Neorickettsia risticii surface-exposed proteins: proteomics identification, recognition by naturally-infected horses, and strain variations
© Gibson et al; licensee BioMed Central Ltd. 2011
Received: 11 March 2011
Accepted: 2 June 2011
Published: 2 June 2011
Neorickettsia risticii is the Gram-negative, obligate, and intracellular bacterial pathogen responsible for Potomac horse fever (PHF): an important acute systemic disease of horses. N. risticii surface proteins, critical for immune recognition, have not been thoroughly characterized. In this paper, we identified the 51-kDa antigen (P51) as a major surface-exposed outer membrane protein of older and contemporary strains of N. risticii through mass spectrometry of streptavidin-purified biotinylated surface-labeled proteins. Western blot analysis of sera from naturally-infected horses demonstrated universal and strong recognition of recombinant P51 over other Neorickettsia recombinant proteins. Comparisons of amino acid sequences for predicted secondary structures of P51, as well as Neorickettsia surface proteins 2 (Nsp2) and 3 (Nsp3) among N. risticii strains from horses with PHF during a 26-year period throughout the United States revealed that the majority of variations among strains were concentrated in regions predicted to be external loops of their β-barrel structures. Large insertions or deletions occurred within a tandem-repeat region in Ssa3. These data demonstrate patterns of geographical association for P51 and temporal associations for Nsp2, Nsp3, and Ssa3, indicating evolutionary trends for these Neorickettsia surface antigen genes. This study showed N. risticii surface protein population dynamics, providing groundwork for designing immunodiagnostic targets for PHF.
Discovered in 1984, Neorickettsia (formerly Ehrlichia) risticii is an obligate intracellular bacterium and the causative agent of Potomac horse fever (PHF) [1–3]. The bacterium uses a digenetic trematode to survive and proliferate in its natural lifecycle [4–9]. It is through accidental ingestion of the metacercarial stage of the digenetic trematode within its insect host that the horse becomes infected with N. risticii and develops PHF . PHF is an acute, severe, and potentially fatal disease of horses, normally contracted during the summer months in North America when aquatic insect larvae infested with N. risticii-infected digenetic trematodes molt and emerge (hatch) from the water as adults [6, 10]. Clinical signs range from mild (anorexia, fever, lethargy, and depression) to life-threatening (laminitis, abortion, and diarrhea followed by severe dehydration) [10, 11]. The administration of tetracyclines at the early stage of infection is effective, in part by inhibiting bacterial protein synthesis and facilitating lysosome fusion with inclusions containing N. risticii [12–15]. Diagnosis of this disease is mainly done by indirect fluorescent-antibody (IFA) test based on N. risticii-infected cells and by nested polymerase chain reaction (PCR) on blood samples [5, 16–22]. The only available vaccines are bacterins using the 1984 N. risticii type strain, which demonstrate inadequate efficacy [23, 24].
It was determined that N. risticii has similar genetic, antigenic, and morphologic characteristics to Neorickettsia helminthoeca [25, 26], which were the major reasons it, as well as Neorickettsia (formerly Rickettsia, Ehrlichia) sennetsu, was regrouped into the genus Neorickettsia . In addition, the bacterial parasite, known as the Stellantchasmus falcatus (SF) agent, isolated from metacercariae in fish from Japan and Oregon [28–30] belongs to this group. N. risticii also consists of a variety of strains, based on PCR and sequencing of 16S RNA and groEL, Western blot analyses using purified bacteria as antigen, and morphology [20, 22, 24, 31].
Little is known about N. risticii surface-exposed proteins, and this missing information is crucial in the understanding of bacterium-host cell interactions. Antigenic and potential surface proteins ranging between 28 and 110-kDa in mass were previously detected by Western blotting, but these proteins were not identified . Immunoprecipitation of N. risticii labeled with I125 and N. risticii immune mouse sera revealed potential surface proteins ranging from 25 to 62-kDa in mass, although these proteins were not identified . Antigenic proteins of 70, 55, 51, and 44-kDa masses have been demonstrated utilizing recombinant proteins; again the proteins were not identified . Two highly-immunodominant proteins in two N. risticii strains were identified as GroEL and the 51-kDa antigen (P51) , but it was not shown whether these proteins were surface exposed. Strain-specific antigen (Ssa) was suggested as a surface immunogenic protein with potential use in vaccine production, although it was not determined to be bacterial surface exposed [24, 36].
