Ehrlichia TRP effectors: moonlighting, mimicry and infection.

Intracellular bacteria have evolved various strategies to evade host defense mechanisms. Remarkably, the obligately intracellular bacterium, Ehrlichia chaffeensis, hijacks host cell processes of the mononuclear phagocyte to evade host defenses through mechanisms executed in part by tandem repeat protein (TRP) effectors secreted by the type 1 secretion system. In the past decade, TRP120 has emerged as a model moonlighting effector, acting as a ligand mimetic, nucleomodulin and ubiquitin ligase. These defined functions illuminate the diverse roles TRP120 plays in exploiting and manipulating host cell processes, including cytoskeletal organization, vesicle trafficking, cell signaling, transcriptional regulation, post-translational modifications, autophagy and apoptosis. This review will focus on TRP effectors and their expanding roles in infection and provide perspective on Ehrlichia chaffeensis as an invaluable model organism for understanding infection strategies of obligately intracellular bacteria.

An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.

Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with In Vivo Infection Assessment.

Ehrlichia chaffeensis, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of E. chaffeensis genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining E. chaffeensis genes critical for its persistent growth in vivo in reservoir and incidental hosts. The method also aided in extending studies focused on vaccine development and immunity. Here, we describe the generation and mapping of 55 new mutations. To define the critical nature of the bacterial genes, infection experiments were carried out in the canine host with pools of mutant organisms. Infection evaluation in the physiologically relevant host by molecular assays and by xenodiagnoses allowed the identification of many proteins critical for the pathogen's persistent in vivo growth. Genes encoding proteins involved in biotin biosynthesis, protein synthesis and fatty acid biosynthesis, DNA repair, electron transfer, and a component of a multidrug resistance (MDR) efflux pump were concluded to be essential for the pathogen's in vivo growth. Three known immunodominant membrane proteins, i.e., two 28-kDa outer membrane proteins (P28/OMP) and a 120-kDa surface protein, were also recognized as necessary for the pathogen's obligate intracellular life cycle. The discovery of many E. chaffeensis proteins crucial for its continuous in vivo growth will serve as a major resource for investigations aimed at defining pathogenesis and developing novel therapeutics for this and related pathogens of the rickettsial family Anaplasmataceae.

Host membrane lipids are trafficked to membranes of intravacuolar bacterium Ehrlichia chaffeensis.

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response.

Ehrlichia chaffeensis is an obligately intracellular bacterium that establishes infection in mononuclear phagocytes through largely undefined reprogramming strategies including modulation of host gene transcription. In this study, we demonstrate that the E. chaffeensis effector TRP47 enters the host cell nucleus and binds regulatory regions of host genes relevant to infection. TRP47 was observed in the nucleus of E. chaffeensis-infected host cells, and nuclear localization was dependent on a variant MYND-binding domain. An electrophoretic mobility shift assay (EMSA) demonstrated that TRP47 directly binds host DNA via its tandem repeat domain. Utilizing chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) with E. chaffeensis-infected cells, TRP47 was found to bind at multiple sites in the human genome (n = 2,051 at p < 10-30). Ontology analysis identified genes involved in functions such as immune response, cytoskeletal organization, and signal transduction. TRP47-bound genes included RNA-coding genes, many of these linked to cell proliferation or apoptosis. Comparison of TRP47 binding sites with those of previously-identified E. chaffeensis nucleomodulins identified multiple genes and gene functional categories in common including intracellular transport, cell signaling, and transcriptional regulation. Further, motif analysis followed by EMSA with synthetic oligonucleotides containing discovered motifs revealed a conserved TRP47 DNA-binding motif. This study reveals that TRP47 is a nucleomodulin that enters the nucleus via a MYND-binding domain and appears to play a role in host cell reprogramming by regulation of transcription.

Exposure to Ticks and their Pathogens in Northeast Missouri.

While the prevalence of human pathogens has been quantified in ticks in Adair County, Missouri, the prevalence of residents acquiring tick-borne diseases and seeking medical treatment has not. A public survey (n=109) revealed that 96% of respondents reported finding attached ticks on their person; of these, 38% developed symptoms post tick bite; of these, 55% reported consultation with a health care provider. Overall, 89% of practitioners surveyed had treated at least one patient for tick-borne disease. Rocky Mountain spotted fever and Lyme disease were the most common illnesses diagnosed, however, the only confirmed cases reported by Missouri Department of Health and Senior Services from 2013-2017 were ehrlichiosis. Results from these surveys indicate that exposure to ticks is common and ehrlichiosis infections are likely underdiagnosed while Rocky Mountain spotted fever and Lyme disease are likely overdiagnosed.

