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

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.

Ehrlichia chaffeensis and Four Anaplasma Species With Veterinary and Public Health Significance Identified in Tibetan Sheep (Ovis aries) and Yaks (Bos grunniens) in Qinghai, China

Tick-borne diseases (TBDs) can cause serious economic losses and are very important to animal and public health. To date, research on TBDs has been limited in Qinghai-Tibet Plateau, China. This epidemiological investigation was conducted to evaluate the distribution and risk factors of Anaplasma spp. and Ehrlichia chaffeensis in livestock in Qinghai. A total of 566 blood samples, including 330 yaks (Bos grunniens) and 236 Tibetan sheep (Ovis aries) were screened. Results showed that A. bovis (33.3%, 110/330) and A. phagocytophilum (29.4%, 97/330) were most prevalent in yaks, followed by A. ovis (1.2%, 4/330), A. capra (0.6%, 2/330), and E. chaffeensis (0.6%, 2/330). While A. ovis (80.9%, 191/236) and A. bovis (5.1%, 12/236) infection was identified in Tibetan sheep. To our knowledge, it is the first time that A. capra and E. chaffeensis have been detected in yaks in China. Apart from that, we also found that co-infection of A. bovis and A. phagocytophilum is common in yaks (28.2%, 93/330). For triple co-infection, two yaks were infected with A. bovis, A. phagocytophilum, and A. capra, and two yaks were infected with A. bovis, A. phagocytophilum, and E. chaffeensis. Risk analysis shows that infection with A. bovis, A. phagocytophilum, and A. ovis was related to region and altitude. This study provides new data on the prevalence of Anaplasma spp. and E. chaffeensis in Qinghai, China, which may help to develop new strategies for active responding to these pathogens.

Functional Characterization of Multiple Ehrlichia chaffeensis Sodium (Cation)/Proton Antiporter Genes Involved in the Bacterial pH Homeostasis

Ehrlichia chaffeensis causes human monocytic ehrlichiosis. Little is known about how this and other related tick-borne rickettsia pathogens maintain pH homeostasis in acidified phagosomes and the extracellular milieu. The membrane-bound sodium (cation)/proton antiporters are found in a wide range of organisms aiding pH homeostasis. We recently reported a mutation in an antiporter gene of E. chaffeensis (ECH_0379) which causes bacterial in vivo attenuation. The E. chaffeensis genome contains 10 protein coding sequences encoding for predicted antiporters. We report here that nine of these genes are transcribed during the bacterial growth in macrophages and tick cells. All E. chaffeensis antiporter genes functionally complemented antiporter deficient Escherichia coli. Antiporter activity for all predicted E. chaffeensis genes was observed at pH 5.5, while gene products of ECH_0179 and ECH_0379 were also active at pH 8.0, and ECH_0179 protein was complemented at pH 7.0. The antiporter activity was independently verified for the ECH_0379 protein by proteoliposome diffusion analysis. This is the first description of antiporters in E. chaffeensis and demonstrates that the pathogen contains multiple antiporters with varying biological functions, which are likely important for the pH homeostasis of the pathogen’s replicating and infectious forms.

Immunoreactive Protein Repertoires of Ehrlichia chaffeensis and E. canis Reveal the Dominance of Hypothetical Proteins and Conformation-dependent Antibody Epitopes

The immunomes of Ehrlichia chaffeensis ( E. ch. ) and E. canis ( E. ca. ) have recently be revised to include immunodominant hypothetical proteins with conformational antibody epitopes. In this study, we examined 216 E. ch. and 190 E. ca. highly antigenic proteins according to ANTIGENpro and also performed a genome-wide hypothetical protein analysis ( E. ch. n=104; E. ca. n=124) for immunoreactivity. Using cell-free protein expression and immunoanalysis, 118 E. ch. and 39 E. ca . proteins reacted with sera from naturally E. ch. -infected patients or E. ca. -infected dogs. Moreover, 22 E. ch. and 18 E. ca. proteins consistently and strongly reacted with a panel of patient or canine sera. A subset of E. ch. (n=18) and E. ca. (n=9) proteins were identified as immunodominant. Consistent with our previous study, most proteins were classified as hypothetical and the antibody epitopes exhibited complete or partial conformation-dependence. The majority (28/40; 70%) of E. ch. and E. ca. proteins contained transmembrane domains and 19 (48%) were predicted to be secreted effectors. The antigenic repertoires of E. ch. and E. ca. were mostly diverse and suggest that the immunomes of these closely related ehrlichiae are dominated by species-specific conformational antibody epitopes. This study reveals a significant group of previously undefined E. ch. and E. ca. antigens and reaffirms the importance of conformation-dependent epitopes as targets of anti- Ehrlichia immune responses. These findings substantially expand our understanding of host- Ehrlichia immune responses, advance efforts to define the molecular features of protective proteins and improve prospects for effective vaccines for the ehrlichioses.

