Natural variation of immune epitopes reveals intrabacterial antagonism.

Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation.

Immunization with pneumococcal elongation factor Tu enhances serotype-independent protection against Streptococcus pneumoniae infection.

Vaccination is an effective strategy to prevent pneumococcal diseases. Currently, licensed vaccines include the pneumococcal polysaccharide vaccine (PPSV) and the pneumococcal conjugate vaccine (PCV), which target some of the most common of the 94 serotypes of S. pneumoniae based on their capsular composition. However, it has been reported that PPSV is not effective in children aged less than 2 years old and PCV induces serotype replacement, which means that the pneumococcal population has changed following widespread introduction of these vaccines, and the non-vaccine serotypes have increased in being the cause of invasive pneumococcal disease. Therefore, it is important that there is development of novel pneumococcal vaccines to either replace or complement current polysaccharide-based vaccines. Our previous study suggested that S. pneumoniae releases elongation factor Tu (EF-Tu) through autolysis followed by the induction of proinflammatory cytokines in macrophages via toll-like receptor 4, that may contribute to the development of pneumococcal diseases. In this study, we investigated the expression of EF-Tu in various S. pneumoniae strains and whether EF-Tu could be an antigen candidate for serotype-independent vaccine against pneumococcal infection. Western blotting and flow cytometry analysis revealed that EF-Tu is a common factor expressed on the surface of all pneumococcal strains tested, as well as intracellularly. In addition, we demonstrate that immunization with recombinant (r) EF-Tu induced the production of inflammatory cytokines and the IgG1 and IgG2a antibodies in mice, and increased the CD4+ T-cells proportion in splenocytes. We also reveal that anti-EF-Tu serum increased the phagocytic activity of mouse peritoneal macrophages against S. pneumoniae infection, independent of their serotypes. Finally, our results indicate that mice immunized with rEF-Tu were significantly and non-specifically protected against lethal challenges with S. pneumoniae serotypes (2 and 15A). Therefore, pneumococcal EF-Tu could be an antigen candidate for the serotype-independent vaccine against pneumococcal infection.

Anti-EF-Tu IgG titers increase with age and may contribute to protection against the respiratory pathogen Haemophilus influenzae.

Non-typeable Haemophilus influenzae (NTHi) is a pathogen that commonly colonizes the nasopharynx of preschool children, causing opportunistic infections including acute otitis media (AOM). Patients suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized with NTHi and occasionally suffer from exacerbations by the bacterium leading to increased morbidity. Elongation-factor thermo unstable (EF-Tu), a protein critical for bacterial protein synthesis, has been found to moonlight on the surface of several bacteria. Here, we show that antibodies against NTHi EF-Tu were present in children already at 18 months of age, and that the IgG antibody titers increased with age. Children harboring NTHi in the nasopharynx also displayed significantly higher IgG concentrations. Interestingly, children suffering from AOM had significantly higher anti-EF-Tu IgG levels when NTHi was the causative agent. Human sera recognized mainly the central and C-terminal part of the EF-Tu molecule and peptide-based epitope mapping confirmed similar binding patterns for sera from humans and immunized mice. Immunization of BALB/c and otitis-prone Junbo (C3H/HeH) mice promoted lower infection rates in the nasopharynx and middle ear, respectively. In conclusion, our results suggest that IgG directed against NTHi EF-Tu may play an important role in the host immune response against NTHi.

Immunogenicity and protective capacity of EF-Tu and FtsZ of Streptococcus suis serotype 2 against lethal infection.

Vaccine development efforts against Streptococcus suis serotype 2 (S. suis 2) are often constrained by strain/serotype antigen variability. Bioinformatics analyses revealed two highly conserved S. suis 2 factors, EF-Tu and FtsZ. Murine immunization with recombinant proteins emulsified in white oil adjuvant or eukaryotic DNA vaccine vectors provided significant protection against lethal S. suis 2 challenge. Immune responses elicited by recombinant protein immunization revealed the robust generation of humoral immune responses, with a mixed induction of Th1-type and Th2-type responses. Furthermore, the antiserum from mice immunized with recombinant proteins significantly inhibited the growth of S. suis 2 in healthy pig whole blood, suggesting the triggering of a strong opsonizing response. Histological examination found that immunizing mice with purified recombinant proteins reduced neutrophil and macrophage accumulation in brain and lung tissues after challenge with virulent S. suis. Taken together, these findings reveal that EF-Tu and FtsZ may be promising targets for subunit and DNA vaccine candidates against S. suis 2 infection.

