Development of effective strategies for the control of JD remains one of the most challenging issues in animal health (15
). Advances in M. avium
genomics and molecular genetics provide an integrated rational approach to solve this problem (1
). In this context, the search for new diagnostic tests can be most effective if combined with novel approaches to vaccination and knowledge of what the test indicates regarding the underlying mechanisms of disease pathogenesis. Targeting of the MAP1152-MAP1156 gene cluster was based on the identification of an attenuating mutation possibly related to the expression of this cluster and a potential role of the encoded proteins as B- or T-cell antigens. An attenuated mutant may serve as a candidate live attenuated vaccine strain and the protein as a subunit vaccine or diagnostic indicator; all relevant aspects are combined into an integrated approach.
In this study, we demonstrated that both MAP1152 and MAP1156 encode reactive B-cell epitopes, a result predicted from the surface localization implied by in silico
analysis (Table ). This finding is also consistent with prior experimental evidence on other PE/PPE proteins. For example, the M. tuberculosis
protein Rv2430c (PE25) was identified as a strong B-cell antigen (12
), and the seroreactivity of MAP3420 c was demonstrated in cattle (36
). In contrast, antigens MAP1518 (Map41; ortholog of Rv1808 [PPE32]) and MAP3184 (Map39; ortholog of Rv3135 [PPE50]), the M. avium
strain 104 MaPE protein, and the cell wall-associated M. tuberculosis
protein Rv3873 (PPE68) serve as T-cell antigens (35
MAP1152 and related PPE proteins MAP1153 and MAP1155 are members of the ancestral PPE sublineage IV (22
). The genes encoding these proteins belong to an ancestral cluster that underwent duplication events from ancestral ESAT-6 clusters but without a concomitant duplication of the ESAT-6 genes and, thus, these paralogs are no longer associated with the ESAT-6 genes. PE/PPE genes are usually organized in operons encompassing at least several PE and/or PPE members (46
). Biochemical evidence indicates that PE and PPE function in pairwise combinations of interacting proteins exposed to the cell surface (18
), with the larger-sized PPE proteins providing a pocket for the PE partner (54
). However, MAP1152 is organized in a cluster devoid of coding sequences for PE proteins. Because the probability of PPE proteins participating in pairwise associations decreases for unlinked PE and PPE genes (46
), the function of MAP1152 may or may not require a PE partner. Instead, MAP1152 may play additional roles in the mechanisms of pathogenesis, as shown for other PE/PPE proteins. For example, M. tuberculosis
transposon mutants of the PE/PPE genes Rv1807 (PPE31), Rv3872 (PPE35), and Rv3873 (PPE68) are attenuated in mice (49
). Likewise, a transposon mutant in the M. avium
PPE gene homologous to Rv1787 (PPE25) displayed impaired growth in macrophages, reduced virulence in mice, and failed to prevent phagosome acidification (26
). Thus, a group of PE and PPE proteins may be necessary for intracellular survival.
MAP1156 is also a B-cell antigen, with stronger reactivity than MAP1152 in Western blot assays, a property also consistent with its reactivity with immune mouse and rabbit sera and inferred surface localization. MAP1156 belongs to the uncharacterized protein family UPF0089. M. tuberculosis
encodes 15 members of this family, which includes proteins with triacylglycerol synthase (TGS) activity (16
). The M. tuberculosis
ORF Rv1425 is the closest homolog to MAP (Table ), but this ORF has been shown not to possess significant TGS activity, at least under the conditions tested. One possible role consistent with a strong humoral reaction is that this ORF “hitchhiked” with M. avium
into a coexpression unit that modulates the immune response. Microorganisms of the M. tuberculosis
complex possess a large number of PE-PGRS proteins that are associated with immunoregulatory roles (4
). However, as indicated above, the M. avium
evolutionary lineage separated prior to the further expansion of the ESAT-6 region V that gave rise to the PE-PGRS and PPE-MPTR sublineage V PE/PPE proteins (22
). Thus, the absence of PE-PGRS proteins may require the recruitment of other proteins, for example, MAP1156, to play this immunomodulatory role. Future experiments will be directed to identify T-cell epitopes in both MAP1152 and MAP1156. These tests are indispensable for determining the major roles of these antigens in immunopathogenesis. For example, it is possible that the PPE protein MAP1152, the weaker B-cell antigen in Western immunoblotting analyses, may elicit a predominant T-cell response early in infection, with MAP1156 exerting a countermodulating B-cell response, most favorable for M. avium
to maintain a chronic infection. This hypothesis is consistent with the increasing MAP1156 seroreactivity observed with serum samples withdrawn as JD progressed from the subclinical to clinical stage (Fig. ). However, a more detailed study of linear and conformational epitopes in these proteins, especially for MAP1152, should be undertaken, as conformational epitopes may not be reactive in Western immunoblot assays.
The B-cell reactivities of both MAP1152 and MAP1156 in Western blot assays and the ELISA with serum samples from M. avium subsp. paratuberculosis-infected cattle are likely associated with private rather than cross-reactive or shared epitopes from related M. avium subsp. paratuberculosis proteins. In this context, the closest paralog to MAP1152 is MAP1518, with 47% identity. Likewise, the closest paralog to MAP1156 is MAP1969c, with 38% identity (see Table S5 in the supplemental material). The low degree of reactivity of MAP1152 in the immunoblot assays against sera from M. bovis-infected animals may be explained in a similar manner, as the closest homolog (Mb1837) shares only 50% identity. In contrast, the low reactivity of MAP1156 against the M. bovis serum, and of MAP1152 and MAP1156 against M. avium hominissuis serum, may be associated with low expression levels in these microorganisms. Otherwise, the corresponding proteins are highly homologous, with 86 to 99% identity (see Table S5) and, thus, likely to be highly cross-reactive, as most epitopes are shared. Moreover, the MAP1152-MAP11156 cluster organization is conserved between M. avium subsp. paratuberculosis and M. avium 104 (a sequenced isolate of M. avium subsp. hominissuis), with both genomes possessing highly homologous genes within this region. However, sequence divergence with M. avium 104 occurs upstream from MAP1152 that could affect gene regulation. Nonetheless, to substantiate these findings for the development of diagnostic tests, it will be necessary to test a larger group of cattle infected with M. bovis, M. avium subspecies, and other environmental species, such as Mycobacterium kansasii.
Interestingly, MAP1152, MAP1156, and the Idexx antigen yielded similar absorbance values against the standardized Idexx positive-control bovine serum included in the Idexx kit, which is from a single naturally infected Holstein cow (Table ). However, the other three seropositive clinical samples reacted more strongly with the Idexx antigen (Fig. ). As indicated above, these results may be due to methodological aspects, as the overall assay optimization was based on the Idexx protocol. Alternatively, or by compounding effects, the increased reactivity in these samples against the Idexx antigen may in part reflect the presence of cross-reactive antibodies against the various M. avium subsp. paratuberculosis proteins present in the Idexx antigen.
In summary, M. avium
-infected cattle mount a humoral response to both MAP1152 and MAP1156. Further research is needed to determine the values of these recombinant proteins as diagnostic capture antigens, subunit vaccines, markers of disease progression, and their suitability for the development of tests for diagnosis of infected from vaccinated animals (DIVA), coupled with the development of live attenuated deletion mutant marker vaccines (13