Comparative Analysis of Genome of Ehrlichia sp. HF, a Model Bacterium to Study Fatal Human Ehrlichiosis

Abstract

Background

The genus Ehrlichia consists of tick-borne obligatory intracellular bacteria that can cause deadly diseases of medical and agricultural importance. Ehrlichia sp. HF, isolated from Ixodes ovatus ticks in Japan [also referred to as I. ovatus Ehrlichia (IOE) agent], causes acute fatal infection in laboratory mice that resembles acute fatal human monocytic ehrlichiosis caused by Ehrlichia chaffeensis. As there is no small laboratory animal model to study fatal human ehrlichiosis, Ehrlichia sp. HF provides a needed disease model. However, the inability to culture Ehrlichia sp. HF and the lack of genomic information have been a barrier to advance this animal model. In addition, Ehrlichia sp. HF has several designations in the literature as it lacks a taxonomically recognized name.

Results

We stably cultured Ehrlichia sp. HF in canine histiocytic leukemia DH82 cells from the HF strain-infected mice, and determined its complete genome sequence. Ehrlichia sp. HF has a single double-stranded circular chromosome of 1,148,904 bp, which encodes 866 proteins with a similar metabolic potential as E. chaffeensis. Ehrlichia sp. HF encodes homologs of all virulence factors identified in E. chaffeensis, including 23 paralogs of P28/OMP-1 family outer membrane proteins, type IV secretion system apparatus and effector proteins, two-component systems, ankyrin-repeat proteins, and tandem repeat proteins. Ehrlichia sp. HF is a novel species in the genus Ehrlichia, as demonstrated through whole genome comparisons with six representative Ehrlichia species, subspecies, and strains, using average nucleotide identity, digital DNA-DNA hybridization, and core genome alignment sequence identity.

Conclusions

The genome of Ehrlichia sp. HF encodes all known virulence factors found in E. chaffeensis, substantiating it as a model Ehrlichia species to study fatal human ehrlichiosis. Comparisons between Ehrlichia sp. HF and E. chaffeensis will enable identification of in vivo virulence factors that are related to host specificity, disease severity, and host inflammatory responses. We propose to name Ehrlichia sp. HF as Ehrlichia japonica sp. nov. (type strain HF), to denote the geographic region where this bacterium was initially isolated.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-020-07309-z.

Background

The incidence of tick-borne diseases has risen dramatically in the past two decades, and continues to rise [1–3]. The 2011 Institute of Medicine report “Critical Needs and Gaps in...Lyme and Other Tick-Borne Diseases” revealed the urgent need for research into tick-borne diseases [4]. Ehrlichia species are tick-borne obligate intracellular bacteria, which are maintained via the natural transmission and infection cycle between particular species of ticks and mammals (Table 1). The genus Ehrlichia belongs to the family Anaplasmataceae in the order Rickettsiales. According to International Code of Nomenclature of Prokaryotes and International Journal of Systematic and Evolutionary Microbiology [46], and following the reorganization of genera in the family Anaplasmataceae based on molecular phylogenetic analysis [47], the genus Ehrlichia currently consists of six taxonomically classified species with validly published names, including E. chaffeensis, E. ewingii, E. canis, E. muris, E. ruminantium, and a recently culture-isolated E. minasensis that is closely related to E. canis (Table 1) [19, 37]. Accidental transmission and infection of domestic animals and humans can cause potentially severe to fatal diseases, and four species (E. chaffeensis, E. ewingii, E. canis, and E. muris) are known to infect humans and cause emerging tick-borne zoonoses [11, 19–21, 34, 48, 49]. In the US, the most common human ehrlichiosis is human monocytic ehrlichiosis (HME) caused by E. chaffeensis, which was discovered in 1986 [12], followed by human Ewingii ehrlichiosis discovered in 1998 [34]. The most recently discovered human ehrlichiosis is caused by E. muris subsp. eauclairensis [originally referred to as E. muris-like agent (EMLA)] [19, 20]. Human infection with E. canis has been reported in South and Central America [21, 22, 49]. Regardless of the Ehrlichia species, clinical signs of human ehrlichiosis include fever, headache, myalgia, thrombocytopenia, leukopenia, and elevated serum liver enzyme levels [20, 21, 34, 48–50].

Table 1.

Biological characteristics of representative Ehrlichia species

Species1 (Type strain)	Diseases	Mammalian Host	Tick Vector/Host	Geographic Distribution	References
Ehrlichia. sp. HF (HF565)	Acute fatal infection of mice (experimental)	Unknown	Ixodes ovatus , I. ricinus, and I. apronophorus ticks	Japan, France, Serbia, Romania	[5–10]
E. chaffeensis (Arkansas)2	Human monocytic ehrlichiosis (HME)	Deer, Human, Dog, Coyote, Fox3	Amblyomma americanum (Lone star tick)	USA, Africa, South America, Europe, Japan	[11–16]
E. muris subsp. muris (AS145)	Murine monocytic ehrlichiosis (chronic systemic infection of mice)	Mouse, Vole	Ticks (Haemaphysalis flava or Ixodes persulcatus)	Japan, Russia4	[17, 18]
E. muris subsp. eauclairensis (Wisconsin)	Human or murine monocytic ehrlichiosis (fatal infection of mice)	Human, Mouse	Ixodes scapularis (black-legged tick)	Wisconsin and Minnesota, USA	[19, 20]
E. canis (Oklahoma)	Canine tropical pancytopenia, Venezuelan Human Ehrlichiosis 5	Dog, Human	Rhipicephalus sanguineus (brown dog tick)	Global	[21–25]
E. ruminantium (Welgevonden)	Heartwater	Ruminants (Cattle, Sheep, Goats, Antelope)	Various Amblyomma species of ticks	Africa, Caribbean6	[26–33]
E. ewingii (Stillwater)	Canine granulocytic ehrlichiosis, Human ewingii ehrlichiosis	Deer, Dog, Human	Amblyomma americanum	USA, Japan	[34–36]
E. minasensis (UFMG-EV)	Ehrlichiosis	Cattle, Deer, Dog 7	Rhipicephalus microplus tick	Brazil, Global	[37–45]

¹Based on International Code of Nomenclature of Prokaryotes, and published in International Journal of Systematic and Evolutionary Microbiology, which lists officially approved list of bacterial classification and nomenclature, the genus Ehrlichia currently consists of six validly published species with correct names (https://lpsn.dsmz.de/genus/ehrlichia)

²Ehrlichia sp. HF, or Ixodes ovatus Ehrlichia (IOE) agent, is a field tick isolate of Ehrlichia species in Fukushima Prefecture, Japan from 1993 to 1994. Ehrlichia sp. HF DNA was also detected in I. ricinus tick from Brittany, France and Serbia, and I. apronophorus tick in Romania

³E. chaffeensis DNA was detected in 71% of free-ranging coyotes in Oklahoma and experimentally infected red foxes

⁴E muris DNA was found in I. persulcatus ticks and small mammals in Russia

⁵Human Infection with E. canis with clinical signs was reported in Venezuela, and E. canis was culture isolated from a VHE patient. In addition, E. canis DNA was detected in human blood bank donors in Costa Rica

⁶Heartwater in Caribbean islands of Guadeloupe was caused E. ruminantium Gardel, which is transmitted by Amblyomma variegatum (Tropical bont tick) and exceptionally virulent in Dutch goats. More heartwater cases in wild and domestic ruminants have been reported in five Caribbean islands, posing an increasing threat to domestic and wild ruminants in the continental US

⁷E. minasensis strain UFMG-EVT was isolated from the haemolymph of engorged Rhipicephalus microplus female ticks in Brazil, whereas strain Cuiaba was isolated from the whole blood of a naturally infected cattle. E. minasensis DNAs have also been reported in ticks, cervids, and dogs from France, Pakistan, Ethiopia, and Israel

HME is a significant, emerging tick-borne disease with serious health impacts with the highest incidence in people over 60 years of age and immunocompromised individuals [48]. Life-threatening complications such as renal failure, adult respiratory distress syndrome, meningoencephalitis, multi-system organ failure, and toxic shock occur in a substantial portion of the patients who are hospitalized and resulting in a case fatality rate of 3% [48]. However, there is no vaccine available for HME [51], and the only drug of choice is doxycycline, which is only effective with early diagnosis and treatment, and is not suitable for all patient groups [48]. In addition, pathogenesis and immunologic studies on human ehrlichiosis have been hampered due to the lack of an appropriate small animal disease model, as E. chaffeensis only transiently infects immunocompetent laboratory mice [52, 53]. E. chaffeensis naturally infects dogs and deer with mild to no clinical signs [53–55]. However, use of these animals is difficult and cost-prohibitive, while not being suitable for pathogenesis studies.