Alpha Enolase 1 Ubiquitination and Degradation Mediated by Ehrlichia chaffeensis TRP120 Disrupts Glycolytic Flux and Promotes Infection

Ehrlichia chaffeensis modulates numerous host cell processes, including gene transcription to promote infection of the mononuclear phagocyte. Modulation of these host cell processes is directed through E. chaffeensis effectors, including TRP120. We previously reported that TRP120 moonlights as a HECT E3 Ub ligase that ubiquitinates host cell transcription and fate regulators (PCGF5 and FBW7) to promote infection. In this study, we identified a novel TRP120 substrate and examined the relationship between TRP120 and α-enolase (ENO1), a metalloenzyme that catalyzes glycolytic pathway substrate dehydration. Immunofluorescence microscopy and coimmunoprecipitation demonstrated interaction between ENO1 and TRP120, and ubiquitination of ENO-1 by TRP120 was detected in vivo and in vitro. Further, ENO-1 degradation was observed during infection and was inhibited by the proteasomal inhibitor bortezomib. A direct role of TRP120 Ub ligase activity in ENO-1 degradation was demonstrated and confirmed by ectopic expression of TRP120 HECT Ub ligase catalytic site mutant. siRNA knockdown of ENO-1 coincided with increased E. chaffeensis infection and ENO-1 knockdown disrupted glycolytic flux by decreasing the levels of pyruvate and lactate that may contribute to changes in host cell metabolism that promote infection. In addition, we elucidated a functional role of TRP120 auto-ubiquitination as an activating event that facilitates the recruitment of the UbcH5 E2 ubiquitin-conjugating enzyme. This investigation further expands the repertoire of TRP120 substrates and extends the potential role of TRP120 Ub ligase in infection to include metabolic reprogramming.

Ehrlichia chaffeensis TRP120 Is a Wnt Ligand Mimetic That Interacts with Wnt Receptors and Contains a Novel Repetitive Short Linear Motif That Activates Wnt Signaling

ABSTRACT Ehrlichia chaffeensis expresses the TRP120 multifunctional effector, which is known to play a role in phagocytic entry, on the surface of infectious dense-cored ehrlichiae, but a cognate host receptor has not been identified. We recently reported that E. chaffeensis activates canonical Wnt signaling in monocytes to promote bacterial uptake and intracellular survival and that TRP120 was involved in this activation event. To identify the specific mechanism of pathway activation, we hypothesized that TRP120 is a Wnt signaling ligand mimetic that initiates Wnt pathway activity through direct interaction with the Wnt pathway Frizzled family of receptors. In this study, we used confocal immunofluorescence microscopy to demonstrate very strong colocalization between E. chaffeensis and Fzd2, 4, 5, 7, and 9 as well as coreceptor LRP5 at 1 to 3 h postinfection. Direct binding between TRP120 and multiple Fzd receptors was further confirmed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Interfering RNA knockdown of Wnt receptors, coreceptors, and signaling pathway components significantly reduced E. chaffeensis infection, demonstrating that complex and redundant interactions are involved in Wnt pathway exploitation. We utilized in silico approaches to identify a repetitive short linear motif (SLiM) in TRP120 that is homologous to Wnt ligands and used mutant SLiM peptides and an α-TRP120-Wnt-SLiM antibody to demonstrate that the TRP120 Wnt SLiM activates the canonical Wnt pathway and promotes E. chaffeensis infection. This study reports the first example of bacterial mimicry of Wnt pathway ligands and highlights a pathogenic mechanism with potential for targeting by antimicrobial therapeutics. IMPORTANCE Upon infecting mammalian hosts, Ehrlichia chaffeensis establishes a replicative niche in microbe-eating immune system cells where it expertly orchestrates infection and spread. One of the ways Ehrlichia survives within these phagocytes is by activating evolutionarily conserved signaling pathways including the Wnt pathway; however, the molecular details of pathway hijacking have not been defined. This study is significant because it identifies an ehrlichial protein that directly interacts with components of the Wnt receptor complex, influencing pathway activity and promoting infection. Consequentially, Ehrlichia serves as a unique tool to investigate the intricacies of how pathogens repurpose human immune cell signaling and provides an opportunity to better understand many cellular processes in health and disease. Furthermore, understanding how this bacterium utilizes its small genome to survive within cells that evolved to destroy pathogens will facilitate the development of antibacterial therapeutics that could target Ehrlichia as well as other intracellular agents of human disease.

Mutations in Ehrlichia chaffeensis Genes ECH_0660 and ECH_0665 Cause Transcriptional Changes in Response to Zinc or Iron Limitation