EF-Tu From Non-typeable Haemophilus influenzae Is an Immunogenic Surface-Exposed Protein Targeted by Bactericidal Antibodies.

Non-typeable Haemophilus influenzae (NTHi), a commensal organism in pre-school children, is an opportunistic pathogen causing respiratory tract infections including acute otitis media. Adults suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized by NTHi. Previous research has suggested that, in some bacterial species, the intracellular elongation factor thermo-unstable (EF-Tu) can moonlight as a surface protein upon host encounter. The aim of this study was to determine whether EF-Tu localizes to the surface of H. influenzae, and if such surface-associated EF-Tu is a target for bactericidal antibodies. Using flow cytometry, transmission immunoelectron microscopy, and epitope mapping, we demonstrated that EF-Tu is exposed at the surface of NTHi, and identified immunodominant epitopes of this protein. Rabbits immunized with whole-cell NTHi produced significantly more immunoglobulin G (IgG) directed against EF-Tu than against the NTHi outer membrane proteins D and F as revealed by enzyme-linked immunosorbent assays. Chemical cleavage of NTHi EF-Tu by cyanogen bromide (CNBr) followed by immunoblotting showed that the immunodominant epitopes were located within the central and C-terminal regions of the protein. Peptide epitope mapping by dot blot analysis further revealed four different immunodominant peptide sequences; EF-Tu41-65, EF-Tu161-185, EF-Tu221-245, and EF-Tu281-305. These epitopes were confirmed to be surface-exposed and accessible by peptide-specific antibodies in flow cytometry. We also analyzed whether antibodies raised against NTHi EF-Tu cross-react with other respiratory tract pathogens. Anti-EF-Tu IgG significantly detected EF-Tu on unencapsulated bacteria, including the Gram-negative H. parainfluenzae, H. haemolyticus, Moraxella catarrhalis and various Gram-positive Streptococci of the oral microbiome. In contrast, considerably less EF-Tu was observed at the surface of encapsulated bacteria including H. influenzae serotype b (Hib) and Streptococcus pneumoniae (e.g., serotype 3 and 4). Removal of the capsule, as exemplified by Hib RM804, resulted in increased EF-Tu surface density. Finally, anti-NTHi EF-Tu IgG promoted complement-dependent bacterial killing of NTHi and other unencapsulated Gram-negative bacteria as well as opsonophagocytosis of Gram-positive bacteria. In conclusion, our data demonstrate that NTHi EF-Tu is surface-exposed and recognized by antibodies mediating host innate immunity against NTHi in addition to other unencapsulated respiratory tract bacteria.

Gallibacterium elongation factor-Tu possesses amyloid-like protein characteristics, participates in cell adhesion, and is present in biofilms.

Gallibacterium, which is a bacterial pathogen in chickens, can form biofilms. Amyloid proteins present in biofilms bind Congo red dye. The aim of this study was to characterize the cell-surface amyloid-like protein expressed in biofilms formed by Gallibacterium strains and determine the relationship between this protein and curli, which is an amyloid protein that is commonly expressed by members of the Enterobacteriaceae family. The presence of amyloid-like proteins in outer membrane protein samples from three strains of G. anatis and one strain of Gallibacterium genomospecies 2 was evaluated. A protein identified as elongation factor-Tu (EF-Tu) by mass spectrometric analysis and in silico analysis was obtained from the G. anatis strain F149T. This protein bound Congo red dye, cross-reacted with anti-curli polyclonal serum, exhibited polymerizing properties and was present in biofilms. This protein also reacted with pooled serum from chickens that were experimentally infected with G. anatis, indicating the in vivo immunogenicity of this protein. The recombinant EF-Tu purified protein, which was prepared from G. anatis 12656-12, polymerizes under in vitro conditions, forms filaments and interacts with fibronectin and fibrinogen, all of which suggest that this protein functions as an adhesin. In summary, EF-Tu from G. anatis presents amyloid characteristics, is present in biofilms and could be relevant for the pathogenesis of G. anatis.

Identification of a new diagnostic antigen for glanders using immunoproteome analysis.

Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of these proteins, GroEL, was cloned and expressed in E. coli and purified using Ni-NTA affinity chromatography. The recombinant GroEL protein was evaluated in ELISA format on a panel of glanders positive (n=49) and negative (n=79) equine serum samples to determine its diagnostic potential. The developed ELISA had a sensitivity and specificity of 96 and 98.7% respectively. The results of this study highlight the potential of GroEL in serodiagnosis of glanders.

Experimental vaccine induces Th1-driven immune responses and resistance to Neisseria gonorrhoeae infection in a murine model.

Female mice were immunized intravaginally with gonococcal outer membrane vesicles (OMVs) plus microencapsulated interleukin-12 (IL-12), and challenged using an established model of genital infection with Neisseria gonorrhoeae. Whereas sham-immunized and control animals cleared the infection in 10-13 days, those immunized with OMV plus IL-12 cleared infection with homologous gonococcal strains in 6-9 days. Significant protection was also seen after challenge with antigenically distinct strains of N. gonorrhoeae, and protective anamnestic immunity persisted for at least 6 months after immunization. Serum and vaginal immunoglobulin G (IgG) and IgA antibodies were generated against antigens expressed by homologous and heterologous strains. Iliac lymph node CD4+ T cells secreted interferon-γ (IFNγ), but not IL-4, in response to immunization, and produced IL-17 in response to challenge regardless of immunization. Antigens recognized by immunized mouse serum included several shared between gonococcal strains, including two identified by immunoproteomics approaches as elongation factor-Tu (EF-Tu) and PotF3. Experiments with immunodeficient mice showed that protective immunity depended upon IFNγ and B cells, presumably to generate antibodies. The results demonstrated that immunity to gonococcal infection can be induced by immunization with a nonliving gonococcal antigen, and suggest that efforts to develop a human vaccine should focus on strategies to generate type 1 T helper cell (Th1)-driven immune responses in the genital tract.

Elongation Factor Tu and Heat Shock Protein 70 Are Membrane-Associated Proteins from Mycoplasma ovipneumoniae Capable of Inducing Strong Immune Response in Mice.

Chronic non-progressive pneumonia, a disease that has become a worldwide epidemic has caused considerable loss to sheep industry. Mycoplasma ovipneumoniae (M. ovipneumoniae) is the causative agent of interstitial pneumonia in sheep, goat and bighorn. We here have identified by immunogold and immunoblotting that elongation factor Tu (EF-Tu) and heat shock protein 70 (HSP 70) are membrane-associated proteins on M. ovipneumonaiea. We have evaluated the humoral and cellular immune responses in vivo by immunizing BALB/c mice with both purified recombinant proteins rEF-Tu and rHSP70. The sera of both rEF-Tu and rHSP70 treated BALB/c mice demonstrated increased levels of IgG, IFN-γ, TNF-α, IL-12(p70), IL-4, IL-5 and IL-6. In addition, ELISPOT assay showed significant increase in IFN-γ+ secreting lymphocytes in the rHSP70 group when compared to other groups. Collectively our study reveals that rHSP70 induces a significantly better cellular immune response in mice, and may act as a Th1 cytokine-like adjuvant in immune response induction. Finally, growth inhibition test (GIT) of M. ovipneumoniae strain Y98 showed that sera from rHSP70 or rEF-Tu-immunized mice inhibited in vitro growth of M. ovipneumoniae. Our data strongly suggest that EF-Tu and HSP70 of M. ovipneumoniae are membrane-associated proteins capable of inducing antibody production, and cytokine secretion. Therefore, these two proteins may be potential candidates for vaccine development against M. ovipneumoniae infection in sheep.

Differentiation between MAMP Triggered Defenses in Arabidopsis thaliana.

A first line of defense against pathogen attack for both plants and animals involves the detection of microbe-associated molecular patterns (MAMPs), followed by the induction of a complex immune response. Plants, like animals, encode several receptors that recognize different MAMPs. While these receptors are thought to function largely redundantly, the physiological responses to different MAMPs can differ in detail. Responses to MAMP exposure evolve quantitatively in natural populations of Arabidopsis thaliana, perhaps in response to environment specific differences in microbial threat. Here, we sought to determine the extent to which the detection of two canonical MAMPs were evolving redundantly or distinctly within natural populations. Our results reveal negligible correlation in plant growth responses between the bacterial MAMPs EF-Tu and flagellin. Further investigation of the genetic bases of differences in seedling growth inhibition and validation of 11 candidate genes reveal substantial differences in the genetic loci that underlie variation in response to these two MAMPs. Our results indicate that natural variation in MAMP recognition is largely MAMP-specific, indicating an ability to differentially tailor responses to EF-Tu and flagellin in A. thaliana populations.

