Negligible Genetic Diversity of Mycobacterium tuberculosis Host Immune System Protein Targets: Evidence of Limited Selective Pressure

Corresponding author: James M. Musser, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th St., Hamilton, MT 59840., jmusser{at}niaid.nih.gov (E-mail)

Communicating editor: Y.-X. FU

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A common theme in medical microbiology is that the amount of amino acid sequence variation in proteins that are targets of the host immune system greatly exceeds that found in metabolic enzymes or other housekeeping proteins. Twenty-four Mycobacterium tuberculosis genes coding for targets of the host immune system were sequenced in 16 strains representing the breadth of genomic diversity in the species. Of the 24 genes, 19 were invariant and only six polymorphic nucleotide sites were identified in the 5 genes that did have variation. The results document the highly unusual circumstance that prominent M. tuberculosis antigenic proteins have negligible structural variation worldwide. The data are best explained by a combination of three factors: (i) evolutionarily recent global dissemination in humans, (ii) lengthy intracellular quiescence, and (iii) active replication in relatively few fully immunocompetent hosts. The very low level of amino acid diversity in antigenic proteins may be cause for optimism in the difficult fight to control global tuberculosis.

IT is conventional wisdom in medical microbiology that pathogen extracellular and surface proteins involved in interaction with the host immune system and other variable environmental factors are highly polymorphic relative to metabolic enzymes or other "house-keeping" proteins (SELANDER et al. 1994 ; LI et al. 1995 ; MATHIESEN et al. 1997 ; YAMAGUCHI and GOJOBORI 1997 ; RICH et al. 1998 ; STOCKBAUER et al. 1998 ). Extensive study of polymorphisms in diverse viral, prokaryotic, and eukaryotic pathogens, including those affecting plants and invertebrate and vertebrate animals, have shown this to be the case. The increased variability of surface and extracellular proteins is usually attributed to antigenic diversity to escape host immune functions.

Sequence analysis of 26 structural genes in 842 strains (2 Mb) of the important human pathogen Mycobacterium tuberculosis identified a striking reduction of silent nucleotide substitutions and unselected amino acid replacements compared with other microbes (SREEVATSAN et al. 1997 ). The lack of variation in structural genes indicated that M. tuberculosis is evolutionarily young and has recently spread globally, perhaps as recently as 20,000 years ago (KAPUR et al. 1994 ). Although many of the genes encoded metabolic enzymes, the analysis included genes for a 16-kD antigen, a 65-kD heat-shock protein, and catalase-peroxidase, three proteins that are targets of the host immune response (ANDERSEN 1994 ; ANDERSEN and BRENNAN 1994 ; LYASHCHENKO et al. 1998 ). The paucity of naturally occurring amino acid polymorphisms in these antigens suggested that variation in proteins that interact with the host immune system was uncommon. Inasmuch as a general lack of variation in M. tuberculosis antigens would be unprecedented and have important implications for vaccine, therapeutics, and evolutionary biology research, we investigated this issue in detail.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Bacterial strains:
All M. tuberculosis isolates used in this study were obtained from the culture collection of J. M. Musser (SREEVATSAN et al. 1997 ). A core group of 16 strains (Table 1) was used for initial sequencing of all target genes (Table 2). Although 12 of the 16 organisms were isolated from ethnically diverse patients in Texas, all three principal genetic groups of M. tuberculosis were represented. The three principal genetic groups were identified on the basis of the combinations of polymorphisms located in codon 463 of the katG gene encoding catalase-peroxidase and codon 95 of gyrA encoding the A subunit of DNA gyrase (SREEVATSAN et al. 1997 ). In addition, the 16 strains have from 4 to 20 copies of IS6110 and a diverse array of spoligotypes (Fig 1; GROENEN et al. 1993 ; VAN EMBDEN et al. 1993 ). Diversity in spoligotype and IS6110 copy number and location in the chromosome have been used extensively as indicators of overall genomic differentiation in M. tuberculosis (GROENEN et al. 1993 ; VAN EMBDEN et al. 1993 ). On the basis of molecular analysis of >5000 strains from global sources, the sample represents the breadth of genomic diversity in M. tuberculosis strict sense, although it may not fully represent all minor branches of the phylogenetic tree. The core sample of 16 strains was supplemented when necessary with additional M. tuberculosis isolates recovered from global sources and maintained in the culture collection of J. M. Musser.

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Spoligotype patterns identified among the 16 M. tuberculosis isolates studied. The arbitrary spoligotype pattern designations (SOINI et al. 2000 ) are shown at left. Solid squares, hybridization with the designated spacer probe; open squares, lack of hybridization.

Selection of genes encoding antigens and culture supernatant proteins:
Extensive study of the host cellular and humoral immune response and characterization of culture supernatants of organisms grown in vitro has identified a relatively large number of proteins (ANDERSEN 1994 ; ANDERSEN and BRENNAN 1994 ; LYASHCHENKO et al. 1998 ). Although not all genes encoding antigenic or supernatant proteins were sequenced in this study, those chosen for analysis represent a random sample of proteins described in the M. tuberculosis literature.

Selection of open reading frames (ORFs) encoding members of the Pro-Glu (PE) and Pro-Pro-Glu (PPE) families:
The H37Rv genome (COLE et al. 1998 ) contains two large families of genes encoding proteins with PE (n = 99) and PPE (n = 68) motifs. Four genes of each class were selected for sequence analysis on the basis of being located in positions arrayed around the M. tuberculosis chromosome. In addition, one member of the PPE gene family (Rv3135) was selected for analysis because alignment of the sequences available for H37Rv and CSU93 (as of June 1999) suggested the gene was variable in size among M. tuberculosis isolates.

DNA sequence analysis and PCR size variation in the Rv3135 gene:
Automated DNA sequencing methods using genomic DNA samples and an Applied Biosystems (Foster City, CA) model 377 instrument have been described previously (SREEVATSAN et al. 1997 ). The sequence data were assembled and edited using EDITSEQ and MEGALIGN programs (DNASTAR, Madison, WI) and the sequences were compared with published data (COLE et al. 1998 ).