Ehrlichia chaffeensis causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s ECH_0660 gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In this study, we describe the characterization of a cluster of seven genes spanning from ECH_0659 to ECH_0665, which contained four genes encoding bacterial phage proteins, including the ECH_0660 gene. Assessment of the promoter region upstream to the first gene of the seven genes (ECH_0659) in Escherichia coli demonstrated transcriptional enhancement under zinc and iron starvation. Further, transcription of the seven genes was significantly higher for E. chaffeensis having a mutation in the ECH_0660 gene compared to the wild-type pathogen under zinc and iron starving conditions. In contrast, transcription from the genes was mostly similar to wild-type or moderately downregulated for the ECH_0665 gene mutant with the function disruption. Recently, we reported that this mutation caused a minimal impact on the pathogen’s in vivo growth, as it persisted similar to wild-type. The current study is the first in describing how zinc and iron contribute to E. chaffeensis biology. Specifically, we demonstrated that the functional disruption in the gene encoding the predicted head-to-tail connector protein in E. chaffeensis results in the enhanced transcription of seven genes including those encoding phage proteins during zinc and iron limitation. IMPORTANCE Ehrlichia chaffeensis, a tick-transmitted bacterium, causes human monocytic ehrlichiosis by replicating within phagosomes of monocytes/macrophages. A function disruption mutation within the pathogen’s gene encoding a phage head-to-tail connector protein resulted in the rapid clearance of the pathogen in vivo, while aiding to induce sufficient immunity in a host to protect against wild-type infection challenge. In the current study, we investigated if the functional disruption in the predicted head-to-tail connector protein gene caused transcriptional changes resulting from metal ion limitations. This is the first study describing how zinc and iron may contribute to E. chaffeensis replication.

Multi-Locus Sequence Typing of Ehrlichia chaffeensis Reveals Extensive Genotypic Variation across the United States

ABSTRACTEhrlichia chaffeensis causes human monocytic ehrlichiosis, and its principal vector is the Amblyomma americanum tick. The most frequently identified cases of ehrlichiosis come from the southeastern and south central states of the United States. In this study, a molecular typing system was developed that allows for the genetic differentiation of E. chaffeensis isolates. This multi-locus typing system included sequencing and analyzing intergenic regions ECH0033–ECH0035 and ECH0217–ECH0218, plus, variable genes variable length PCR target, 28-kDa, 120-kDa, and hemE. We examined a total of 31 unique isolates from humans and white-tailed deer, and eight DNA samples extracted from infected A. americanum collected from multiple states. This is the largest evaluation of E. chaffeensis isolates and their genotypes. Our findings show that when sequences of all six loci were concatenated and compared, the 39 samples could be separated into 23 genotypes and further grouped into six phylogenetic clades. The data in this study show no clear pattern between the geographic alignment with the genetic differentiation between the strains. As a result, this poses a challenge to understanding the spread of E. chaffeensis in the United States. Interestingly, our findings indicate that multiple strains from distant geographic origins share the same mutations, which suggests that the strains are being moved from one site to another by their hosts or vectors. In addition, we are seeing a northward shift in the lone star tick distribution in the United States. Last, some data also suggest minimal genetic mutations have occurred over time among strains that are within geographical proximity.

ЗАРАЖЕНІСТЬ КЛІЩІВ У ПАРКАХ ТЕРНОПОЛЯ

Кліщі при багатьох зоонозних хворобах є переносниками чи резервуарами патогенів. До таких інфекцій належать бореліози, туляремія, кліщовий вірусний та весняно-літній енцефаліти, Кримська-Конго геморагічна гарячка, окремі рикетсіози, гранулоцитарний анаплазмоз, бабезіоз, ерліхіоз, гарячка Ку та інші. Ці захворювання об’єднані в групу так званих іксодових кліщових інфекцій. На сьогодні верифікується тільки частина з них. Реальна захворюваність перевищує кількість офіційно зареєстрованих випадків у декілька разів. Мета роботи – виявити патогени, що циркулюють у парках м. Тернополя в умовах антропозно змінених екосистем, шляхом дослідження кліщів як їх складової. Оцінити епідемічні ризики щодо окремих хвороб з трансмісивним механізмом передачі, особливо тих, що дотепер не реєструються. Напрацювати епідеміологічні дані для використання в діагностиці кліщових хвороб поряд з клінічними та лабораторними критеріями, а також для проведення комплексу профілактичних заходів. Матеріали і методи. Ентомологічні: збір кліщів проводився методом прапора та удосконаленими і запатентованими нами засобами. Вид кліщів встановлювали за допомогою визначника. У польові сезони 2018-2019 рр. було зібрано 358 кліщів, 2 з них D. reticulatus, решта – I. ricinus. Було сформовано 63 пули кліщів. Лабораторні: дослідження пулів кліщів на зараженість патогенами методом полімеразної ланцюгової реакції в режимі реального часу з використанням тест-системи виробництва Vector-Best. Тест система дозволяла визначати фрагменти ДНК B. burgdorferi s. l., B. miyamotoi, B. abesia sp., Anaplasma phagocytophilum, Ehrlichia muris та Ehrlichia chaffeensis. Результати. При дослідженні виявлені фрагменти ДНК B. burgdorferi s. l. – 43,9 % від числа позитивних результатів, B. miyamotoi – 10,6 %, Babesia sp. – 22,7 %, A. phagocytophilum – 22,7 %, E. muris і E. chaffeensis – 0 % від виявлених. Висновки. У парках м. Тернополя домінує I. ricinus. В популяціях кліщів циркулюють: B. burgdorferi s. l., B. miyamotoi, Babesia sp., A. phagocytophilum. Серед виявлених патогенів переважає B. burgdorferi s. l. В окремих пулах одночасно виявлялися декілька патогенів.