Kumar et al, 2013;Rikihisa, 2015Rikihisa, , 2017Lin et al, 2016;Sharma et al, 2017;Teymournejad et al, 2017;Yan et al, 2018). Of the remaining ∼330 genes, most encode hypothetical proteins or proteins with unknown functions.…”

“…All Ehrlichia spp. sequenced so far have multiplicated genes encoding immunodominant outer membrane proteins P28/OMP-1 (Ohashi et al, 1998b(Ohashi et al, , 2001Rikihisa, 2015), some of which were demonstrated to have porin activity in E. chaffeensis (Kumagai et al, 2008), and genes encoding type IV secretion apparatus proteins VirB/D (Rikihisa, 2010(Rikihisa, , 2017Gillespie et al, 2016). Of 23 paralogs of p28/omp-1 of the HF strain, insertion was detected in four p28/omp-1s: EHF_0066, EHF_0067, EHF_0045 (two different insertions), and EHF_0048 (four different insertions), showing not all P28/OMP-1s are required for growth in macrophages.…”

“…No known pathogen-associated molecular patterns (PAMPs) including endotoxin, peptidoglycan, flagella, common pili, or exotoxin has been detected in Ehrlichia spp., yet they induce acute and/or chronic inflammatory cytokines production in MyD88-dependent, but Toll-like receptors (TLR)-independent manner (Koh et al, 2010;Miura et al, 2011;Rikihisa, 2015). Several virulence factors of E. chaffeensis, which are required for bacterial entry, survival and proliferation of macrophages, have been demonstrated, including ehrlichial invasin that binds host cell receptor and triggers its entry into host cells, three type IV secretion system effectors, three pairs of two-component regulatory system, and outer membrane porins for nutrient acquisition (Kumagai et al, 2006(Kumagai et al, , 2008Mohan Kumar et al, 2013;Rikihisa, 2015Rikihisa, , 2017Lin et al, 2016;Sharma et al, 2017;Teymournejad et al, 2017;Yan et al, 2018). However, whether Ehrlichia spp.…”