The identification of Neorickettsia proteins is now achievable with the availability of whole genome sequencing data on both the type strain (Miyayama) of N. sennetsu  and the type strain (Illinois) of N. risticii . In this paper, we determined 1) major surface proteins by proteomics analysis on N. risticii, 2) horse immune recognition of N. risticii surface proteins, and 3) strain variations in aligned sequences of these major surface proteins with respect to their predicted secondary structures.
Materials and methods
Culturing and isolation of N. risticii strains
N. risticii IllinoisT  and a Pennsylvania strain (PA-1)  were cultured in P388D1 cells in 75-cm2 flasks containing RPMI 1640 (Mediatech, Inc., Herdon, VA, USA) supplemented with 5-10% fetal bovine serum (FBS) (U.S. Biotechnologies, Inc., Pottstown, PA, USA) and 4-6 mM L-glutamine (Invitrogen, Carlsbad, CA, USA) at 37°C under 5% CO2. N. risticii was isolated from highly-infected P388D1 cells as previously described for N. sennetsu MiyayamaT .
Biotinylation and streptavidin-affinity purification of N. risticii surface proteins
Biotinylation of purified N. risticii Illinois and PA-1 from twenty-five 75-cm2 flasks using EZ Link Sulfo-NHS-SS-Biotin (Pierce Biotechnology, Rockford, IL, USA) and subsequent bacterial lysis and collection of solubilized bacterial proteins were performed as previously described . Streptavidin purification of Sulfo-NHS-SS-Biotinylated N. risticii proteins was then performed, followed by SDS-polyacrylamide gel electrophoresis (PAGE) and fixation and GelCode blue (Pierce) staining of the gel . Proteins from seven bands from N. risticii Illinois and proteins from four bands or band collections from PA-1 were identified by capillary-liquid chromatography-nanospray tandem mass spectrometry (Nano-LC/MS/MS) as previously described .
Western blotting using recombinant proteins
Recombinant P51 (rP51, 57 kDa), cloned from N. risticii Illinois (NRI_0235), and rNsp2 (35 kDa) and rNsp3 (28 kDa), cloned from N. sennetsu Miyayama (NSE_0873 and NSE_0875, respectively), were expressed by transformed BL21(DE3) cells using isopropyl-β-D-thiogalactopyranoside induction and His-tag purified as described previously [30, 39]. Recombinant GroEL (55 kDa), derived from N. sennetsu Miyayama (NSE_0642), was acquired from stored aliquots . Fifty μg of each recombinant protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, and cut into strips. Western blotting was then performed on these strips using 1:500 dilutions of known positive horse sera samples as determined by IFA [16, 21]. The membrane was subsequently incubated with a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-horse (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA) as secondary antibody. Enhanced chemiluminescence (ECL) LumiGLO chemiluminescent reagent (Pierce) and a LAS3000 image documentation system (FUJIFILM Medical Systems USA, Stamford, CT, USA) were used to visualize the protein bands with 300 s exposure. Bands were aligned using Precision Plus prestained protein standards (Bio-Rad Laboratories, Hercules, CA, USA).
Polymerase chain reaction, sequencing, and sequence alignment
DNA was purified from buffy coats of PHF-positive horses or cultures of N. risticii in P388D1 cells using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA), according to manufacturer's instructions. PCR amplification was then performed using either Phusion or Taq DNA polymerase (New England BioLabs, Ipswich, MA, USA) and primers designed for conserved regions through alignment of multiple Neorickettsia spp. and/or N. risticii strains (see Additional file 1). Sequencing was performed by The Ohio State University Plant-Microbe Genomics Facility. Sequences containing whole genes or gene fragments were translated and aligned mainly through the CLUSTAL W (slow/accurate) method in the MegAlign program of DNAStar (DNAStar, Madison, WI, USA); P51 was first aligned by CLUSTAL V (PAM250) method, and Ssa3 was aligned both by CLUSTAL W and manually. External loops were also aligned separately by CLUSTAL W for both P51 and Nsp3. Amino acid (aa) variations in N. risticii strains and other Neorickettsia spp. for all proteins were determined in relation to N. risticii Illinois. Protein alignments of the same size (including deletions as dashes) were analyzed by PHYLIP (v3.66) to obtain bootstrap values for 1000 replicates (using the programs SeqBoot, Protdist, Neighbor, and Consense) and to create dendrograms (using the programs Protdist, Neighbor, and Drawgram) . Protein properties, including antigenicity profiles and β-sheet predictions were determined using the Protean program (DNAStar). Gene and protein sequence homologies were also demonstrated using Basic Local Alignment Search Tool (BLAST) algorithms, including blastn, protein-protein blastp, and blastp [43, 44].