Size variation in the Rv3135 gene was studied by PCR with the following primers: forward, 5'-TCGACTGCCATACAACCTG-3' and reverse, 5'-GTGCTGGTCGAGAACTGAATG-3', located 210 bp upstream from the Rv3135 start site and 23 bp downstream from the stop codon, respectively. These primers amplify a 632-bp product from strain H37Rv.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Invariant antigen genes:
Of the 24 genes encoding proteins known to be targets of the host immune system, 19 were invariant in the core group of 16 M. tuberculosis isolates sequenced. The 19 invariant genes encoded the following proteins: 10-kD culture filtrate protein (CFP10) (BERTHET et al. 1998 ); ESAT-6 antigen (SORENSEN et al. 1995 ; RAVN et al. 1999 ); 14-kD antigen (MATTHEWS et al. 1985 ; ENGERS et al. 1986 ; JACKETT et al. 1988 ; VERBON et al. 1992 ); MPT63 (16-kD antigen) (HORWITZ et al. 1995 ; LEE and HORWITZ 1995 , LEE and HORWITZ 1999 ); MPT83 (HEWINSON et al. 1996 ; WIKER et al. 1998 ); 38-kD lipoprotein antigen (ANDERSEN and HANSEN 1989 ; ESPITIA et al. 1992 ; HARBOE and WIKER 1992 ); 8.4-kD antigen (COLER et al. 1998 ); CFP17, CFP21, CFP22, CFP25, and CFP29 (ROSENKRANDS et al. 1998 ; WELDINGH et al. 1998 ); MTC28 (28-kD antigen) (MANCA et al. 1997 ); superoxide dismutase (23-kD antigen) (YOUNG et al. 1985 ; ZHANG et al. 1991 ; KANG et al. 1998 ); antigen 85A (HAVLIR et al. 1991 ; WIKER and HARBOE 1992 ; HORWITZ et al. 1995 ; GARBE et al. 1996 ; HUYGEN et al. 1996 ; BELISLE et al. 1997 ; LOZES et al. 1997 ; JACKSON et al. 1999 ); MPT70 antigen (MATSUMOTO et al. 1995 ); 35-kD antigen (BAIRD et al. 1988 ; BILLMAN-JACOBE et al. 1990 ); GroES (10-kD antigen) (COHEN et al. 1987 ); and heparin-binding hemagglutinin (MENOZZI et al. 1996 , MENOZZI et al. 1998 ; DELOGU and BRENNAN 1999 ).

Variable antigen genes:
The remaining five antigen-encoding genes (discussed in detail below) had nucleotide polymorphisms in the 16 M. tuberculosis core isolates. One of the nucleotide changes was silent (synonymous substitution), and six would result in amino acid replacements (nonsynonymous substitutions).

45/47-kD secreted antigen complex (MPT32): The 45/47-kD secreted antigen complex is present in culture filtrates of virulent M. tuberculosis isolates (LAQUEYRERIE et al. 1995 ). The antigen complex was initially purified and characterized because of its ability to react with antibodies present in the sera of guinea pigs immunized with live M. tuberculosis (LAQUEYRERIE et al. 1995 ). DIAGBOUGA et al. 1997 reported that 40% of sera obtained from smear-positive tuberculosis patients in Burkina Faso reacted with the 45/47-kD antigen complex. We identified one nonsynonymous substitution that resulted in a Phe136Leu amino acid replacement in all principal genetic group 1 isolates and virtually all group 2 organisms. All genetic group 3 M. tuberculosis had Phe136.

19-kD antigen: The 19-kD antigen is a highly expressed surface-associated glycolipoprotein that is a dominant antigen in infected humans (ANDERSEN and BRENNAN 1994 ; HARRIS et al. 1994 ; LATHIGRA et al. 1996 ; ERB et al. 1998 ; MAHENTHIRALINGAM et al. 1998 ). Its role in human-pathogen interactions is not known. LATHIGRA et al. 1996 suggested that the protein is a virulence factor, but inactivation of the homologous Mycobacterium intracellulare gene failed to demonstrate an essential role in growth and virulence in BALB/c mice (MAHENTHIRALINGAM et al. 1998 ). Our analysis identified two amino acid replacements, Gly115Arg and Cys158Ser, both occurring in a distinct phylogenetic lineage of principal genetic group 2 organisms. The Cys158Ser replacement was found in strain HN1489; both the Gly115Arg and Cys158Ser amino acid changes were identified in strain HN1426. The Cys158Ser amino acid residue is located in a region of the molecule containing a mouse linear B-cell epitope (HARRIS et al. 1994 ). However, neither polymorphism is located in a region known to be recognized by human sera or in T-cell epitopes (LATHIGRA et al. 1996 ; ERB et al. 1998 ). Sequence analysis of the 19-kD antigen gene in 47 additional principal genetic group 2 M. tuberculosis strains confirmed that these amino acid variants were confined to clonally related organisms having similar IS6110 profiles and four or six hybridizing copies of this insertion element.

MPT64 (23.5-kD antigen): MPT64 is a 23.5-kD secreted antigen that is identical to a protein initially purified from culture filtrates of Mycobacterium bovis BCG Tokyo and designated MPB64 (HARBOE et al. 1986 ; OETTINGER and ANDERSEN 1994 ). Humans mount an immune response to MPT64 during tuberculosis disease. T-cell and linear B-cell epitope mapping has been conducted with recombinant proteins (OETTINGER et al. 1995 ). A putative delayed-type hypersensitivity-inducing epitope is located within amino acid residues Gly-173 to Ala-187 (OETTINGER et al. 1995 ; ELHAY et al. 1998 ). Our analysis identified a Thr35Ala amino acid replacement in one strain from Peru (NHN390) that is a member of principal genetic group 2. We note that OETTINGER and ANDERSEN 1994 reported that monoclonal antibody C24b3 defined an epitope composed of two structural domains located in the sequences Ala-1 to Leu-43 and Ala-108 to Ser-152.

