Faculty Bibliography

Human tuberculosis (TB) is caused by the bacillus Mycobacterium tuberculosis, a subspecies of the M. tuberculosis complex (MTC) of mycobacteria. Postgenomic dissection of the M. tuberculosis proteome is ongoing and critical to furthering our understanding of factors mediating M. tuberculosis pathobiology. Towards this end, a 32-kDa putative glyoxalase in the culture filtrate (CF) of growing M. tuberculosis (originally annotated as Rv0577 and hereafter designated CFP32) was identified, cloned, and characterized. The cfp32 gene is MTC restricted, and the gene product is expressed ex vivo as determined by the respective Southern and Western blot testing of an assortment of mycobacteria. Moreover, the cfp32 gene sequence is conserved within the MTC, as no polymorphisms were found in the tested cfp32 PCR products upon sequence analysis. Western blotting of M. tuberculosis subcellular fractions localized CFP32 predominantly to the CF and cytosolic compartments. Data to support the in vivo expression of CFP32 were provided by the serum recognition of recombinant CFP32 in 32% of TB patients by enzyme-linked immunosorbent assay (ELISA) as well as the direct detection of CFP32 by ELISA in the induced sputum samples from 56% of pulmonary TB patients. Of greatest interest was the observation that, per sample, sputum CFP32 levels (a potential indicator of increasing bacterial burden) correlated with levels of expression in sputum of interleukin-10 (an immunosuppressive cytokine and a putative contributing factor to disease progression) but not levels of gamma interferon (a key cytokine in the protective immune response in TB), as measured by ELISA. Combined, these data suggest that CFP32 serves a necessary biological function(s) in tubercle bacilli and may contribute to the M. tuberculosis pathogenic mechanism. Overall, CFP32 is an attractive target for drug and vaccine design as well as new diagnostic strategies

Efforts to devise immunoassays for tuberculosis (TB) that can be adapted to rapid formats are ongoing. The present study was aimed at determining whether urinary anti-Mycobacterium tuberculosis antibodies are present in patients with TB, to evaluate the feasibility of developing a urine antibody-based diagnostic test. Urinary antibodies directed against the culture filtrate proteins of M. tuberculosis, MPT 32, and the 81-kDa GlcB protein were detectable in patients with TB, although the sensitivity of antibody detection was lower (53%-64%), compared with serum antibodies (68%-77%). Surprisingly, with all 3 antigens, the use of paired serum and urine samples provided higher sensitivities of antibody detection than either single specimen, and anti-GlcB antibodies were present in the serum and/or urine of 39 (90%) of 43 smear-positive patients with TB. Although, with the current methods and antigens, the level of sensitivity is insufficient to design a urinary antibody diagnostic test, these studies provide the foundation for further studies on the development of a urine antibody-based immunoassay for TB

Arabinomannan (AM) is a polysaccharide antigen of the mycobacterial capsule. However, it is uncertain whether AM constitutes an immunologically distinct fraction of Mycobacterium tuberculosis. In this study, we analyzed the repertoire and specificity of antibodies to AM by using AM-binding murine monoclonal antibodies (MAbs) and human serum samples. Murine MAbs were found to be diverse in their specificity to AM and cross-reactivity with other arabinose-containing mycobacterial polysaccharides, with MAb 9d8 binding exclusively to AM. Human antibodies to AM were detected in serum samples from patients with pulmonary tuberculosis (TB), as well as in those from healthy, purified protein derivative-negative controls, with significantly higher titers among patients. The binding of human antibodies to AM was inhibited by MAb 9d8 in three patients with TB but not in controls. MAb 5c11, which recognizes other mycobacterial arabinose-containing carbohydrates in addition to AM, inhibited the binding of serum samples from 75% of patients and 76% of controls. Analysis of human antibodies with murine MAbs to human V(H) determinants demonstrated diversity among antibodies to AM with qualitative and quantitative differences compared with antibodies to lipoarabinomannan. In summary, our study suggests that antibodies to AM are diverse and heterogeneous with respect to antigen recognition and V(H) determinant expression, with human serum samples containing different subsets of antibodies to AM with the specificities of AM-binding murine MAbs. One MAb and a subset of human antibodies bind AM specifically, suggesting that this polysaccharide is antigenically distinct and is expressed in human infection

Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. It is not clear how M. tuberculosis avoids the destructive action of macrophages, but this ability is fundamental in the pathogenicity of tuberculosis. A gene previously identified in M. tuberculosis, designated eis, was found to enhance intracellular survival of Mycobacterium smegmatis in the human macrophage-like cell line U-937 (J. Wei et al., J. Bacteriol. 182:377-384, 2000). When eis was introduced into M. smegmatis on a multicopy vector, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the appearance of a unique 42-kDa protein band corresponding to the predicted molecular weight of the eis gene product. This band was electroeluted from the gel with a purity of >90% and subjected to N-terminal amino acid sequencing, which demonstrated that the 42-kDa band was indeed the protein product of eis. The Eis protein produced by M. tuberculosis H37Ra had an identical N-terminal amino acid sequence. A synthetic polypeptide corresponding to a carboxyl-terminal region of the deduced eis protein sequence was used to generate affinity-purified rabbit polyclonal antibodies that reacted with the 42-kDa protein in Western blot analysis. Hydropathy profile analysis showed the Eis protein to be predominantly hydrophilic with a potential hydrophobic amino terminus. Phase separation of M. tuberculosis H37Ra lysates by the nonionic detergent Triton X-114 revealed the Eis protein in both the aqueous and detergent phases. After fractionation of M. tuberculosis by differential centrifugation, Eis protein appeared mainly in the cytoplasmic fraction but also in the membrane, cell wall, and culture supernatant fractions as well. Forty percent of the sera from pulmonary tuberculosis patients tested for anti-Eis antibody gave positive reactions in Western blot analysis. Although the function of Eis remains unknown, evidence presented here suggests it associates with the cell surface and is released into the culture medium. It is produced during human tuberculosis infection and therefore may be an important M. tuberculosis immunogen.

The goals of the present study were twofold: (i) to compare the repertoires of antigens in culture filtrates of in vitro-grown Mycobacterium tuberculosis that are recognized by antibodies from noncavitary and cavitary tuberculosis (TB) patients and (ii) to determine the extent of variation that exists between the antigen profiles recognized by individual TB patients. Lipoarabinomannan-free culture filtrate proteins of M. tuberculosis were fractionated by one-dimensional (1-D) and 2-D polyacrylamide gel electrophoresis, and the Western blots were probed with sera from non-human immunodeficiency virus (non-HIV)-infected cavitary and noncavitary TB patients and from HIV-infected, noncavitary TB patients. In contrast to earlier studies based on recombinant antigens of M. tuberculosis which suggested that antibody responses in TB patients were heterogeneous (K. Lyashchenko et al., 1998, Infect. Immun. 66:3936-3940, 1998), our studies with native culture filtrate proteins show that the antibody responses in TB patients show significant homogeneity in being directed against a well-defined subset of antigens. Thus, there is a well-defined subset of culture filtrate antigens that elicits antibodies during noncavitary and cavitary disease. In addition, another set of antigens is recognized primarily by cavitary TB patients. The mapping with individual patient sera presented here suggests that serodiagnostic tests based on the subset of antigens recognized during both noncavitary and cavitary TB will enhance the sensitivity of antibody detection in TB patients, especially in difficult-to-diagnose, smear-negative, noncavitary TB patients

Four antigens of Mycobacterium tuberculosis that are expressed in vivo after aerosol infection but prior to the development of clinical tuberculosis (TB) in rabbits were identified by immunoscreening of an expression library of M. tuberculosis genomic DNA with sera obtained 5 weeks postinfection. Three of the proteins identified, PirG (Rv3810), polymorphic GC-repetitive sequence (PE-PGRS; Rv3367), and proline-threonine repetitive protein (PTRP) (Rv0538), have multiple tandem repeats of unique amino acid sequences and have characteristics of surface or secreted proteins. The fourth protein, MtrA (Rv3246c), is a response regulator of a putative two-component signal transduction system, mtrA-mtrB, of M. tuberculosis. All four antigens were recognized by pooled sera from TB patients and not from healthy controls, confirming their in vivo expression during active infection in humans. Three of the antigens (PE-PGRS, PTRP, and MtrA) were also recognized by retrospective preclinical TB sera obtained, prior to the clinical manifestation of TB, from human immunodeficiency virus-TB patients, suggesting that they are potential candidates for devising diagnostic tests for active, preclinical TB