Ehrlichia chaffeensis and Its Invasin EtpE Block Reactive Oxygen Species Generation by Macrophages in a DNase X-Dependent Manner.

The obligatory intracellular pathogen Ehrlichia chaffeensis lacks most genes that confer resistance to oxidative stress but can block reactive oxygen species (ROS) generation by host monocytes-macrophages. Bacterial and host molecules responsible for this inhibition have not been identified. To infect host cells, Ehrlichia uses the C terminus of its surface invasin, entry-triggering protein of Ehrlichia (EtpE; EtpE-C), which directly binds the mammalian cell surface receptor glycosylphosphatidylinositol-anchored protein DNase X. We investigated whether EtpE-C binding to DNase X blocks ROS production by mouse bone marrow-derived macrophages (BMDMs). On the basis of a luminol-dependent chemiluminescence assay, E. chaffeensis inhibited phorbol myristate acetate (PMA)-induced ROS generation by BMDMs from wild-type, but not DNase X-/-, mice. EtpE-C is critical for inhibition, as recombinant EtpE-C (rEtpE-C)-coated latex beads, but not recombinant N-terminal EtpE-coated or uncoated beads, inhibited PMA-induced ROS generation by BMDMs from wild-type mice. DNase X is required for this inhibition, as none of these beads inhibited PMA-induced ROS generation by BMDMs from DNase X-/- mice. Previous studies showed that E. chaffeensis does not block ROS generation in neutrophils, a cell type that is a potent ROS generator but is not infected by E. chaffeensis Human and mouse peripheral blood neutrophils did not express DNase X. Our findings point to a unique survival mechanism of ROS-sensitive obligate intramonocytic bacteria that involves invasin EtpE binding to DNase X on the host cell surface. This is the first report of bacterial invasin having such a subversive activity on ROS generation.IMPORTANCEEhrlichia chaffeensis preferentially infects monocytes-macrophages and causes a life-threatening emerging tick-transmitted infectious disease called human monocytic ehrlichiosis. Ehrlichial infection, and hence the disease, depends on the ability of this bacterium to avoid or overcome powerful microbicidal mechanisms of host monocytes-macrophages, one of which is the generation of ROS. Our findings reveal that an ehrlichial surface invasin, EtpE, not only triggers bacterial entry but also blocks ROS generation by host macrophages through its host cell receptor, DNase X. As ROS sensitivity is an Achilles' heel of this group of pathogens, understanding the mechanism by which E. chaffeensis rapidly blocks ROS generation suggests a new approach for developing effective anti-infective measures. The discovery of a ROS-blocking pathway is also important, as modulation of ROS generation is important in a variety of ailments and biological processes.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector.

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Ehrlichia chaffeensis Tandem Repeat Effector Targets Differentially Influence Infection.

Ehrlichia chaffeensis infects mononuclear phagocytes and survives intracellularly by exploiting host cell processes to evade host defenses. The mechanisms involved are not fully defined, but appear to rely largely on a subset of tandem repeat proteins (TRP) effectors. E. chaffeensis TRPs are type 1 secreted effectors that interact with a functionally diverse group of host cell targets associated with various biological processes. In this study, we investigated the influence of TRP host target proteins on ehrlichial infection by RNA interference. In total, 138 TRP-interacting host proteins identified by yeast two-hybrid were targeted by siRNA and the infection level determined by real-time qPCR. Knockdown of 124 (89%) TRP target proteins had significant influence on infection either by inhibiting (85%) or promoting (15%) ehrlichial infection. Notably, knockdown of 18 host proteins which interacted with TRP120 promoted the infection, suggesting that these targets may be degraded to promote infection. Host proteins that interact with TRPs are involved in cellular processes, including cell signaling, vesicle trafficking and intracellular transport, transcriptional regulation, metabolism, protein posttranslational modification, and apoptosis. Selected host targets were examined by immunofluorescent microscopy during infection and were found to localize with the morulae, or in the host cell cytoplasm adjacent to morulae. This study confirms that the majority of host proteins known to interact with TRP effectors influence infection and further extends the current knowledge that E. chaffeensis TRPs participate in a complex array of host protein interactions in order to reprogram the host cell and promote intracellular survival.

