Restriction of major surface protein 2 (MSP2) variants during tick transmission of the ehrlichia Anaplasma marginale.
ABSTRACT: Anaplasma marginale is an ehrlichial pathogen of cattle that establishes lifelong persistent infection. Persistence is characterized by rickettsemic cycles in which new A. marginale variant types, defined by the sequence of the expressed msp2 transcripts, emerge. The polymorphic msp2 transcripts encode structurally distinct MSP2 proteins and result in an antigenically diverse and continually changing A. marginale population within the blood. In this manuscript, we used sequence analysis of msp2 transcripts to show that a restricted repertoire of variant types, designated SGV1 and SGV2, is expressed within the tick salivary gland. The same SGV1 and SGV2 variant types were expressed in ticks regardless of the variant types expressed in the blood of infected cattle at the time of acquisition feeding by the ticks. Importantly, subsequent tick transmission to susceptible cattle resulted in acute rickettsemia composed of organisms expressing only the same SGV1 and SGV2 variant types. This indicates that the msp2 expressed by organisms within the tick salivary gland predicts the variant type responsible for acute rickettsemia and disease. This restriction of transmitted A. marginale variant types, in contrast to the marked diversity within persistently infected cattle, supports development of MSP2 vaccines to prevent acute rickettsemia in tick-transmitted infections.
Project description:The rickettsial pathogen Anaplasma marginale expresses a variable immunodominant outer membrane protein, major surface protein 2 (MSP2), involved in antigenic variation and long-term persistence of the organism in carrier animals. MSP2 contains a central hypervariable region of about 100 amino acids that encodes immunogenic B-cell epitopes that induce variant-specific antibodies during infection. Previously, we have shown that MSP2 is encoded on a polycistronic mRNA transcript in erythrocyte stages of A. marginale and defined the structure of the genomic expression site for this transcript. In this study, we show that the same expression site is utilized in stages of A. marginale infecting tick salivary glands. We also analyzed the variability of this genomic expression site in Oklahoma strain A. marginale transmitted from in vitro cultures to cattle and between cattle and ticks. The structure of the expression site and flanking regions was conserved except for sequence that encoded the MSP2 hypervariable region. At least three different MSP2 variants were encoded in each A. marginale population. The major sequence variants did not change on passage of A. marginale between culture, acute erythrocyte stage infections, and tick salivary glands but did change during persistent infections of cattle. The variant types found in tick salivary glands most closely resembled those present in bovine blood at the time of acquisition of infection, whether infection was acquired from an acute or from a persistent rickettsemia. These variations in structure of an expression site for a major, immunoprotective outer membrane protein have important implications for vaccine development and for obtaining an improved understanding of the mechanisms of persistence of ehrlichial infections in humans, domestic animals, and reservoir hosts.
Project description:Anaplasma marginale is an ehrlichial pathogen of cattle, in the order Rickettsiales, that establishes persistent cyclic rickettsemia in the infected host. Within each rickettsemic cycle, A. marginale expressing antigenically variant major surface protein 2 (MSP2) emerge. By cloning 17 full-length msp2 transcripts expressed during cyclic rickettsemia, we determined that emergent variants have a single, central hypervariable region encoding variant B-cell epitopes. The N- and C-terminal regions are highly conserved among the expressed A. marginale variants, and similar sequences define the MSP2 homologues in the agent of human granulocytic ehrlichiosis (HGE). This is in contrast to the MSP2 homologues in ehrlichial genogroup I pathogens, Ehrlichia chaffeensis, Ehrlichia canis, and Cowdria ruminantium, that have multiple hypervariable regions. By defining the variable and conserved regions, we were able to show that the single hypervariable region of A. marginale MSP2 encodes epitopes that are immunogenic and induce variant-specific antibody responses during persistent infection. These findings demonstrate that the MSP2 structural variants that emerge during each cycle of persistent rickettsemia are true antigenic variants, consistent with MSP2 antigenic variation as a mechanism of A. marginale persistence.
Project description:The antigenically variant major surface protein 2 (MSP2) of Anaplasma marginale is expressed from a 3.5-kb operon that contains, in a 5'-to-3' direction, four open reading frames, opag3, opag2, opag1, and msp2. This operon structure was shown to be conserved among genotypically and phenotypically distinct A. marginale, A. ovis, and A. centrale strains. The individual OpAG amino acid sequences are highly conserved among A. marginale strains, with identities ranging from 95 to 99%. OpAG2 and OpAG3 were expressed by all examined A. marginale strains during the acute rickettsemia in the mammalian host and, like MSP2, localize to the bacterial surface. OpAG2 and OpAG3 were also expressed in an infected Ixodes scapularis tick cell line. In contrast, the same A. marginale strains expressed only OpAG2 in two different Dermacentor spp. during transmission feeding. OpAG1 expression was not detected in the infected mammalian host, the infected tick cell line, or within infected Dermacentor ticks. The differential expression of outer membrane proteins from within an operon is a novel finding in tick-transmitted bacteria, and the regulation of expression may be broadly applicable to understanding how the pathogen adapts to the mammalian host-tick vector transition.
