Abstract
A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. Our laboratories recently showed that different stages of disease were distinguishable via two-dimensional Western blot analyses of Mycobacterium tuberculosis culture filtrate proteins. However, this methodology is not suitable for high throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease states. Techniques were established for separation of native M. tuberculosis cytosol and culture filtrate proteins, resulting in 960 unique protein fractions that were used to generate protein microarrays. Evaluation of serological reactivity from 42 patients in three tuberculosis disease states and healthy purified protein derivative-positive individuals demonstrated that human immunodeficiency virus (HIV)-negative cavitary and noncavitary tuberculosis (TB) patients' sera recognized 126 and 59 fractions, respectively. Sera from HIV patients coinfected with TB recognized 20 fractions of which five overlapped with those recognized by non-HIV TB patients' sera and 15 were unique to the HIV+TB+ disease state. Identification of antigens within the reactive fractions yielded 11 products recognized by both cavitary and noncavitary TB patients' sera and four proteins (HspX, MPT64, PstS1, and TrxC) specific to cavitary TB patients. Moreover four novel B cell antigens (BfrB, LppZ, SodC, and TrxC) of human tuberculosis were identified.
Highlights
A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease
Multidimensional Protein Fractionation Resulted in the Reduced Complexity of Mycobacterial Protein Pools—Recent advances in proteomics have resulted in the ability to separate and identify individual proteins or peptides from complex biological samples
Those fractions containing 500 g of protein were excluded from further separation, and fractions containing between 500 g and 1 mg were pooled with neighboring fractions
Summary
Mtb strain H37Rv was expanded from a 1-ml frozen stock (ϳ108 colony-forming units/ml) to 24 liters of late log culture in glycerolalanine salts medium [17]. The Mtb H37Rv cells (88.9 g, wet weight) were washed three times with PBS (pH 7.4), frozen at Ϫ70 °C, and inactivated with 24 kilograys of ␥-irradiation. Lysis of these cells was achieved by suspending them in 44 ml of TSE buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA) containing 0.06% DNase, 0.06% RNase, 0.07% pepstatin, 0.05% leupeptin, and 20 M PMSF and passing them through a French press five times at 1,500 p.s.i. The resulting lysate was diluted with 1 volume of TSE buffer and centrifuged at 2,000 ϫ g for 5 min to remove unbroken cells. The protein concentrations of the cytosol and CFP were determined with the bicinchoninic acid (BCA) protein assay [20]
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