In an attempt to determine the pathogens harbored by Ixodes ovatus ticks prevalent in Japan, Fujita and Watanabe inoculated tick homogenates into the intraperitoneal cavity of laboratory mice, followed by serial passage through naïve mice using homogenized spleens from infected mice [5]. From 1983 to 1994, twelve “HF strains” were isolated from I. ovatus ticks in this manner, with the strain named after the scientist Hiromi Fujita who first discovered and isolated this bacterium [5]. Electron micrographs of HF326 showed the typical ultrastructure of Ehrlichia in the mouse liver [5]. A few years later, analysis of the 16S rRNA gene of the HF strains showed that four isolates (HF565, HF568-1, HF568-2, and HF639-2) from Fukushima, and two isolates (HF642 and HF652) from Aomori, northern Japan, were identical and closely related to Ehrlichia spp. [6]. The phylogenetic comparison of 16S rRNA and GroEL protein sequences of HF565 with those of members of the family Anaplasmataceae, and electron micrographs of HF565 verified that the HF strain belongs to the genus Ehrlichia [6]. Recent studies indicated that DNA sequences of Ehrlichia sp. HF have been detected not only in I. ovatus ticks throughout Japan, but also in Ixodes ricinus ticks in France [7] and Serbia [8], and Ixodes apronophorus ticks in Romania [9].

Unlike E. muris, HF565 does not induce splenomegaly but is highly virulent in mice, as intraperitoneal inoculation kills immunocompetent laboratory mice in 6-10 days [5, 6, 10, 56]. HF565 (the HF strain described here) was requested by and distributed to several US laboratories, where the strain was dubbed as I. ovatus Ehrlichia (IOE) agent. Using the HF strain-infected mouse spleen homogenate as the source of HF bacterium, pathogenesis studies in inoculated mice revealed that these bacteria induce a toxic shock-like cytokine storm, involving cytotoxic T-cells, NKT cells, and neutrophils similar to those reported in fatal HME [57–68]. Therefore, Ehrlichia sp. HF has been increasingly serving as a needed immunocompetent mouse model for studying fatal ehrlichiosis.

The major barriers for advancing research on Ehrlichia sp. HF, however, have been the inability to stably culture it in a mammalian macrophage cell line and lack of genome sequence and analysis data. Previously, it was cultured in monkey endothelial RF/6A cells and Ixodes scapularis tick embryo ISE6 cells [69]. To facilitate studies using Ehrlichia sp. HF, we stably cultured the HF strain in a canine histiocytic leukemia cell line DH82, and obtained the complete whole genome sequence (GenBank accession NZ_CP007474). Despite many studies being conducted with Ehrlichia sp. HF, this bacterium has not been classified into any species, causing confusion in the literature with several different names (IOE agent, Ehrlichia sp. HF, the HF strain). Comparative core genome alignment and phylogenetic analysis reveal that Ehrlichia sp. HF is a new species that is most closely related to E. muris and E. chaffeensis, justifying the formal nomenclature of this species. The genome sequencing and analysis, including comparative virulence factor analysis of Ehrlichia sp. HF, provides important insights, resources, and validation for advancing the research on emerging human ehrlichioses.

Results and Discussion

Culture Isolation of Ehrlichia sp. HF and purification of Ehrlichia genomic DNA

To obtain sufficient amounts of bacterial DNA free from host cell DNA, we stably cultured Ehrlichia sp. HF in DH82 cells. Spleen and blood samples were collected from Ehrlichia sp. HF-infected mice euthanized at an acute stage of illness (8 d post inoculation) (Fig. S1A). Diff-Quik staining showed that the bacteria were present in blood monocytes (Fig. S1B). After 2 - 3 weeks co-culturing with infected spleen homogenates, large vacuoles (inclusions) containing numerous bacteria (known as morulae) were observed in the cytoplasm of DH82 (Fig. S1C) and RF/6A cells (Fig. S1D). Ehrlichia sp. HF could also be successfully passaged from DH82 cells to ISE6 cells (Fig. S1E). Morulae of Ehrlichia sp. HF in cell cultures were like those seen in the tissue sections of the thymus and the lungs of infected mice [6], and in the endothelial cells of most organs of infected mice [10]. Ehrlichia sp. HF cultured in DH82 cells infects and kills mice at 7 – 10 days post intraperitoneal inoculation, similar to those inoculated with the infected mouse spleen homogenate, demonstrating that Ehrlichia sp. HF culture isolate maintains mouse virulence [56]. The mouse LD₅₀ of Ehrlichia sp. HF cultured in DH82 cells is approximately 100 bacteria [56].

General features of the Ehrlichiasp. HF genome

The complete genome of Ehrlichia sp. HF was sequenced using both Illumina and PacBio platforms, and the reads from both platforms were combined at multiple levels in order to obtain a reliable assembly. The genome was rotated to the replication origin of Ehrlichia sp. HF (Fig. 1), which was predicted to be the region between hemE (uroporphyrinogen decarboxylase, EHF_0001) and tlyC (hemolysin or related HlyC/CorC family transporter, EHF_0999) as described for other members in the family Anaplasmataceae [70]. Annotation of the finalized genome assembly was generated using the IGS prokaryotic annotation pipeline [71]. The completed genome of Ehrlichia sp. HF is a single double-stranded circular chromosome of 1,148,904 bp with an overall G+C content of ~30%, which is similar to those of E. chaffeensis Arkansas [72], E. muris subsp. eauclairensis Wisconsin [19], and E. muris AS145^T [73] (Table 2).

Fig. 1.

Circular representation of Ehrlichia sp. HF genome. From outside to inside, the first circle represents predicted protein coding sequences (ORFs) on the plus and minus strands, respectively. The second circle represent RNA genes, including tRNAs (black), rRNAs (red), tmRNAs (blue), and ncRNAs (orange). The third circle represents GC skew values [(G-C)/(G+C)] with a windows size of 500 bp and a step size of 250 bp. Colors indicate the functional role categories of ORFs - black: hypothetical proteins or proteins with unknown functions; gold: amino acid and protein biosynthesis; sky blue: purines, pyrimidines, nucleosides, and nucleotides; cyan: fatty acid and phospholipid metabolism; light blue: biosynthesis of cofactors, prosthetic groups, and carriers; aquamarine: central intermediary metabolism; royal blue: energy metabolism; pink: transport and binding proteins; dark orange: DNA metabolism and transcription; pale green: protein fate; tomato: regulatory functions and signal transduction; peach puff: cell envelope; pink: cellular processes; maroon: mobile and extrachromosomal element functions

Table 2.

Genome properties of representative Ehrlichia species

Ehrlichia Species1	EHF	ECH	EMU	EmCRT2	ECA	ERW
NCBI RefSeq	NZ_CP007474	NC_007799	NC_023063	NZ_LANU01000001	NC_007354	NC_005295
Size (bp)	1,148,904	1,176,248	1,196,717	1,148,958	1,315,030	1,516,355
GC (%)	29.6	30.1	29.7	29.8	29.0	27.5
Protein	866	892	874	866	933	934
tRNA	36	37	37	36	36	36
rRNA	3	3	3	3	3	3
Other RNA	4	3	3	4	3	4
Pseudogene	11	17	24	15	10	18
Total Gene	920	952	941	924	985	995

¹Abbreviations: EHF Ehrlichia sp. HF (HF565), EMU E. muris subsp. muris AS145, EmCRT E. muris subsp. eauclairensis Wisconsin, ECH E. chaffeensis Arkansas, ECA E. canis Jake, ERW E. ruminantium Welgevonden

²The genome of E. muris subsp. eauclairensis Wisconsin is incomplete, consisting of 3 contigs, NZ_LANU01000001, NZ_LANU01000002, and NZ_LANU01000003

The Ehrlichia sp. HF genome encodes one copy each of the 5S, 16S, and 23S rRNA genes, which are separated in 2 locations with the 5S and 23S rRNA being adjacent (Fig. 1, red bars in the middle circle) as in other sequenced members in the family Anaplasmataceae [72, 74]. Thirty-six tRNA genes are identified with cognates for all 20 amino acids (AA) (Table 2 and Fig. 1, black bars in the middle circle), similar to other Ehrlichia spp. (36 – 37 genes, Table 2).

