One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of Ϸ6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.One-third of the world's population is infected with Mycobacterium tuberculosis, and 3 million human deaths annually are attributed to the organism (1, 2). Although there is a very large global pool of infected individuals and considerable chromosomal heterogeneity based on restriction fragment length polymorphism (RFLP) patterns generated by probing with mobile insertion elements (3, 4), studies of drug resistance and pathogenesis have raised the possibility that synonymous (silent) nucleotide substitutions in structural genes may be limited (5). To investigate this apparent discrepancy from the perspective of molecular population genetics, we sequenced two megabases in 26 structural genes or loci in strains of M. tuberculosis and the three closely related members of the M. tuberculosis complex (M. africanum, M. bovis, and M. microti) collected worldwide. MATERIALS AND METHODSBacterial Isolates. The study is based on a sample of 842 M. tuberculosis complex isolates recovered from diverse geographic localities. The organisms include M. tuberculosis (n ϭ 715), M. bovis (n ϭ 109), and M. africanum and M. microti (n ϭ 9 each). M. tuberculosis isolates were recovered from diseased patients in the United States (five states), Latin America (Mexico, Honduras, Ecuador, Peru, Venezuela, Brazil, and Chile), Europe (Portugal, Spain, The Netherlands, Belgium, Germany, Switzerland, Italy, former Yugoslavia, and Romania), Africa (Kenya, Rwanda, Guinea, Algeria, Som...

Ethambutol (EMB), a frontline antituberculous drug, targets the mycobacterial cell wall, a unique structure among prokaryotes which consists of an outer layer of mycolic acids covalently bound to peptidoglycan via the arabinogalactan. EMB inhibits the polymerization of cell wall arabinan, and results in the accumulation of the lipid carrier decaprenol phosphoarabinose, which suggests that the drug interferes with the transfer of arabinose to the cell wall acceptor. Unfortunately, resistance to EMB has been described in up to 4% of clinical isolates of Mycobacterium tuberculosis and is prevalent among isolates from patients with multidrug-resistant tuberculosis. We used resistance to EMB as a tool to identify genes participating in the biosynthesis of the mycobacterial cell wall. This approach led to the identification of the embCAB gene cluster, recently proposed to encode for mycobacterial arabinosyl transferases. Resistance to EMB results from an accumulation of genetic events determining overexpression of the Emb protein(s), structural mutation in EmbB, or both. Further characterization of these proteins might provide information on targets for new chemotherapeutic agents and might help development of diagnostic strategies for the detection of resistant M. tuberculosis.

The Bordetella BvgAS sensory transduction system has traditionally been viewed as controlling a transition between two distinct phenotypic phases: the Bvg+ or virulent phase and the Bvg− or avirulent phase. Recently, we identified a phenotypic phase of Bordetella bronchiseptica that displays reduced virulence in a rat model of respiratory infection concomitant with increased ability to survive nutrient deprivation. Characterization of this phase, designated Bvg‐intermediate (Bvgi), indicated the presence of antigens that are maximally, if not exclusively, expressed in this phase and therefore suggested the existence of a previously unidentified class of Bvg‐regulated genes. We now report the identification and characterization of a Bvgi phase protein, BipA (Bvg‐intermediate phase protein A), and its structural gene, bipA. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis indicates that bipA is expressed maximally under Bvgi phase conditions and thus represents the first identified Bvgi phase gene. bipA encodes a 1578‐amino‐acid protein that shares amino acid sequence similarity at its N‐terminus with the proposed outer membrane localization domains of intimin (Int) of enteropathogenic and enterohaemorrhagic Escherichia coli and invasin (Inv) of Yersinia spp. Although not apparent at the amino acid level, BipA is also similar to Int and Inv in that the proposed membrane‐spanning domain is followed by several 90‐amino‐acid repeats and a distinct C‐terminal domain. Localization studies using an antibody directed against the C‐terminus of BipA indicated that its C‐terminus is exposed on the bacterial cell surface. Western blot analysis with this same antibody indicated that BipA homologues are expressed in Bvgi phase Bordetella pertussis and Bordetella parapertussis. Comparison of a ΔbipA strain with wild‐type B. bronchiseptica indicated that BipA is not required for Bvgi phase‐specific aggregative adherence to rat lung epithelial cells in vitro or for persistent colonization of the rabbit respiratory tract in vivo. However, our data are consistent with the hypothesis that BipA, and the Bvgi phase in general, play an important role in the Bordetella infectious cycle, perhaps by contributing to aerosol transmission.

