Molecular characterization of Ehrlichia interactions with tick cells and macrophages

Roman Reddy Ganta¹, Lalitha Peddireddi¹, Gwi-Moon Seo¹, Sarah Elizabeth DeDonder ¹, Chuanmin Cheng¹, and Stephen Keith Chapes²

1Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, ²Division of Biology, College of Arts and Sciences, Kansas State University, Manhattan, KS66506

TABLE OF CONTENTS

1. Abstract

2. Background

2.1. Tick-borne illnesses caused by Anaplasmataceae family pathogens

2.2. E. chaffeensis, an emerging pathogen

2. 3. Rickettsial pathgen persistence

3. Molecular characterization of Ehrlichia interactions with tick cells and macrophages

3.1. Host-specific differences in E. chaffeensis protein expression assessed at protein and RNA levels

3.2. Host-specific differences in E. chaffeensis morphology assessed by transmission electron microscopy

3.3. Tick cell-derived E. chaffeensis clearance by mice is delayed and associated with an altered immune response

4. Conclusions and perspectives

5. Acknowledgments

6. References

1. ABSTRACT

Several tick-transmitted Anaplasmataceae family rickettsiales of the genera Ehrlichia and Anaplasma have been discovered in recent years. Some species are classified as pathogens causing emerging diseases with growing health concern for people. They include human monocytic ehrlichiosis, human granulocytic ewingii ehrlichiosis and human granulocytic anaplasmosis which are caused by Ehrlichia chaffeensis, E. ewingii and Anaplasma phagocytophilum, respectively. Despite the complex cellular environments and defense systems of arthropod and vertebrate hosts, rickettsials have evolved strategies to evade host clearance and persist in both vertebrate and tick host environments. For example, E. chaffeensis growing in vertebrate macrophages has distinct patterns of global host cell-specific protein expression and differs considerably in morphology compared with its growth in tick cells. Immunological studies suggest that host cell-specific differences in Ehrlichia gene expression aid the pathogen, extending its survival. Bacteria from tick cells persist longer when injected into mice compared with mammalian macrophage-grown bacteria, and the host response is also significantly different. This review presents the current understanding of tick-Ehrlichia interactions and implications for future research in devising effective control methods.

2. BACKGROUND

2.1. Tick-borne illnesses caused by Anaplasmataceae family pathogens

The family Anaplasmataceae within alpha-proteobacteria contains several species of obligate, intracellular pathogens of the genera Ehrlichia and Anaplasma that infect a wide range of vertebrate host species (1-3). In recent years, several new Ehrlichia and Anaplasma species responsible for causing potentially fatal human diseases have been reported (4-6). These species include E. chaffeensis, the causative agent of human monocytic ehrlichiosis (HME) in 1987 (7-9), A. phagocytophilum, the human granulocytic anaplasmosis (HGA) agent (previously known as the HGE agent) in 1994 (10) and E. ewingii, the agent of human ewingii (granulocytic) ehrlichiosis in 1999 (11). E. ewingii is first identified as the canine pathogen responsible for granulocytic ehrlichiosis (3,5,12). Ehrlichia phagocytophilum and Ehrlichia equi have long been known as the causal agents of bovine and equine ehrlichiosis, respectively (1-3,13). Following the discovery that human granulocytic ehrlichiosis (HGE) is caused by a pathogen highly homologous to bovine and equine ehrlichiosis pathogens in 1993 and subsequent molecular reevaluation, the HGE agent together with E. phagocytophilum and E. equi are grouped as one pathogen with a newly assigned name, Anaplasma phagocytophilum (1). Infections in people with E. canis, the canine monocytic ehrlichiosis agent (3,5), are also reported from Venezuela (14,15). E. ruminantium is the agent of an economically important disease, heartwater, in domestic and wild ruminants in sub-Saharan Africa and in some Caribbean islands (16-18). Infections in ruminant populations with E. ruminantium can reach upto 80% mortality, particularly when the pathogen is introduced into a nonendemic area (18). Reports from South Africa suggest that E. ruminantium may be associated with infections and disease in people (19,20). A novel Amblyomma americanum tick-transmitted Ehrlichia is identified in Panola Mountain State Park, Georgia, USA (21). This Ehrlichia, referred to as the Panola Mountain Ehrlichia (PME), is closely related to E. ruminantium. Infections with this agent are originally reported in goats and white-tailed deer, Odocoileus virginianus (21,22). Human infections with PME have also been documented (23). However, the potential for PME to cause severe and potentially fatal infections in people remains unclear.