Project description:Specific major surface protein 2 (MSP2) variants are expressed by Anaplasma marginale within the tick salivary gland and, following transmission, are expressed during acute rickettsemia. In previous work, we have shown that a restricted pattern of MSP2 variants is expressed in the salivary glands of Dermacentor andersoni ticks infected with the South Idaho strain of A. marginale. Now we demonstrate that the identical restriction does not apply to two other strains of A. marginale, and that different variants are also expressed when the same strain is transmitted by different Dermacentor spp. This indicates that antigenic diversity among strains is maintained in tick transmission and may be a significant constraint to MSP2 vaccine development.
Project description:Anaplasma marginale, an intraerythrocytic ehrlichial pathogen of cattle, establishes persistent infections in both vertebrate (cattle) and invertebrate (tick) hosts. The ability of A. marginale to persist in cattle has been shown to be due, in part, to major surface protein 2 (MSP2) variants which are hypothesized to emerge in response to the bovine immune response. MSP2 antigenic variation has not been studied in persistently infected ticks. In this study we analyzed MSP2 in A. marginale populations from the salivary glands of male Dermacentor variabilis persistently infected with A. marginale after feeding successively on one susceptible bovine and three sheep. New MSP2 variants appeared in each A. marginale population, and sequence alignment of the MSP2 variants revealed multiple amino acid substitutions, insertions, and deletions. These results suggest that selection pressure on MSP2 occurred in tick salivary glands independent of the bovine immune response.
Project description:Antigenic variation of major surface proteins is considered an immune-evasive maneuver used by pathogens as divergent as bacteria and protozoa. Likewise, major surface protein 2 (Msp2) of the tick-borne pathogen, Anaplasma marginale, is thought to be involved in antigenic variation to evade the mammalian host immune response. However, this dynamic process also works in the tick vector in the absence of immune selection pressure. We examined Msp2 variants expressed during infection of four tick and two mammalian cell-lines to determine if the presence of certain variants correlated with specific host cell types. Anaplasma marginale colonies differed in their development and appearance in each of the cell lines (P<0.001). Using Western blots probed with two Msp2-monospecific and one Msp2-monoclonal antibodies, we detected expression of variants with differences in molecular weight. Immunofluorescence-assay revealed that specific antibodies bound from 25 to 60% of colonies, depending on the host cell-line (P<0.001). Molecular analysis of cloned variant-encoding genes demonstrated expression of different predominant variants in tick (V1) and mammalian (V2) cell-lines. Analysis of the putative secondary structure of the variants revealed a change in structure when A. marginale was transferred from one cell-type to another, suggesting that the expression of particular Msp2 variants depended on the cell-type (tick or mammalian) in which A. marginale developed. Similarly, analysis of the putative secondary structure of over 200 Msp2 variants from ticks, blood samples, and other mammalian cells available in GenBank showed the predominance of a specific structure during infection of a host type (tick versus blood sample), demonstrating that selection of a possible structure also occurred in vivo. The selection of a specific structure in surface proteins may indicate that Msp2 fulfils an important role in infection and adaptation to diverse host systems. Supplemental Abstract in Spanish (File S1) is provided.
Project description:BACKGROUND: The cattle pathogen, Anaplasma marginale, undergoes a developmental cycle in ticks that begins in gut cells. Transmission to cattle occurs from salivary glands during a second tick feeding. At each site of development two forms of A. marginale (reticulated and dense) occur within a parasitophorous vacuole in the host cell cytoplasm. However, the role of tick genes in pathogen development is unknown. Four genes, found in previous studies to be differentially expressed in Dermacentor variabilis ticks in response to infection with A. marginale, were silenced by RNA interference (RNAi) to determine the effect of silencing on the A. marginale developmental cycle. These four genes encoded for putative glutathione S-transferase (GST), salivary selenoprotein M (SelM), H+ transporting lysosomal vacuolar proton pump (vATPase) and subolesin. RESULTS: The impact of gene knockdown on A. marginale tick infections, both after acquiring infection and after a second transmission feeding, was determined and studied by light microscopy. Silencing of these genes had a different impact on A. marginale development in different tick tissues by affecting infection levels, the densities of colonies containing reticulated or dense forms and tissue morphology. Salivary gland infections were not seen in any of the gene-silenced ticks, raising the question of whether these ticks were able to transmit the pathogen. CONCLUSION: The results of this RNAi and light microscopic analyses of tick tissues infected with A. marginale after the silencing of genes functionally important for pathogen development suggest a role for these molecules during pathogen life cycle in ticks.
Project description:Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.
Project description:BACKGROUND: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus)microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. RESULTS: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. CONCLUSIONS: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.
Project description:The rickettsia Anaplasma marginale is the most prevalent tick-borne livestock pathogen worldwide and is a severe constraint to animal health. A. marginale establishes lifelong persistence in infected ruminants and these animals serve as a reservoir for ticks to acquire and transmit the pathogen. Within the mammalian host, A. marginale generates antigenic variants by changing a surface coat composed of numerous proteins. By sequencing and annotating the complete 1,197,687-bp genome of the St. Maries strain of A. marginale, we show that this surface coat is dominated by two families containing immunodominant proteins: the msp2 superfamily and the msp1 superfamily. Of the 949 annotated coding sequences, just 62 are predicted to be outer membrane proteins, and of these, 49 belong to one of these two superfamilies. The genome contains unusual functional pseudogenes that belong to the msp2 superfamily and play an integral role in surface coat antigenic variation, and are thus distinctly different from pseudogenes described as byproducts of reductive evolution in other Rickettsiales.