A Variable-Length PCR Target Protein of Ehrlichia chaffeensis Contains Major Species-Specific Antibody Epitopes in Acidic Serine-Rich Tandem Repeats

ABSTRACT Ehrlichia chaffeensis and E. canis have a small subset of tandem repeat (TR)-containing proteins that elicit strong host immune responses and are associated with host-pathogen interactions. In a previous study, we molecularly characterized a highly conserved 19-kDa major immunoreactive protein (gp19) of E. canis and identified the corresponding TR-containing ortholog variable-length PCR target (VLPT) protein in E. chaffeensis. In this study, the native 32-kDa VLPT protein was identified and the immunodeterminants defined in order to further understand the molecular basis of the host immune response to E. chaffeensis. Synthetic and/or recombinant polypeptides corresponding to various regions of VLPT were used to localize major antibody epitopes to the TR-containing region. Major antibody epitopes were identified in three nonidentical repeats (R2, R3, and R4), which reacted strongly with antibodies in sera from an E. chaffeensis-infected dog and human monocytotropic ehrlichiosis patients. VLPT-R3 and VLPT-R2 reacted most strongly with antibody, and the epitope was further localized to a nearly identical proximal 17-amino-acid region common between these repeats that was species specific. The epitope in R4 was distinct from that of R2 and R3 and was found to have conformational dependence. VLPT was detected in supernatants from infected cells, indicating that the protein was secreted. VLPT was localized on both reticulate and dense-core cells, and it was found extracellularly in the morula fibrillar matrix and associated with the morula membrane.

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Tyrosine-Phosphorylated Ehrlichia chaffeensis and Ehrlichia canis Tandem Repeat Orthologs Contain a Major Continuous Cross-Reactive Antibody Epitope in Lysine-Rich Repeats

Infection and Immunity ◽

10.1128/iai.01347-10 ◽

2011 ◽

Vol 79 (8) ◽

pp. 3178-3187 ◽

Cited By ~ 17

Author(s):

Jere W. McBride ◽

Xiaofeng Zhang ◽

Abdul Wakeel ◽

Jeeba A. Kuriakose

Keyword(s):

Amino Acids ◽

Tandem Repeat ◽

Tandem Repeats ◽

Ehrlichia Canis ◽

Small Subset ◽

Ehrlichia Chaffeensis ◽

Whole Cell ◽

Content Type ◽

Cell Lysates ◽

Species Specific

ABSTRACTA small subset of major immunoreactive proteins have been identified inEhrlichia chaffeensisandEhrlichia canis, including three molecularly and immunologically characterized pairs of immunoreactive tandem repeat protein (TRP) orthologs with major continuous species-specific epitopes within acidic tandem repeats (TR) that stimulate strong antibody responses during infection. In this study, we identified a fourth major immunoreactive TR-containing ortholog pair and defined a major cross-reactive epitope in homologous nonidentical 24-amino-acid lysine-rich TRs. Antibodies from patients and dogs with ehrlichiosis reacted strongly with recombinant TR regions, and epitopes were mapped to the N-terminal TR region (18 amino acids) inE. chaffeensisand the complete TR (24 amino acids) inE. canis. Two less-dominant epitopes were mapped to adjacent glutamate/aspartate-rich and aspartate/tyrosine-rich regions in the acidic C terminus ofE. canisTRP95 but not inE. chaffeensisTRP75. Major immunoreactive proteins inE. chaffeensis(75-kDa) andE. canis(95-kD) whole-cell lysates and supernatants were identified with TR-specific antibodies. Consistent with other ehrlichial TRPs, the TRPs identified in ehrlichial whole-cell lysates and the recombinant proteins migrated abnormally slow electrophoretically a characteristic that was demonstrated with the positively charged TR and negatively charged C-terminal domains.E. chaffeensisTRP75 andE. canisTRP95 were immunoprecipitated with anti-pTyr antibody, demonstrating that they are tyrosine phosphorylated during infection of the host cell.