2.2. E. chaffeensis, an emerging pathogen

In 1987, monocytic infection with an Ehrlichia organism highly homologous to the canine ehrlichiosis agent, E. canis, is reported in a clinically ill patient with a history of tick bites (7,8,24). Subsequent molecular characterization confirmed that the human infection is the result of a new species, E. chaffeensis (24). Later, the transmitting tick vector, A. americanum, and the natural reservoir host of the pathogen, white-tailed deer, are identified (25-27). E. chaffeensis is now well recognized as the agent of HME (24). Initially, HME cases are reported from southeastern United States where the A. americanum tick is the most abundant species (28,29). Later, widespread distribution of the disease is documented from all parts of the USA and also from many regions of the world (30,31). As of 2006, there are approximately 600 documented cases in the USA per year (32). However, the true incidence is likely significantly more than reported because active surveillance studies revealed much higher infections rates. For example, a study conducted in the state of Missouri revealed 0.02%- 0.06% E. chaffeensis infection rates in people and a similar study in the state of Tennessee revealed even a higher incidence of 0.3%-0.4% (30). Together, these numbers translate to ~25,000 HME cases in Missouri and Tennessee alone. A. americanum has a much broader distribution than the foci of the disease indicates, spanning several southern and midwestern states. Consistent with this hypothesis, HME cases have been documented from many regions of the south central, southeastern and mid-Atlantic states, where A. americanum tick and white-tailed deer are prevalent (31).

E. chaffeensis is maintained in nature by transmission between its reservoir host and tick vectors, white-tailed deer and A. americanum, respectively. E. chaffeensis infections have also been identified in several other vertebrate animals such as dogs, coyotes, and raccoons (33-35). The availability of additional hosts for E. chaffeensis may contribute to the maintenance of this pathogen in nature. Ticks acquire an E. chaffeensis infection after feeding on an infected animal during their larval or nymphal stages. The bacteria acquired at larval or nymphal stage remain in the ticks as they transform (transstadial transmission) to nymphal or adult stages, respectively. Infected nymphs and adult ticks can then transmit E. chaffeensis to people and other vertebrates during blood feeding on a host (31,36) Disease manifestation of HME in people varies significantly from an asymptomatic infection to a severe, life-threatening disease with about 3% mortality rate (31). HME patients are typically more than 50 years old (37) and exhibit clinical symptoms that may include fever, headache, myalgia and malaise, thrombocytopenia, leucopenia, and hepatic injury (37-40).

2. 3. Rickettsial pathgen persistence

Tick-borne pathogenic bacteria reside in the complex cellular environments of ticks and vertebrates in spite of the sophisticated systems of defense found in these environments. The Anaplasmataceae pathogens evolved strategies to persist in both tick and vertebrate hosts in support of their lifecycle. Persistence in a host is particularly important for a pathogen, such as E. chaffeensis, which cannot be transovarially transmitted from an infected adult female tick to its offspring (41-43). Because ticks have a low probability of finding a host for a blood meal, a pathogen transfer from a tick to a vertebrate host would be significantly low. It is, therefore, crucial for a tick-transmitted pathogen to survive in its vertebrate host for extended periods of time. Understanding how tick-transmitted rickettsiales, such as E. chaffeensis, remain in tick and vertebrate hosts for long periods is important for devising effective methods of controlling the disease spread. There are many potential evasion mechanisms that allow tick-borne pathogens to persist in vertebrate hosts altering host responses, host protein mimicry, varying expressed antigens relative to time postinfection in a vertebrate host and vector- and host-specific changes in protein expression. Ticks are heterothermic, and vertebrate hosts are homeothermic, thus it is likely that the pathogens have evolved to express unique proteins and other macromolecules in support of their growth in dual-host environments.