Antigen-85B and antigen-85C proteins: The antigen-85 complex is formed by three major proteins—designated Ag85A, Ag85B, and Ag85C—that are expressed by actively replicating organisms (HORWITZ et al. 1995 ). Ag85-complex proteins are mycolyltransferases that participate in the final stages of cell wall synthesis (WIKER and HARBOE 1992 ; BELISLE et al. 1997 ; JACKSON et al. 1999 ). Their expression is upregulated in response to isoniazid treatment in vitro (HARRIS et al. 1994 ; BARDOU et al. 1996 ). Ag85-complex proteins induce cellular and humoral immune responses in infected laboratory animals and humans (WIKER and HARBOE 1992 ). Levels of anti-Ag85 antibodies are usually low in healthy purified protein-derivative (PPD) positive individuals but increase in patients with active tuberculosis (HAVLIR et al. 1991 ; WIKER and HARBOE 1992 ; LYASHCHENKO et al. 1998 ). Active immunization with purified Ag85-complex proteins or DNA encoding Ag85B can induce protective immunity and a positive therapeutic effect in laboratory animals (HUYGEN et al. 1996 ; LOZES et al. 1997 ; LOWRIE et al. 1999 ). The precise role of these proteins in M. tuberculosis host-pathogen interactions is not yet clear, but they are known to be upregulated in response to growth in macrophages in vitro (LEE and HORWITZ 1995 ). Recently, LEE and HORWITZ 1999 mapped T-cell epitopes in outbred guinea pigs immunized with purified Ag85A and Ag85B.

One synonymous (silent) nucleotide change was found in codon 238 (CCC CCA; Pro Pro) of the Ag85B gene in HN1543. In addition, all five organisms belonging to principal genetic group 3 had a G A nucleotide substitution located at position -3 relative to the start codon. Sequence analysis of 20 additional isolates found that all 14 members of principal genetic group 3, regardless of IS6110 pattern or spoligotype, also had this upstream polymorphism. In contrast, this nucleotide substitution was not present in members of the other two principal genetic groups. All 16 core isolates had the same sequence for the gene encoding Ag85C except strain HN1305, which had a G A mutation at position -63 upstream of the start codon and a nucleotide change resulting in a Glu103Asp amino acid replacement.

PE and PPE genes:
The M. tuberculosis H37Rv genome contains two large families of genes encoding glycine-rich proteins designated PE (Pro-Glu) and PPE (Pro-Pro-Glu) (COLE et al. 1998 ). The H37Rv genome contains 99 members of the PE family and 68 members of the PPE family. The cell location and biologic function of these proteins are unknown, but it has been suggested that they participate in antigenic variation or interfere with host immune responses by inhibiting antigen processing (COLE et al. 1998 ). Recently, DILLON et al. 1999 reported that a 39-kD member of the PPE family elicited strong T-cell proliferative and gamma-interferon responses in peripheral blood mononuclear cells from PPD-positive individuals. No nucleotide variation was identified in five (Rv0285, Rv0388, Rv1169, Rv1788, and Rv2430) of the eight PE and PPE genes chosen for sequence analysis. Two isolates belonging to principal genetic group 1 (HN1343 and HN1489) had a C T nucleotide polymorphism located at position -78 relative to the start codon of Rv3477. One group 2 (HN1305) and one group 3 (HN1339) organism had a Pro190Ser amino acid replacement in Rv1790.

Rv3135, a member of the PPE family of proteins, was uniquely variable. PCR analysis of the Rv3135 gene in the core group of 16 isolates identified four distinct sizes of products, and sequence analysis revealed them to be 322 bp, 632 bp, 1021 bp, and 1973 bp long, respectively (Fig 2). Isolates assigned to principal genetic group 1 had the 1973-bp or 1021-bp sequences, those belonging to group 2 had the 632-bp or 322-bp sequences, and those belonging to group 3 had the 632-bp variant. To more fully investigate the extent and phylogenetic distribution of Rv3135 size variation, PCR analysis was also conducted on 141 M. tuberculosis isolates from global sources. Four additional PCR fragment sizes were found, and sequence analysis identified products of 200 bp, 460 bp, 499 bp, and 533 bp, respectively (Fig 2). As observed for the 16 core isolates, all group 3 organisms had the 632-bp product and group 2 organisms had either the 322-bp or 632-bp sequence. In contrast, group 1 organisms had a broad range of Rv3135 gene sizes, including 200 bp, 460 bp, 499 bp, 533 bp, 1021 bp, and 1973 bp. Inspection of all Rv3135 data found that, in principle, each variant could be linked to one or more of the other variants by a single molecular step, suggesting that the Rv3135 polymorphisms were generated by rare deletion or insertion events. On the basis of the inferred amino acid sequences, not all isolates would express a protein product (Fig 3). Some of the gene variants had premature stop codons resulting in truncated proteins relative to the H37Rv product. In addition, two of the variants lacked upstream regions encoding putative regulatory sequences and, hence, would probably not express the Rv3135 protein.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Schematic representation of variation identified in the Rv3135 gene encoding a member of the Pro-Pro-Glu (PPE) family of M. tuberculosis proteins. The thin horizontal line represents DNA found in the 1973-bp segment of HN1489 but absent from other strains. The genetic group was defined by polymorphisms present in katG codon 463 and gyrA codon 95 (SREEVATSAN et al. 1997 ). The Rv3135 gene was obtained by PCR from representative isolates. The sizes (in base pairs) shown are the lengths of the PCR products obtained for each variant. The lengths of the longest open reading frame for each variant are shown at right. Strains HN1250, HN1489, HN1525, and HN1543 are described in Table 1. Strains HN1036, HN1088, and HN1430, recovered from patients in Houston, Texas, are principal genetic group 1 (SREEVATSAN et al. 1997 ) and have spoligotype S1 (SOINI et al. 2000 ).