Comparative genomic analysis of Ehrlichia sp. HF with other Ehrlichia species

Previous studies have shown that some Anaplasma spp. and Ehrlichia spp. have a single large-scale symmetrical inversion (X-alignment) near the replication origin, which may have resulted from recombination between duplicated, but not identical rho termination factors [72, 75, 76]. All genomes of the sequenced Ehrlichia spp. encode duplicated rho genes. Whole genome alignments demonstrate that the Ehrlichia sp. HF genome exhibits almost complete synteny with other Ehrlichia spp., including E. muris, E. canis, and E. ruminantium, without any significant genomic rearrangements or inversions despite these genomes being oriented in the opposite directions (Fig. 2). However, Ehrlichia sp. HF has a single large-scale symmetrical inversion relative to E. chaffeensis at the duplicated rho genes (Fig. 2b). Large scale inversion was also reported in other bacteria such as Yersinia and Legionella species when genomes of closely related species are compared [77]. However, the biological meaning and evolutionary implications of such process, if any, are largely unknown.

Fig. 2.

Whole genome alignment between Ehrlichia sp. HF and three Ehrlichia spp. Genome sequences were aligned between Ehrlichia sp. HF and E. muris subsp. muris AS145 a, E. chaffeensis Arkansas b, E. canis Jake c, or E. ruminantium Gardel d using MUGSY program with default parameters, and the graphs were generated using GMAJ. Ehrlichia sp. HF genome has a single large-scale symmetrical inversion with E. chaffeensis, but exhibits almost complete synteny with other Ehrlichia spp

In order to compare the protein ortholog groups among four closely-related Ehrlichia spp., including Ehrlichia sp. HF, E. muris subsp. eauclairensis, E. muris AS145, and E. chaffeensis Arkansas, 4-way comparisons were performed using reciprocal Blastp algorithm with E-value < 1e^-10 (Fig. 3). The four-way comparison showed that the core proteome, defined as the set of proteins present in all four genomes, consists of 823 proteins representing 94.9% of the total 867 protein-coding ORFs in Ehrlichia sp. HF (Fig. 3 and Table 3). Among these conserved proteins, the majority are associated with housekeeping functions and are likely essential for Ehrlichia survival (Table 3).

Fig. 3.

Numbers of protein homologs conserved among representative Ehrlichia spp. A Venn diagram was constructed showing the comparison of conserved and unique genes between Ehrlichia spp. as determined by reciprocal Blastp algorithm using an E-value of < 1e^-10. Numbers within the intersections of different circles indicate protein homologs conserved within 2, 3, or 4 organisms. Species indicated in the diagram are abbreviated as follows: EHF a, Ehrlichia sp. HF; ECH b, E. chaffeensis Arkansas; EMU c, E. muris subsp. muris AS145; EmCRT d, E. muris subsp. eauclairensis Wisconsin.

Table 3.

Role category breakdown of protein coding genes in Ehrlichia species

Role Category1	EHF	ECH	EMU	EmCRT	Unique in EHF2
Amino acid biosynthesis	22	23	23	22
Biosynthesis of cofactors, prosthetic groups, and carriers	64	60	65	61
Cell envelope	53	51	51	48	1
Cellular processes	42	41	42	41
Central intermediary metabolism	3	3	5	3
DNA metabolism	41	44	41	42
Energy metabolism	84	82	80	83
Fatty acid and phospholipid metabolism	20	19	21	21
Mobile elements	4	4	4	4
Protein fate	79	78	77	78
Protein synthesis	108	108	107	107
Nucleotide biosynthesis	35	35	35	35
Regulatory functions	14	15	13	14
Transcription	21	21	19	19
Transport and binding proteins	33	33	32	33
Hypothetical proteins or proteins with unknown functions	244	276	268	255	8
Total Assigned Functions:	623	617	615	611
Total Proteins	867	893	883	866

¹Abbreviations: EHF Ehrlichia sp. HF, ECH E. chaffeensis Arkansas, EMU E. muris subsp. muris AS145, EmCRT E. muris subsp. eauclairensis Wisconsin.

²Proteins specific to Ehrlichia sp. HF are based on 4-way comparison analysis among four Ehrlichia spp. by Blastp (E < 1e-10)

By 4-way comparison, a hypothetical protein (EHF_RS02845 or MR76_RS01735) is found only in Ehrlichia sp. HF and E. muris subsp. muris, the two strains that do not infect humans, but not in E. chaffeensis and E. muris subsp. eauclairensis, which both infect humans [11, 12, 78] (Table S1). On the other hand, the human-infecting strains of E. chaffeensis and E. muris subsp. eauclairensis have genes encoding a bifunctional DNA-formamidopyrimidine glycosylase/DNA-(apurinic or apyrimidinic site) lyase protein, MutM (ECH_RS02515 or EMUCRT_RS01070) (Table S1). In addition, transposon mutagenesis studies have identified intragenic insertions of genes encoding DNA mismatch repair proteins MutS and MutL in Ehrlichia sp. HF [56]. Biological relationship between MutM and the human infectivity remains to be investigated.

E. muris subsp. muris, E. muris subsp. eauclairensis, and Ehrlichia sp. HF cause persistent or lethal infection in mice, whereas immunocompetent mice clear E. chaffeensis infection within 10 – 16 days [79–81]. A metallophosphoesterase (ECH_RS03950/ECH_0964), which may function as a phosphodiesterase or serine/threonine phosphoprotein phosphatase, was found only in E. chaffeensis but not in the other three Ehrlichia spp. (Table S2).

Except for 28 E. chaffeensis-specific proteins, there are less than 10 species-specific proteins present in Ehrlichia sp. HF, E. muris subsp. muris AS145, or E. muris subsp. eauclairensis (Table S2), all of which are hypothetical proteins without any known functions or domains. Potentially, these proteins may be involved in differential pathogenesis of these Ehrlichia species.

Two-way comparisons identified further proteins that are unique to Ehrlichia sp. HF, but absent in other Ehrlichia spp. (Table S3). Several of these proteins are involved in DNA metabolism, mutation repairs, or regulatory functions that were only found in Ehrlichia sp. HF (Table S3). For example, compared to Ehrlichia sp. HF proteomes, E. chaffeensis lacks a patatin-like phospholipase family protein (ECH_RS03820, a pseudogene with internal frameshift at AA¹⁸⁰), which has phospholipase A₂ activity catalyzing the nonspecific hydrolysis of phospholipids, glycolipids, and other lipid acyl hydrolase activities [82–84]. E. muris subsp. muris lacks CckA protein, a histidine kinase that can phosphorylate response regulator CtrA and regulate the DNA segregation and cell division of E. chaffeensis [85, 86]. However, the absence of these proteins needs to be further validated since sequencing errors and mis-annotations can frequently confound such analyses. For example, although the homolog to E. chaffeensis TRP120 was not identified in E. muris subsp. eauclairensis, Tblastn searches indicated that this ORF is split into two pseudogenes (EMUCRT_0995 and EMUCRT_0731) in two separate contigs of the draft genome sequences. In addition, RpoB/C were misannotated in E. muris subsp. eauclairensis genome as a concatenated pseudogene EMUCRT_RS04655, whereas several genes encoding GyrA, PolI, AtpG, and CckA of E. muris AS145 were annotated as pseudogenes due to frameshifts in homopolymeric tracts (Table S3).