Tick-transmitted Ehrlichia and Anaplasma species establish persistent infections in the vertebrates they infect (26,41-49). Molecular evidence on A. marginale and A. phagocytophilum suggest that the pathogens vary their antigens during persistent infection in vertebrate hosts, a possible mechanism for evading host clearance (50-53). To date, no evidence has been presented about antigenic variation in Ehrlichia species as one of the strategies employed in support of evading host clearance. Differential protein expression may be an important adaptation mechanism evolved by several members of the arthropod-borne pathogens to support their growth and persistence in invertebrate and vertebrate host-cell environments. For example, differential antigen expression is reported for tick-transmitted pathogens such as A. marginale and Borrelia burgdorferi (54-59). Differential expression in B. burgdorferi aids in the adaptation of the pathogen transition between the arthropod vector and mammalian hosts (55,56,58,59). Ehrlichia species may have evolved common strategies of host-pathogen adaptation that may be distinct from those of Anaplasma species. Ehrlichia species share extensive genetic homology and have several expressed proteins with similar immunogenic epitopes (10,60). We reported that Ehrlichia species vary many expressed proteins in a host cell-specific manner. We also presented evidence suggesting that mice take longer to clear E. chaffeensis originating from a tick cell compared with those grown in a vertebrate host environment (described in detail in the following section).

3. MOLECULAR CHARACTERIZATION OF EHRLICHIA INTERACTIONS WITH TICK CELLS AND MACROPHAGES

3.1. Host specific differences in E. chaffeensis protein expression assessed at protein and RNA levels

We utilized proteomic approaches to demonstrate novel host cell-specific protein expression differences in E. chaffeensis (61-63). Two-dimensional gel electrophoresis (2DE) analysis revealed protein expression differences between E. chaffeensis cultivated in macrophages and tick cells (Figure 1) (61). E. chaffeensis grown in macrophages expresses numerous proteins that are resolved on pH gradient between 7 and 9, whereas bacteria grown in tick cells expressed considerably more proteins that resolved between pH 5 and 7. Immunoblots of 2DE proteome gels revealed host cell-specific differences in E. chaffeensis expressed proteins (Figure 1 inset). The majority of the immunoreactive proteins in E. chaffeensis grown in macrophage cells resolved in two rows between 28 to 30 kDa with pH ranging from 4.5 to 5.5. The immunodominant proteins of the tick cell-grown E. chaffeensis resolved as one row in the 30 kDa region within the same pH range. Mass spectrometric analysis by LC-MS/MS established these immunodominant proteins as the products of p28-Omp multigene locus genes 19 and 20 in macrophage-derived E. chaffeensis and p28-Omp14 in tick cell-derived E. chaffeensis (61). The expression of p28-Omp14 protein by E. chaffeensis growing in tick cells in vitro is consistent with the gene expression in E. chaffeensis-infected A. americanum ticks (45).

The E. chaffeensis p28-Omp multigene locus contains 22 tandemly arranged paralogous genes that encode for immunodominant 28 kDa outer membrane proteins. E. canis and E. ruminantium, two other closely related species of E. chaffeensis, also have multigene loci that are homologous to the p28-Omp locus of E. chaffeensis (Figure 2) (64-71). Recently, Kumagai et al. (72) reported that the p28-Omp gene products form porin-like structures and activities on the membrane of E. chaffeensis. Macrophage- and tick cell-specific expression has also been documented from the p28/p30-Omp multigene loci from several E. chaffeensis isolates and in E. canis (61,62). Macrophage-specific expression in E. chaffeensis is primarily from p28-Omp 19 and from its paralog, p30, in E. canis. In E. ruminantium infected ruminants, the host induced B-cell response is against the protein made from major antigenic protein 1 (MAP1) gene (71,73-75) which is located at the 3' end of the alpha-region of the multigene locus similar to p28-Omp 19 and p30 of E. chaffeensis and E. canis, respectively (Figure 2). In tick cells, E. canis expresses only one protein, p30-10, from the p30-Omp multigene locus (62). This E. canis gene is positioned at the 5' end of the alpha-region, similar to the p28-Omp 14 gene in E. chaffeensis (70). The protein expression data of p28-Omp 14 and p30-10 in E. chaffeensis and E. canis, respectively, are consistent with the RT-PCR data for gene expression of E. chaffeensis and E. canis in infected ticks (45,76). Bekker et al. (77) presented evidence for the predominant expression of the E. ruminantium MAP1-1 gene in infected tick cells. This gene is also positioned within the multigene locus homologous to the p28-Omp locus of E. chaffeensis (71). The E. ruminantium MAP1-1 is located at the 5' end of alpha-region similar to that reported for p28-Omp 14 and p30-10 in E. chaffeensis and E. canis, respectively (Figure 2) (71).

