Differential Expression and Sequence Conservation of the Anaplasma marginale msp2 Gene Superfamily Outer Membrane Proteins

ABSTRACT Bacterial pathogens in the genera Anaplasma and Ehrlichia encode a protein superfamily, pfam01617, which includes the predominant outer membrane proteins (OMPs) of each species, major surface protein 2 (MSP2) and MSP3 of Anaplasma marginale and Anaplasma ovis, Anaplasma phagocytophilum MSP2 (p44), Ehrlichia chaffeensis p28-OMP, Ehrlichia canis p30, and Ehrlichia ruminantium MAP1, and has been shown to be involved in both antigenic variation within the mammalian host and differential expression between the mammalian and arthropod hosts. Recently, complete sequencing of the A. marginale genome has identified an expanded set of genes, designated omp1-14, encoding new members of this superfamily. Transcriptional analysis indicated that, with the exception of the three smallest open reading frames, omp2, omp3, and omp6, these superfamily genes are transcribed in A. marginale-infected erythrocytes, tick midgut and salivary glands, and the IDE8 tick cell line. OMPs 1, 4, 7 to 9, and 11 were confirmed to be expressed as proteins by A. marginale within infected erythrocytes, with expression being either markedly lower (OMPs 1, 4, and 7 to 9) or absent (OMP11) in infected tick cells, which reflected regulation at the transcript level. Although the pfam01617 superfamily includes the antigenically variable MSP2 and MSP3 surface proteins, analysis of the omp1-14 sequences throughout a cycle of acute and persistent infection in the mammalian host and tick transmission reveals a high degree of conservation, an observation supported by sequence comparisons between the St. Maries strain and Florida strain genomes.

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Simultaneous Variation of the Immunodominant Outer Membrane Proteins, MSP2 and MSP3, during Anaplasma marginale Persistence In Vivo

Infection and Immunity ◽

10.1128/iai.71.11.6627-6632.2003 ◽

2003 ◽

Vol 71 (11) ◽

pp. 6627-6632 ◽

Cited By ~ 32

Author(s):

Kelly A. Brayton ◽

Patrick F. M. Meeus ◽

Anthony F. Barbet ◽

Guy H. Palmer

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Bacterial Pathogen ◽

Anaplasma Marginale ◽

Surface Proteins ◽

Mammalian Host ◽

Surface Coat ◽

Vector Borne

ABSTRACT Vector-borne bacterial pathogens persist in the mammalian host by varying surface antigens to evade the existing immune response. To test whether the model of surface coat switching and immune evasion can be extended to a vector-borne bacterial pathogen with multiple immunodominant surface proteins, we examined Anaplasma marginale, a rickettsia with two highly immunogenic outer membrane proteins, major surface protein 2 (MSP2) and MSP3. The simultaneous clearance of variants of the two most immunodominant surface proteins of A. marginale followed by emergence of unique variants indicates that the switch rates and immune selection for MSP2 and MSP3 are sufficiently similar to explain the cyclic bacteremia observed during infection in the immunocompetent host.

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Composition of the Surface Proteome of Anaplasma marginale and Its Role in Protective Immunity Induced by Outer Membrane Immunization

Infection and Immunity ◽

10.1128/iai.00008-08 ◽

2008 ◽

Vol 76 (5) ◽

pp. 2219-2226 ◽

Cited By ~ 58

Author(s):

Susan M. Noh ◽

Kelly A. Brayton ◽

Wendy C. Brown ◽

Junzo Norimine ◽

Gerhard R. Munske ◽

...

Keyword(s):