Metabolic and Biosynthetic Potential

The metabolic potential of Ehrlichia sp. HF (Table 3) was analyzed by functional role categories using Genome Properties [87], Kyoto Encyclopedia of Genes and Genomes (KEGG) [88], and Biocyc [89]. In addition, by two and four-way comparisons between Ehrlichia sp. HF and E. chaffeensis (Fig. 3 and Table 3), results indicated that Ehrlichia sp. HF possesses similar metabolic pathways as previously described for E. chaffeensis [72]. Ehrlichia sp. HF genome encodes pathways for aerobic respiration to produce ATP, including pyruvate metabolism, the tricarboxylic acid (TCA) cycle, and the electron transport chain, but lacks critical enzymes for glycolysis and gluconeogenesis. Similar to E. chaffeensis, Ehrlichia sp. HF can synthesize fatty acids, nucleotides, and cofactors, but has very limited capabilities for amino acid biosynthesis, and is predicted to make only glycine, glutamine, glutamate, aspartate, arginine, and lysine. Ehrlichia sp. HF encodes very few enzymes related to central intermediary metabolism (Table 3) and partially lacks genes for glycerophospholipid biosynthesis, rendering this bacterium dependent on the host for its nutritional needs, like E. chaffeensis [90, 91].

Ehrlichia species, including the HF strain and E. chaffeensis, are deficient in biosynthesis pathways of typical pathogen-associate molecular patterns (PAMPs), including lipopolysaccharide, peptidoglycan, common pili, and flagella. Nevertheless, both E. chaffeensis and Ehrlichia sp. HF induce acute and/or chronic inflammatory cytokines production in a MyD88-dependent, but Toll-like receptors (TLR)-independent manner [92–94]. Similar to acute severe cases of HME, Ehrlichia sp. HF causes an acute toxic shock-like syndrome in mice involving many inflammatory factors and kills mice in 10 days [56, 61, 66, 67], suggesting that Ehrlichia species have unique, yet to be identified inflammatory molecules.

Two-component regulatory systems

A two-component regulatory system (TCS) is a bacterial signal transduction system, generally composed of a sensor histidine kinase and a cognate response regulator, which allows bacteria to sense and respond rapidly to environmental changes [95]. Our previous studies showed that E. chaffeensis encodes three pairs of TCSs, including CckA/CtrA, PleC/PleD, and NtrX/NtrY, and that the histidine kinase activities were required for bacterial infection [85, 86]. Analysis showed that all three histidine kinases were identified in four species of Ehrlichia including Ehrlichia sp. HF (Table 4). However, the response regulator cckA gene of E. muris subsp. muris AS145 was annotated as a pseudogene due to an internal frameshift (Table 4). Since CckA regulates the critical biphasic developmental cycle of Ehrlichia, which converts between infectious compact dense-cored cell (DC) and replicative larger reticulate cell (RC) form [85], the mutation of cckA in E. muris AS145 needs to be further validated to rule out sequencing error in a homopolymeric tract.

Table 4.

Potential pathogenic genes in Ehrlichia sp. HF, E. chaffeensis, E. muris subsp. muris, and E. muris subsp. eauclairensis

Organisms1	EHF	ECH	EMU	EmCRT
Outer Membrane Proteins:
Omp-1/P28 family proteins	23	22	20	20
Omp85	+	+	+	+
OmpH	+	+	+	+
OmpA family protein	+	+	+	+
EtpE	+	+	+	+
Type IV Secretion System:
VirB1/B5	-	-	-	-
VirB2	+ (5)	+ (4)	+ (4)	+ (5)
VirB3	+	+	+	+
VirB4	+ (2)	+ (2)	+ (2)	+ (2)
VirB6	+ (4)	+ (4)	+ (4)	+ (4)
VirB7	+	+	+	+
VirB8	+ (2)	+ (2)	+ (2)	+ (2)
VirB9	+ (2)	+ (2)	+ (2)	+ (2)
VirB10/B11/D4	+	+	+	+
Putative T4SS Effectors:
Etf-1	+	+	+	+
Etf-22	±	+	±	±
Etf-3	+	+	+	+
Type I Secretion System3	+	+	+	+
Twin-arginine Translocation (TAT) Pathway4	+	+	+	+
TRP Proteins
TRP32	+ (94 aa)	+ (198 aa)	+ (112 aa)	+ (105 aa)
TRP47	+ (255 aa)	+ (316 aa)	+ (228 aa)	+ (252 aa)
TRP120	+ (584 aa)	+ (548 aa)	+ (1288 aa)	+5
Ankyrin-repeat domain proteins	5	5	5	5
Two-Component Regulatory Systems:
PleC/PleD	+	+	+	+
NtrY/NtrX	+	+	+	+
CckA/CtrA	+	+	±6	+

¹Abbreviations: EHF, Ehrlichia sp. HF; EMU, E. muris subsp. muris AS145; ECH, E. chaffeensis Arkansas; EmCRT, E. muris subsp. eauclairensis Wisconsin. Numbers inside parentheses indicate the copy number of the gene; or else, only a single copy exists. +, genes present; -, homolog of the gene not identified based on Blast searches.

²In addition to Etf-2 (ECH_0261, 264 aa), E. chaffeensis encodes six paralogs of Etf-2 with protein sizes range from 190 ~ 350 AA (ECH_0243, 293 aa; ECH_0246, 285 aa; ECH_0247, 316 aa; ECH_0253, 189 aa; ECH_0255, 352 aa; and ECH_0257, 226 aa). However, only low homologies (26 ~ 32% AA sequence identity) to E. chaffeensis Etf-2 were identified in other Ehrlichia spp. (indicated by ±)

³Type I Secretion System is consisting of an outer membrane channel protein TolC, a membrane fusion protein HlyD, and an ATPase HlyB. All are present in these Ehrlichia spp

⁴Both twin-arginine translocase subunits TatA and TatC were identified in all Ehrlichia spp

⁵Tblastn search indicates that that the homolog of E. chaffeensis TRP120 in E. muris subsp. eauclairensis Wisconsin is split into two pseudogenes (EMUCRT_0995 and EMUCRT_0731) present in two separate contigs (NZ_LANU01000002 and NZ_LANU01000003) of the incomplete genome sequences

⁶Gene encoding CtrA protein was identified in E. muris subsp. muris AS145 genome. However, cckA gene is annotated as a pseudogene due to an internal deletion, causing frameshift at 1,123 bp

Ehrlichia Outer Membrane Proteins (Omps)

Ehrlichia spp. encode 14 – 23 tandemly-arrayed paralogous Omp-1/P28 major outer membrane family proteins in a >26 kb genomic region [52, 93, 96–98]. This polymorphic multigene family is located downstream of tr1, a putative transcription factor, and upstream of secA gene [97]. Compensating for incomplete metabolic pathways, the major outer membrane proteins P28 and Omp-1F of E. chaffeensis possess porin activities for nutrient uptake from the host, which allow the passive diffusion of l-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose as determined by a proteoliposome swelling assay [99]. The Ehrlichia sp. HF genome has 23 paralogous omp-1/p28 family genes, named omp-1.1 to omp-1.23 (Fig. 4), and similarly flanked by tr1 and secA genes. Comparing with the E. chaffeensis Omp-1/P28 proteins by the best matches from Blastp search, the HF genome lacks orthologs of E. chaffeensis Omp-1Z, C, D, F, and P28-2, but has duplicated Omp-1H and 6 copies of Omp-1E (Fig. 4). Since P28 and OMP-1F of E. chaffeensis showed different solute diffusion rates [99], the divergence of Ehrlichia sp. HF Omp-1 protein family could affect the effectiveness of nutrient acquisition by these bacteria.

Fig. 4.

Gene structures of Omp-1/P28 family outer membrane proteins. E. chaffeensis Arkansas encodes 22 copies of Omp-1/P28 major outer membrane proteins clustered in tandem. Ehrlichia sp. HF encodes 23 copies, which are named Omp-1.1 to Omp-1.23 consecutively. However, it lacks homologs to E. chaffeensis Omp-1Z, C, D, F, and P28-2, but has duplicated Omp-1H and 6 copies of Omp-1E (based on best Blastp matches to E. chaffeensis Omp-1/P28 proteins). Note: omp-1.1 of Ehrlichia sp. HF (EHF_0067, ortholog of E. chaffeensis omp-1m) was initially annotated as a pseudogene by NCBI automated annotation pipeline. New start site was determined based on homolog to E. chaffeensis omp-1m. Grey bars indicate non-omp-1 genes within Ehrlichia omp-1/p28 gene clusters

Gram-negative bacteria encode a conserved outer membrane protein Omp85 (or YaeT) for outer membrane protein assembly [100, 101], and a molecular chaperone OmpH that interacts with unfolded proteins as they emerge in the periplasm from the Sec translocation machinery [102, 103]. The outer membrane lipoprotein OmpA of E. chaffeensis is highly expressed [104–106], and OmpA family proteins in other gram-negative bacteria are well characterized for their roles in porin functions, bacterial pathogenesis, and immunity [107]. All three outer membrane proteins were identified in Ehrlichia sp. HF, and highly conserved in these Ehrlichia spp. (Table 4), suggesting their essential roles in bacterial infection and survival.