Detection of Borrelia miyamotoi and other tick-borne pathogens in human clinical specimens and Ixodes scapularis ticks in New York State, 2012-2015.

Borrelia miyamotoi (Bm) is a recently emerging bacterial agent transmitted by several species of ixodid ticks. Diagnosis of Bm infection can be challenging, as the organism is not easily cultivable. We have developed and validated a multiplex real-time PCR to simultaneously identify Bm infection and the agents causing human granulocytic anaplasmosis and human monocytic ehrlichiosis, Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. The assay is 100% specific; highly sensitive, detecting 11 gene copies of Bm DNA in both whole blood and cerebral spinal fluid; and provides rapid results in less than two hours. A retrospective study of 796 clinical specimens collected between the years 2012 and 2014 and a prospective study of 366 clinical specimens were performed utilizing this novel assay to evaluate the frequency of Bm infection in New York State (NYS). Eight clinical specimens (1%) were found to be positive for Bm, 216 were positive for A. phagocytophilum, and 10 were positive for E. chaffeensis. Additionally, we tested 411 I. scapularis ticks collected in NYS during 2013 and 2014 in a separate multiplex real-time PCR to determine the prevalence of Bm, A. phagocytophilum, Borrelia burgdorferi s.s., and Borrelia species. Our results indicated rates of 1.5%, 27%, 19.7%, and 8.8% respectively. The ability to monitor both the frequency and geographic distribution of Bm cases and the prevalence and geographic distribution of Bm in ticks will help create a better understanding of this emerging tick-borne pathogen.

Ehrlichia chaffeensis TRP120 Activates Canonical Notch Signaling To Downregulate TLR2/4 Expression and Promote Intracellular Survival.

UNLABELLED: Ehrlichia chaffeensis preferentially targets mononuclear phagocytes and survives through a strategy of subverting innate immune defenses, but the mechanisms are unknown. We have shown E. chaffeensis type 1 secreted tandem repeat protein (TRP) effectors are involved in diverse molecular pathogen-host interactions, such as the TRP120 interaction with the Notch receptor-cleaving metalloprotease ADAM17. In the present study, we demonstrate E. chaffeensis, via the TRP120 effector, activates the canonical Notch signaling pathway to promote intracellular survival. We found that nuclear translocation of the transcriptionally active Notch intracellular domain (NICD) occurs in response to E. chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes, and hey Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway. We linked Notch pathway activation specifically to the TRP120 effector, which directly interacts with the Notch metalloprotease ADAM17. Using pharmacological inhibitors and small interfering RNAs (siRNAs) against γ-secretase enzyme, Notch transcription factor complex, Notch1, and ADAM17, we demonstrated that Notch signaling is required for ehrlichial survival. We studied the downstream effects and found that E. chaffeensis TRP120-mediated activation of the Notch pathway causes inhibition of the extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways required for PU.1 and subsequent Toll-like receptor 2/4 (TLR2/4) expression. This investigation reveals a novel mechanism whereby E. chaffeensis exploits the Notch pathway to evade the host innate immune response for intracellular survival. IMPORTANCE: E. chaffeensis is an obligately intracellular bacterium and the etiologic agent of human monocytotropic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection and avoids innate host defenses are not understood, but functionally relevant host-pathogen interactions with type 1 secreted TRP effectors are essential for the ehrlichial cellular reprogramming strategy. This study provides further insight into the molecular strategies used by obligately intracellular pathogens such as E. chaffeensis, which have small genomes and a limited number of effector proteins and exploit evolutionarily conserved host cell programs such as Notch signaling to promote infection and intracellular survival.

Comparative Genomics of the Zoonotic Pathogen Ehrlichia chaffeensis Reveals Candidate Type IV Effectors and Putative Host Cell Targets.

