Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenases show strong B cell response and distinguish vaccinated controls from TB patients

Abstract

Proteins released from Mycobacterium tuberculosis (Mtb) during late logarithmic growth phase are often considered candidate components of immunogenic or autolysis markers. One such protein is isocitrate dehydrogenase (ICD), a key regulatory enzyme in the citric acid cycle. We have evaluated the immunogenic properties of two isoforms of Mtb ICD and compared them with the control antigens heat-shock protein 60 and purified protein derivative (PPD). PPD lacks the sensitivity to distinguish between bacillus Calmette-Guérin (BCG)-vaccinated and tuberculosis (TB)-infected populations, and, therefore, epidemiological relevance of PPD in BCG-vaccinated regions is debatable. We show that Mtb ICDs elicit a strong B cell response in TB-infected populations and can differentiate between healthy BCG-vaccinated populations and those with TB. The study population (n = 215) was categorized into different groups, namely, patients with fresh infection (n = 42), relapsed TB cases (n = 32), patients with extrapulmonary TB (n = 35), clinically healthy donors (n = 44), nontuberculous mycobacteria patients (n = 30), and non-TB patients (culture negative for acid-fast bacteria but carrying other infections, n = 32). The Mtb ICDs showed statistically significant antigenic distinction between healthy BCG-vaccinated controls and TB patients (P < 0.0001) and those with other infections. Although extrapulmonary infections could not be discriminated from healthy controls by heat-shock protein 60 (P = 0.2177), interestingly, the Mtb ICDs could significantly (P < 0.0001) do so. Our results highlight the immunodominant, immunosensitive, and immunospecific nature of Mtb ICDs and point to an unusual property of this tricarboxylic acid energy cycle enzyme.

Fig. 1.

Affinity purification of Mtb ICD-1 and Mtb ICD-2. His-tagged recombinant protein was purified by nickel column chromatography under native conditions and stained with Coomassie blue after 10% SDS/PAGE. Lanes: 1 and 2, Mtb ICD-2; M, protein molecular mass markers (200, 116, 97, 66, 45, 31, and 21.5 kDa); 3 and 4, Mtb ICD-1.

Fig. 2.

Mtb ICD-1 and ICD-2 show high B cell reactivity to sera from TB-infected patients from different groups as opposed to bacillus Calmette–Guérin-vaccinated healthy controls. The humoral immune responses directed against the recombinant proteins Mtb ICD-1 (A) and Mtb ICD-2 (B) and control antigens HSP 60 (C) and PPD (D) were compared among different categories of patients and healthy controls. Group 1, fresh infections; Group 2, relapsed infection; Group 3, extrapulmonary TB. The respective sample numbers and P values are shown.

Fig. 3.

Mtb ICDs are more immunogenic than HSP 60. The ELISA reactivity to Mtb ICD-1, Mtb ICD-2, and control antigen HSP 60 was compared in different patient groups. Horizontal bands represent the mean reactivity or average levels of humoral response in each category.

Fig. 4.

Mtb ICDs could significantly distinguish TB-infected sera from NTMs and non-TB patient sera. Recombinant Mtb ICD-1 and Mtb ICD-2 and HSP 60 were tested against sera of NTM (A) and non-TB (B) patients. The respective humoral responses were compared with those for TB-infected sera, the P values for which are shown. HSP 60 could not distinguish TB-infected patients from either NTM or non-TB patients significantly. Horizontal bands represent the mean reactivity in each category.