Our previous studies showed that E. chaffeensis uses its outer membrane invasin EtpE to bind host cell receptor DNase X, and regulates signaling pathways required for entry and concomitant blockade of reactive oxygen species production for successful infection of host monocytes [108–111]. Analysis showed that the homologs of EtpE were present in Ehrlichia sp. HF as well as other Ehrlichia (Table 4), suggesting these bacteria might use similar mechanisms for entry and infection of their host cells.

Protein secretion systems

Ehrlichia sp. HF encodes all major components for the Sec-dependent protein export system to secrete proteins across the membranes. In addition, intracellular bacteria often secrete effector molecules into host cells via Sec-independent pathways, which regulate host cell physiological processes, thus enhancing bacterial survival and/or causing diseases [112]. Analysis of the Ehrlichia sp. HF genome identifies the Sec-independent Type I secretion system (T1SS), which can transport target proteins with a C-terminal secretion signal across both inner and outer membranes into the extracellular medium, and twin-arginine dependent translocation (TAT) pathway, which can transport folded proteins across the bacterial cytoplasmic membrane by recognizing N-terminal signal peptides harboring a distinctive twin-arginine motif (Table 4) [113].

The Type IV secretion system (T4SS) is a protein secretion system of Gram-negative bacteria that can translocate bacterial effector molecules into host cells and plays a key role in pathogen-host interactions [90, 114]. Except for VirB1 and VirB5, all key components of the T4SS apparatus were identified in Ehrlichia sp. HF, similar to those of E. chaffeensis (Table 4). The minor pilus subunit VirB5 is absent in all Rickettsiales [115]. VirB1, which is involved in murein degradation, is not present in Ehrlichia spp., likely due to the lack of peptidoglycan. These virB/D genes encoding T4SS apparatus are split into three major operons as well as single genes in three separate loci that encode VirB7 and duplicated VirB8/9 proteins (Table 4 and Fig. S2). Genes encoding VirB4 are also duplicated, which are clustered with multiple paralogs of virB2 and virB6 genes (Table 4 and Fig. S2). Ehrlichia sp. HF encodes four tandem functionally uncharacterized VirB6-like paralogs (800 – 1,942 AA), which have increasing masses and are three- to six-fold larger than Agrobacterium tumefaciens VirB6 (~300 AA), with extensions found at both N- and C-terminus [116].

In A. tumefaciens, VirB2 is the major T-pilus component that forms the main body of this extracellular structure, which is believed to initiate cell-cell contact with plant cells prior to the initiation of T-complex transfer [117, 118]. A yeast two-hybrid screen identified interaction partners in Arabidopsis thaliana, suggesting that Agrobacterium VirB2 directly contacts the host cell during the substrate translocation process [114, 119, 120]. Compared to E. chaffeensis and E. muris subsp. muris AS145 that encode four VirB2 paralogs, both Ehrlichia sp. HF and E. muris subsp. eauclairensis encode five VirB-2 paralogs at ~120 AA (Table 4 and Fig. 5). Most virB2 genes are clustered in tandem except for virB2-1, which is separated from the rest. VirB2 paralogs are quite divergent and only share 26% identities despite their similar sizes and domain architecture among Rickettsiales [115, 121]. Phylogenetic analysis of VirB2 paralogs in representative Ehrlichia species showed that VirB2-1 proteins are clustered in a separate branch; whereas the rest of VirB2 paralogs are more divergent (Fig. S3). A. tumefaciens VirB2 undergoes a novel head-to-tail cyclization reaction and polymerizes to form the T-pilus [116], and mature VirB2 integrates into the cytoplasmic membrane via two hydrophobic α-helices [122, 123]. Analysis of Ehrlichia sp. HF VirB2-4 showed that it possesses a signal peptide (cleavage site between residues 29 and 30) and two hydrophobic transmembrane α-helices (Fig. S4A). Alignment of these VirB2 paralogs showed that two hydrophobic α-helices are completely conserved, although they are more divergent on the N- and C-terminus (Fig. S4B), suggesting that Ehrlichia VirB2s could form the secretion channels for mature T4SS pili as in Agrobacterium [121]. Our previous study confirmed that VirB2 is expressed on the surface of a closely related bacterium Neorickettsia risticii [124]. Studies indicated that VirB2 paralogs of Anaplasma phagocytophilum are differentially expressed in tick and mammalian cells [125], and an outer membrane vaccine of Anaplasma marginale containing VirB2 can protect against the disease and persistent infection [126, 127]. Therefore, the expression of VirB2 paralogs could be specific to the host environment, and their highly divergent C-terminus may offer antigenic variations for protection from host adaptive immunity.

Fig. 5.

Gene structures of Ehrlichia VirB2 paralogs. All Ehrlichia spp. encodes 3 - 5 copies of VirB2 paralogs. Ehrlichia sp. HF and E. muris subsp. eauclairensis encode five VirB2 paralogs at ~120 AA, whereas E. chaffeensis and E. muris subsp. muris subsp. muris AS145 encode four VirB2. E. canis only encodes only 3 copies of VirB2 paralogs, and E. ruminantium encodes 4 copies with larger gaps between each virB2 paralogs. Except for E. ruminantium, most virB2 genes are clustered in tandem with virB2-1 separated from the rest.

Putative T4SS Effectors

In contrast to other intracellular pathogens with enormous numbers of effectors (i.e. Legionella pneumophila), E. chaffeensis encodes much fewer but versatile effectors [128]. Three E. chaffeensis T4SS effectors have been experimentally characterized, namely Ehrlichia translocated factor (Etf)-1, -2, and -3 [129]. These T4SS effectors are essential for infection of host cells, through inhibition of host apoptosis by Etf-1 [129], acquisition of host nutrients by Etf-1-induced autophagosomal pathways [90], or maintenance of the bacterial replication compartments by Etf-2-mediated inhibition of endosome maturation [130]. Homologs of Etf-1 and Etf-3 were identified in all Ehrlichia spp., and they are highly conserved with percent protein identities over 77% and 85%, respectively. Etf-2 proteins are more divergent among Ehrlichia spp., and E. chaffeensis encodes five paralogs of Etf-2 with protein lengths range from 190 ~ 350 AA; however, only low homologies (26 ~ 32% protein identity) to E. chaffeensis Etf-2 were identified in Ehrlichia sp. HF and other Ehrlichia species (Table 4). Whether these proteins contain a T4SS motif and can be secreted into the host cell cytoplasm remains to be studied.

Ankyrin-repeat containing proteins

Ankyrin-repeats (Ank) are structural repeating motifs that consist of 33-AA with two anti-parallel α-helices connected to the next repeat via a loop region [131]. Ank proteins are more common in eukaryotes, which mediate protein–protein interactions involved in a multitude of host processes including cytoskeletal motility, tumor suppression, and transcriptional regulation [131]. AnkA of A. phagocytophilum is one of a few known T4SS effectors, which can be translocated into the host cells, tyrosine-phosphorylated, and plays an important role in facilitating intracellular infection by regulating host signaling pathways [132–134]. A. phagocytophilum AnkA can also be translocated to the cell nucleus and bind to transcriptional regulatory regions of the CYBB locus to suppress host-cell innate immune response [135, 136]. The AnkA homolog in E. chaffeensis, Ank200, also contains tyrosine kinase phosphorylation sites and can be tyrosine-phosphorylated in the infected host cells [137, 138]. E. chaffeensis Ank200 interacts with Alu-Sx elements to regulate several genes associated with ehrlichial pathobiology [139]. A homolog of E. chaffeensis Ank200 was identified in Ehrlichia sp. HF (EHF_0607), which also contains two putative tyrosine kinase phosphorylation sites and SH3 domains in addition to 14 Ank repeats [133] (Fig. 6). Our analysis identified four additional Ank-repeat containing proteins in these representative Ehrlichia spp. (Table 4). In Ehrlichia sp. HF, these proteins range from ~150 to over 3,000 AA in length and contain 2 - 14 copies of Ank repeats (Fig. 6). It remains to be elucidated if any of the ankyrin repeat-containing proteins in Ehrlichia sp. HF can be secreted, and whether these proteins regulate host cell signaling to benefit intracellular ehrlichial infection.