During infection, some intracellular pathogenic bacteria use a dedicated multiprotein complex known as the type IV secretion system to deliver type IV effector (T4E) proteins inside the host cell. These T4Es allow the bacteria to evade host defenses and to subvert host cell processes to their own advantage. Ehrlichia chaffeensis is a tick-transmitted obligate intracellular pathogenic bacterium, which causes human monocytic ehrlichiosis. Using comparative whole genome analysis, we identified the relationship between eight available E. chaffeensis genomes isolated from humans and show that these genomes are highly conserved. We identified the candidate core type IV effectome of E. chaffeensis and some conserved intracellular adaptive strategies. We assigned the West Paces strain to genetic group II and predicted the repertoires of T4Es encoded by E. chaffeensis genomes, as well as some putative host cell targets. We demonstrated that predicted T4Es are preferentially distributed in gene sparse regions of the genome. In addition to the identification of the two known type IV effectors of Anaplasmataceae, we identified two novel candidates T4Es, ECHLIB_RS02720 and ECHLIB_RS04640, which are not present in all E. chaffeensis strains and could explain some variations in inter-strain virulence. We also identified another novel candidate T4E, ECHLIB_RS02720, a hypothetical protein exhibiting EPIYA, and NLS domains as well as a classical type IV secretion signal, suggesting an important role inside the host cell. Overall, our results agree with current knowledge of Ehrlichia molecular pathogenesis, and reveal novel candidate T4Es that require experimental validation. This work demonstrates that comparative effectomics enables identification of important host pathways targeted by the bacterial pathogen. Our study, which focuses on the type IV effector repertoires among several strains of E. chaffeensis species, is an original approach and provides rational putative targets for the design of alternative therapeutics against intracellular pathogens. The collection of putative effectors of E. chaffeensis described in our paper could serve as a roadmap for future studies of the function and evolution of effectors.

An animal model of a newly emerging human ehrlichiosis.

BACKGROUND: Human ehrlichioses are emerging life-threatening diseases transmitted by ticks. Animal models have been developed to study disease development; however, there is no valid small animal model that uses a human ehrlichial pathogen. The objective of this study was to develop a mouse model for ehrlichiosis with the newly discovered human pathogen, Ehrlichia muris-like agent (EMLA). METHODS: Three strains of mice were inoculated with different doses of EMLA by the intravenous, intraperitoneal, or intradermal route and evaluated for clinical and pathologic changes during the course of infection. RESULTS: EMLA infected C57Bl/6, BALB/c, and C3H/HeN mice and induced lethal or persistent infection in a route- and dose-dependent manner. The clinical chemistry and hematologic changes were similar to those of human infection by Ehrlichia chaffeensis or EMLA. Bacterial distribution in tissues differed after intradermal infection, compared with the distribution after intravenous or intraperitoneal injection. Lethal infection did not cause remarkable pathologic changes, but it caused fluid imbalance. EMLA infection of endothelium and mononuclear cells likely plays a role in the severe outcome. CONCLUSIONS: The EMLA mouse model mimics human infection and can be used to study pathogenesis and immunity and for development of a vector transmission model of ehrlichiosis.

Identifying requirements for the invasion of a tick species and tick-borne pathogen through TICKSIM.

Ticks and tick-borne diseases have been on the move throughout the United State over the past twenty years. We use an agent-based model, TICKSIM, to identify the key parameters that determine the success of invasion of the tick and if that is successful, the succees of the tick-borne pathogen. We find that if an area has competent hosts, an initial population of ten ticks is predicted to always establish a new population. The establishment of the tick-borne pathogen depends on three parameters: the initial prevalence in the ten founding ticks, the probability that a tick infects the longer-lived hosts and the probability that a tick infects the shorter-lived hosts. These results indicate that the transmission rates to hosts in the newly established area can be used to predict the potential risk of disease to humans.

Targeted and random mutagenesis of Ehrlichia chaffeensis for the identification of genes required for in vivo infection.

Ehrlichia chaffeensis is a tick transmitted pathogen responsible for the disease human monocytic ehrlichiosis. Research to elucidate gene function in rickettsial pathogens is limited by the lack of genetic manipulation methods. Mutational analysis was performed, targeting to specific and random insertion sites within the bacterium's genome. Targeted mutagenesis at six genomic locations by homologous recombination and mobile group II intron-based methods led to the consistent identification of mutants in two genes and in one intergenic site; the mutants persisted in culture for 8 days. Three independent experiments using Himar1 transposon mutagenesis of E. chaffeensis resulted in the identification of multiple mutants; these mutants grew continuously in macrophage and tick cell lines. Nine mutations were confirmed by sequence analysis. Six insertions were located within non-coding regions and three were present in the coding regions of three transcriptionally active genes. The intragenic mutations prevented transcription of all three genes. Transposon mutants containing a pool of five different insertions were assessed for their ability to infect deer and subsequent acquisition by Amblyomma americanum ticks, the natural reservoir and vector, respectively. Three of the five mutants with insertions into non-coding regions grew well in deer. Transposition into a differentially expressed hypothetical gene, Ech_0379, and at 18 nucleotides downstream to Ech_0230 gene coding sequence resulted in the inhibition of growth in deer, which is further evidenced by their failed acquisition by ticks. Similarly, a mutation into the coding region of ECH_0660 gene inhibited the in vivo growth in deer. This is the first study evaluating targeted and random mutagenesis in E. chaffeensis, and the first to report the generation of stable mutants in this obligate intracellular bacterium. We further demonstrate that in vitro mutagenesis coupled with in vivo infection assessment is a successful strategy in identifying genomic regions required for the pathogen's in vivo growth.

