Benfang Lei

Genome sequence of a serotype M3 strain of group A Streptococcus: Phage-encoded toxins, the high-virulence phenotype, and clone emergence

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group A Streptococcus (GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares ≈1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A2 (designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are made in vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encoded speK and sla genes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.

Action Mechanism of Antitubercular Isoniazid ACTIVATION BY MYCOBACTERIUM TUBERCULOSIS KatG, ISOLATION, AND CHARACTERIZATION OF InhA INHIBITOR

Activation of the antitubercular isoniazid (INH) by the Mycobacterium tuberculosis KatG produces an inhibitor for enoyl reductase (InhA). The mechanism for INH activation remains poorly understood, and the inhibitor has never been isolated. We have purified the InhA-inhibitor complex generated in the M. tuberculosis KatG-catalyzed INH activation. The complex exhibited a 278-nm absorption peak and a shoulder around 326 nm with a characteristicA 326/A 278 ratio of 0.16. The complex was devoid of enoyl reductase activity. The inhibitor noncovalently binds to InhA with a K d < 0.4 nm and can be dissociated from denatured InhA for chromatographic isolation. The free inhibitor showed absorption peaks at 326 (ε326 6900 m −1cm−1) and 260 nm (ε260 27,000m −1 cm−1). The inactive complex can be reconstituted from InhA and the isolated inhibitor. The InhA inhibitor from the KatG-catalyzed INH activation was identical to that from a slow, KatG-independent, Mn2+-mediated reaction based on high pressure liquid chromatography analysis and absorption and mass spectral characteristics. By monitoring the formation of the InhA-inhibitor complex, we have found that manganese is not essential to the INH activation by M. tuberculosis KatG. Furthermore, the formation of the InhA inhibitor in the KatG reaction was independent of InhA.

Genome-wide protective response used by group A Streptococcus to evade destruction by human polymorphonuclear leukocytes

Group A Streptococcus (GAS) evades polymorphonuclear leukocyte (PMN) phagocytosis and killing to cause human disease, including pharyngitis and necrotizing fasciitis (flesh-eating syndrome). We show that GAS genes differentially regulated during phagocytic interaction with human PMNs comprise a global pathogen-protective response to innate immunity. GAS prophage genes and genes involved in virulence, oxidative stress, cell wall biosynthesis, and gene regulation were up-regulated during PMN phagocytosis. Genes encoding novel secreted proteins were up-regulated, and the proteins were produced during human GAS infections. We discovered an essential role for the Ihk-Irr two-component regulatory system in evading PMN-mediated killing and promoting host–cell lysis, processes that would facilitate GAS pathogenesis. Importantly, the irr gene was highly expressed during human GAS pharyngitis. We conclude that a complex pathogen genetic program circumvents human innate immunity to promote disease. The gene regulatory program revealed by our studies identifies previously undescribed potential vaccine antigens and targets for therapeutic interventions designed to control GAS infections.

Identification of New Candidate Vaccine Antigens Made by Streptococcus pyogenes: Purification and Characterization of 16 Putative Extracellular Lipoproteins

Putative extracellular lipoproteins made by group A Streptococcus (GAS) are the focus of this study, which was designed to identify new candidate vaccine antigens. Bioinformatic analysis of a serotype M1 GAS strain identified 30 open-reading frames encoding putative lipoproteins. The genes encoding the mature form of 29 of these proteins were cloned, and 16 recombinant proteins were overexpressed in Escherichia coli and purified to apparent homogeneity. The genes encoding these 16 proteins were highly conserved in GAS strains for which genome sequence data are available (serotypes M1, M3, M5, M12, M18, and M28). Mice inoculated subcutaneously with GAS and humans with GAS pharyngitis and invasive infections seroconverted to most of the 16 recombinant proteins, which indicates that these lipoproteins were produced during infection. The blood of mice actively immunized with 5 of the 16 recombinant proteins had significantly (P<.05) increased growth-inhibitory activity, compared with the blood of unimmunized mice, which identified these proteins as potential new vaccine candidates.

Mechanism of reduced flavin transfer from Vibrio harveyi NADPH-FMN oxidoreductase to luciferase.

