Recombinant surface proteomics as a tool to analyze humoral immune responses in bovines infected by Mycoplasma mycoides subsp. mycoides small colony type

Abstract

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.

Fig. 1.

Antibody kinetics. Incubation of protein R816 with four CBPP-positive (▴) and three negative (■) control sera. This example shows that longer incubation times of sera with the bead mixture prior to bead-based assay analysis increase separation between strong, moderate, and weak signals in the analyses. A linear correlation between signal (MFI) and incubation time for sera was observed up to 3 h (R² = 0.89–0.99; please note the log scale on the y axis). In total, 30 proteins were analyzed with similar results.

Fig. 2.

Overview of 242 analyzed sera. Log₂-transformed signals from the analysis of 242 sera (Table I) toward 51 Mycoplasma proteins and the His₆ABP fusion protein per se (ID 58) as a control are displayed as a heat map. Color intensity denotes signal intensity (white, low; orange, medium; red, high). A serum sample dendrogram is displayed on top, an antigen dendrogram is displayed on the left, and protein IDs are on the right. The top colored line indicates sample type as CBPP-affected (red), CBPP-free (blue), uncertain disease status (pale yellow), and serum-free blanks (green). Serum samples clustered into two groups: one lacking or having low antibody levels that bind the recombinant M. mycoides SC proteins and one with medium to high titers for most proteins. Proteins clustered into three major groups, presumably of poor, medium, and high immunogenicity.

Fig. 3.

IgG profiles. A shows IgG binding profiles for 64 recombinant surface proteins and a control (*), displayed as bar charts for two CBPP-positive sera (black bars, 1MUK15A; gray bars, 2MUK15A; top chart), a CBPP-negative serum (black bars) and a serum-free control (gray bars). Whiskers denote S.D. in replicate samples. The mean intensities for the CBPP-positive and -negative sera were 1071 and 53 MFI, respectively. Protein 58 (denoted *) is His₆ABP, the fusion tag present in all proteins. The binding profiles for the CBPP-positive sera were used to select seven recombinant proteins with high and three with low signal intensities for a comparison with Western blot analysis shown in B. Here, binding to selected proteins and the fusion tag as a negative control is shown for both CBPP-positive sera.

Fig. 4.

Time course study. IgG, IgM, and IgA responses to 64 proteins were studied with a series of samples collected over time for eight animals from a vaccine trial as exemplified in A for a T1-44-vaccinated animal. Each line represents signals obtained by one protein at the different time points. All Ig classes show specific protein responses both after vaccination (Vacc.) and following CBPP exposure. In B, protein-specific IgG responses are exemplified by data from an intubated bovine, one vaccinated with T1SR, and an untreated control animal.

Fig. 5.

Protein size. To monitor the influence of protein size on signal intensity, the recombinant proteins were divided into three groups according to molecular mass (containing 32, 19, and 14 proteins, respectively), and their mean IgG responses in all 116 time course sera were summarized as box plots. Here, the bold line represents the median signal, the boxes comprise 50% of the data set, and the whiskers extend to the furthest data point within 1.5 times the box length. Although there is a shift in the median from the first group, no obvious correlation to protein size was found.