Prediction of secondary structures
Predictions for Nsp2 and Nsp3 were based on a combination of the programming algorithm in the PRED-TMBB web server , hydrophobicity and hydrophobic movement profiles , and DNAStar MegAlign (DNAStar, Madison, WI, USA) alignment and analyses of all available N. risticii strain and Neorickettsia spp. sequences.
GenBank Accession Numbers
Sequences amplified for Neorickettsia.
Fragment size (bp)b
nsp2 (p), nsp3
nsp2 (p), nsp3
nsp2 (p), nsp3
nsp2, nsp3 (p)
nsp2, nsp3 (p)
nsp2 (p), nsp3 (p)
nsp2 (p), nsp3 (p)
nsp2 (p), nsp3 (p)
nsp2 (p), nsp3
GenBank P51 sequences used in this study.
N. risticii IllinoisT
N. sennetsu MiyayamaT
Nano-LC/MS/MS of streptavidin-affinity purified surface proteins
Proteomics-identified proteins for two N.risticii strains.
Mol Mass (kDa)
% (query) peptide coverage b
N. risticii Illinois T
51-kDa antigen (P51)
Heat shock protein 60 (GroEL)
Neorickettsia surface protein 3 (Nsp3)
Conserved hypothetical protein
Heat shock protein 70 (DnaK)
ATP synthase F1, alpha subunit (AtpA)
Strain-specific surface antigen 3 (Ssa3)
2.36 or 4.72 d (2)
Immune recognition of major surface antigens by PHF-positive horse sera
PHF-positive sera from naturally-infected horses and negative sera.
A, F, De, Deh, C
A, F, De, C, L, Et, EUTH
Grove City, OH
A, F, De, Deh, L, Et, EUTH
A, De, F
A, De, Deh, F, C, L
A, F, C, L, EUTH
A, Di, De, Deh, F, L
A, F, Di, De, Deh
A, F, Di
A, F, C
Oak Hill, OH
Sequence variation in P51
Sequence variation in Nsp2
Sequence variation in Nsp3
Sequence variation in Ssa3
Sequence variation in Ssa1
Ssa1 sequences of N. risticii, other than that of N. risticii Illinois have not been determined. Given the strongest similarities between ssa1 of N. risticii Illinois and the unknown ssa s from N. risticii strains 25-D (isolated in 1984) and 90-12 (isolated in 1990) , two ssa1 fragments were amplified, sequenced, and translated from PA-1 and OH07-1. PA-1 (aa 11-249) and OH07-1 (aa 1-239) Ssa1 fragments were aligned with corresponding regions from N. risticii Illinois Ssa1 (aa 246-469) and the Ssas from 25-D (aa 287-507) and 90-12 (aa 579-817). Ssa1 fragments from PA-1 and OH07-1, which are both post-2000 strains, clustered with the 90-12 Ssa, rather than with the 1980s isolates N. risticii Illinois Ssa1 and 25-D Ssa, suggesting a chronological trend (Figure 6b).
The genes p51, nsp2, nsp3, and ssa3 are uniquely evolved in Neorickettsia spp. The gene p51 is a single copy gene and demonstrates only loose associations with other proteins of the family Anaplasmataceae [37, 38]. The nsp s and ssa s are both potential operons, consisting of three genes tandemly arranged . The nsp s belong to pfam01617, and similar to Ehrlichia chaffeensis omp-1 (p28) genes (also from pfam01617) , the proteins encoded by nsp2 and nsp3 were strain variable. As seen in the ssa s, other members of the family Anaplasmataceae have genes encoding proteins containing strain-variable tandem repeats (involving amino acid variation and changes in the numbers of tandem repeats), including Trp120 (formerly gp120), Trp47 (formerly gp47), and VLPT (variable-length PCR target) from E. chaffeensis and Trp140 (formerly gp140), Trp36 (formerly gp36), and gp19 from Ehrlichia canis [50–52]. Of note, the proteins encoded by the ssa s are not homologous to any proteins of the family Anaplasmataceae by blastp. Among p51, the nsp s, and the ssa s, there have been no signs of intragenomic recombination events, which are seen in the Anaplasma p44/msp2 expression locus [53, 54].
Proteomics results performed on two strains of N. risticii established that P51 is a dominant surface-expressed protein. The recognition of recombinant P51 by PHF horse sera, even by 1:80 IFA titer sera suggests P51 is expressed and highly recognized within the present day naturally-infected horses. Despite P51 amino acid sequence variation among N. risticii strains, this strong universal recognition by horse immune sera suggests rP51 may serve as a defined serodiagnostic antigen. Furthermore, the study suggests that there are immunodominant conserved peptide sequences within P51 which might serve as even more specific PHF diagnostic antigens.
