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.

Iron robbery by intracellular pathogen via bacterial effector–induced ferritinophagy

Iron is essential for survival and proliferation of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how Ehrlichia acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the Ehrlichia genome) Ehrlichia translocated factor-3 (Etf-3), a previously predicted effector of the Ehrlichia type IV secretion system (T4SS), is secreted into the host cell cytoplasm. Secreted Etf-3 directly bound ferritin light chain with high affinity and induced ferritinophagy by recruiting NCOA4, a cargo receptor that mediates ferritinophagy, a selective form of autophagy, and LC3, an autophagosome biogenesis protein. Etf-3−induced ferritinophagy caused ferritin degradation and significantly increased the labile cellular iron pool, which feeds Ehrlichia. Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced Ehrlichia proliferation, whereas knockdown of Etf-3 in Ehrlichia via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited Ehrlichia proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both Ehrlichia and host cells. However, during Ehrlichia proliferation, we observed concomitant up-regulation of Ehrlichia Fe-superoxide dismutase, which is an integral component of Ehrlichia T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in Ehrlichia-infected cells compared with uninfected cells. Thus, Ehrlichia safely robs host cell iron sequestered in ferritin. Etf-3 is a unique example of a bacterial protein that induces ferritinophagy to facilitate pathogen iron capture.

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.

Detection of a Rickettsia sp. and an Ehrlichia chaffeensis-like organism in ticks parasitizing the endangered Florida Grasshopper Sparrow (Ammodramus savannarum floridanus)

Between 2013 and 2015, 163 resident endangered Florida Grasshopper Sparrows (Ammodramus savannarum floridanus) and four migratory Eastern Grasshopper Sparrows (A. savannarum pratensis) were examined for the presence of ticks in peninsular Florida. Thirteen Amblyomma maculatum and seven Haemaphysalis chordeilis ticks were removed from 13 Florida Grasshopper Sparrows. Two A. maculatum were discovered on two Eastern Grasshopper Sparrows. Polymerase chain reaction (PCR) and sequencing of resultant amplicons of some of the tick specimens were performed to determine if ticks were infected with pathogenic bacteria. Salivary gland and midgut contents of five of six (83%) of the H. chordeilis tested positive for a novel Rickettsia closely related to, but distinct from, Rickettsia aeschlimannii (causative agent of Mediterranean spotted fever-like illness), an infectious zoonotic bacterium that has not been previously reported in the United States. Four of 14 (29%) of the A. maculatum tested positive for an agent most closely related to an uncultured Ehrlichia previously isolated from Oriental house rats (Rattus tanezumi; 97.5% identity to GenBank KM817187), which is genetically similar to Ehrlichia chaffeensis (causative agent of human monocytic ehrlichiosis), another infectious zoonotic bacterium. Blood from 16 Florida Grasshopper Sparrows and one Eastern Grasshopper Sparrow tested negative for spotted fever group rickettsiae, Anaplasma spp. and Ehrlichia spp. We recommend that additional collections and screening of ticks and blood from Florida Grasshopper Sparrows be undertaken to determine the rates of infection with rickettsiae and ehrlichiae in these imperiled songbirds.

Verification of mono- and mixed contaminations transmitting agents tick-borne infections

In the territory of the Primorsky region, the nosoareas of causative agents of tick-borne infections often coincide and lead to mixed infection of ticks, which causes the mixed pathology in humans . We investigated ixodid ticks taken from people during the epidemic season of 2019 for the spectrum of pathogens of transmissible infections to determine the degree of their mono- and mixed infection. 651 specimens of ixodid ticks investigated. RNA/DNA pathogens was determined by the method of polymerase chain reaction (PCR), TBEV antigen by ELISA. The presence in ticks of pathogens of the following diseases was detected: Lyme borreliosis (35.3%), tick-borne replasing fever (11.3%), human granulocytic anaplasmosis (2.6%), human monocytic ehrlichiosis (2.4%), tick-borne encephalitis (1.8%), tick-borne ricketsiosis (1.0%). As agents of mono-infections, the virus of TBE, ehrlichia, anaplasma and rickettsia in ticks occurred less frequently than in combination with other pathogens. A high frequency of occurrence B. burgdorferi s.l. in mono-infection detected (58.7%). B. miyamotoi has met evenly at ticks with mono- and mixed infections. Total mixed infection of ticks was 23.1%. Borrelia have been identified in all mixed cases. Variants of double combinations (95.5 % of cases from all mixed infections) were as follows: B. burgdorferi s. l. + A. phagocytophilum, B. burgdorferi s.l. + E. chaffeensis / E. muris, B. burgdorferi s.l. + TBEV, B. burgdorferi s.l. + R. heilongjiangensis, B. burgdorferi s.l. + B. miyamotoi. The most frequent combination was B. burgdorferi s.l. + B. miyamotoi (37.8% of all mixed infections). In triple infection of B. burgdorferi s.l. + A. phagocytophilum + TBEV and B. burgdorferi s.l. + A. phagocytophilum + B. miyamotoi, the frequency of occurrence for each combination was 2.2%. Given the above, a complex laboratory diagnosis of tick-borne infections in individuals who have been bitten by a tick is necessary, including the detection of all pathogens common in focal areas.