Transcriptional analysis data of the p28-Omp 14 and 19 genes by Northern blot and diplex real time RT-PCR are in agreement with the protein expression data for these genes (Figures 3 and 4). The presence of multiple p28-Omp gene transcripts using non-quantitative RT-PCR methods have also been reported (45,78,79). One possible explanation for the presence of transcripts from multiple genes is that there may have been quantitative differences in the protein expression from the p28-Omp locus. For example, Ehrlichia species may induce higher levels of transcription leading to the synthesis of detectable protein from a subset of genes, whereas the gene activity from other genes is limited only to minimal levels of transcription that may be difficult to visualize with direct RNA detection methods. Diplex real time RT-PCR and large scale mass-spectrometric analysis of the total protein repertoire support this hypothesis (62,63). Using real time RT-PCR analysis, we noted minor expression of transcripts from gene 14 relative to gene 19 in macrophage-grown E. chaffeensis and similar minor expression of gene 19 relative to gene 14 in the bacteria derived from tick cells (Figure 4) (62). E. chaffeensis total protein expression is assessed using a shotgun proteomic approach. The analysis revealed the expression of 18 of the 22 p28-Omp proteins in macrophage-grown E. chaffeensis and p28-Omp 1 and 14 in the tick cell-grown pathogen (63). Nevertheless, the major expressed proteins in macrophage and tick cells are still the p28-Omp 19 and 14 gene products, respectively (63). These data are consistent with the identification of multiple p28-Omp proteins being expressed by E. chaffeensis by Ge and Rikihisa (80) and the identification of immunoglobulins specific for all 22 proteins of the p28-Omp locus in dogs experimentally infected with E. chaffeensis (81). It is not clear why E. chaffeensis expresses nearly all 22 proteins when it is grown in macrophages with dominant expression by one or two genes. One hypothesis is that the dominant expression of p28-Omp 19 is critical for the pathogen's survival in vertebrate hosts and the expression of other genes at low levels may serve as non-essential decoy targets to confuse the host immune system. This strategy allows the pathogen to evade host defenses and survive longer in the vertebrate. Conservation of host cell-specific protein expression from similar genomic regions of three different Ehrlichia species suggest that the pathogens have conserved this decoy strategy by inducing the expression of multiple proteins in support of their growth in vertebrate cells.

Expressed p28-Omp proteins in E. chaffeensis and E. canis have post-translational modifications: phosphorylation and glycosylation (61,62). Phosphorylation is reported for the first time in an Ehrlichia species, whereas glycosylation has been documented for several membrane-expressed proteins of both Ehrlichia and Anaplasma species (82-84). Prokaryotic glycoproteins may be involved in maintaining cell shape, protein stability, protection against proteolysis and/or adherence to host cells (82,83,85). MSP1a is a glycosylated outer surface protein of A. marginale. It appears to be involved in adhesion of the bacteria to host cells because chemical deglycosylation significantly reduces the bacteria's adhesive properties (82). As with p28-Omp proteins in Ehrlichia species, MSP1a is differentially expressed when it is grown in vertebrate and tick cells (57). Phosphorylation of proteins in bacteria can affect a wide variety of cellular activities ranging from activation of membrane protein transport to alteration of a protein's function (86). However, little is known about the contributions of either glycosylation or phosphorylation to the growth and persistence of Ehrlichia species in vertebrate and tick hosts. It is not clear how those different protein forms modified bacterial function or recognition by a host. This is clearly a nascent field for Ehrlichia and Anaplasma research.