The mechanisms of reduced flavin transfer in biological systems are poorly understood at the present. The Vibrio harveyi NADPH-FMN oxidoreductase (FRP) and the luciferase pair were chosen as a model for the delineation of the reduced flavin transfer mechanism. FRP, which uses FMN as a cofactor to mediate the reduction of the flavin substrate by NADPH, exhibited a ping-pong kinetic pattern with a Km, FMN of 8 microM and a Km,NADPH of 20 microM in a single-enzyme spectrophotometric assay monitoring the NADPH oxidation. However, the kinetic mechanism of FRP was changed to a sequential pattern with a Km,FMN of 0.3 microM and a Km,NADPH of 0.02 microM in a luciferase-coupled assay measuring light emission. In contrast, the Photobacterium fischeri NAD(P)H-FMN oxidoreductase FRG showed the same ping-pong mechanism in both the single-enzyme spectrophotometric and the luciferase-coupled assays. Moreover, for the FRP, FMN at concentrations over 2 microM significantly inhibited the coupled reaction in both light intensity and quantum yield, and showed apparent noncompetitive and competitive inhibition patterns against NADPH and luciferase, respectively. No inhibition of the NADPH oxidation was detected under identical conditions. These results are consistent with a scheme that the reduced flavin cofactor of FRP is preferentially utilized by luciferase for light emission, the reduced flavin product generated by the reductase is primarily channeled into a dark oxidation, and luciferase competes against flavin substrate in reacting with the FRP reduced flavin cofactor. An FRP derivative containing 2-thioFMN as the cofactor was also used to further examine the mechanism of flavin transfer. Results again indicate a preferential utilization of the reductase reduced flavin cofactor by luciferase for the bioluminescence reaction.

Isoniazid Activation Defects in Recombinant Mycobacterium tuberculosis Catalase-Peroxidase (KatG) Mutants Evident in InhA Inhibitor Production

ABSTRACT Mycobacterium tuberculosis KatG catalyzes the activation of the antitubercular agent isoniazid to yield an inhibitor targeting enoyl reductase (InhA). However, no firm biochemical link between many KatG variants and isoniazid resistance has been established. In the present study, six distinct KatG variants identified in clinical Mycobacterium tuberculosis isolates resistant to isoniazid were generated by site-directed mutagenesis, and the recombinant mutant proteins (KatGA110V, KatGA139P, KatGS315N, KatGL619P, KatGL634F, and KatGD735A) were purified and characterized with respect to their catalase-peroxidase activities (in terms of kcat/Km), rates of free-radical formation from isoniazid oxidation, and, moreover, abilities to activate isoniazid. The A110V amino acid replacement did not result in significant alteration of KatG activities except that the peroxidase activity was enhanced. The other mutations, however, resulted in modestly reduced catalase and peroxidase catalytic efficiencies and, for the four mutants tested, significantly lower activities to oxidize isoniazid. Compared to the wild-type enzyme, the ability of the KatGL634F, KatGA139P, and KatGD735A variants to activate isoniazid decreased by 36%, 76%, and 73%, respectively, whereas the KatGS315N and KatGL619P variants completely lost their abilities to convert isoniazid into the InhA inhibitor. In addition, the inclusion of exogenous Mn2+ to the isoniazid activation reaction mix significantly improved the ability of wild-type and KatG mutants to produce the InhA inhibitor.

Opsonophagocytosis-inhibiting Mac protein of group A Streptococcus: Identification and characteristics of two genetic complexes