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Major Species-Specific Antibody Epitopes of the Ehrlichia chaffeensis p120 and E. canis p140 Orthologs in Surface-Exposed Tandem Repeat Regions

Clinical and Vaccine Immunology ◽

10.1128/cvi.00048-09 ◽

2009 ◽

Vol 16 (7) ◽

pp. 982-990 ◽

Cited By ~ 29

Author(s):

Tian Luo ◽

Xiaofeng Zhang ◽

Jere W. McBride

Keyword(s):

Tandem Repeat ◽

Posttranslational Modifications ◽

Molecular Characteristics ◽

Small Subset ◽

Ehrlichia Chaffeensis ◽

Flight Mass Spectrometry ◽

Major Species ◽

Molecular Masses ◽

Species Specific ◽

Antibody Epitopes

ABSTRACT Ehrlichia chaffeensis and E. canis have a small subset of tandem repeat (TR)-containing protein orthologs, including p120/p140, which elicit strong antibody responses. The TR regions of these protein orthologs are immunoreactive, but the molecular characteristics of the p120/p140 epitopes have not been determined. In this study, the immunodeterminants of the E. chaffeensis p120 and E. canis p140 were identified and molecularly defined. Major antibody epitope-containing regions of both p120 and p140 were localized to the TR regions, which reacted strongly by Western immunoblotting with antibodies in sera from E. chaffeensis-infected dogs or patients and E. canis-infected dogs, respectively. Single continuous species-specific major epitopes within the E. chaffeensis p120 and E. canis p140 TRs were mapped to homologous surface-exposed glutamate/aspartate-rich regions (19 to 22 amino acids). In addition, minor cross-reactive epitopes were localized to homologous N- and C-terminal regions of p120 and p140. Furthermore, although the native and recombinant p120 and p140 proteins exhibited higher-than-predicted molecular masses, posttranslational modifications were not present on abnormally migrating p120 and p140 TR recombinant proteins as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

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Ehrlichia canis gp200 Contains Dominant Species-Specific Antibody Epitopes in Terminal Acidic Domains

Infection and Immunity ◽

10.1128/iai.00041-07 ◽

2007 ◽

Vol 75 (10) ◽

pp. 4900-4908 ◽

Cited By ~ 19

Author(s):

Kimberly A. Nethery ◽

C. Kuyler Doyle ◽

Xiaofeng Zhang ◽

Jere W. McBride

Keyword(s):

Amino Acids ◽

Specific Antibody ◽

Amino Acid Content ◽

Ehrlichia Canis ◽

Biophysical Properties ◽

B Cell Epitopes ◽

Immunoreactive Protein ◽

Recombinant Polypeptides ◽

Species Specific ◽

Antibody Epitopes

ABSTRACT Species-specific antibody epitopes within several major immunoreactive protein orthologs of Ehrlichia species have recently been identified and molecularly characterized. In this study, dominant B-cell epitopes within the acidic (pI 5.35) ankyrin repeat-containing 200-kDa major immunoreactive protein (gp200) of Ehrlichia canis were defined. The E. canis gp200 gene (4,263 bp; 1,421 amino acids) was cloned and expressed as four (N-terminal, 1,107 bp; N-internal, 910 bp; C-internal, 1,000 bp; and C-terminal, 1,280 bp) overlapping recombinant proteins. The N-terminal, C-internal, and C-terminal polypeptides (369, 332, and 426 amino acids, respectively) were strongly recognized by antibody, and the major epitope(s) in these polypeptides was mapped to four polypeptide regions (40 to 70 amino acids). Smaller overlapping recombinant polypeptides (14 to 15 amino acids) spanning these regions identified five strongly immunoreactive species-specific epitopes that exhibited conformational dependence. The majority of the epitopes (four) were located in two strongly acidic (pI 4 to 4.9) domains in the distal N- and C-terminal regions of the protein flanking the centralized ankyrin domain-containing region. The amino acid content of the epitope-containing domains included a high proportion of strongly acidic amino acids (glutamate and aspartate), and these domains appear to have important biophysical properties that influence the antibody response to gp200.