View larger version (81K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Alignment of amino acid residues in Rv3135 variants. Aligned are the inferred amino acid sequences for each of the six Rv3135 protein variants putatively expressed by M. tuberculosis isolates and identified in this study. Two of the Rv3135 gene PCR variants (200 bp and 322 bp, see Fig 2) may not result in expression of a protein product due to structural alterations (apparent deletions) of the presumed upstream regulatory sequence. The inferred amino acid sequence for NHN653 is shown at the bottom of the figure because the amino terminus does not align with the other proteins. Strains HN1489 and HN1543 are described in Table 1. Strains HN1088 and HN1430, recovered from patients in Houston, Texas, are principal genetic group 1 (SREEVATSAN et al. 1997 ) and have spoligotype S1 (SOINI et al. 2000 ).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Most of the 26 M. tuberculosis structural genes previously studied (SREEVATSAN et al. 1997 ) for allelic variation encode intracellular metabolic enzymes that are not known to directly interact with the host. However, allelic variation was also restricted in three genes encoding proteins (16-kD antigen, 65-kD heat-shock protein, and catalase-peroxidase) that are host immune system targets (ANDERSEN 1994 ; ANDERSEN and BRENNAN 1994 ; LYASHCHENKO et al. 1998 ). Inasmuch as an important common theme in infectious disease research is that pathogen surface and other proteins involved in direct interaction with the host immune system are highly polymorphic relative to metabolic housekeeping enzymes (SELANDER et al. 1994 ; LI et al. 1995 ; MATHIESEN et al. 1997 ; YAMAGUCHI and GOJOBORI 1997 ; RICH et al. 1998 ; STOCKBAUER et al. 1998 ), we thought it critical to more fully investigate the level of antigen gene variation. The analysis was also motivated by the substantial interest in protein-subunit vaccine strategies and other new therapeutic and diagnostic methods (ANDERSEN 1994 ; HORWITZ et al. 1995 ; HUYGEN et al. 1996 ; TASCON et al. 1996 ; LOZES et al. 1997 ; HARTH and HORWITZ 1999 ). In addition, since many of the proteins released into the extracellular medium from growing M. tuberculosis are immunologically recognized during infection in animals and early stages of human pulmonary tuberculosis (PAL and HORWITZ 1992 ; ORME et al. 1993 ; ANDERSEN 1994 ; ANDERSEN and BRENNAN 1994 ; HORWITZ et al. 1995 ), the failure of tuberculosis patients to uniformly respond to many of those same proteins raised the possibility that amino acid sequence variation in the target molecules is a contributing factor.

Our analysis demonstrated that an absence or very low frequency of structural polymorphism is a general characteristic of genes encoding prominent targets for host B-cell and T-cell immune responses. There are several hypotheses that may account for this observation: (i) the diversification of antigens is restricted by the intracellular niche, (ii) the organism has a low spontaneous mutation rate, (iii) humans have little effective immunity to the target proteins so far characterized, (iv) major protective immune targets have not yet been identified, and (v) the widespread dissemination of M. tuberculosis is evolutionarily very recent.

In principle the intracellular lifestyle of M. tuberculosis may restrict antigen variation by sequestering the pathogen from the host immune response, especially the humoral defense mechanism. However, there is no evidence that restricted antigen gene variation is a common theme for intracellular pathogens, including obligate parasites in the extreme case (WEIDMANN et al. 1997 ; BRISSE et al. 1998 ; STOTHARD et al. 1998 ). For example, abundant levels of nucleotide and amino acid polymorphisms have been described in Chlamydia trachomatis (STOTHARD et al. 1998 ), Salmonella enterica (SELANDER et al. 1994 ; LI et al. 1995 ), Listeria monocytogenes (WEIDMANN et al. 1997 ), and Trypanosoma cruzi (BRISSE et al. 1998 ). In addition, evidence indicates that humoral immune processes participate in host defense against M. tuberculosis (GLATMAN-FREEDMAN and CASADEVALL 1998 ; TEITELBAUM et al. 1998 ). Hypothesis ii is ruled out because M. tuberculosis does not have an unusually low spontaneous mutation frequency (DAVID and NEWMAN 1971 ). On the contrary, spontaneous drug resistance mutants can be selected rapidly in the laboratory and in human patients (DAVID and NEWMAN 1971 ; BIFANI et al. 1996 ; RAMASWAMY and MUSSER 1998 ). In regard to hypothesis iii, immunity to M. tuberculosis is complex, involving innate factors (BELLAMY et al. 1998 ) and acquired humoral and cellular responses. Without documented in vitro or animal model correlates of human protective immunity, it is difficult to speculate about the potential contributions of hypotheses iii and iv, above. However, generation of a protective or therapeutic response to tuberculosis by immunization with several of the antigens we studied has been well described in animal models, which reduces the likelihood that these two hypotheses alone are responsible. Evidence has also been presented that patients with pulmonary tuberculosis have depressed effector immune function, which can be long lasting (HIRSCH et al. 1996 , HIRSCH et al. 1999A , HIRSCH et al. 1999B ). Effective immunity could also be altered by the magnitude of posttranslational modification of target proteins by, for example, glycosylation. According to this hypothesis, glycosylation would alter amino acid residues encompassed in a region of the molecule commonly recognized as foreign. This modification would then alter host immune system recognition of the target protein. In this regard, it was recently shown that deglycosylation of the 45/47-kD antigen complex significantly decreased its capacity to elicit in vivo and in vitro cellular immune responses (HORN et al. 1999 ; ROMAIN et al. 1999 ). We also note that HERRMANN et al. 1996 postulated that glycosylation functions to regulate the relative amounts of cell-associated and soluble forms of protein antigens. In addition, effective immunity may depend on response to nonprotein antigens, and TEITELBAUM et al. 1998 reported that a monoclonal antibody specific for arabinomannan conferred partial protection on mice challenged by the aerosol route. Finally, certain M. tuberculosis isolates may not be rapidly recognized as foreign early in the course of host-pathogen interactions (MANCA et al. 1999 ).

It is highly unlikely that an immunologic selection hypothesis alone accounts for our observations. One strong argument favoring hypothesis v (the very recent origin and widespread dissemination of M. tuberculosis) is the absence or very low level of silent nucleotide substitutions in all genes characterized to date, which in our laboratory now number ~80 genes characterized in large numbers of strains from global sources. Moreover, comparison of the genome sequences available for H37Rv and CSU93 indicate that although several large insertion and deletion events differentiate the genomes, none of the roughly 4000 ORFs is strongly polymorphic at the nucleotide level. The lack of significant variation in genes encoding proteins that are host immune system targets indicates that in all likelihood M. tuberculosis has spread globally even more recently than roughly 20,000 years ago (KAPUR et al. 1994 ; SREEVATSAN et al. 1997 ). If, as the data indicate, interaction of humans with M. tuberculosis has been frequent for only a brief evolutionary period, then immunogenetics may play an even more prominent role in determining the outcome of the host-pathogen relationship. The occurrence of epidemic waves of tuberculosis in many populations in recent centuries and in certain inbred and historically isolated human populations (SOUSA et al. 1997 ) is consistent with this idea.