Transfusion-transmitted anaplasmosis from leukoreduced red blood cells.

BACKGROUND: Human granulocytic anaplasmosis (HGA) is a tick-borne rickettsial infectious disease. To date four cases of transfusion-transmitted anaplasmosis (TTA) have been described in the literature, and only one from leukoreduced red blood cells (RBCs). CASE REPORT: A 64-year-old patient with acute gastrointestinal blood loss was admitted to the hospital and received 5 units of prestorage leukoreduced RBCs. He was stabilized and discharged. He developed headache, fever, and chills 2 days after discharge and was readmitted. On Day 5 of his second admission polymorphonuclear leukocytes containing morulae consistent with HGA were reported in the peripheral smear. RESULTS: Samples from the recipient tested positive by polymerase chain reaction (PCR) for Anaplasma phagocytophilum, the causative agent of HGA and a segment from one of the five donors tested positive by both serology and PCR. CONCLUSION: Leukoreduction theoretically reduces the risk of TTA but does not interdict all infections. TTA requires consideration in recipients of RBC transfusion with unexplained fever.

Ehrlichia chaffeensis TRP32 interacts with host cell targets that influence intracellular survival.

Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes and survives by evading host cell defense mechanisms. Recently, molecular interactions of E. chaffeensis tandem repeat proteins 47 and 120 (TRP47 and -120) and the eukaryotic host cell have been described. In this investigation, yeast two-hybrid analysis demonstrated that an E. chaffeensis type 1 secretion system substrate, TRP32, interacts with a diverse group of human proteins associated with major biological processes of the host cell, including protein synthesis, trafficking, degradation, immune signaling, cell signaling, iron metabolism, and apoptosis. Eight target proteins, including translation elongation factor 1 alpha 1 (EF1A1), deleted in azoospermia (DAZ)-associated protein 2 (DAZAP2), ferritin light polypeptide (FTL), CD63, CD14, proteasome subunit beta type 1 (PSMB1), ring finger and CCCH-type domain 1 (RC3H1), and tumor protein p53-inducible protein 11 (TP53I11) interacted with TRP32 as determined by coimmunoprecipitation assays, colocalization with TRP32 in HeLa and THP-1 cells, and/or RNA interference. Interactions between TRP32 and host targets localized to the E. chaffeensis morulae or in the host cell cytoplasm adjacent to morulae. Common or closely related interacting partners of E. chaffeensis TRP32, TRP47, and TRP120 demonstrate a molecular convergence on common cellular processes and molecular cross talk between Ehrlichia TRPs and host targets. These findings further support the role of TRPs as effectors that promote intracellular survival.

Ehrlichia type IV secretion effector ECH0825 is translocated to mitochondria and curbs ROS and apoptosis by upregulating host MnSOD.

Ehrlichia chaffeensis infects monocytes/macrophages and causes human monocytic ehrlichiosis. To determine the role of type IV secretion (T4S) system in infection, candidates for T4S effectors were identified by bacterial two-hybrid screening of E. chaffeensis hypothetical proteins with positively charged C-terminus using E. chaffeensis VirD4 as bait. Of three potential T4S effectors, ECH0825 was highly upregulated early during exponential growth in a human monocytic cell line. ECH0825 was translocated from the bacterium into the host-cell cytoplasm and localized to mitochondria. Delivery of anti-ECH0825 into infected host cells significantly reduced bacterial infection. Ectopically expressed ECH0825 also localized to mitochondria and inhibited apoptosis of transfected cells in response to etoposide treatment. In double transformed yeast, ECH0825 localized to mitochondria and inhibited human Bax-induced apoptosis. Mitochondrial manganese superoxide dismutase (MnSOD) was increased over ninefold in E. chaffeensis-infected cells, and the amount of reactive oxygen species (ROS) in infected cells was significantly lower than that in uninfected cells. Similarly, MnSOD was upregulated and the ROS level was reduced in ECH0825-transfected cells. These data suggest that, by upregulating MnSOD, ECH0825 prevents ROS-induced cellular damage and apoptosis to allow intracellular infection. This is the first example of host ROS levels linked to a bacterial T4S effector.