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Immunoreactive Protein Repertoires of Ehrlichia chaffeensis and E. canis Reveal the Dominance of Hypothetical Proteins and Conformation-dependent Antibody Epitopes

Infection and Immunity ◽

10.1128/iai.00224-21 ◽

2021 ◽

Author(s):

Tian Luo ◽

Jignesh G. Patel ◽

Xiaofeng Zhang ◽

David H. Walker ◽

Jere W. McBride

Keyword(s):

Immune Responses ◽

Hypothetical Protein ◽

Ehrlichia Chaffeensis ◽

Hypothetical Proteins ◽

Significant Group ◽

Molecular Features ◽

A Genome ◽

Species Specific ◽

Protective Proteins ◽

Antibody Epitopes

The immunomes of Ehrlichia chaffeensis ( E. ch. ) and E. canis ( E. ca. ) have recently be revised to include immunodominant hypothetical proteins with conformational antibody epitopes. In this study, we examined 216 E. ch. and 190 E. ca. highly antigenic proteins according to ANTIGENpro and also performed a genome-wide hypothetical protein analysis ( E. ch. n=104; E. ca. n=124) for immunoreactivity. Using cell-free protein expression and immunoanalysis, 118 E. ch. and 39 E. ca . proteins reacted with sera from naturally E. ch. -infected patients or E. ca. -infected dogs. Moreover, 22 E. ch. and 18 E. ca. proteins consistently and strongly reacted with a panel of patient or canine sera. A subset of E. ch. (n=18) and E. ca. (n=9) proteins were identified as immunodominant. Consistent with our previous study, most proteins were classified as hypothetical and the antibody epitopes exhibited complete or partial conformation-dependence. The majority (28/40; 70%) of E. ch. and E. ca. proteins contained transmembrane domains and 19 (48%) were predicted to be secreted effectors. The antigenic repertoires of E. ch. and E. ca. were mostly diverse and suggest that the immunomes of these closely related ehrlichiae are dominated by species-specific conformational antibody epitopes. This study reveals a significant group of previously undefined E. ch. and E. ca. antigens and reaffirms the importance of conformation-dependent epitopes as targets of anti- Ehrlichia immune responses. These findings substantially expand our understanding of host- Ehrlichia immune responses, advance efforts to define the molecular features of protective proteins and improve prospects for effective vaccines for the ehrlichioses.

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Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

Infection and Immunity ◽

10.1128/iai.01494-06 ◽

2006 ◽

Vol 75 (1) ◽

pp. 74-82 ◽

Cited By ~ 38

Author(s):

Jere W. McBride ◽

C. Kuyler Doyle ◽

Xiaofeng Zhang ◽

Ana Maria Cardenas ◽

Vsevolod L. Popov ◽

...

Keyword(s):