An Entry-Triggering Protein of Ehrlichia Is a New Vaccine Candidate against Tick-Borne Human Monocytic Ehrlichiosis

ABSTRACT Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. E. chaffeensis outer membrane protein entry triggering protein of Ehrlichia (EtpE) is necessary for bacterial entry into human cells. We investigated the role of EtpE in transmission of the bacteria from tick to human cells and whether or not vaccination with EtpE can prevent transmission of ehrlichiae from ticks to mammals. An antiserum against the recombinant C terminus of EtpE (rEtpE-C), which binds a mammalian cell-surface receptor and triggers bacterial entry, significantly inhibited E. chaffeensis transmission from infected tick cells to human monocytes in culture. Each of five specific-pathogen-free dogs were vaccinated with rEtpE-C along with an immunostimulating complex or were sham vaccinated with the complex alone. Dogs vaccinated with rEtpE-C developed high antibody titers against rEtpE-C and produced interferon-γ-secreting cells, as assessed with the ELISpot assay. All 10 dogs were challenged with A. americanum adult ticks infected as nymphs by syringe inoculation with E. chaffeensis. Upon challenge, both the vaccinated and control dogs became infected by day 1 post-tick attachment, but the majority of rEtpE-C-vaccinated dogs rapidly cleared the infection from the bloodstream as soon as day 7, whereas most of sham-vaccinated dogs remained infected at day 35. Peripheral blood leukocytes from vaccinated dogs had significantly elevated interferon-γ mRNA levels and secreted significantly elevated interferon-γ soon after tick attachment. Thus, the EtpE-C vaccine represents the first ehrlichial protein vaccine demonstrated to reduce bacterial infection in mammals upon challenge with infected ticks. IMPORTANCE The incidence of tick-borne diseases has risen dramatically in the past two decades and continues to rise. Discovered in 1986 and designated a nationally notifiable disease in 1998 by the Centers for Disease Control and Prevention, human monocytic ehrlichiosis, which is caused by the bacterium Ehrlichia chaffeensis, is one of the most prevalent, life-threatening, emerging tick-borne zoonoses in the United States. We investigated the role of the E. chaffeensis protein EtpE in transmission of the bacterium from tick to human cells and in vaccinated dogs with EtpE to assess the efficacy of vaccination against E. chaffeensis-infected tick challenge. Our results help fill gaps in our understanding of E. chaffeensis-derived protective antigens that could be used in a candidate vaccine for immunization of humans to counter tick-transmitted ehrlichiosis.

Results of Epizootiological Survey on Plague and Other Zoonotic Infections in the Northern Provinces of the Socialist Republic of Vietnam During Spring Months of 2019

Objective of the study is to detect circulation of plague agent and agents of other zoonotic infections (tularemia, pseudotuberculosis, leptospirosis, West Nile fever, Lassa fever, Dengue fever, Chikungunya fever, CrimeanCongo hemorrhagic fever, Q fever, Hantaviruses, tick-borne encephalitis, human monocytic ehrlichiosis, granulocytic anaplasmosis, and borreliosis) among small mammals and their ectoparasites in the territory of seven northern provinces of the Socialist Republic of Vietnam. Materials and methods. We have carried out epizootiological survey of seven northern provinces of the Socialist Republic of Vietnam (Dien Bien, Lai Chau, Lào Cai, Hà Giang, Lạng Sơn, Cao Bằng, and Quảng Ninh). Over the period of the study, 3400 trap/nights were accumulated, 179 specimens of small mammals caught, belonging to 10 species, 213 fleas of seven different species – collected, and 143 specimens of gamaside ticks falling under two species. The material gathered was investigated using enzyme immunoassay and polymerase chain reaction at the premises of mobile laboratory for monitoring and diagnostics. Results and discussion. Two-fold testing of 136 blood samples from small mammals revealed antibodies to F1 of Y. pestis in 14 (10.3 %) of them. Investigation of 158 samples of lung and kidney suspensions of small mammals showed that 22 (13.9 %) samples contained 16S rRNA of pathogenic leptospira, Leptospira spp. Analysis of 60 brain samples for the presence of Leptospira spp. revealed three (5.0 %) positive ones. 25 samples of gamaside ticks were tested for the presence of the DNA of Q fever, plague, tularemia and granulocytic anaplasmosis agents, and for the RNA of tick-borne encephalitis, human monocytic ehrlichiosis, and borreliosis agents. One sample (4 %) of gamaside ticks, Laelaps echidninus, contained RNA of Borrelia.

mTORC1-mediated polarization of M1 macrophages and their accumulation in the liver correlate with immunopathology in fatal ehrlichiosis

A polarized macrophage response into inflammatory (M1) or regenerative/anti-inflammatory (M2) phenotypes is critical in host response to multiple intracellular bacterial infections. Ehrlichia is an obligate Gram-negative intracellular bacterium that causes human monocytic ehrlichiosis (HME): a febrile illness that may progress to fatal sepsis with multi-organ failure. We have shown that liver injury and Ehrlichia-induced sepsis occur due to dysregulated inflammation. Here, we investigated the contribution of macrophages to Ehrlichia-induced sepsis using murine models of mild and fatal ehrlichiosis. Lethally-infected mice showed accumulation of M1 macrophages (iNOS-positive) in the liver. In contrast, non-lethally infected mice showed polarization of M2 macrophages and their accumulation in peritoneum, but not in the liver. Predominance of M1 macrophages in lethally-infected mice was associated with expansion of IL-17-producing T, NK, and NKT cells. Consistent with the in vivo data, infection of bone marrow-derived macrophages (BMM) with lethal Ehrlichia polarized M0 macrophages into M1 phenotype under an mTORC1-dependent manner, while infection with non-lethal Ehrlichia polarized these cells into M2 types. This work highlights that mTORC1-mediated polarization of macrophages towards M1 phenotype may contribute to induction of pathogenic immune responses during fatal ehrlichiosis. Targeting mTORC1 pathway may provide a novel aproach for treatment of HME.