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Ethambutol [(S,S')-2,2'-(ethylenediimino)di-1-butanol; EMB], is a first-line drug used to treat tuberculosis. To gain insight into the molecular basis of EMB resistance, we characterized the 10-kb embCAB locus in 16 EMB-resistant and 3 EMB-susceptible genetically distinct Mycobacterium tuberculosis strains from diverse localities by automated DNA sequencing and single-stranded conformation polymorphism analysis. All 19 organisms had virtually identical sequences for the entire 10-kb region. Eight EMB-resistant organisms had mutations located in codon 306 of embB that resulted in the replacement of the wild-type Met residue with Ile or Val. Automated sequence analysis of the 5' region (1,892 bp) of embB in an additional 69 EMB-resistant and 30 EMB-susceptible M. tuberculosis isolates from diverse geographic localities and representing 70 distinct IS6110 fingerprints confirmed the unique association of substitutions in amino acid residue 306 of EmbB with EMB resistance. Six other embB nucleotide substitutions resulting in four amino acid replacements were uniquely found in resistant strains. Sixty-nine percent of epidemiologically unassociated EMB-resistant organisms had an amino acid substitution not found in susceptible strains, and most (89%) replacements occurred at amino acid residue 306 of EmbB. For strains with the Met306Leu or Met306Val replacements EMB MICs were generally higher (40 microg/ml) than those for organisms with Met306Ile substitutions (20 microg/ml). The data are consistent with the idea that amino acid substitutions in EmbB alter the drug-protein interaction and thereby cause EMB resistance.

Two genes (rpsL and rrs) with mutations associated with streptomycin resistance in Mycobacterium tuberculosis were characterized in 78 streptomycin-resistant and 61 streptomycin-susceptible isolates recovered from patients living in the United States, South America, Europe, Africa, and Asia. Fifty-four percent of the 78 resistant organisms had missense mutations in codon 43 of rpsL resulting in a K-43-->R substitution. Mutations in codon 88 of rpsL were also identified in four Asian isolates.

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The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin ␣ v ␤ 3 (also known as the vitronectin receptor) or ␣ IIb ␤ 3 (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin ␣ IIb ␤ 3 . Defined ␤ 3 mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing ␣ v ␤ 3 and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of hostpathogen interactions.Group A Streptococcus (GAS) is a human pathogenic bacterium that causes diverse infections ranging in severity from relatively mild pharyngitis to life-threatening toxic shock syndrome and necrotizing fasciitis (1). GAS is composed of a heterogeneous array of chromosomal genotypes, and substantial levels of allelic variation also exist in genes encoding putative and proven virulence factors that mediate hostpathogen interactions (2-5). With the exception of M protein, whose structural variation helps GAS evade the type-specific immune response of the host (6), there has been little investigation of the potential ramifications of GAS allelic variation for host-pathogen interactions.GAS isolates produce a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB) (reviewed in ref. 5). SpeB is initially expressed as a 40-kDa zymogen but then is converted to a 28-kDa active protease by proteolytic truncation (5, 7). SpeB is a critical virulence factor in two mouse models of invasive disease (8, 38) and human infections (5). Purified SpeB causes a cytopathic effect on cultured human endothelial cells (3) and has been shown to activate a host matrix metalloprotease (9).Integrins are heterodimeric membrane proteins located on the surface of mammalian cells that participate in ce...