Mycobacterium Tuberculosis Proteome Microarray for Global Studies of Protein Function and Immunogenicity

Abstract

Poor understanding of the basic biology of Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis, hampers development of much-needed drugs, vaccines, and diagnostic tests. Better experimental tools are needed to expedite investigations of this pathogen at the systems level. Here, we present a functional MTB proteome microarray covering most of the proteome and an ORFome library. We demonstrate the broad applicability of the microarray by investigating global protein-protein interactions, small-molecule-protein binding, and serum biomarker discovery, identifying 59 PknG-interacting proteins, 30 bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding proteins, and 14 MTB proteins that together differentiate between tuberculosis (TB) patients with active disease and recovered individuals. Results suggest that the MTB rhamnose pathway is likely regulated by both the serine/threonine kinase PknG and c-di-GMP. This resource has the potential to generate a greater understanding of key biological processes in the pathogenesis of tuberculosis, possibly leading to more effective therapies for the treatment of this ancient disease.

Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays

“…Rv2031c (hspX) was also previously identified using a Mtb proteomic level microarray with printing of unpurified Mtb proteins generated in E. coli lysate (6). Interestingly, we did not identify shared candidate proteins with an independent recent microarray study that was based on printing proteins generated in a Saccharomyces cerevisiae system (47). Overall, we have validated the value of some previously known serological biomarkers and further identified some new TB biomarker candidates.…”

Latently and uninfected healthcare workers exposed to TB make protective antibodies against Mycobacterium tuberculosis

“…Antibodies from five donors from this study were shipped to TB Healthcare (China), which commercialized the assay. The assay was run as previously described (29). Briefly, the Mtb proteome microarrays were blocked for 1 h at RT with blocking buffer (3% BSA in 1× PBS, 0.1% Tween-20, pH 7.4) in a shaker.…”

“…To determine if the antigen targets of the protective antibodies were proteinaceous in nature, we panned antibodies from three protective donors and two nonprotective donors (24 and 26) against an Mtb proteome chip (29). The five pools of antibodies made multiple detectable binding responses against a variety of recombinantly expressed Mtb antigens (Dataset S1).…”

Advances in Drug Discovery and Development for Pediatric Tuberculosis

“…The field as a whole has a promising outlook with broad chemical diversity being displayed in newly identified lead compounds. Phenotypic screening is by and large the most successful tool in this renaissance of discovery [112, 151, 152], empowered greatly by advances in genomic and structural knowledge of druggable targets [153]. The most common targets identified in TB via typical phenotypic whole-cell screening are Mmpl3, DprE1 and QcrB [150, 154, 155] and it comes as no surprise that many of the advanced drugs under review and undergoing optimization hit these molecular targets.…”