Fig. 6.

Domain structures of Ankyrin-repeat containing proteins in Ehrlichia sp. HF. Ehrlichia sp. HF encodes 5 Ank-repeat containing proteins, including E. chaffeensis Ank200 homolog (EHF_0607). Ank-repeat domains were determined by NCBI Conserved Domains Database (CDD, https://www.ncbi.nlm.nih.gov/Structure/cdd) [140, 141], and eukaryotic phosphorylation sites were determined by Scansite 4.0 (https://scansite4.mit.edu/) [142]. In addition to 14 Ank repeats, Ank200 (EHF_0607) contains two tyrosine kinase phosphorylation sites (red bars), two SH3 domains (blue), and one Ser/Thr kinase site (green). Domain abbreviations: Ank, Ankyrin repeat; DUF5401, family of unknown function initially found in Chromadorea like Caenorhabditis elegans; NtpI, Archaeal/vacuolar-type H⁺-ATPase subunit I/STV1.

Tandem-repeat containing proteins (TRPs)

Using a heterologous Escherichia coli T1SS apparatus, studies have identified four potential E. chaffeensis T1SS effectors, including ankyrin-repeat containing protein Ank200, and three tandem-repeat containing proteins (TRPs), TRP47, TRP120, and TRP32 [138]. TRP120 protein also contains a motif that is rich in glycine and aspartate and relates to the repeats-in-toxins (RTX) family of exoproteins [93, 138, 143]. Our current analysis identified homologs of E. chaffeensis TRP proteins in Ehrlichia sp. HF and other representative Ehrlichia spp. (Table 4). In E. chaffeensis, all three TRP proteins contain various numbers of tandem repeats with repeat lengths ranging from 19 ~ 80 AA. However, bioinformatic analysis of TRP homologs in Ehrlichia spp. indicated that these proteins are highly variable, and the length and numbers of repeats are different among all Ehrlichia spp. (Fig. 7, Table S4). Unlike E. chaffeensis TRP32 and TRP47, no repeats or variable-length PCR target (VLPT) domains were detected in homologs of those in Ehrlichia sp. HF and other Ehrlichia spp. (Fig. 7a - b). Interestingly, TRP120 homolog of Ehrlichia sp. HF has tandem repeats with longer length (100-AA), whereas that of E. muris AS145 encodes a very large protein at 1,288 AA with over 12 repeats that are highly enriched in glutamic acid (Fig. 7c, Table S4). TRP120 homolog is also identified in E. muris subsp. eauclairensis, which is split into two ORFs in two separate contigs of the incomplete genome sequences, and has a total of ~11 repeats (Fig. 7c, Table S4). Previous studies have indicated that E. chaffeensis TRP proteins are highly immunogenic in infected patients and animals [144], and could play important roles in host–pathogen interactions [143, 145–152]. Our recent study using Himar1 transposon mutagenesis of Ehrlichia sp. HF recovered a mutant with insertion within TRP120 gene from DH82 cells, indicating that TRP120 is not essential for survival and infection of Ehrlichia sp. HF in DH82 cells [56]. As targeted mutagenesis of Ehrlichia is still unavailable, future studies using the cloned TRP120 mutant will benefit functional analysis of TRP120. In addition, it remains to be studied if any of TRPs of Ehrlichia sp. HF can be secreted by the T1SS, and whether these proteins regulate host cell signaling to benefit intracellular ehrlichial infection or pathogenicity.

Fig. 7.

Analysis of Ehrlichia tandem repeat proteins TRP-32/47/120. TRP Homologs of E. chaffeensis Arkansas were first identified using Blastp among Ehrlichia spp., and the internal repeats were determined by XSTREAM (https://amnewmanlab.stanford.edu/xstream/). Colored boxes indicated different repeat sequences and lengths, and were drawn to scale with the protein lengths. TRP proteins are highly variable, and the length and numbers of repeats are different among all Ehrlichia spp. a E. chaffeensis TRP32 (ECH_0170) protein (or variable length PCR target/VLPT, 198 AA) contains 4 consecutive VLPT repeats (30-AA). However, no repeats or VLPT domains were detected in Ehrlichia sp. HF (EHF_0893/EHF_RS04015, only 90 AA with 45% identity matched to the C-terminus of ECH0170), E. muris subsp. eauclairensis (EMUCRT_RS02860, 105 AA), and E. muris subsp. muris (EMUR_00520/MR76_RS00500, 112 AA). b E. chaffeensis TRP47 protein (ECH_0166, 316 AA) contains eight consecutive 19-AA repeats at its C-terminus. TRP47 homologs in Ehrlichia sp. HF (EHF_0897/EHF_RS04625, annotation revised based on Tblastn against Ehrlichia sp. HF genome) encodes a smaller protein (255 AA) with 40% identity, mostly conserved in N-terminus. However, no repeat sequences were identified in TRP47 homologs in Ehrlichia sp. HF, E. muris subsp. eauclairensis (EMUCRT_0637/ EMUCRT_RS04575, 252 AA), and E. muris subsp. muris (EMUR_00500/MR76_RS04630, 228 AA). c E. chaffeensis TRP120 protein (ECH_0039, 548 AA) contains 4^1/3 consecutive 80-AA repeats. TRP120 homolog in Ehrlichia sp. HF (EHF_0897/EHF_RS04625, 584 AA) contains 4¼ consecutive 100-AA repeats. A much larger protein was identified in E. muris subsp. muris AS145 (EMUR_0035/MR76_RS00035, 1,288 AA) with 12^1/3 repeats (8 repeats with 67-AA length and 4^1/3 repeats of 56-AA length). Two ORFs (EMUCRT_0995 and EMUCRT_09731) in E. muris subsp. eauclairensis that match to E. chaffeensis TRP120 at the N- and C-terminus respectively, were identified in two contigs (NZ_LANU01000002 and NZ_LANU01000003) of the incomplete genome sequences. Nine repeats of 65-AA length were identified in both proteins, whereas two shorter repeats of 38-AA length were found in EMUCRT_0995 only.

Ehrlichia sp. HF is a new Ehrlichia species based on genome and proteome phylogenetic analysis

To classify Ehrlichia sp. HF in the genus Ehrlichia, we conducted phylogenetic analyses of Ehrlichia sp. HF by using nucleotide-based core genome alignment of Ehrlichia sp. HF and 6 representative Ehrlichia species, subspecies, and strains by using three different parameters: (1) average nucleotide identity (ANI) [153], (2) digital DNA-DNA hybridization (dDDH) [154], and (3) core genome alignment sequence identity (CGASI) [155]. ANI values are calculated by first splitting the genome of one organism into 1 kbp fragments, which are then searched against the genome of the other organism. ANI is then calculated by taking the average sequence identity of all matches spanning >70% of their length with >60% sequence identity [153]. dDDH values are calculated by using the sequence similarity of conserved regions between two genomes and taking the sum of all identities found in matches divided by the overall match length [154]. CGASI values between genomes are calculated by generating a core genome alignment, consisting of all positions present in all analyzed genomes, and calculating the sequence identities between them [155].

Using the core genome alignment used to calculate CGASI values, the maximum-likelihood phylogenetic tree of the seven recognized species in the genus Ehrlichia showed that Ehrlichia sp. HF is a sister taxon to E. muris (Fig. 8a), being most closely related to E. muris subsp. muris AS145. However, between the two genomes, ANI, dDDH, and CGASI values are 91.8%, 43.2%, and 95.7%, respectively, all below the species cutoffs (95%, 70%, and 96.8%, respectively) [155]. Additionally, the current species designations for these 7 Ehrlichia genomes are supported by all three parameters, with the exception of two subspecies in E. muris (Fig. 8b).

Fig. 8.