To identify the expressed proteins, we analyzed total, membrane and immunogenic proteomes of E. chaffeensis grown in macrophage and tick cell cultures (63). Total proteins resolved by one-dimensional gel electrophoresis and individually picked immunogenic membrane and total membrane proteins subjected to mass-spectrometry. These analyses identified 191 and 128 proteins from macrophage- and tick cell-derived E. chaffeensis, respectively (63). These represent a total of 278 distinct E. chaffeensis proteins. Genes encoding for normal physiological functions of a cell, (e.g., those involved in protein synthesis, energy metabolism and biosynthesis of building blocks (amino acids, nucleic acids and lipids.)) are among the commonly expressed proteins (63). Macrophage- and tick cell-specific proteins also included many hypothetical, cell envelope proteins and proteins with unknown function. Proteins identified in the unknown function group included many novel proteins such as ankyrin repeat proteins, GTP-binding proteins, zinc finger-like domain proteins and metallopseudopeptide glycoprotease. The ankyrin-repeat protein homologue from A. phagocytophilum has recently been described as a secretory protein (87,88) and may play an important role in modulating host resistance, possibly by interfering with host gene expression (89). GTP-binding proteins and zinc finger proteins play important roles in regulating cell function and gene expression (90,91). Expression of these proteins in E. chaffeensis suggests that they may allow the bacteria to control cellular processes within macrophage and tick cells.

The outer membrane is the primary contact between Ehrlichia and the parasitized cell. Membrane proteins, therefore, are likely targets of the host response (65,92,93). It would be to a pathogen's advantage to alter its membrane makeup to avoid clearance and to support its adaptation to host environments. E. chaffeensis membrane protein analysis by mass-spectrometry revealed the identity of 63 expressed proteins (63) (Table 1). Macrophage-derived E. chaffeensis membrane proteins included several that are not detected in tick cell-derived bacteria. Mass-spectrometric analysis of total and immunogenic membrane proteins from tick cell-derived E. chaffeensis also confirmed the expression of host cell-specific p28-Omp protein expression reported earlier (61,62). In addition, total membrane protein analysis aided in identifying several more membrane-associated proteins (Table 1). These novel data suggest that the E. chaffeensis membrane is very complex and differs considerably in bacteria originating from macrophage and tick cells.

3.2. Host specific differences in E. chaffeensis morphology assessed by transmission electron microscopy

Transmission electron microscopic analysis revealed considerable differences in E. chaffeensis originating from macrophages and tick cells (DeDonder and Ganta, unpublished results). An important difference noted is that the pathogen in macrophage phagosomes typically grows in a synchronized form, whereas in tick cells both reticulate and dense core bodies are seen (Figure 5). Also, the reticulate bodies in tick cells are pleomorphic and larger in size than those observed in macrophages.

3.3. Tick cell-derived E. chaffeensis clearance by mice is delayed and associated with an altered immune response

Borrelia burgdorferi, a tick-transmitted spirochete, varies protein expression between tick and vertebrate hosts and the differences in protein expression appear to contribute to the spirochete's dual host adaptation (55,56,58). For example, B. burgdorferi, expresses outer surface protein A (OspA) when it infects the tick, Ixodes scapularis, and continues to produce abundant quantities of OspA while growing in the resting tick (references in (55)). During the transmission from tick to mammals and while it is growing in the vertebrate the predominant outer membrane protein expression is switched from OspA to OspC. To address whether host-specific differences in protein expression by E. chaffeensis similarly contribute to the pathogen's adaptation, we conducted experimental infection studies in C57Bl/6J mice.