Identification of proteins derived from Listeria monocytogenes inducing human dendritic cell maturation.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that can promote antitumor immunity when pulsed with tumor antigens and then matured by stimulatory agents. Despite apparent progress in DC-based cancer immunotherapy, some discrepancies were reported in generating potent DCs. Listeria monocytogenes as an intracellular microorganism is able to effectively activate DCs through engaging pattern-recognition receptors (PRRs). This study aimed to find the most potent components derived from L. monocytogenes inducing DC maturation. The preliminary results demonstrated that the ability of protein components is higher than DNA components to promote DC maturation and activation. Protein lysate fractionation demonstrated that fraction 2 HIC (obtained by hydrophobic interaction chromatography) was able to efficiently mature DCs. F2HIC-matured DCs are able to induce allogeneic CD8(+) T cells proliferation better than LPS-matured DCs and induce IFN-γ producing CD8(+) T cells. Mass spectrometry results showed that F2HIC contains 109 proteins. Based on the bioinformatics analysis for these 109 proteins, elongation factor Tu (EF-Tu) could be considered as a PRR ligand for stimulating DC maturation.

Borrelia burgdorferi elongation factor EF-Tu is an immunogenic protein during Lyme borreliosis.

Borrelia burgdorferi, the etiological agent of Lyme disease, does not produce lipopolysaccharide but expresses a large number of lipoproteins on its cell surface. These outer membrane lipoproteins are highly immunogenic and have been used for serodiagnosis of Lyme disease. Recent studies have shown that highly conserved cytosolic proteins such as enolase and elongation factor Tu (EF-Tu) unexpectedly localized on the surface of bacteria including B. burgdorferi, and surface-localized enolase has shown to contribute to the enzootic cycle of B. burgdorferi. In this study, we studied the immunogenicity, surface localization, and function of B. burgdorferi EF-Tu. We found that EF-Tu is highly immunogenic in mice, and EF-Tu antibodies were readily detected in Lyme disease patients. On the other hand, active immunization studies showed that EF-Tu antibodies did not protect mice from infection when challenged with B. burgdorferi via either needle inoculation or tick bites. Borrelial mouse-tick cycle studies showed that EF-Tu antibodies also did not block B. burgdorferi migration and survival in ticks. Consistent with these findings, we found that EF-Tu primarily localizes in the protoplasmic cylinder of spirochetes and is not on the surface of B. burgdorferi. Taken together, our studies suggest that B. burgdorferi EF-Tu is not surfaced exposed, but it is highly immunogenic and is a potential serodiagnostic marker for Lyme borreliosis.

Proteomic analysis of the interaction of Bifidobacterium longum NCC2705 with the intestine cells Caco-2 and identification of plasminogen receptors.

To identify proteins with a potential role in the interaction of Bifidobacterium longum with intestinal epithelial cells, we profiled the protein response of B. longum NCC2705 following interaction with Caco-2 cells. Thirty-one protein spots, belonging to a total of 23 proteins, which exhibited a change in abundance of at least 3-fold were identified in B. longum NCC2705 following co-culture with Caco-2 cells, and were subsequently identified. Changes in expression were confirmed at the transcriptional level for a selection of these proteins. Enolase (Eno) and elongation factor Tu (EF-Tu) were amongst the proteins that showed the most prominent increase in abundance. Interaction of these proteins with plasminogen (Plg) was analyzed by Plg overlay assays, glutathione S-transferase (GST)-pull down, and western blot analysis. The results suggested that EF-Tu and Eno serve as surface receptors for B. longum NCC2705 binding to human plasminogen. Purified GST-EF-Tu and GST-Eno inhibited adhesion of B. longum NCC2705 to Caco-2 cells. Collectively, our data suggest that Eno and EF-Tu moonlight as adhesions, and are possibly involved in the protective role played by B. longum NCC2705 in defense against enteric pathogens. Biological significance The interaction of bifidobacteria with the human host plasminogen/plasmin system confirms the existence of a new component in the molecular cross-talk between bacteria and the host. Our study analyzed proteins EF-Tu and Eno with Plg binding activity, and they can inhibit adhesion of B. longum NCC2705 to Caco-2 cells, suggesting their role in the bacterial adherent to the enterocyte surface.

Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity.

The rapid production of reactive oxygen species (ROS) burst is a conserved signaling output in immunity across kingdoms. In plants, perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) activates the NADPH oxidase RBOHD by hitherto unknown mechanisms. Here, we show that RBOHD exists in complex with the receptor kinases EFR and FLS2, which are the PRRs for bacterial EF-Tu and flagellin, respectively. The plasma-membrane-associated kinase BIK1, which is a direct substrate of the PRR complex, directly interacts with and phosphorylates RBOHD upon PAMP perception. BIK1 phosphorylates different residues than calcium-dependent protein kinases, and both PAMP-induced BIK1 activation and BIK1-mediated phosphorylation of RBOHD are calcium independent. Importantly, phosphorylation of these residues is critical for the PAMP-induced ROS burst and antibacterial immunity. Our study reveals a rapid regulatory mechanism of a plant RBOH, which occurs in parallel with and is essential for its paradigmatic calcium-based regulation.

Repression of sucrose/ultraviolet B light-induced flavonoid accumulation in microbe-associated molecular pattern-triggered immunity in Arabidopsis.

Recognition of microbe-associated molecular patterns (MAMPs) leads to the generation of MAMP-triggered immunity (MTI), which restricts the invasion and propagation of potentially infectious microbes. It has been described that the perception of different bacterial and fungal MAMPs causes the repression of flavonoid induction upon light stress or sucrose application. However, the functional significance of this MTI-associated signaling output remains unknown. In Arabidopsis (Arabidopsis thaliana), FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR act as the pattern recognition receptors for the bacterial MAMP epitopes flg22 (of flagellin) and elf18 (of elongation factor [EF]-Tu), respectively. Here, we reveal that reactive oxygen species spiking and callose deposition are dispensable for the repression of flavonoid accumulation by both pattern recognition receptors. Importantly, FLS2-triggered activation of PATHOGENESIS-RELATED (PR) genes and bacterial basal defenses are enhanced in transparent testa4 plants that are devoid of flavonoids, providing evidence for a functional contribution of flavonoid repression to MTI. Moreover, we identify nine small molecules, of which eight are structurally unrelated, that derepress flavonoid accumulation in the presence of flg22. These compounds allowed us to dissect the FLS2 pathway. Remarkably, one of the identified compounds uncouples flavonoid repression and PR gene activation from the activation of reactive oxygen species, mitogen-activated protein kinases, and callose deposition, corroborating a close link between the former two outputs. Together, our data imply a model in which MAMP-induced repression of flavonoid accumulation serves a role in removing the inherent inhibitory action of flavonoids on an MTI signaling branch.

An elongation factor-like protein (EF-Tu) elicits a humoral response in infiltrating ductal breast carcinomas: an immunoproteomics investigation.

OBJECTIVES: In the current study, we have used an immunoproteomics approach to identify proteins that commonly elicit a humoral response in patients with infiltrating ductal carcinomas of the breast. DESIGN AND METHODS: Sera obtained at the time of diagnosis from 40 patients with invasive breast cancer and 42 healthy controls were screened for the presence of IgG antibodies to MCF-7 cell line proteins using a serological proteomics-based approach. RESULTS: An immunoreactive protein detected in sera from 21 of 40 patients was isolated and subsequently identified as elongation factor-Tu. CONCLUSIONS: The immunoproteomic approach implemented here offers a powerful tool for determining novel tumor antigens that induce a humoral immune response in cancer patients. From our findings, the immunoreactive EF-Tu protein and/or the related circulating antibodies may display clinical usefulness as potential diagnostic markers and provide a means for a better understanding of the molecular mechanisms underlying breast cancer development.

TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu.

The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent Francisella novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant. EF-Tu was immunodominant in mice vaccinated and protected from virulent F. novicida. Moreover, recombinant EF-Tu induced macrophages to produce inflammatory cytokines in a TLR4 dependent manner. This study shows immune stimulatory properties of a cytoplasmic protein EF-Tu expressed on the membrane of virulent Francisella strains.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu.

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.