Early in the course of host-pathogen interaction M. tuberculosis is sequestered in the macrophage. Most individuals control the organism without detrimental effects. The pathogen then resides in a quiescent state with relatively little replication until cell-mediated immunity is compromised, commonly by HIV infection or aging. Most other pathogens are frequently confronted with very labile environments in which there is a substantial premium on variation and adaptation. In contrast, the life cycle of M. tuberculosis lessens the extent of exposure to wide fluctuations in variable host and environmental factors and minimizes DNA replication cycles. Together, these two factors tend to constrain species diversity. Importantly, emergence from the quiescent state usually occurs when the host is relatively immunocompromised, which means that the pathogen usually is not exposed to strong immune selective pressure. Indeed, it well may be that this aspect of the M. tuberculosis infection cycle is a primary cause of the lack of variation in the protein antigens studied, in spite of being responsible for 3 million deaths yearly. These considerations, coupled with the evolutionarily recent introduction of M. tuberculosis into humans from nonhuman hosts living in close association, such as cattle, goats, or water buffalo, may contribute to the lack of amino acid polymorphism in this pathogen.

PE and PPE variation:
COLE et al. 1998 speculated that the PE and PPE protein families are a source of antigenic variation in M. tuberculosis. Of note, we found that the gene encoding PPE protein Rv3135 varied in size, although not all of the allelic variants would result in a protein product. Size variants were not found in high frequency in isolates recovered from the same patient over time, suggesting that the gene is not hypervariable or hypermutable (S. RAMASWAMY and J. M. MUSSER, unpublished data). The nonrandom association of Rv3135 variants with distinct genetic groups also is consistent with a lack of a hypervariable or hypermutable phenotype.

There are 167 PE and PPE gene family members in the H37Rv chromosome (COLE et al. 1998 ). Variation was identified in the coding sequence of 1 of the 8 PE and PPE genes analyzed, suggesting by extrapolation that roughly 20 PE and PPE members would be polymorphic in M. tuberculosis. Alignment of additional members of the PE and PPE families in H37Rv and CSU93 indicates that other examples of size polymorphism exist. Notwithstanding the important recent observation that strains may lack certain PE or PPE genes (GORDON et al. 1999 ), the results suggest that a relatively small percentage of these proteins are variable. However, this issue requires more extensive investigation at the gene and protein level in large samples of isolates.

Implications for development of new therapeutics and diagnostics:
The current global tuberculosis epidemic and the spread of multidrug-resistant strains make development of new therapeutics an important priority. Recently, HARTH and HORWITZ 1999 demonstrated that an inhibitor of exported M. tuberculosis glutamine synthetase selectively blocked pathogen growth in axenic culture and human monocytes. Their findings suggest that extracellular proteins made by M. tuberculosis are potential novel drug targets; a similar idea was advanced for the Ag85-complex mycolyltransferase (BELISLE et al. 1997 ). Our findings, together with data for the 26 genes previously reported (SREEVATSAN et al. 1997 ), indicate that prominent M. tuberculosis antigenic proteins and potential novel drug and diagnostic targets are likely to have very little structural variation worldwide. If the apparent lack of host immune pressure can be abrogated by induction of enhanced effector function, then the lack of diversity in protein targets may be cause for optimism in the difficult fight to control global tuberculosis.

	FOOTNOTES

This article is dedicated to Professor R. K. Selander on the occasion of his retirement.

	ACKNOWLEDGMENTS

We thank colleagues who generously contributed strains. We are indebted to A. Casadevall, M. L. Gennaro, B. N. Kreiswirth, L. Schlesinger, and R. K. Selander for reading the manuscript and suggesting improvements. This research was supported by U.S. Public Health Services grants AI-41168, AI-37004, and DA-09238 to J.M.M.

Manuscript received September 15, 1999; Accepted for publication January 12, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ANDERSEN, P., 1994  Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect. Immun. 62:2536-2544[Abstract/Free Full Text].

ANDERSEN, Å. B., and P. BRENNAN, 1994 Proteins and antigens of Mycobacterium tuberculosis, pp. 307–332 in Tuberculosis: Pathogenesis, Protection, and Control, edited by B. R. BLOOM. American Society for Microbiology, Washington, DC.

ANDERSEN, Å. B. and E. B. HANSEN, 1989  Structure and mapping of antigenic domains of protein antigen b, a 38,000-molecular-weight protein of Mycobacterium tuberculosis.. Infect. Immun. 57:2481-2488[Abstract/Free Full Text].

BAIRD, P. N., L. M. C. HALL, and A. R. M. COATES, 1988  A major antigen from Mycobacterium tuberculosis which is homologous to the heat shock proteins groES from E. coli and the htpA gene product of Coxiella burneti.. Nucleic Acids Res. 16:9047[Free Full Text].

BARDOU, F., A. QUÉMARD, M.-A. DUPONT, C. HORN, and G. MARCHAL et al., 1996  Effects of isoniazid on ultrastructure of Mycobacterium aurum and Mycobacterium tuberculosis and on production of secreted proteins. Antimicrob. Agents Chemother. 40:2459-2467[Abstract].

BELISLE, J. T., V. D. VISSA, T. SIEVERT, K. TAKAYAMA, and P. J. BRENNAN et al., 1997  Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 276:1420-1422[Abstract/Free Full Text].

BELLAMY, R., C. RUWENDE, T. CORRAH, K. P. MCADAM, and H. C. WHITTLE et al., 1998  Variations in the NRAMP1 gene and susceptibility to tuberculosis in West Africans. N. Engl. J. Med. 338:640-644[Abstract/Free Full Text].

BERTHET, F.-X., P. B. RASMUSSEN, I. ROSENKRANDS, P. ANDERSEN, and B. A. GICQUEL, 1998  Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10). Microbiol. 144:3195-3203[Abstract].

BIFANI, P., B. B. PLIKAYTIS, V. KAPUR, K. STOCKBAUER, and X. PAN et al., 1996  Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosis clone family. J. Am. Med. Assoc. 275:452-457[Abstract].

BILLMAN-JACOBE, H., A. J. RADFORD, J. S. ROTHEL, and P. R. WOOD, 1990  Mapping of the T and B cell epitopes of the Mycobacterium bovis protein, MPB70. Immunol. Cell. Biol. 68:359-365.

BRISSE, S., C. BARNABÉ, A. L. BAÑULS, I. SIDIBÉ, and S. NOËL et al., 1998  A phylogenetic analysis of the Trypanosoma cruzi genome project CL Brener reference strain by multilocus enzyme electrophoresis and multiprimer random amplified polymorphic DNA fingerprinting. Mol. Biochem. Parasitol. 92:253-263[Medline].