E. chaffeensis grown in macrophages is cleared by mice in about two weeks, and optimal resolution requires macrophage activation, MHCII molecules, CD4⁺ helper T-cell responses and antibody production (94-96). The rapid clearance of Ehrlichia in mice contrasts persistent infections in hosts infected from a tick bite (42,48,49). Therefore, because Ehrlichia protein expression is influenced by the parasitized cell, and possibly by the way the bacteria are spread, we assessed the effect of the origin of the bacteria on host resistance and immune function (95). Although the bacteria grown in tick cells or macrophages are cleared by mice, the infection by E. chaffeensis grown in tick cells takes longer to cure (Figure 6). Those mice exhibited higher rickettsemia and resolved peritoneal bacteremia about 9 days later than mice injected with bacteria grown in macrophages (Figure 6). This appears to be, in part, a result of the poorer macrophage response as judged from the nitric oxide and IL-6 secretion by the peritoneal macrophages (95). E. chaffeensis grown in macrophages and tick cells induced a similar range of macrophage and T-cell cytokines. However, the concentrations of cytokines made in response to infection with tick cell-derived E. chaffeensis were lower than those induced by macrophage-derived bacteria (95). The host response against bacteria originating from tick cells also included a steady rise in antibody production with distinct antibody specificities compared with that observed for macrophage-grown bacteria (Figure 7) (95). The suppressed cytokine responses in mice challenged with tick cell-grown bacteria along with their distinct humoral responses have significant implications. Importantly, the host cell environment (macrophage vs. tick cells) plays a role in activating the host immune response. The transition of host cell-specific protein expression (e.g. p28-Omp 14 to Omp19) is a slow process for pathogens moving from tick cells to mammalian macrophages under both in vitro and in vivo conditions (Figure 8) (95). Therefore, the delayed clearance of tick cell-derived E. chaffeensis with distinct immune responses may reflect the host's inability to alter its immune response to the changing antigenic makeup of the bacteria. The differential protein expression might be one of the important virulence mechanisms used by E. chaffeensis to persist longer in vertebrate hosts.

4. CONCLUSIONS AND PERSPECTIVES

In summary, studies aimed at understanding the evasion strategies employed by pathogens are particularly important in devising effective intervention measures. We discovered that E. chaffeensis protein expression for the pathogen grown in tick cells differs considerably from that of the pathogen grown in macrophages (61-63). These differences caused a delay in curing the infection and a subdued immune response to tick cell-derived E. chaffeensis (95). The data suggest that E. chaffeensis protein expression pattern differences in tick cells and macrophages influence the way animals respond to infection. Although, some of the differences in protein expression are confirmed in vivo, whether the proteomic differences observed in vitro for macrophage and tick cell-grown Ehrlichia reflect those in the bacteria in ticks and vertebrate hosts remains to be determined. Importantly, Ehrlichia protein expression changes during the pathogen developmental cycle in a vertebrate host and in a tick at salivary glands and midgut may differ considerably and the differences may play crucial role in the pathogen's survival. Nonetheless, our novel findings of host-specific differences in the pathogen proteomes and morphology, and differences in host responses point out that the pathogen emerging from the tick cell environment has evolved a natural ability to evade the host response. Importantly, the early host immune response to Ehrlichia growing in tick cells is likely to be significantly different than that for bacteria growing in macrophages. Because the antigenic make up on Ehrlichia originating from tick cells is likely to represent antigens that a mammalian host sees during the early stages of a natural infection from an infected tick, future studies should focus on understanding differences in Ehrlichia in ticks and the importance of host-specific, differentially expressed proteins to the pathogen's growth and how they allow for the pathogen's survival in response to vertebrate host immunity. Such studies are important for learning what actually happens in nature and how to develop effective methods of controlling tick-borne illnesses.

5. ACKNOWLEDGMENTS

This work was supported in part by the National Institutes of Health grants AI050785, AI055052, AI070908, AI052206, RR016475, RR017686, and NASA grant NAG2-1274, and the Kansas Agricultural Experiment Station. We thank V. K. Singu, K. R. Sirigireddy, E. C. Miller and other members of our research group for their contributions through the years. The manuscript is a Kansas Agricultural Experiment Station contribution, number 09-094-J.

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Key Words: Tick-Transmitted Pathogen, Macrophage, Ehrlichia, Anaplasma, Rickettsial, Intra-Phagosomal, Host-Pathogen Interactions, Review