Phylogenetic analysis and determination of ANI, dDDH, and CGASI values of 7 representative Ehrlichia species. a A maximum-likelihood phylogenetic tree with 1,000 bootstraps was generating using the core genome alignment used to calculate CGASI values. Bootstrap values are indicated next to their respective nodes. b The values of ANI, dDDH, and CGASI are calculated between 7 Ehrlichia genomes and plotted as a heatmap. The respective values for each pairwise comparison are shown in each cell. Colored circles next to each strain name indicate whether each genome belongs to the same species, with circles of the same color indicating genomes are of the same species according to either ANI, dDDH, or CGASI below the species cutoffs of 95%, 70%, and 96.8%, respectively. Abbreviations and GenBank Accession numbers: EHF, Ehrlichia sp. HF (NZ_CP007474.1); EchA, E. chaffeensis Arkansas (NC_007799.1); EmuA, E. muris subsp. muris AS145 (NC_023063.1); EmuW, E. muris subsp. eauclairensis Wisconsin (NZ_LANU01000001, NZ_LANU01000002, and NZ_LANU01000003); EcaJ, E. canis Jake (NC_007354.1); EruW, E. ruminantium Welgevonden (NC_005295.2); EruG, E. ruminantium Gardel (NC_006831.1).

Similar results are observed in a phylogenetic analyses based on the 16S rRNA sequences (Fig. S5A) or eight concatenated protein sequences (3,188 AA total) consisting of five conserved housekeeping proteins (TyrB/Mdh/Adk/FumC/GroEL) and three more divergent surface proteins like major outer membrane or T4SS apparatus proteins (P28/VirB2-1/VirB6-1) (Fig. S5B). However, the nodes on the phylogenetic tree generated using the core nucleotide alignment consistently have higher bootstrap support values than those of 16S rRNAs or concatenated proteins (Fig. 8a and S5). Based on these analyses, we proposed the following new classification of Ehrlichia sp. HF.

Description of Ehrlichia japonica sp. nov. (japonica, N.L. fem. adj. japonica from Japan)

The distances observed between Ehrlichia sp. HF and other Ehrlichia species by whole genome sequence-based phylogenetic analysis indicate that Ehrlichia sp. HF represents a new species in the genus Ehrlichia. This species is therefore named as Ehrlichia japonica sp. nov. to denote the geographic region where this bacterium was initially isolated. The type strain, HF^T, was named after the scientist Hiromi Fujita who first discovered and isolated this bacterium [5].

To date all E. japonica was found in various Ixodes species of ticks in Japan, France, Serbia, and Romania. This species is highly pathogenic to mice. E. japonica can be distinguished by PCR of 16S RNA using Ehrlichia sp. HF-specific primer pair HF51f/HF954r (923 bp target size, Table S5, Fig. S6) from other Ehrlichia species [156]. E. japonica HF^T can be stably cultured in DH82 cells, which is available from BEI Resources (Deposit ID# NR-46450, Manassas, VA) and Collection de Souches de l’Unité des Rickettsies (CSUR Q1926, Marseille, France).

Conclusions

By comparing with closely related Ehrlichia spp., this study indicates that the genome of Ehrlichia sp. HF encodes all homologs to virulence factors of E. chaffeensis required to infect host cells, including outer membrane proteins, protein secretion systems and effectors, supporting that this species can serve as a model bacteria to study in vivo pathogenesis and immune responses for fatal ehrlichiosis. Whole genome alignment and phylogenetic analyses indicate that Ehrlichia sp. HF can be classified as a new species in the genus Ehrlichia, and we propose to name it as Ehrlichia japonica sp. nov. Availability of this bacterial strain in macrophage cultures and complete whole genome sequence data will greatly advance ehrlichiosis researches, including in vivo virulence factors, therapeutic interventions, and vaccine studies.

Methods

Culture isolation of Ehrlichia sp. HF

Two C57BL/6 mice (Envigo, Indianapolis, IN) were intraperitoneally inoculated with mouse spleen homogenates containing Ehrlichia sp. HF in RPMI-1640 (Mediatech, Manassas, VA) freezing medium containing 20% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 10% DMSO (Millipore Sigma, Burlington, MA), which are stored in liquid nitrogen at approximate 0.35 ml, equivalent to ½ of an infected spleen. Clinical signs and body weight were monitored daily. Moribund mice at 8 day post inoculation were euthanized by CO₂ inhalation and cervical dislocation. Blood samples were collected by cardiac puncture, and buffy coat was separated by centrifugation at 1,000 × g. The presence of Ehrlichia sp. HF in monocytes in the blood smear was confirmed by Diff-Quik staining (Thermo Fisher Scientific, Waltham, MA). The spleen was aseptically excised and a single-cell suspension was prepared in 0.7-ml of RPMI-1640 media after lysing red blood cells with ammonium chloride. DH82 cells were cultured in DMEM (Dulbecco minimal essential medium; Mediatech) supplemented with 5% FBS and 2 mM l-glutamine (l-Gln; GIBCO, Waltham, MA) at 37°C under 5% CO₂ in a humidified atmosphere as described previously [157]. RF/6A cells (ATCC) were cultured in advanced minimal essential medium (AMEM, Gibco) supplemented with 5% FBS and 2 mM l-glutamine. The ISE6 cell line, derived from the Ixodes scapularis tick embryo, was cultured in L15C300 medium at 34°C as described previously [158]. Half of buffy coat cells and spleen cell suspension from one mouse were overlaid on DH82 and RF/6A cells in respective culture media, and cultured with the addition of 0.1 μg/mL cycloheximide (Millipore Sigma). To assess the degree of Ehrlichia infection in host cells, a drop of infected cells was centrifuged onto a slide in a Shandon Cytospin 4 cytocentrifuge (Thermo Fisher), and the presence of Ehrlichia-containing inclusions was examined in both cell types by Diff-Quik staining every 3 – 4 days. Ehrlichia sp. HF was continuously passaged in DH82 cells with the addition of 0.1 μg/mL cycloheximide.

Culture and purification of host cell-free Ehrlichia sp. HF and bacterial genomic DNA

Twelve T175 flasks of Ehrlichia sp. HF-infected DH82 cells (>80% infectivity) at 3 d post infection (pi) were homogenized in 30 ml of 1× SPK buffer (0.2 M sucrose and 0.05 M potassium phosphate, pH 7.4) for 30 times with type A tight-fitting pestle in a dounce homogenizer (Wheaton, Millville, NJ). After centrifugation at 700 × g (Sorvall 6000D, Thermo Fisher), the pellet was further homogenized for additional 30 times. Homogenates were combined and step-wise centrifuged at 700, 1,000, and 1,500 × g for 10 min without using the break function of the centrifuge to avoid disturbing the loosely-packed pellets, then passed through 5.0- and 2.7-μm filters, and centrifuged at 10,000 × g for 10 min (Sorvall RC 5C Plus using SS-34 rotor). The purity of bacteria was determined by Diff-Quik staining (Fig. S6A). Genomic DNA samples were prepared using Qiagen genomic tips (Qiagen, Germantown, MD) according to the manufacturer’s instructions, and resuspended in TE buffer. The quantity and quality of genomic DNA were determined by Nanodrop (8.41 μg total DNA; Thermo Fisher) as well as 0.9% agarose gel electrophoresis with BioLine markers (Fig. S6B). The purity of bacterial genomic DNA was confirmed by PCR and agarose gel electrophoresis using specific primers targeting Ehrlichia sp. HF 16S rRNA gene (HF51f/HF954r) and canine G3PDH DNA (Table S5 and Fig. S6) [156, 159]. The contamination of host DNA was estimated to be satisfactorily low for shotgun sequencing to obtain complete genome sequence (Fig. S6B).

Sequencing and annotation

Indexed Illumina mate pair libraries were prepared following the mate pair library v2 sample preparation guide (Illumina, San Diego, CA), with two modifications. First, the shearing was performed with the Covaris E210 (Covaris, Wobad, MA) using the following conditions: duty cycle, 10; time, 120 sec; intensity 4; and cycles per burst, 200. The DNA was purified between enzymatic reactions and the size selection of the library was performed with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA).

Paired-end genomic DNA libraries for sequencing using Illumina platform were constructed using the KAPA library preparation kit (Kapa Biosystems, Woburn, MA). DNA was fragmented with the Covaris E210 and the libraries were prepared using a modified version of manufacturer’s protocol. The DNA was purified between enzymatic reactions and the size selection of the library was performed with AMPure XT beads (Beckman Coulter Genomics), using 33.3 μl beads for 50 μl purified ligation product. For indexed samples, the PCR amplification step was performed with primers containing a six-nucleotide index sequence.