COHEN, M. L., L. W. MAYER, H. S. RUMSCHLAG, M. A. YAKRUS, and W. D. JONES, JR. et al., 1987  Expression of proteins of Mycobacterium tuberculosis in Escherichia coli and potential of recombinant genes and proteins for development of diagnostic reagents. J. Clin. Microbiol. 25:1176-1180[Abstract/Free Full Text].

COLE, S. T., R. BROSCH, J. PARKHILL, T. GARNIER, and C. CHURCHER et al., 1998  Deciphering the biology of Mycobacterium tuberculosis from the complete genomc sequence. Nature 393:537-544[Medline].

COLER, R. N., Y. A. W. SKEIKY, T. VEDVICK, T. BEMENT, and P. OVENDALE et al., 1998  Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis.. J. Immunol. 161:2356-2364[Abstract/Free Full Text].

DAVID, H. L. and C. M. NEWMAN, 1971  Some observations on the genetics of isoniazid resistance in the tubercle bacilli. Am. Rev. Respir. Dis. 104:508-515[Medline].

DELOGU, G. and M. J. BRENNAN, 1999  Functional domains present in the mycobacterial hemagglutinin, HBHA. J. Bacteriol. 181:7464-7469[Abstract/Free Full Text].

DIAGBOUGA, S., F. FUMOUX, A. ZOUBGA, P. T. SANOU, and G. MARCHAL, 1997  Immunoblot analysis for serodiagnosis of tuberculosis using a 45/47-kilodalton antigen complex of Mycobacterium tuberculosis.. Clin. Diag. Lab. Immunol. 4:334-338[Abstract].

DILLON, D. C., M. R. ALDERSON, C. H. DAY, D. M. LEWINSOHN, and R. COLER et al., 1999  Molecular characterization and human T-cell responses to a member of a novel Mycobacterium tuberculosis mtb39 gene family. Infect. Immun. 67:2941-2950[Abstract/Free Full Text].

ELHAY, M. J., T. OETTINGER, and P. ANDERSEN, 1998  Delayed-type hypersensitivity responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the guinea pig. Infect. Immun. 66:3454-3456[Abstract/Free Full Text].

ENGERS, H. D., V. HOUBA, J. BENNEDSEN, T. M. BUCHANAN, and S. D. CHAPARAS et al., 1986  Results of a World Health Organization-sponsored workshop to characterize antigens recognized by mycobacterium-specific monoclonal antibodies. Infect. Immun. 51:718-720[Free Full Text].

ERB, K. J., J. KIRMAN, L. WOODFIELD, T. WILSON, and D. M. COLLINS et al., 1998  Identification of potential CD8⁺ T-cell epitopes of the 19 kD and AhpC proteins of Mycobacterium tuberculosis. No evidence for CD8⁺ T-cell priming against the identified peptides after DNA-vaccination of mice. Vaccine 16:692-697[Medline].

ESPITIA, C., M. ELINOS, R. HERNÁNDEZ-PANDO, and R. MANCILLA, 1992  Phosphate starvation enhances expression of the immunodominant 38-kilodalton protein antigen of Mycobacterium tuberculosis: demonstration by immunogold electron microscopy. Infect. Immun. 60:2998-3001[Abstract/Free Full Text].

GARBE, T. R., N. S. HIBLER, and V. DERETIC, 1996  Isoniazid induces expression of the antigen 85 complex in Mycobacterium tuberculosis.. Antimicrob. Agents Chemother. 40:1754-1756[Abstract].

GLATMAN-FREEDMAN, A. and A. CASADEVALL, 1998  Serum therapy for tuberculosis revisited: reappraisal of the role of antibody-mediated immunity against Mycobacterium tuberculosis.. Clin. Microbiol. Rev. 11:514-532[Abstract/Free Full Text].

GORDON, S. V., R. BROSCH, A. BILLAULT, T. GARNIER, and K. EIGLMEIER et al., 1999  Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol. Microbiol. 32:643-655[Medline].

GROENEN, P. M. A., A. E. BUNSCHOTEN, D. VAN SOOLINGEN, and J. D. A. VAN EMBDEN, 1993  Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis: application for strain differentiation by a novel typing method. Mol. Microbiol. 10:1057-1065[Medline].

HARBOE, M. and H. G. WIKER, 1992  The 38-kD protein of Mycobacterium tuberculosis: a review. J. Infect. Dis. 166:874-884[Medline].

HARBOE, M., S. NAGAI, M. E. PATARROYO, M. L. TORRES, and C. RAMIREZ et al., 1986  Properties of proteins MPB64, MPB70, and MPB80 of Mycobacterium bovis BCG. Infect. Immun. 52:293-302[Abstract/Free Full Text].

HARRIS, D. P., H.-M. VORDERMEIER, S. J. BRETT, G. PASVOL, and C. MORENO et al., 1994  Epitope specificity and isoforms of the mycobacterial 19-kilodalton antigen. Infect. Immun. 62:2963-2972[Abstract/Free Full Text].

HARTH, G. and M. A. HORWITZ, 1999  An inhibitor of exported Mycobacterium tuberculosis glutamine synthetase selectively blocks the growth of pathogenic mycobacteria in axenic culture and in human monocytes: extracellular proteins as potential novel drug targets. J. Exp. Med. 189:1425-1435[Abstract/Free Full Text].

HAVLIR, D. V., R. S. WALLIS, W. H. BOOM, T. M. DANIEL, and K. CHERVENAK, 1991  Human immune response to Mycobacterium tuberculosis antigens. Infect. Immun. 59:665-670[Abstract/Free Full Text].

HERRMANN, J. L., P. O'GAORA, A. GALLAGHER, J. E. R. THOLE, and D. B. YOUNG, 1996  Bacterial glycoproteins: a link between glycosylation and proteolytic cleavage of a 19 kD antigen from Mycobacterium tuberculosis.. EMBO J. 15:3547-3554[Medline].

HEWINSON, R. G., S. L. MICHELL, W. P. RUSSELL, R. A. MCADAM, and W. R. JACOBS, JR., 1996  Molecular characterization of MPT83: a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70. Scand. J. Immunol. 43:490-499[Medline].