Amino Acids ◽

Specific Antibody ◽

Tandem Repeats ◽

Ehrlichia Canis ◽

Small Subset ◽

Amino Acid Homology ◽

Amino Terminal ◽

Neutral Sugars ◽

Carboxyl Terminal ◽

Species Specific

ABSTRACT Ehrlichia canis has a small subset of major immunoreactive proteins that includes a 19-kDa protein that elicits an early Ehrlichia-specific antibody response in infected dogs. We report herein the identification and molecular characterization of this highly conserved 19-kDa major immunoreactive glycoprotein (gp19) ortholog of the Ehrlichia chaffeensis variable-length PCR target (VLPT) protein. E. canis gp19 has substantial carboxyl-terminal amino acid homology (59%) with E. chaffeensis VLPT and the same chromosomal location; however, the E. chaffeensis VLPT gene (594 bp) has tandem repeats that are not present in the E. canis gp19 gene (414 bp). Consistent with other ehrlichial glycoproteins, the gp19 protein exhibited a larger-than-predicted mass (∼3 kDa), O-linked glycosylation sites were predicted in an amino-terminal serine/threonine/glutamate (STE)-rich patch (26 amino acids), carbohydrate was detected on the recombinant gp19 protein, and the neutral sugars glucose and galactose were detected on the recombinant amino-terminal polypeptide. E. canis gp19 composition consists of five predominant amino acids, cysteine, glutamate, tyrosine, serine, and threonine, concentrated in the STE-rich patch and a carboxyl-terminal domain predominated by cysteine and tyrosine (55%). The amino-terminal STE-rich patch contained a major species-specific antibody epitope strongly recognized by serum from an E. canis-infected dog. The recombinant glycopeptide epitope was substantially more reactive with antibody than the synthetic (nonglycosylated) peptide, and periodate treatment of the recombinant glycopeptide epitope reduced its immunoreactivity, demonstrating the importance of a carbohydrate immunodeterminant(s). The gp19 protein was present on reticulate and dense-cored cells, and it was found extracellularly in the fibrillar matrix and associated with the morula membrane, the host cell cytoplasm, and the nucleus.

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Characterization of the Major Antigenic Protein 2 of Ehrlichia canis and Ehrlichia chaffeensis and Its Application for Serodiagnosis of Ehrlichiosis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.4.520-524.2003 ◽

2003 ◽

Vol 10 (4) ◽

pp. 520-524 ◽

Cited By ~ 7

Author(s):

Tamece T. Knowles ◽

A. Rick Alleman ◽

Heather L. Sorenson ◽

David C. Marciano ◽

Edward B. Breitschwerdt ◽

...

Keyword(s):

Blot Analysis ◽

Single Copy ◽

Ehrlichia Canis ◽

Specific Method ◽

Ehrlichia Chaffeensis ◽

Antigenic Protein ◽

Canine Monocytic Ehrlichiosis ◽

Copy Gene ◽

Species Specific

ABSTRACT Canine monocytic ehrlichiosis, caused by Ehrlichia canis or Ehrlichia chaffeensis, can result in clinical disease in naturally infected animals. Coinfections with these agents may be common in certain areas of endemicity. Currently, a species-specific method for serological diagnosis of monocytic ehrlichiosis is not available. Previously, we developed two indirect enzyme-linked immunosorbent assays (ELISAs) using the major antigenic protein 2 (MAP2) of E. chaffeensis and E. canis. In this study, we further characterized the conservation of MAP2 among various geographic isolates of each organism and determined if the recombinant MAP2 (rMAP2) of E. chaffeensis would cross-react with E. canis-infected dog sera. Genomic Southern blot analysis using digoxigenin-labeled species-specific probes suggested that map2 is a single-copy gene in both Ehrlichia species. Sequences of the single map2 genes of seven geographically different isolates of E. chaffeensis and five isolates of E. canis are highly conserved among the various isolates of each respective ehrlichial species. ELISA and Western blot analysis confirmed that the E. chaffeensis rMAP2 failed to serologically differentiate between E. canis and E. chaffeensis infections.

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A species-specific satellite DNA from the entomopathogenic nematode Heterorhabditis indicus

Genome ◽

10.1139/g98-005 ◽

1998 ◽

Vol 41 (2) ◽

pp. 148-153 ◽

Cited By ~ 8

Author(s):

Monique Abadon ◽

Eric Grenier ◽

Christian Laumond ◽

Pierre Abad

Keyword(s):