ABSTRACT Recently, it was reported that a streptococcal Mac protein (designated Mac5005) made by serotype M1 group A Streptococcus (GAS) is a homologue of human CD11b that inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear leukocytes (PMNs) (B. Lei, F. R. DeLeo, N. P. Hoe, M. R. Graham, S. M. Mackie, R. L. Cole, M. Liu, H. R. Hill, D. E. Low, M. J. Federle, J. R. Scott, and J. M. Musser, Nat. Med. 7:1298-1305, 2001). To study mac variation and expression of the Mac protein, the gene in 67 GAS strains representing 36 distinct M protein serotypes was sequenced. Two distinct genetic complexes were identified, and they were designated complex I and complex II. Mac variants in each of the two complexes were closely related, but complex I and complex II variants differed on average at 50.66 ± 5.8 amino acid residues, most of which were located in the middle one-third of the protein. Complex I Mac variants have greater homology with CD11b than complex II variants. GAS strains belonging to serotypes M1 and M3, the most abundant M protein serotypes responsible for human infections in many case series, have complex I Mac variants. The mac gene was cloned from representative strains assigned to complexes I and II, and the Mac proteins were purified to apparent homogeneity. Both Mac variants had immunoglobulin G (IgG)-endopeptidase activity. In contrast to Mac5005 (complex I), Mac8345 (complex II) underwent autooxidation of its cysteine residues, resulting in the loss of IgG-endopeptidase activity. A Mac5005 Cys94Ala site-specific mutant protein was unable to cleave IgG but retained the ability to inhibit IgG-mediated phagocytosis by human PMNs. Thus, the IgG-endopeptidase activity was not essential for the key biological function of Mac5005. Although Mac5005 and Mac8345 each have an Arg-Gly-Asp (RGD) motif, the proteins differed in their interactions with human integrins αvβ3 and αIIbβ3. Binding of Mac5005 to integrins αvβ3 and αIIbβ3 was mediated primarily by the RGD motif in Mac5005, whereas binding of Mac8345 involved the RGD motif and a region in the middle one-third of the molecule whose sequence is different in Mac8345 and Mac5005. Taken together, the data add to the emerging theme in GAS pathogenesis that allelic variation in virulence genes contributes to fundamental differences in host-pathogen interactions among strains.

Characterization of an extracellular virulence factor made by group A Streptococcus with homology to the Listeria monocytogenes internalin family of proteins.

ABSTRACT Leucine-rich repeats (LRR) characterize a diverse array of proteins and function to provide a versatile framework for protein-protein interactions. Importantly, each of the bacterial LRR proteins that have been well described, including those of Listeria monocytogenes, Yersinia pestis, and Shigella flexneri, have been implicated in virulence. Here we describe an 87.4-kDa group A Streptococcus (GAS) protein (designated Slr, for streptococcal leucine-rich) containing 10 1/2 sequential units of a 22-amino-acid C-terminal LRR homologous to the LRR of the L. monocytogenes internalin family of proteins. In addition to the LRR domain, slr encodes a gram-positive signal secretion sequence characteristic of a lipoprotein and a putative N-terminal domain with a repeated histidine triad motif (HxxHxH). Real-time reverse transcriptase PCR assays indicated that slr is transcribed abundantly in vitro in the exponential phase of growth. Flow cytometry confirmed that Slr was attached to the GAS cell surface. Western immunoblot analysis of sera obtained from 80 patients with invasive infections, noninvasive soft tissue infections, pharyngitis, and rheumatic fever indicated that Slr is produced in vivo. An isogenic mutant strain lacking slr was significantly less virulent in an intraperitoneal mouse model of GAS infection and was significantly more susceptible to phagocytosis by human polymorphonuclear leukocytes. These studies characterize the first GAS LRR protein as an extracellular virulence factor that contributes to pathogenesis and may participate in evasion of the innate host defense.

Toward a genome-scale understanding of group A Streptococcus pathogenesis

Recent significant contributions have been made to the understanding of Group A Streptococcus (GAS) pathogenesis. New regulatory pathways have been discovered, insight into the molecular basis of epidemics of serotype M1 disease has been obtained, the crystal structures of four toxins have been reported and a genome sequence of one GAS strain has been determined. Genome-scale approaches to the study of GAS pathogenesis are now rapidly emerging and will advance our fundamental understanding of the molecular basis of host-pathogen interactions.

Histidine and aspartic acid residues important for immunoglobulin G endopeptidase activity of the group A Streptococcus opsonophagocytosis-inhibiting Mac protein

ABSTRACT The secreted Mac protein made by serotype M1 group A Streptococcus (GAS) (designated Mac5005) inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear neutrophils. This protein also has cysteine endopeptidase activity against human immunoglobulin G (IgG). Site-directed mutagenesis was used to identify histidine and aspartic acid residues important for Mac IgG endopeptidase activity. Replacement of His262 with Ala abolished Mac5005 IgG endopeptidase activity. Asp284Ala and Asp286Ala mutant proteins had compromised enzymatic activity, whereas 21 other Asp-to-Ala mutant proteins cleaved human IgG at the apparent wild-type level. The results suggest that His262 is an active-site residue and that Asp284 and Asp286 are important for the enzymatic activity or structure of Mac protein. These Mac mutants provide new information about structure-activity relationships in this protein and will assist study of the mechanism of inhibition of opsonophagocytosis and killing of GAS by Mac.