HIRSCH, C. S., R. HUSSAIN, Z. TOOSSI, G. DAWOOD, and F. SHAHID et al., 1996  Cross-modulation by transforming growth factor ß in human tuberculosis: suppression of antigen-driven blastogenesis and interferon production. Proc. Natl. Acad. Sci. USA 93:3193-3198[Abstract/Free Full Text].

HIRSCH, C. S., Z. TOOSSI, C. OTHIENO, J. L. JOHNSON, and S. K. SCHWANDER et al., 1999a  Depressed T-cell interferon- responses in pulmonary tuberculosis: analysis of underlying mechanisms and modulation with therapy. J. Infect. Dis. 180:2069-2073[Medline].

HIRSCH, C. S., Z. TOOSSI, G. VANHAM, J. L. JOHNSON, and P. PETERS et al., 1999b  Apoptosis and T cell hyporesponsiveness in pulmonary tuberculosis. J. Infect. Dis. 179:945-953[Medline].

HORN, C., A. NAMANE, P. PESCHER, M. RIVIERE, and F. ROMAIN et al., 1999  Decreased capacity of recombinant 45/47-kD molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern. J. Biol. Chem. 274:32023-32030[Abstract/Free Full Text].

HORWITZ, M. A., B.-W. E. LEE, B. J. DILLON, and G. HARTH, 1995  Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis.. Proc. Natl. Acad. Sci. USA 92:1530-1534[Abstract/Free Full Text].

HUYGEN, K., J. CONTENT, O. DENIS, D. L. MONTGOMERY, and A. M. YAWMAN et al., 1996  Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat. Med. 2:893-898[Medline].

JACKETT, P. S., G. H. BOTHAMLEY, H. V. BATRA, A. MISTRY, and D. B. YOUNG et al., 1988  Specificity of antibodies to immunodominant mycobacterial antigens in pulmonary tuberculosis. J. Clin. Microbiol. 26:2313-2318[Abstract/Free Full Text].

JACKSON, M., C. RAYNAUD, M.-A. LANÉELLE, C. GUILHOT, and C. LAURENT-WINTER et al., 1999  Inactivation of the antigen 85C gene profoundly affects the mycolate content and alters the permeability of the Mycobacterium tuberculosis cell envelope. Mol. Microbiol. 31:1573-1587[Medline].

KANG, S.-K., Y.-J. JUNG, C.-H. KIM, and C.-Y. SONG, 1998  Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG. Clin. Diag. Lab. Immunol. 5:784-789[Abstract/Free Full Text].

KAPUR, V., T. S. WHITTAM, and J. M. MUSSER, 1994  Is Mycobacterium tuberculosis 15,000 years old? J. Infect. Dis. 170:1348-1349[Medline].

LAQUEYRERIE, A., P. MILITZER, F. ROMAIN, K. EIGLMEIER, and S. COLE et al., 1995  Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex. Infect. Immun. 63:4003-4010[Abstract].

LATHIGRA, R., Y. ZHANG, M. HILL, M.-J. GARCIA, and P. S. JACKETT et al., 1996  Lack of production of the 19-kD glycolipoprotein in certain strains of Mycobacterium tuberculosis.. Res. Microbiol. 147:237-249[Medline].

LEE, B.-Y. and M. A. HORWITZ, 1995  Identification of macrophage and stress-induced proteins of Mycobacterium tuberculosis.. J. Clin. Invest. 96:245-249.

LEE, B.-Y. and M. A. HORWITZ, 1999  T-cell epitope mapping of the three most abundant extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs. Infect. Immun. 67:2665-2670[Abstract/Free Full Text].

LI, J., H. OCHMAN, E. A. GROISMAN, E. F. BOYD, and F. SOLOMON et al., 1995  Relationship between evolutionary rate and cellular location among the Inv/Spa invasion proteins of Salmonella enterica.. Proc. Natl. Acad. Sci. USA 92:7252-7256[Abstract/Free Full Text].

LOWRIE, D. B., R. E. TASCON, V. L. D. BONATO, V. M. F. LIMA, and L. H. FACCIOLI et al., 1999  Therapy of tuberculosis in mice by DNA vaccination. Nature 400:269-271[Medline].

LOZES, E., K. HUYGEN, J. CONTENT, O. DENIS, and D. L. MONTGOMERY et al., 1997  Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine 15:830-833[Medline].

LYASHCHENKO, K., R. COLANGELI, M. HOUDE, H. AL JAHDALI, and D. MENZIES et al., 1998  Heterogeneous antibody responses in tuberculosis. Infect. Immun. 66:3936-3940[Abstract/Free Full Text].

MAHENTHIRALINGAM, E., B.-I. MARKLUND, L. A. BROOKS, D. A. SMITH, and G. BANCROFT et al., 1998  Site-directed mutagenesis of the 19-kilodalton lipoprotein antigen reveals no essential role for the protein in the growth and virulence of Mycobacterium tuberculosis.. Infect. Immun. 66:3626-3634[Abstract/Free Full Text].

MANCA, C., K. LYASHCHENKO, R. COLANGELI, and M. L. GENNARO, 1997  MTC28, a novel 28-kilodalton proline-rich secreted antigen specific for the Mycobacterium tuberculosis complex. Infect. Immun. 65:4951-4957[Abstract].

MANCA, C., L. TSENOVA, C. E. BARRY, III, A. BERGTOLD, and S. FREEMAN et al., 1999  Mycobacterium tuberculosis CDC1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates. J. Immunol. 162:6740-6746[Abstract/Free Full Text].

MATHIESEN, D. A., J. H. OLIVER, JR., C. P. KOLBERT, E. D. TULLSON, and B. J. JOHNSON et al., 1997  Genetic heterogeneity of Borrelia burgdorferi in the United States. J. Infect. Dis. 175:98-107[Medline].

MATSUMOTO, S., T. MATSUO, N. OHARA, H. HOTOKEZAKA, and M. NAITO et al., 1995  Cloning and sequencing of a unique antigen MPT70 from Mycobacterium tuberculosis H37Rv and expression in BCG using E. coli-mycobacteria shuttle vector. Scand. J. Immunol. 41:281-287[Medline].

MATTHEWS, R., A. SCOGING, and A. D. M. REES, 1985  Mycobacterial antigen-specifc human T-cell clones secreting macrophage activating factors. Immunology 54:17-23[Medline].