Outer Membrane ◽

Protective Immunity ◽

Vaccine Development ◽

Bacterial Pathogen ◽

Protein Composition ◽

Anaplasma Marginale ◽

Surface Proteins ◽

Mammalian Host ◽

Surface Complexes ◽

Surface Proteome

ABSTRACT Surface proteins of tick-borne, intracellular bacterial pathogens mediate functions essential for invasion and colonization. Consequently, the surface proteome of these organisms is specifically relevant from two biological perspectives, induction of protective immunity in the mammalian host and understanding the transition from the mammalian host to the tick vector. In this study, the surface proteome of Anaplasma marginale, a tick-transmitted bacterial pathogen, was targeted by using surface-specific cross-linking to form intermolecular bonds between adjacent proteins. Liquid chromatography and tandem mass spectroscopy were then employed to characterize the specific protein composition of the resulting complexes. The surface complexes of A. marginale isolated from erythrocytes of the mammalian host were composed of multiple membrane proteins, most of which belong to a protein family, pfam01617, which is conserved among bacteria in the genus Anaplasma and the closely related genus Ehrlichia. In contrast, the surface proteome of A. marginale isolated from tick cells was much less complex and contained a novel protein, AM778, not identified within the surface proteome of organisms from the mammalian host. Immunization using the cross-linked surface complex induced protection against high-level bacteremia and anemia upon A. marginale challenge of cattle and effectively recapitulated the protection induced by immunization with whole outer membranes. These results indicate that a surface protein subset of the outer membrane is capable of inducing protective immunity and serves to direct vaccine development. Furthermore, the data support that remodeling of the surface proteome accompanies the transition between mammalian and arthropod hosts and identify novel targets for blocking transmission.

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Proteomic analysis of Proteus mirabilis outer membrane proteins reveals differential expression in vivo vs. in vitro conditions

FEMS Immunology & Medical Microbiology ◽

10.1111/j.1574-695x.2011.00839.x ◽

2011 ◽

Vol 63 (2) ◽

pp. 174-182 ◽

Cited By ~ 5

Author(s):

Bruno D'Alessandro ◽

Leticia M. S. Lery ◽

Wanda M. A. Krüger ◽

Analía Lima ◽

Claudia Piccini ◽

...

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Differential Expression ◽

Proteus Mirabilis ◽

Proteomic Analysis ◽

Outer Membrane Proteins

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The search forBrachyspiraouter membrane proteins that interact with the host

Animal Health Research Reviews ◽

10.1079/ahrr200112 ◽

2001 ◽

Vol 2 (1) ◽

pp. 19-30 ◽

Cited By ~ 27

Author(s):

Darren J. Trott ◽

David P. Alt ◽

Richard L. Zuerner ◽

Michael J. Wannemuehler ◽

Thaddeus B. Stanton

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Membrane Vesicle ◽

Outer Membrane Proteins ◽

Indirect Evidence ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Surface Proteins ◽

Brachyspira Pilosicoli ◽

Variable Surface

AbstractLittle is known about the outer membrane structure ofBrachyspira hyodysenteriae and Brachyspira pilosicolior the role of outer membrane proteins (OMPs) in host colonization and the development of disease. The isolation of outer membrane vesicles fromB. hyodysenteriaehas confirmed that cholesterol is a significant outer membrane constituent and that it may impart unique characteristics to the lipid bilayer structure, including a reduced density. Unique proteins that have been identified in theB. hyodysenteriaeouter membrane include the variable surface proteins (Vsp) and lipoproteins such as SmpA and BmpB. While the function of these proteins remains to be determined, there is indirect evidence to suggest that they may be involved in immune evasion. These data may explain the ability of the organism to initiate chronic infection. OMPs may be responsible for the unique attachment ofB. pilosicolito colonic epithelial cells; however, the onlyB. pilosicoliOMPs that have been identified to date are involved in metabolism. In order to identify furtherB. pilosicoliOMPs we have isolated membrane vesicle fractions from porcine strain 95–1000 by osmotic lysis and isopycnic centrifugation. The fractions were free of contamination by cytoplasm and fla-gella and contained outer membrane. Inner membrane contamination was minimal but could not be completely excluded. An abundant 45-kDa, heat-modifiable protein was shown to have significant homology withB. hyodysenteriaeVsp, and monoclonal antibodies were produced that reacted with fiveB. pilosicoli-specificmembrane protein epitopes. The first of these proteins to be characterized is a unique surface-exposed lipoprotein.

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Identification of Novel Antigenic Proteins in a Complex Anaplasma marginale Outer Membrane Immunogen by Mass Spectrometry and Genomic Mapping

Infection and Immunity ◽

10.1128/iai.73.12.8109-8118.2005 ◽

2005 ◽

Vol 73 (12) ◽

pp. 8109-8118 ◽

Cited By ~ 79

Author(s):

Job E. Lopez ◽

William F. Siems ◽

Guy H. Palmer ◽

Kelly A. Brayton ◽

Travis C. McGuire ◽

...