Dna Sequence ◽

Satellite Dna ◽

Tandem Repeats ◽

Sequence Data ◽

Entomopathogenic Nematode ◽

Consensus Sequence ◽

Repeated Sequence ◽

Nucleotide Sequence Analysis ◽

Specific Sequence ◽

Species Specific

An AluI satellite DNA family has been cloned from the entomopathogenic nematode Heterorhabditis indicus. This repeated sequence appears to be an unusually abundant satellite DNA, since it constitutes about 45% of the H. indicus genome. The consensus sequence is 174 nucleotides long and has an A + T content of 56%, with the presence of direct and inverted repeat clusters. DNA sequence data reveal that monomers are quite homogeneous. Such homogeneity suggests that some mechanism is acting to maintain the homogeneity of this satellite DNA, despite its abundance, or that this repeated sequence could have appeared recently in the genome of H. indicus. Hybridization analysis of genomic DNAs from different Heterorhabditis species shows that this satellite DNA sequence is specific to the H. indicus genome. Considering the species specificity and the high copy number of this AluI satellite DNA sequence, it could provide a rapid and powerful tool for identifying H. indicus strains.Key words: AluI repeated DNA, tandem repeats, species-specific sequence, nucleotide sequence analysis.

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Going Wild: Lessons from Naturally Occurring T-Lymphotropic Lentiviruses

Clinical Microbiology Reviews ◽

10.1128/cmr.00009-06 ◽

2006 ◽

Vol 19 (4) ◽

pp. 728-762 ◽

Cited By ~ 167

Author(s):

Sue VandeWoude ◽

Cristian Apetrei

Keyword(s):

Immune Responses ◽

Natural Populations ◽

Host Immune Responses ◽

Experimental Systems ◽

Naturally Occurring ◽

Immunodeficiency Virus ◽

New Directions ◽

Species Specific ◽

Lentiviral Infections ◽

Hiv Aids

SUMMARY Over 40 nonhuman primate (NHP) species harbor species-specific simian immunodeficiency viruses (SIVs). Similarly, more than 20 species of nondomestic felids and African hyenids demonstrate seroreactivity against feline immunodeficiency virus (FIV) antigens. While it has been challenging to study the biological implications of nonfatal infections in natural populations, epidemiologic and clinical studies performed thus far have only rarely detected increased morbidity or impaired fecundity/survival of naturally infected SIV- or FIV-seropositive versus -seronegative animals. Cross-species transmissions of these agents are rare in nature but have been used to develop experimental systems to evaluate mechanisms of pathogenicity and to develop animal models of HIV/AIDS. Given that felids and primates are substantially evolutionarily removed yet demonstrate the same pattern of apparently nonpathogenic lentiviral infections, comparison of the biological behaviors of these viruses can yield important implications for host-lentiviral adaptation which are relevant to human HIV/AIDS infection. This review therefore evaluates similarities in epidemiology, lentiviral genotyping, pathogenicity, host immune responses, and cross-species transmission of FIVs and factors associated with the establishment of lentiviral infections in new species. This comparison of consistent patterns in lentivirus biology will expose new directions for scientific inquiry for understanding the basis for virulence versus avirulence.

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Pathogenesis of Gram-Negative Bacteremia

Clinical Microbiology Reviews ◽

10.1128/cmr.00234-20 ◽

2021 ◽

Vol 34 (2) ◽

Author(s):

Caitlyn L. Holmes ◽

Mark T. Anderson ◽

Harry L. T. Mobley ◽

Michael A. Bachman

Keyword(s):

