Extensive Antibody Cross-reactivity among Infectious Gram-negative Bacteria Revealed by Proteome Microarray Analysis

Abstract

Antibodies provide a sensitive indicator of proteins displayed by bacteria during sepsis. Because signals produced by infection are naturally amplified during the antibody response, host immunity can be used to identify biomarkers for proteins that are present at levels currently below detectable limits. We developed a microarray comprising ϳ70% of the 4066 proteins contained within the Yersinia pestis proteome to identify antibody biomarkers distinguishing plague from infections caused by other bacterial pathogens that may initially present similar clinical symptoms. We first examined rabbit antibodies produced against proteomes extracted from Y. pestis, Burkholderia mallei, Burkholderia cepecia, Burkholderia pseudomallei, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella flexneri, and Escherichia coli, all pathogenic Gram-negative bacteria. These antibodies enabled detection of shared cross-reactive proteins, fingerprint proteins common for two or more bacteria, and signature proteins specific to each pathogen. Recognition by rabbit and non-human primate antibodies involved less than 100 of the thousands of proteins present within the Y. pestis proteome. Further antigen binding patterns were revealed that could distinguish plague from anthrax, caused by the Gram-positive bacterium Bacillus anthracis, using sera from acutely infected or convalescent primates. Thus, our results demonstrate potential biomarkers that are either specific to one strain or common to several species of pathogenic bacteria. Molecular & Cellular Proteomics 8:924 -935, 2009.

Characterization of monoclonal antibody's binding kinetics using oblique-incidence reflectivity difference approach

“…However, previous studies demonstrated that many commercially available mAbs cross-react extensively with other cellular antigens, and some may not even recognize their purported targets. [4][5][6][7] To analyze the specificity of antibodies generated against viral, 8 microbial 9,10 and mammalian proteins, 11-14 a protein microarray-based approach has been previously reported to determine potential cross-reactivity. For example, protein microarrays, composed of protein epitope signature tags (PrESTs), have been implemented as part of the Human Protein Atlas projects, one of the several ongoing large-scale efforts to systematically identify high-grade antibodies against much of the human proteome.…”

Cell-free Determination of Binary Complexes That Comprise Extended Protein-Protein Interaction Networks of Yersinia pestis

“…Binary Protein-Protein Interactions-We utilized a protein microarray (18) that comprised Ͼ85% of the predicted Y. pestis proteome to identify protein interaction partners (Fig. 1A).…”

“…1A). The microarrayed proteins were expressed in vitro as recombinant GST-fusions from DNA sequence-confirmed plasmid clones and printed on glass slides that were coated with a thin layer of nitrocellulose, as previously described (18). Because the proteomes of Y. pestis and E. coli share ϳ50% of proteins with Ͼ60% amino acid identity (28, 29), we examined two reported AP-MS data sets of E. coli PPi (6, 7) to select protein probes that were common to both species.…”

“…pestis Proteome Microarrays-The Y. pestis protein microarray was constructed as described previously (18). Briefly, 3968 Y. pestis ORFs cloned into pENTR221 were sequenced, fully characterized, and recombined into the expression vector pEXP7-DEST GST using Gateway cloning methods (Life Technologies, Carlsbad, CA).…”

“…Proteins that passed quality control criteria of quantity, solubility, and stability (3552 proteins out of 4146 total ORFs), representing ϳ85% total proteome coverage, were printed in microarrays on glass slides coated with a thin film of nitrocellulose (Gentel Biosciences, Madison, WI). The microarrays passing additional quality control measures for printing, as previously described (18), were cryo-preserved (Ϫ20°C) until use. Larger quantities of recombinant Y. pestis proteins were produced in E. coli BL21-AI cells (Life Technologies) for probes to determine PPi.…”