Keyword(s):

Mass Spectrometry ◽

Membrane Proteins ◽

Outer Membrane ◽

Vaccine Development ◽

Outer Membrane Proteins ◽

Serum Antibody ◽

Elongation Factor ◽

Anaplasma Marginale ◽

Antigenic Proteins ◽

Associated Proteins

ABSTRACT Immunization with purified Anaplasma marginale outer membranes induces complete protection against infection that is associated with CD4+ T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of the A. marginale genome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membrane-vaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of the A. marginale genome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in the A. marginale genome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development.

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Linkage between Anaplasma marginale Outer Membrane Proteins Enhances Immunogenicity but Is Not Required for Protection from Challenge

Clinical and Vaccine Immunology ◽

10.1128/cvi.00600-12 ◽

2013 ◽

Vol 20 (5) ◽

pp. 651-656 ◽

Cited By ~ 11

Author(s):

Susan M. Noh ◽

Joshua E. Turse ◽

Wendy C. Brown ◽

Junzo Norimine ◽

Guy H. Palmer

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Protective Immunity ◽

Bacterial Infections ◽

Vaccine Development ◽

Outer Membrane Proteins ◽

Anaplasma Marginale ◽

Anamnestic Response ◽

Covalent Bonds ◽

Content Type

ABSTRACTThe prevention of bacterial infections via immunization presents particular challenges. While outer membrane extracts are often protective, they are difficult and expensive to isolate and standardize and thus are often impractical for development and implementation in vaccination programs. In contrast, individual proteins, which are easily adapted for use in subunit vaccines, tend to be poorly protective. Consequently, identification of the specific characteristics of outer membrane-based immunogens, in terms of the antigen contents and contexts that are required for protective immunity, represents a major gap in the knowledge needed for bacterial vaccine development. Using as a modelAnaplasma marginale, a persistent tick-borne bacterial pathogen of cattle, we tested two sets of immunogens to determine whether membrane context affected immunogenicity and the capacity to induce protection. The first immunogen was composed of a complex of outer membrane proteins linked by covalent bonds and known to be protective. The second immunogen was derived directly from the first one, but the proteins were individualized rather than linked. The antibody response induced by the linked immunogen was much greater than that induced by the unlinked immunogen. However, both immunogens induced protective immunity and an anamnestic response. These findings suggest that individual proteins or combinations of proteins can be successfully tested for the ability to induce protective immunity with less regard for overall membrane context. Once protective antigens are identified, immunogenicity could be enhanced by cross-linking to allow a reduced immunogen dose or fewer booster vaccinations.

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Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0406656102 ◽

2004 ◽

Vol 102 (3) ◽

pp. 844-849 ◽

Cited By ~ 185

Author(s):

K. A. Brayton ◽

L. S. Kappmeyer ◽

D. R. Herndon ◽

M. J. Dark ◽

D. L. Tibbals ◽

...

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Genome Sequencing ◽

Complete Genome ◽

Outer Membrane Proteins ◽

Anaplasma Marginale ◽

Complete Genome Sequencing

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Flagellin and Outer Surface Proteins from Borrelia burgdorferi Are Not Glycosylated

Journal of Bacteriology ◽

10.1128/jb.01885-07 ◽

2008 ◽

Vol 190 (7) ◽

pp. 2619-2623 ◽

Cited By ~ 7

Author(s):

Ján Štěrba ◽

Marie Vancová ◽

Nataliia Rudenko ◽

Maryna Golovchenko ◽

Tammy-Lynn Tremblay ◽

...

Keyword(s):

Membrane Proteins ◽

Borrelia Burgdorferi ◽

Outer Membrane ◽

Outer Surface ◽

Culture Medium ◽

Outer Membrane Proteins ◽

Surface Proteins ◽

Outer Surface Proteins ◽

Flagellar Proteins

ABSTRACT We investigated the presence of glycoproteins in Borrelia burgdorferi. We did not find any evidence for glycosylation of the major outer membrane proteins OspA and OspB or the structural flagellar proteins FlaB and FlaA. We suggest that glycoproteins present on the surface of B. burgdorferi may be tightly bound culture medium glycoproteins.

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