Immune Responses ◽

Global Economy ◽

Human Infection ◽

Model Systems ◽

Small Subset ◽

Gram Negative ◽

Content Type ◽

Capsule Production ◽

Gram Negative Bacteremia ◽

Species Specific

SUMMARY Gram-negative bacteremia is a devastating public health threat, with high mortality in vulnerable populations and significant costs to the global economy. Concerningly, rates of both Gram-negative bacteremia and antimicrobial resistance in the causative species are increasing. Gram-negative bacteremia develops in three phases. First, bacteria invade or colonize initial sites of infection. Second, bacteria overcome host barriers, such as immune responses, and disseminate from initial body sites to the bloodstream. Third, bacteria adapt to survive in the blood and blood-filtering organs. To develop new therapies, it is critical to define species-specific and multispecies fitness factors required for bacteremia in model systems that are relevant to human infection. A small subset of species is responsible for the majority of Gram-negative bacteremia cases, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The few bacteremia fitness factors identified in these prominent Gram-negative species demonstrate shared and unique pathogenic mechanisms at each phase of bacteremia progression. Capsule production, adhesins, and metabolic flexibility are common mediators, whereas only some species utilize toxins. This review provides an overview of Gram-negative bacteremia, compares animal models for bacteremia, and discusses prevalent Gram-negative bacteremia species.

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Iterons Homologous to Helper Geminiviruses Are Essential for Efficient Replication of Betasatellites

Journal of Virology ◽

10.1128/jvi.01532-18 ◽

2018 ◽

Vol 93 (5) ◽

Cited By ~ 2

Author(s):

Xiongbiao Xu ◽

Yajuan Qian ◽

Yaqin Wang ◽

Zhenghe Li ◽

Xueping Zhou

Keyword(s):

Tandem Repeats ◽

Repeated Sequence ◽

Iterative Sequence ◽

Binding Motif ◽

Sequence Motif ◽

High Affinity ◽

Efficient Replication ◽

Tobacco Curly Shoot Virus ◽

Species Specific ◽

Origin Recognition

ABSTRACTBetasatellites associated with geminiviruses can be replicated promiscuously by distinct geminiviruses but exhibit a preference for cognate helper viruses. However, theciselements responsible for betasatellite origin recognition have not been characterized. In this study, we identified an iteron-like repeated sequence motif, 5′-GAGGACC-3′, in a tobacco curly shoot betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV). Competitive DNA binding assays revealed that two core repeats (5′-GGACC-3′) are required for specific binding to TbCSV Rep; TbCSB iteron mutants accumulated to greatly reduced levels and lost the cognate helper-mediated replication preference. Interestingly, TbCSV also contains identical repeated sequences that are essential for specific Rep binding andin vivoreplication. In order to gain insight into the mechanism by which TbCSB has acquired the cognate iterons, we performed a SELEX (systematic evolution of ligands by exponential enrichment) assay to identify the high-affinity Rep binding ligands from a large pool of randomized sequences. Analysis of SELEX winners showed that all of the sequences contained at least one core iteron-like motif, suggesting that TbCSB has evolved to contain cognate iterons for high-affinity Rep binding. Further analyses of various betasatellite sequences revealed a region upstream of the satellite conserved region replete with iterative sequence motifs, including species-specific repeats and a general repeat (5′-GGTAAAT-3′). Remarkably, the species-specific repeats in many betasatellites are homologous to those in their respective cognate helper begomoviruses, whereas the general repeat is widespread in most of the betasatellite molecules analyzed. These data, taken together, suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for efficient replication mediated by cognate helper viruses.IMPORTANCEThe geminivirus-encoded replication initiator protein (Rep) binds to repeated sequence elements (also known as iterons) in the origin of replication that serve as essentialciselements for specific viral replication. Betasatellites associated with begomoviruses can be replicated by cognate or noncognate helper viruses, but theciselements responsible for betasatellite origin recognition have not been characterized. Using a betasatellite (TbCSB) associated with tobacco curly shoot virus (TbCSV) as a model, we identify two tandem repeats (iterons) in the Rep-binding motif (RBM) that are required for specific Rep binding and efficient replication, and we show that identical iteron sequences present in TbCSV are also necessary for Rep binding and the replication of helper viruses. Extensive analysis of begomovirus/betasatellite sequences shows that many betasatellites contain iteron-like elements homologous to those of their respective cognate helper begomoviruses. Our data suggest that many betasatellites have evolved to acquire homologous iteron-like sequences for efficient replication mediated by cognate helper viruses.

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