Micmbiology (1 998), 144,1197-1 203 Printed in Great Britain Characterization of novel irnrnunodorninant antigens of Mycobacterium tuberculosis Rama Rao Amara, S. Shanti and Vijaya Satchidanandam Author for correspondence: Vijaya Satchidanandam. Tel : + 91 80 3092685. Fax : + 91 80 3341683 or 3444697. e-mail : vijaya@cge.iisc.ernet.in Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 56001 2, India Seven novel antigens of Mycobacterium tubersulosis, which had previously been identified based on reactivity to sera from patients with tuberculosis, were characterized. Nucleotide sequence analysis of the genes encoding these seven antigens identified one of them as the FtsH and a second as the aminoimidazole ribotide synthase of M. tuberculosis. Antisera raised to the recombinant forms of each of these seven antigens were used to study the distribution of these proteins within mycobacterial species as well as to determine their subcellular localization and hydrophobicity. Four of the seven antigens were conserved only among pathogenic strains of mycobacteria. Of the seven proteins studied, FtsH and a second protein of unknown identity were localized in membranes. Two were cytosolic, while two others, which had a high proline content, were tightly associated with the cell wall. One protein was secreted. This secreted protein could be identified by serum from a majority of tuberculosis patients but not BCG-vaccinatedindividuals, suggesting its potential use in the immunodiagnosis of tuberculosis. Keywords : M. tuberculosis, tuberculosis patient sera, immunodiagnosis, FtsH INTRODUCTION Owing to the resurgence of tuberculosis, especially of multi-drug resistant (MDR) strains, there is today a greater need than ever before to identify antigens of Mycobacterium tuberculosis that can serve as im- munodiagnostic agents and/or vaccine candidates. Sev- eral reports documenting the unique capability of live mycobacteria to protect mice from a challenge dose of the organism (Mackaness, 1967; Orme, 1988; Orme & Collins, 1984; Rook et al., 1986) have suggested that bacteria actively replicating in vivo synthesize a set of antigens capable of eliciting protective immunity. Se- creted antigens have been implicated as dominant members of this class by several investigators, an assumption largely validated by the impressive ability of purified culture filtrate antigens to protect mice and guinea pigs against a challenge with live virulent mycobacteria (Andersen, 1994; Horwitz et al., 1995; Pal & Horwitz, 1992). To enhance the probability of identifying protective antigens unique to living myco- bacteria, we screened a genomic DNA expression library of M. tuberculosis with sera from tuberculosis (TB) patients in advanced stage of the disease (Amara & Satchidanandam, 1996). Preliminary nucleotide se- quence analysis revealed that the majority of these antigens were encoded by hitherto unreported genes of the organism. Interestingly, several of these antigens were able to elicit antibodies only when derived from live bacilli, but not when sonicated preparations of M. tuberculosis, which contained significant quantities of these same antigens (Amara & Satchidanandam, 1997), were used to immunize mice and guinea pigs. In this communication we report further characterization of seven of these differentially immunogenic proteins of M. tuberculosis with respect to their subcellular locali- zation, hydrophobicity, distribution among myco- bacterial species and reactivity to sera from individual TB patients. The significance of these findings is discussed. METHODS Mycobacterial strains. M. tuberculosis strain NTI-83949 was isolated from the sputum of a patient with pulmonary tuberculosis and identified as reported previously (Amara & Satchidanandam, 1996). All other mycobacterial strains were obtained from the Mycobacterial Repository Centre, Central Jalma Institute for Leprosy, Agra, India. Mycobacteria were grown in Middlebrook 7H9 medium supplemented with 05 ' / o albumin and 0.75 '/o dextrose. .............................. , .................................................................................... , ............ , ................ Abbreviation : TB, tuberculosis. Serum samples. Serum samples were obtained from inmates of the Shantabai Devarao Shivaram Tuberculosis and Chest ~ 0002-2222 0 1998 SGM 1197