Concentration and fragment size of libraries were determined using the DNA High Sensitivity Assay on the LabChip GX (Perkin Elmer, Waltham, MA) and qPCR using the KAPA Library Quantification Kit (Complete, Universal) (Kapa Biosystems, Woburn, MA). The mate pair libraries were sequenced on an Illumina HiSeq 2500 (Illumina), producing 23.8 M reads (4.8G bases), while the paired-end libraries were sequenced on an Illumina MiSeq (Illumina), producing 1.6 M reads (826.2M bases).

DNA samples for PacBio sequencing were sheared to 8 kbp using the Covaris gTube (Woburn, MA). Sequencing libraries were constructed and prepared for sequencing using the SMRTbell Express Template Prep Kit 2.0 (3kbp - 10kbp) and the DNA/Polymerase Binding Kit 2.0 (Pacific Biosciences. Menlo Park, CA). Libraries were loaded onto v2 SMRT Cells, and sequenced with the DNA Sequencing Kit 2.0 (Pacific Biosciences), producing 81,741 reads (388.8M bases). All sequence reads were deposited at NCBI Sequence Read Archive (SRA, BioProject accession number PRJNA187357).

Five assemblies were generated with various combinations of the data and assembly algorithms: (1) Celera Assembler v7.0 of only PacBio data, (2) Celera Assembler v7.0 of PacBio data with correction using Illumina paired-end data, (3) HGAP assembly of only PacBio data, (4) MaSuRCA 1.9.2 assembly of Illumina paired-end data subsampled to 50× coverage, and (5) MaSuRCA 1.9.2 assembly of Illumina paired-end data subsampled to 80× coverage. The first assembly was the optimal assembly, namely the one generated with Celera Assembler v7.0 with only the PacBio data. The data set was subsampled to ~22× coverage of the longest reads using an 8 Kbp minimum read length cutoff, with the remainder of the reads used for the error correction step. The resulting single-contig assembly totaled ~89.4 Kbp with 41.68% GC-content. The genome was trimmed to remove overlapping sequences, oriented, circularized, and rotated to the predicted origin of replication. Annotation for this finalized genome assembly was generated using the IGS prokaryotic annotation pipeline [71] and deposited in GenBank (accession number NZ_CP007474.1).

ANI, dDDH, and CGASI calculations

ANI values were calculated using OrthiANIu v1.2 [153] paired with USEARCH v9.2.64 [160] run with default settings. dDDH values calculated using the web service GGDC v2.1 (ggdc.dsmz.de) [161] paired with the BLAST+ alignment tool [162] with default settings. For CGASI calculations, core genome alignments were constructed using Mugsy v1r2.2 [163] and mothur v1.40.4 [164]. CGASI values were calculated from the core genomes using the PairwiseAlignments function of the R package Biostrings. A maximum-likelihood phylogenetic tree was generated and plotted using IQTree v1.6.2 [165], with ModelFinder [166] and 100 ultra-fast bootstraps [167], and iTOL v5.5.1 [168], respectively.

Bioinformatic Analysis

For phylogenetic analysis, 16S rRNA genes from seven representative Ehrlichia spp., including Ehrlichia sp. HF, E. chaffeensis Arkansas, E. muris subsp. muris AS145, E. muris subsp. eauclairensis Wisconsin, E. canis Jake, E. ruminantium Welgevonden, and E. ruminantium Gardel, were aligned individually using MegAlign program of DNAStar Lasergene 12 (Madison, WI) with MUSCLE algorithm. Alternatively, eight concatenated proteins [169], including 5 conserved housekeeping proteins (aspartate aminotransferase [TyrB], malate dehydrogenase [Mdh], adenylate kinase [Adk], fumarate hydratase [FumC], and 60 kDa chaperon [GroEL]), and 3 divergent outer membranes proteins (P28/VirB2-1/VirB6-1) from these Ehrlichia spp. were aligned individually using MegAlign program with CLUSTAL OMEGA. The evolutionary analyses were inferred by using the Maximum Likelihood method and Tamura-Nei model for 16S rRNA [170] or JTT matrix-based model for concatenated proteins [171], and bootstrap values for 1,000 replicates were obtained in MEGA X software [172]. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach, and then selecting the topology with superior log likelihood value. Phylogenetic trees were drawn to scale with branch lengths shown under each branch, and the highest log likelihood is shown. The percentage of trees in which the associated taxa clustered together is shown above the branches.

The GC-skew was calculated as (C-G)/(C+G) in windows of 500 bp with step size of 250 bp along the chromosome. Whole genome alignments between Ehrlichia spp. were generated using Mugsy program with default parameters [163], and the graphs were generated using GMAJ (http://globin.bx.psu.edu/dist/gmaj/).

To determine protein orthologs conserved among Ehrlichia spp., and Ehrlichia species-specific genes compared to other related organisms, orthologous clusters were determined by using the reciprocal Basic Local Alignment Search Tool (BLAST) algorithm Blastp with an E-value of < 1e^–10.

Metabolic pathways and transporters were compared across genomes using (1) the Protein homologs generated with reciprocal Blastp, (2) Genome Properties [87], (3) TransportDB [173], (4) Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.kegg.jp), and (5) Biocyc [174]. Signal peptides and membrane proteins were predicted using the pSort-B algorithm (http://psort.org/psortb/) [175], and lipoproteins were predicted by LipoP 1.0 (http://www.cbs.dtu.dk/services/LipoP) [176].

NCBI GenBank Accession numbers and abbreviations of bacteria

Ehrlichia sp. HF (EHF), NZ_CP007474.1; E. chaffeensis Arkansas (ECH), NC_007799.1; E. muris subsp. muris AS145 (EMU), NC_023063.1; E. muris subsp. eauclairensis Wisconsin (EmCRT, three contigs), NZ_LANU01000001, NZ_LANU01000002, and NZ_LANU01000003; E. canis Jake (ECA), NC_007354.1; E. ruminantium Welgevonden (ERU), NC_005295.2.

Notes

During the review process of the present paper, Wang et al. [152] reported (published online on August 3, 2020) that an Himar1 transposon insertion mutant in TRP120 gene of E. chaffeensis was recovered in DH82 cells. This mutant had an initial lag phase but recovered afterwards in DH82 cell culture; however, it could not infect dogs when mixtures of E. chaffeensis transposon mutants were inoculated into dogs. Future availability of TRP120 mutants of multiple Ehrlichia species will help comparative functional analysis of TRP120.

Abbreviations

AA

Amino acid

ANI

Average nucleotide identity

BLAST

Basic local alignment search tool

bp

Base pair

CGASI

Core genome alignment sequence identity

dDDH

Digital DNA-DNA hybridization

Etf

Ehrlichia translocated factor

HME

Human monocytic ehrlichiosis

ORF

Open reading frame

pi

Post infection

Omp

Outer membrane protein

T1SS

Type I secretion system

T4SS

Type IV secretion system

TCS

Two-component regulatory system

TRP

Tandem-repeat containing protein

Authors’ contributions

ML, YR, and JCDH designed research; ML and QX performed research; SD, SN, NS, SQ, and AG contributed to genome sequencing and annotation; LT, LS, CMF, and JCDH oversaw sequencing project management; ML, JCDH, MC, and YR analyzed data; and ML and YR wrote the paper. All authors have read and approved the manuscript for publication.

Funding

This project has been funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH), Department of Health and Human Services under contract number HHSN272200900009C, and NIH R01 AI 047885. The funding agency played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

The datasets supporting the results of this article are included within the article and supplementary information. The genome sequence has been deposited in GenBank with Accession number NZ_CP007474, Ehrlichia japonica strain HF is available from BEI Resources (Deposit ID# NR-46450) and CSUR (Q1926).

Ethics approval and consent to participate

All animal experiments were performed in accordance with the Ohio State University Institutional Animal Care and Use Committee guidelines and approved protocol. The university program has full continued accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care International under 000028, dated 9 June 2000, and has Public Health Services assurance renewal A3261-01, dated 6 February 2019 through 28 February 2023. The program is licensed by the USDA, number 31-R-014, and is in full compliance with Animal Welfare Regulations.

Consent for publication

All authors have read and approved the manuscript for publication.

Competing interests

No potential conflicts of interest were disclosed.