Anaplasma marginale type IV secretion system proteins VirB2, VirB7, VirB11, and VirD4 are immunogenic components of a protective bacterial membrane vaccine

Abstract

Anaplasma and related Ehrlichia spp. are important tick-borne, Gram-negative bacterial pathogens of livestock and humans that cause acute infection and disease and can persist. Immunization of cattle with an Anaplasma marginale fraction enriched in outer membranes (OM) can provide complete protection against disease and persistent infection. Serological responses of OM vaccinees to the OM proteome previously identified over 20 antigenic proteins, including three type IV secretion system (T4SS) proteins, VirB9-1, VirB9-2, and VirB10. Subsequent studies showed that these three proteins also stimulated CD4(+) T-cell responses in OM vaccinees. The T4SS, composed of a complex of proteins spanning the inner and outer membranes of certain bacteria, is an important virulence factor but is relatively unexplored as a vaccine target. The goal of this study was to determine if additional T4SS proteins are immunogenic for animals immunized with the protective OM fraction of A. marginale. T4SS proteins expressed by in vitro transcription and translation were screened for stimulating proliferation of T cells from OM vaccinees, and immunogenic proteins were expressed as recombinant proteins in Escherichia coli and their immunogenicity was verified. VirB2, a putative VirB7, VirB11, and VirD4 were immunogenic for OM vaccinees expressing several common major histocompatibility complex (MHC) class II haplotypes. VirB2 is encoded by multiple genes that share a conserved central region, and epitope mapping revealed T-cell epitopes in this region. The discovery of novel immunogenic T4SS proteins recognized by outbred individuals with common MHC haplotypes further justifies evaluating the T4SS as a potential vaccine candidate for pathogenic bacteria.

FIG. 1.

Expression and relative purity of the T4SS recombinant proteins. Proteins were separated using SDS-PAGE and stained with Coomassie blue (A) or transferred to nitrocellulose membranes and probed with anti-FLAG MAb (B) or anti-6× His MAb (C), and bands were detected by chemiluminescence. Wells received 1.5 μg purified VirB2 (lane 1), VirB7 (lane 2), VirB11 (lane 3), VirD4F1 (lane 4), VirD4F2 (lane 5), or VirD4 (lane 6) or 2 μg MSA1 (lane 7). VirB2 and VirB7 are ∼19 kDa, VirB11 is ∼45 kDa, VirD4F1 is ∼39 kDa, VirD4F2 and MSA1 are ∼55 kDa, and VirD4 is ∼100 kDa, as indicated by asterisks.

FIG. 2.

Short-term T-cell lines established from OM vaccinees proliferate in response to recombinant T4SS proteins. T cells from cattle 04B90, 04B91, 04B92, 4848, and 5982 were tested for proliferation to 1 (white bars) and 10 (black bars) μg/ml VirB9-1, VirB9-2, and VirB10 (A) or VirD4F1, VirD4F2, VirB11, and VirB2 (B). Responses are presented as the SI compared to negative control MSA1 ± 1 SD for triplicate cultures. Responses significantly greater than those for the same concentration of MSA1 are indicated by asterisks.

FIG. 3.

Alignment of a putative A. marginale VirB7 with VirB7 homologs from other bacteria. The A. marginale strain St. Maries (AM_StM) putative VirB7 (AM306) amino acid sequence was aligned with Com7 from C. jejuni strain 81-176 (CJ_81-176) and H. pylori strain Shi170 (HP_Shi170) and with VirB7 from Agrobacterium tumefaciens strain Bo542 (AT_Bo542). Alignment was performed with ClustalW (AlignX) and was followed by manual editing. Residues identical in three or more sequences are shown as white text on a black background, while conserved residues are shown as black text on a gray background.

FIG. 4.

Alignment of Anaplasmataceae VirB2 predicted amino acid sequences. ClustalW alignment of 12 VirB2 sequences from A. marginale (AM), six (of eight) sequences from A. phagocytophilum (APH), and representative sequences from Ehrlichia canis (ECAJ), E. chaffeensis (ECH), E. ruminantium (Erum), Wolbachia pipientis wMel (WD), and Neorickettsia sennetsu (NSE) are shown. Identical residues in all sequences are shown as white text on a black background, while blocks of identical residues are shown as black text on a gray background. Conserved cysteine residues are indicated with an asterisk. The positions of A. marginale peptides 1 through 6 designed against the AM030 sequence are indicated as numbered horizontal lines above the sequences.

FIG. 5.

T-cell epitopes in VirB2 localize to the central conserved region. T-cells from animals 04B90, 04B92, 4848, and 5982 were tested for proliferation to 1 (white bars) and 10 (black bars) μg/ml of VirB2 and overlapping peptides. Responses are represented as the SI in relation to medium. Responses to antigen that are significantly greater than those to the medium or negative control MSA1 (for VirB2) or RAP-1 P1 (for VirB2 peptides) are indicated by asterisks.

FIG. 6.

RT-PCR analysis of VirB2, VirB7, VirB11, and VirD4. RT-PCR was performed using RNA derived from A. marginale St. Maries strain-infected red blood cells to amplify T4SS cDNAs. PCR products for VirB2 (lanes 1 and 2), VirB7 (lanes 3 and 4), VirB11F1 (lanes 5 and 6), and VirD4F2 (lanes 7 and 8), performed without (lanes 1, 3, 5, and 7) or with (lanes 2, 4, 6, and 8) RT, are shown. L represents the 1-kb ladder. All RT-PCR products failed to produce bands and served as negative controls.