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Serotype M1 group A Streptococcus strains cause epidemic waves of human infections long thought to be mono- or pauciclonal. The gene encoding an extracellular group A Streptococcus protein (streptococcal inhibitor of complement) that inhibits human complement was sequenced in 1,132 M1 strains recovered from population-based surveillance of infections in Canada, Finland and the United States. Epidemic waves are composed of strains expressing a remarkably heterogeneous array of variants of streptococcal inhibitor of complement that arise very rapidly by natural selection on mucosal surfaces. Thus, our results enhance the understanding of pathogen population dynamics in epidemic waves and infectious disease reemergence.

The skeletal muscle Ca2+ release channel (RYR1), which plays a critical role in excitation-contraction coupling, is a homotetramer with a subunit molecular mass of 565 kDa. Oxidation of the channel increases its activity and produces intersubunit cross-links within the RYR1 tetramer (Aghdasi, B., Zhang, J., Wu, Y., Reid, M. B., and Hamilton, S. L. (1997) J. Biol. Chem. 272, 3739-3748). Alkylation of hyperreactive sulfhydryls on RYR1 with N-ethylmaleimide (NEM) inhibits channel function and blocks the intersubunit cross-linking. We used calpain and tryptic cleavage, two-dimensional SDS-polyacrylamide gel electrophoresis, N-terminal sequencing, sequence-specific antibody Western blotting, and [14C]NEM labeling to identify the domains involved in these effects. Our data are consistent with a model in which 1) diamide, an oxidizing agent, simultaneously produces an intermolecular cross-link between adjacent subunits within the RYR1 tetramer and an intramolecular cross-link within a single subunit; 2) all of the cysteines involved in both cross-links are in either the region between amino acids approximately 2100 and 2843 or the region between amino acids 2844 and 4685; 3) oxidation exposes a new calpain cleavage site in the central domain of the RYR1 (in the region around amino acid 2100); 4) sulfhydryls that react most rapidly with NEM are located in the N-terminal domain (between amino acids 426 and 1396); 5) alkylation of the N-terminal cysteines completely inhibits the formation of both inter- and intrasubunit cross-links. In summary, we present evidence for interactions between the N-terminal region and the putatively cytoplasmic central domains of RYR1 that appear to influence subunit-subunit interactions and channel activity.

In order to investigate the role of MBL gene polymorphisms in susceptibility to tuberculosis (TB) we genotyped 487 TB cases and 232 controls. Among African-American individuals, the frequency of B allele was lower among controls than cases (p < 0.01), but we found no differences between cases and controls of white and Hispanic ethnicity.

Thirty-seven multidrug-resistant and 13 pan-susceptible isolates of Mycobacterium tuberculosis were analysed for the diversity of genotypes associated with known drug-resistance mechanisms. The isolates were obtained from patients attending a university tuberculosis clinic in Monterrey, Mexico. A total of 25 IS6110-RFLP patterns were obtained from the multidrug-resistant tuberculosis (MDR-TB) isolates. Approximately 65 % of the MDR-TB isolates were attributed to secondary resistance. Different drug-susceptibility patterns were seen with the clustered isolates. The percentage of isolates resistant to isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STR) was 100, 97.3, 48.7 and 67.6, respectively. The most common resistance-associated polymorphisms for the four drugs were as follows: INH, Ser315Thr (67.6 %) in katG; RIF, Ser450Leu (41.7 %) in rpoB; EMB, Met306Ile/Val/Leu (66.7 %) in embB; and STR, Lys43Arg (24 %) in rpsL. Drug-resistance-associated mutations were similar to changes occurring in isolates from other areas of the world, but unique, previously unreported, mutations in katG (n = 5), rpoB (n = 1) and rrs (n = 3) were also identified.

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. Previous studies have identified resistance-associated mutations in katG , inhA , kasA , ndh , and the oxyR-ahpC intergenic region. DNA microarray-based experiments have shown that INH induces several genes in Mycobacterium tuberculosis that encode proteins physiologically relevant to the drug's mode of action. To gain further insight into the molecular genetic basis of INH resistance, 20 genes implicated in INH resistance were sequenced for INH resistance-associated mutations. Thirty-eight INH-monoresistant clinical isolates and 86 INH-susceptible isolates of M. tuberculosis were obtained from the Texas Department of Health and the Houston Tuberculosis Initiative. Epidemiologic independence was established for all isolates by IS 6110 restriction fragment length polymorphism analysis. Susceptible isolates were matched with resistant isolates by molecular genetic group and IS 6110 profiles. Spoligotyping was done with isolates with five or fewer IS 6110 copies. A major genetic group was established on the basis of the polymorphisms in katG codon 463 and gyrA codon 95. MICs were determined by the E-test. Semiquantitative catalase assays were performed with isolates with mutations in the katG gene. When the 20 genes were sequenced, it was found that 17 (44.7%) INH-resistant isolates had a single-locus, resistance-associated mutation in the katG , mabA , or Rv1772 gene. Seventeen (44.7%) INH-resistant isolates had resistance-associated mutations in two or more genes, and 76% of all INH-resistant isolates had a mutation in the katG gene. Mutations were also identified in the fadE24 , Rv1592c , Rv1772 , Rv0340 , and iniBAC genes, recently shown by DNA-based microarray experiments to be upregulated in response to INH. In general, the MICs were higher for isolates with mutations in katG and the isolates had reduced catalase activities. The results show that a variety of single nucleotide polymorphisms in multiple genes are found exclusively in INH-resistant clinical isolates. These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH.

Streptococcal inhibitor of complement (Sic) is a highly polymorphic extracellular protein made predominantly by serotype M1 group A Streptococcus (GAS). New variants of the Sic protein frequently appear in M1 epidemics as a result of positive natural selection. To gain further understanding of the molecular basis of M1 epidemics, the sic gene was sequenced from 471 pharyngitis and 127 pyogenic and blood isolates recovered from 598 patients living in metropolitan Helsinki, Finland, during a 37-month population-based surveillance study. Most M1 GAS subclones recovered from pyogenic infections and blood were abundantly represented in the pool of subclones causing pharyngitis. Alleles shared among the pharyngitis, pyogenic, and blood samples were identified in throat isolates a mean of 9.8 months before their recovery from pyogenic infections and blood, which indicates that selection of most sic variants occurs on mucosal surfaces. In contrast, no variation was identified in the emm and covR/covS genes.

Serotype M1 group A Streptococcus, the most common cause of invasive disease in many case series, generally have resisted extensive molecular subtyping by standard techniques (e.g., multilocus enzyme electrophoresis, pulsed-field gel electrophoresis). We used automated sequencing of the sic gene encoding streptococcal inhibitor of complement and of a region of the chromosome with direct repeat sequences to unambiguously differentiate 30 M1 isolates recovered from 28 patients in Texas with invasive disease episodes temporally clustered and thought to represent an outbreak. Sequencing of the emm gene was less useful for M1 strain differentiation, and restriction fragment length polymorphism analysis with IS1548 or IS1562 as Southern hybridization probes did not provide epidemiologically useful subtyping information. Sequence polymorphism in the direct repeat region of the chromosome and IS1548 profiling data support the hypothesis that M1 organisms have two main evolutionary lineages marked by the presence or absence of the speA2 allele encoding streptococcal pyrogenic exotoxin A2.