Cyclic dimeric GMP signaling regulates intracellular aggregation, sessility, and growth of Ehrlichia chaffeensis.

Cyclic dimeric GMP (c-di-GMP), a bacterial second messenger, is known to regulate bacterial biofilm and sessility. Replication of an obligatory intracellular pathogen, Ehrlichia chaffeensis, is characterized by formation of bacterial aggregates called morulae inside membrane-bound inclusions. When E. chaffeensis matures into an infectious form, morulae become loose to allow bacteria to exit from host cells to infect adjacent cells. E. chaffeensis expresses a sensor kinase, PleC, and a cognate response regulator, PleD, which can produce c-di-GMP. A hydrophobic c-di-GMP antagonist, 2'-O-di(tert-butyldimethysilyl)-c-di-GMP (CDGA) inhibits E. chaffeensis internalization into host cells by facilitating degradation of some bacterial surface proteins via endogenous serine proteases. In the present study, we found that PleC and PleD were upregulated synchronously during exponential growth of bacteria, concomitant with increased morula size. While CDGA did not affect host cells, when infected cells were treated with CDGA, bacterial proliferation was inhibited, morulae became less compact, and the intracellular movement of bacteria was enhanced. Concurrently, CDGA treatment facilitated the extracellular release of bacteria with lower infectivity than those spontaneously released from sham-treated cells. Addition of CDGA to isolated inclusions induced dispersion of the morulae, degradation of an inclusion matrix protein TRP120, and bacterial intrainclusion movement, all of which were blocked by a serine protease inhibitor. These results suggest that c-di-GMP signaling regulates aggregation and sessility of E. chaffeensis within the inclusion through stabilization of matrix proteins by preventing the serine protease activity, which is associated with bacterial intracellular proliferation and maturation.

Ehrlichia chaffeensis tandem repeat proteins and Ank200 are type 1 secretion system substrates related to the repeats-in-toxin exoprotein family.

Ehrlichia chaffeensis has type 1 and 4 secretion systems (T1SS and T4SS), but the substrates have not been identified. Potential substrates include secreted tandem repeat protein (TRP) 47, TRP120, and TRP32, and the ankyrin repeat protein, Ank200, that are involved in molecular host-pathogen interactions including DNA binding and a network of protein-protein interactions with host targets associated with signaling, transcriptional regulation, vesicle trafficking, and apoptosis. In this study we report that E. chaffeensis TRP47, TRP32, TRP120, and Ank200 were not secreted in the Agrobacterium tumefaciens Cre recombinase reporter assay routinely used to identify T4SS substrates. In contrast, all TRPs and the Ank200 proteins were secreted by the Escherichia coli complemented with the hemolysin secretion system (T1SS), and secretion was reduced in a T1SS mutant (ΔTolC), demonstrating that these proteins are T1SS substrates. Moreover, T1SS secretion signals were identified in the C-terminal domains of the TRPs and Ank200, and a detailed bioinformatic analysis of E. chaffeensis TRPs and Ank200 revealed features consistent with those described in the repeats-in-toxins (RTX) family of exoproteins, including glycine- and aspartate-rich tandem repeats, homology with ATP-transporters, a non-cleavable C-terminal T1SS signal, acidic pIs, and functions consistent with other T1SS substrates. Using a heterologous E. coli T1SS, this investigation has identified the first Ehrlichia T1SS substrates supporting the conclusion that the T1SS and corresponding substrates are involved in molecular host-pathogen interactions that contribute to Ehrlichia pathobiology. Further investigation of the relationship between Ehrlichia TRPs, Ank200, and the RTX exoprotein family may lead to a greater understanding of the importance of T1SS substrates and specific functions of T1SS in the pathobiology of obligately intracellular bacteria.