Sequence comparison of these surface-exposed proteins of N. risticii strains, with respect to the predicted protein secondary structure, the majority of which are clinical isolates, indicates there are hot spots within the genes with greater strain divergence. These include external loops 2 and 4 in P51, external loop 4 in Nsp2, external loop 2 in Nsp3, and the repeated region of Ssa3. P51 showed strong geographical association; and Nsp2, Nsp3, and Ssa3 showed temporal association. Importantly, N. risticii Illinois (upon which vaccines for PHF are produced) is distinct from most East/Midwest US strains (P51) and most post-2000 strains (Nsp2, Nsp3, and Ssa3), which may be a contributing factor in PHF vaccine failure [24, 55].
There are outlier strains which do not fit the geographical and temporal patterns. These include 081 [20, 22], the Kentucky strain OV , and the Kentucky strain Herodia. Unique sequences in other N. risticii strains, such as TN02-1 (P51, Nsp2, and Nps3), KY03-3 (Nsp2), IL01-1 (Nsp3), and OH10-1 (Ssa3), suggest that variation contrary to the popular geographical and temporal influences may be more widespread. When additional contemporary sequences and sequences from more varied geographic regions become available, these analyses are expected to improve.
Possible explanations for extensive DNA sequence variation within Neorickettsia include the defective DNA repair systems in both N. risticii and N. sennetsu [37, 38]. This would result in higher mutation rates for Neorickettsia , which would agree with the temporal changes and the production of outlier strains of N. risticii. P51 variation showed substantial geographical association, suggesting these variations were selected under local environmental pressures. It is possible that geographical association of N. risticii sequence variation is due to N. risticii strains being selected within essential reservoir trematode populations. In addition, diverse N. risticii strains may have emerged due to selective pressures inflicted on the infected trematodes and/or on the trematodes' hosts [4–9, 57–59]. Humoral immunity would thus not play any direct role in creating genetic diversity within N. risticii populations. Since Neorickettsia spp. are known (N. risticii and N. helminthoeca) and suspected to be vertically transmitted within their trematode hosts [8, 13, 60], mammalian infection is not expected to be required for maintaining Neorickettsia in the natural environment.
Regardless the cause, this genetic variation would result in increased N. risticii survival as a species. N. risticii surface protein genetic diversity revealed in the present study will help in understanding variations in PHF virulence and clinical signs. It may also be possible to use this new molecular knowledge for vaccine development. It would, however necessitate taking into account that the pathogen is an obligate intracellular pathogen, indicating that not only humoral immune responses, but also cell-mediated immunity would play an active role in preventing bacterial infection [61–63].
Genes encoding the two original Ssas, called P85 (90-12) and P50 (25-D) are most related to ssa1 from N. risticii Illinois [24, 31, 38, 55], but they also show similarities to ssa2 and the non-coding region between ssa1 and ssa2 using blastn. Although both are Maryland isolates, the 25-D strain was isolated six years earlier than the 90-12 strain , suggesting both temporal variation and the potential development of chimeras of multiple Ssas and non-coding regions in P50, P85, and post-2000 Ssa1 (due to the similarities of PA-1 and OH07-1 Ssa1 fragments to P85). It is possible that the high variability of Ssa1 may have prevented PA-1 Ssa1 from being identified by proteomics. However, there is the obvious lack of large numbers of peptides identified by proteomics for Ssas in N. risticii Illinois using the isogenic Illinois strain sequence data and in N. sennetsu using Miyayama isogenic strain data . It is likely that Ssas are not a dominant surface protein in mammalian cells.
In conclusion, our data demonstrate the variety present within major surface proteins of N. risticii, and they suggest conservation among geographical regions and time periods. In addition, P51 is implicated as the major surface antigen of N. risticii. These data will be valuable in developing better diagnostic methods and may help in the development of more efficacious vaccines.
We would like to thank the members of the Mass Spectrometry and Proteomics Facility, including Dr Kari Green-Church for their expertise and assistance. We would like to thank Dr Mingqun Lin for his technical advice and assistance and Dr Koshiro Miura for his assistance in training GP. This work was funded by grant R01AI047885 from the National Institutes of Health. KEG was partially supported by T32 RR0070703, and GP and SM were funded by T35 RR021310 from the National Institutes of Health.
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