SummaryIdentification of the novel PE multigene family was an unexpected finding of the genomic sequencing of Mycobacterium tuberculosis . Presently, the biological role of the PE and PE_PGRS proteins encoded by this unique family of mycobacterial genes remains unknown. In this report, a representative PE_PGRS gene (Rv1818c/ PE_PGRS33 ) was selected to investigate the role of these proteins. Cell fractionation studies and fluorescence analysis of recombinant strains of Mycobacterium smegmatis and M. tuberculosis expressing green fluorescent protein (GFP)-tagged proteins indicated that the Rv1818c gene product localized in the mycobacterial cell wall, mostly at the bacterial cell poles, where it is exposed to the extracellular milieu. Further analysis of this PE_PGRS protein showed that the PE domain is necessary for subcellular localization. In addition, the PGRS domain, but not PE, affects bacterial shape and colony morphology when Rv1818c is overexpressed in M. smegmatis and M. tuberculosis . Taken together, the results indicate that PE_PGRS and PE proteins can be associated with the mycobacterial cell wall and influence cellular structure as well as the formation of mycobacterial colonies. Regulated expression of PE genes could have implications for the survival and pathogenesis of mycobacteria within the human host and in other environmental niches.

The Heparin-Binding Haemagglutinin (HBHA) is a mycobacterial adhesin involved in the dissemination of Mycobacterium tuberculosis from the site of primary infection and a potential candidate for the development of a new vaccine against tuberculosis. Methylation of HBHA is a novel post-translational event that imparts important immunological properties to the protein. Since recombinant HBHA expressed in Escherichia coli is not methylated, we investigated the possibility of producing recombinant methylated HBHA in fast growing mycobacteria for use in immunological and biochemical studies. The complete coding sequence of HBHA was cloned in the plasmid pMV206, under the control of a strong promoter (hsp60) or its own promoter. The constructs generated were electroporated into Mycobacterium smegmatis and the recombinant strains obtained were analyzed for the presence of the HBHA protein using the anti-HBHA monoclonal antibodies D2 and E4. Our results indicate that expression of high amounts of intact protein can be toxic for the mycobacteria, that methylated HBHA can be obtained in M. smegmatis only when using a promoter sequence weaker than hsp60 and that the expression of the complete structural gene is required in order to obtain methylated HBHA. We constructed a recombinant M. smegmatis strain (pMV3-38) that expresses a histidine-tagged methylated HBHA that can be easily purified. The use of fast-growing strains of M. smegmatis to obtain significant amounts of purified HBHA protein within a short timeframe, should be an effective strategy for the evaluation of a new HBHA-based vaccine candidate for tuberculosis.

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Reactivities of human sera against selected recombinant Mycobacterium tuberculosis antigens were assessed by enzyme-linked immunosorbent assay. The results obtained indicate that patients with tuberculosis (TB) do not develop a strong humoral response against PE_PGRS and PPE proteins or against the Ag85B and heparin-binding hemagglutinin (HBHA) recombinant antigens. Conversely, purified methylated HBHA was strongly recognized by sera obtained from TB patients compared to controls.Tuberculosis (TB) is a major infectious disease that kills almost 2 million people every year, mostly in developing countries, where it poses a major health, social, and economic burden (16). The development of improved, clinically sensitive, rapid, and economical diagnostic tests may provide a powerful tool to better control the epidemic. Recently, PCR-based methods and automatic culture systems have been made available, and these methods are in extensive regular use in laboratories in developed countries (1, 3). Moreover, these diagnostic systems are not suited for field use. The idea to develop a test for the diagnosis of TB through a serological assay has been pursued for many decades, but the results so far have been poor. Many of the antigens did not have an adequate sensitivity or specificity, and these assays could not properly distinguish Mycobacterium bovis BCG-vaccinated and purified protein derivative (PPD)-positive individuals from those with active TB.Recently, it has been demonstrated that several mycobacterial proteins undergo a process of posttranslational modification in mycobacteria that provides important immunological properties (15). Among these proteins is heparin-binding hemagglutinin (HBHA), which undergoes a process of methylation involving the lysine residues present at the C terminus (13). Since the recombinant proteins obtained in Escherichia coli cannot be properly processed, the use of these antigens in serological assays is precluded by the cumbersome procedures required to purify the native antigens. Recently, a rapid and effective system for the purification of methylated HBHA has been developed, and the use of these proteins in such assays is now feasible (5).In this study, the humoral responses developed by human subjects against selected Mycobacterium tuberculosis antigens were evaluated to assess the potential use of some of these antigens in a serodiagnostic test to discriminate between infected healthy subjects and TB patients.A total number of 179 sera were analyzed: 52 sera were obtained from PPD-negative individuals, 38 sera were obtained from PPD-positive healthy subjects (BCG-immunized and M. tuberculosis-infected persons), and 111 sera were collected from patients with active TB. Both controls and samples from TB patients were obtained from the Clinic of Infectious Diseases, Institute of Tisiology, University of Sassari, and the Institute of Tisiology A.S.L. no. 3 of Nuoro, Italy.Recombinant histidine-tagged proteins were purified by nickel chromatography as indicated previously (6). The rec...