OBJECTIVE: The biological phenotype of HIV-1 has been associated with various aspects of its infectivity, including syncytium formation and coreceptor usage. Adhesion molecules, present on both the target cell and the virus, have also been shown to play a role in the infectious process. A possible correlation between the presence of adhesion molecules in the envelope of HIV-1 with the biological phenotype of the virus is examined. DESIGN: The envelopes of 56 isolates of HIV-1 of known biological phenotype were analyzed for the presence of lymphocyte function-related molecule 1 (LFA-1) and major histocompatibility complex (MHC) class II molecules. METHODS: The coreceptor usage of each isolate was determined in a GHOST cell or a U87.CD4 infectivity assay. The presence of LFA-1 and MHC class II in each virus envelope was then determined using a virus-binding enzyme-linked immunosorbent assay (ELISA). RESULTS: Viruses using the chemokine receptor CCR5 have relatively higher levels of MHC class II than LFA-1 in their envelopes compared with those using CXCR4. CONCLUSIONS: The finding that there is a differential incorporation of MHC class II and LFA-1 molecules by CXCR4- and CCR5-using viruses augments the list of properties contributing to the biological phenotype of HIV-1. This may explain, in part, how CXCR4-using viruses are able to bind to and infect a broader range of cell types than CCR5-using viruses, and why CXCR4-using viruses are associated with a more aggressive disease course

Our studies of the humoral responses of tuberculosis (TB) patients have defined the repertoire of culture filtrate antigens of Mycobacterium tuberculosis that are recognized by antibodies from cavitary and noncavitary TB patients and demonstrated that the profile of antigens recognized changes with disease progression (K. Samanich et al., J. Infect. Dis. 178:1534-1538, 1998). We have identified several antigens with strong serodiagnostic potential. In the present study we have evaluated the reactivity of cohorts of human immunodeficiency virus (HIV)-negative, smear-positive; HIV-negative, smear-negative; and HIV-infected TB patients, with three of the candidate antigens, an 88-kDa protein, antigen (Ag) 85C, and MPT32, and compared the reactivity of the same patient cohort with the 38-kDa antigen and Ag 85A. We have also compared the reactivity of native Ag 85C and MPT32 with their recombinant counterparts. The evaluation of the reactivity was done by a modified enzyme-linked immunosorbent assay described earlier (S. Laal et al., Clin. Diag. Lab. Immunol. 4:49-56, 1997), in which all sera are preadsorbed against Escherichia coli lysates to reduce the levels of cross-reactive antibodies. Our results demonstrate that (i) antigens identified on the basis of their reactivity with TB patients' sera provide high sensitivities for serodiagnosis, (ii) recombinant Ag 85C and MPT32, expressed in E. coli, show reduced reactivity with human TB sera, and (iii) of the panel of antigens tested, the 88-kDa protein is the most promising candidate for serodiagnosis of TB in HIV-infected individuals. Moreover, these results reaffirm that both the extent of the disease and the bacterial load may play a role in determining the antigen profile recognized by antibodies

Both HIV-1 primary isolates and laboratory strains incorporate cell-derived molecules into their envelopes depending on the host cell in which they are grown. This incorporation is not random and, specifically, HIV-1 has been shown to select against the incorporation into its surface of CD4, its main receptor. In this study, we have looked at the incorporation of HIV coreceptors CXCR4, CCR5, and CCR3 into the HIV envelope. For this purpose, we grew HIV-1 primary isolate BZ167 in several cell lines and PBMCs, and the envelope profiles of the resulting viruses were determined with a virus-binding ELISA. While the virus particle gained several molecules when passed through the different cell lines (e.g., ICAM-3, LFA-1, ICAM-1, or MHC class II), BZ167 never incorporated significant levels of CXCR4, CCR5, or CCR3 into its envelope even though some or all of the cell lines in which it was grown expressed them. These results show that HIV-1 selects against the incorporation of these chemokine receptors into its envelope molecule, as it does against the incorporation of CD4

This study was undertaken to define the antigens in culture filtrates of actively replicating Mycobacterium tuberculosis that are recognized by antibodies from tuberculosis (TB) patients. Two-dimensional Western blots were probed with sera from healthy controls and TB patients that were preabsorbed with Escherichia coli lysates to deplete cross-reactive antibodies. Antibodies from TB patients recognized 26 of the >100 culture filtrate proteins, and the repertoire changed with disease progression. Only 12 of 26 antigens, including 3 proteins implicated in colonization and invasion by mycobacteria (MPT51, MPT32, and 85C), and 9 (as yet undefined proteins) were reactive with sera from TB patients with early noncavitary or cavitary disease. Eight additional antigens, including 4 undefined proteins, were recognized only by sera from a subset of patients with advanced cavitary disease. Studies suggest that 3 of the antigens recognized by sera from patients with early TB (85C, MPT32, and a 88-kDa protein) have strong serodiagnostic potential