MENOZZI, F. D., J. H. ROUSE, M. ALAVI, M. LAUDE-SHARP, and J. MULLER et al., 1996  Identification of a heparin-binding hemagglutinin present in mycobacteria. J. Exp. Med. 184:993-1001[Abstract/Free Full Text].

MENOZZI, F. D., R. BISCHOFF, E. FORT, M. J. BRENNAN, and C. LOCHT, 1998  Molecular characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesin. Proc. Natl. Acad. Sci. USA 95:12625-12630[Abstract/Free Full Text].

OETTINGER, T. and Å. B. ANDERSEN, 1994  Cloning and B-cell-epitope mapping of MPT64 from Mycobacterium tuberculosis H37Rv. Infect. Immun. 62:2058-2064[Abstract/Free Full Text].

OETTINGER, T., A. HOLM, I. M. MTONI, Å. B. ANDERSEN, and K. HASLØV, 1995  Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis.. Infect. Immun. 63:4613-4618[Abstract].

ORME, I. M., P. ANDERSEN, and W. H. BOOM, 1993  T cell response to Mycobacterium tuberculosis.. J. Infect. Dis. 167:1481-1497[Medline].

PAL, P. G. and M. A. HORWITZ, 1992  Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis. Infect. Immun. 60:4781-4792[Abstract/Free Full Text].

RAMASWAMY, S. and J. M. MUSSER, 1998  Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update. Tuberc. Lung Dis. 79:3-29[Medline].

RAVN, P., A. DEMISSIE, T. EGUALE, H. WONDWOSSON, and D. LEIN et al., 1999  Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis.. J. Infect. Dis. 179:637-645[Medline].

RICH, S. M., M. C. LICHT, R. R. HUDSON, and F. J. AYALA, 1998  Malaria's Eve: evidence of a recent population bottleneck throughout the world populations of Plasmodium falciparum.. Proc. Natl. Acad. Sci. USA 95:4425-4430[Abstract/Free Full Text].

ROMAIN, F., C. HORN, P. PESCHER, A. NAMANE, and M. RIVIERE et al., 1999  Deglycosylation of the 45/47-kilodalton antigen complex of Mycobacterium tuberculosis decreases its capacity to elicit in vivo or in vitro cellular immune responses. Infect. Immun. 67:5567-5572[Abstract/Free Full Text].

ROSENKRANDS, I., P. B. RASMUSSEN, M. CARNIO, S. JACOBSEN, and M. THEISEN et al., 1998  Identification and characterization of a 29-kilodalton protein from Mycobacterium tuberculosis culture filtrate recognized by mouse memory effector cells. Infect. Immun. 66:2728-2735[Abstract/Free Full Text].

SELANDER, R. K., J. LI, E. F. BOYD, F.-S. WANG and K. NELSON, 1994 DNA sequence analysis of the genetic structure of populations of Salmonella enterica and Escherichia coli, pp. 17–49 in Bacterial Diversity and Systematics, edited by F. G. PRIEST, A. RAMOS-CORMENZANA and B. J. TINDALL. Plenum, New York.

SOINI, H., X. PAN, A. AMIN, E. A. GRAVISS, and A. SIDDIQUI et al., 2000  Characterization of Mycobacterium tuberculosis isolates from patients in Houston, Texas, by spoligotyping. J. Clin. Microbiol. 38:669-676[Abstract/Free Full Text].

SØRENSEN, A. L., S. NAGAI, G. HOUEN, P. ANDERSEN, and Å. B. ANDERSEN, 1995  Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.. Infect. Immun. 63:1710-1717[Abstract].

SOUSA, A. O., J. I. SALEM, F. K. LEE, M. C. VERCOSA, and P. CRUAUD et al., 1997  An epidemic of tuberculosis with a high rate of tuberculin anergy among a population previously unexposed to tuberculosis, the Yanomami Indians of the Brazilian Amazon. Proc. Natl. Acad. Sci. USA 94:13227-13232[Abstract/Free Full Text].

SREEVATSAN, S., X. PAN, K. E. STOCKBAUER, N. D. CONNELL, and B. N. KREISWIRTH et al., 1997  Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc. Natl. Acad. Sci. USA 94:9869-9874[Abstract/Free Full Text].

STOCKBAUER, K. E., D. GRIGSBY, X. PAN, Y.-X. FU, and L. M. PEREA MEJIA et al., 1998  Hypervariability generated by natural selection in an extracellular complement-inhibiting protein of serotype M1 strains of group A Streptococcus.. Proc. Natl. Acad. Sci. USA 95:3128-3133[Abstract/Free Full Text].

STOTHARD, D. R., G. BOGUSLAWSKI, and R. B. JONES, 1998  Phylogenetic analysis of the Chlamydia trachomatis major outer membrane protein and examination of potential pathogenic determinants. Infect. Immun. 66:3618-3625[Abstract/Free Full Text].

TASCON, R. E., M. J. COLSTON, S. RAGNO, E. STAVROPOULOS, and D. GREGORY et al., 1996  Vaccination against tuberculosis by DNA injection. Nat. Med. 2:888-891[Medline].

TEITELBAUM, R., A. GLATMAN-FREEDMAN, B. CHEN, J. B. ROBBINS, and E. UNANUE et al., 1998  A mAb recognizing a surface antigen of Mycobacterium tuberculosis enhances host survival. Proc. Natl. Acad. Sci. USA 95:15688-15693[Abstract/Free Full Text].

VAN EMBDEN, J. D. A., M. D. CAVE, J. T. CRAWFORD, J. W. DALE, and K. D. EISENACH et al., 1993  Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J. Clin. Microbiol. 31:406-409[Abstract/Free Full Text].

VERBON, A., R. A. HARTSKEERL, A. SCHUITEMA, A. H. J. KOLK, and D. B. YOUNG et al., 1992  The 14,000-molecular-weight antigen of Mycobacterium tuberculosis is related to the alpha-crystallin family of low-molecular-weight heat shock proteins. J. Bacteriol. 174:1352-1359[Abstract/Free Full Text].

WEIDMANN, M., J. L. BRUCE, C. KEATING, A. E. JOHNSON, and P. L. MCDONOUGH et al., 1997  Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65:2707-2716[Abstract].

WELDINGH, K., I. ROSENKRANDS, S. JACOBSEN, P. B. RASMUSSEN, and M. J. ELHAY et al., 1998  Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purific