Results for week 1 Dr Mike Dyall-Smith

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Mike Dyall-Smith Review of week 1 results Why

Typing schemes: M, SOF, T antigens emm sequence

(aerotolerant not aerobic) Gram +ve cocci,

) haemolytic on HBA Catalase -ve Lancefield Group

streptococci using phenotypic tests Gram stain

1 Lecture 2. emm sequence typing of GAS Results for week 1 Dr Mike Dyall-Smith Review of week 1 results Why (sero)group and (sero)type streptococci? Typing schemes: M, SOF, T antigens emm sequence typing Beall et al. paper (1996) Classical microbiological identification All isolates were: Grow in air (aerotolerant not aerobic) Gram +ve cocci, forming chains, size (?) haemolytic on HBA Catalase -ve Lancefield Group A (Streptex test) Bacitracin sensitive Penicillin sensitive (others?) Throat swab plate Identification of streptococci using phenotypic tests Gram stain Genus level Species level Mucoid GAS strain Antibiotic Sensitivity Tests Bacitracin resistant Group G Streptococcus sp. Sensitive to all antibiotics Set Up Isolates showing resistance to erythromycin and tetracycline 1

oup streptococci? Group A Group G Lancefield gr

the presence of group A antigen corresponds to S. pyogenes.

2 Lancefield Grouping Tests Why serogroup streptococci? Group A Group G Lancefield grouping was to aid identification Easy, quick test (e.g. Streptex ) Correlates well with species definitions for GAS and GBS (not so well with others) Know structure and location of A antigen In almost all cases the presence of group A antigen corresponds to S. pyogenes. Identification of streptococci using phenotypic tests Genus level Species level Why serotype streptococci? S. pyogenes isolates all belong to Lancefield Group A, but can be further classified into > 100 M-TYPES on the basis on their different M proteins. The method was developed to aid epidemiology, research, and vaccine efforts. Can you associate M-types with diseases? How do you track outbreaks? Antibodies to M protein are protective. M-SEROTYPING of S. pyogenes Classical M-serotyping (M-typing) Before DNA sequencing, M proteins were typed using antibodies. How was this done? Take cells from plate/broth culture Make crude extract of surface material by heating at low ph. Inject into animals to obtain an antiserum Antigen Cross-absorb antiserum to remove crossreactive antibodies. Do precipitation tests by adding cell wall extract and antiserum. Need many different antisera (>100). Ab + Ag 2

3 Classical M-serotyping (M-typing) Ab to strain 1 Ab to strain 2 Ab to strain 3 Ab to strain Strains 1,2,3 are different serotypes. Strains 3,4 are the same serotype. Ag Ab + - Ag Ab Classical M-serotyping (M-typing) ** PROBLEMS ** 1. Cross-reactions! because the antigen is a crude mixture of cell wall molecules, not pure M- proteins. 2. Many serotypes, so need to prepare >100 different antisera, absorb out cross-reacting antibodies from each, and test they are specific. 3. Many strains are M-untypable, ie. Do NOT react with any of the antisera! (particularly sof+) M-like M T sof Classical M-serotyping: was there a better way?? M protein ** PROBLEMS ** While it was possible to M-serotype S.pyogenes isolates, there were no commercially available kits or antibodies. You either made them yourself (very expensive!) or asked a reference centre to give you some (not likely). They didn t cover all the known types. Even if you had most of the typing sera, many were often of weak strength (titre) or showed cross-reactions, or didn t react with certain isolates (M-nontypeable strains) M-like M T sof Type-specific Ab Cross-reactive Ab M-SEROTYPING: a better way?? People looked for a better way to type the M proteins of S. pyogenes One idea was to use PCR amplification and DNA sequencing of the emm gene. But would the known M-serotypes match the sequence-types predicted by DNA sequencing? And how do you define new emm types? M protein - a long way to the top.. M protein is synthesized inside the cell Ends up right on the outside surface Covalently attached at C-terminus to cell wall Highly structured (long, helical filament) DNA sequence does not directly reflect the mature protein. 3

4 Synthesis, export and maturation of M protein N-terminal signal sequence: transport through general secretory pathway (Sec) virr emm5 enn5 scpa 5 3 mrna Overview N-terminus NH3- N-terminal signal peptide removed M-protein Export from cell C-terminus COOH C-terminus modified and attached to cell wall DETAIL Leader peptidase Economou, TIM 1999 N-terminal signal sequence: transport through general secretory pathway (Sec) N-terminal signal sequence: transport through general secretory pathway (Sec) Signal sequence underlined: cleavage b/n aa 41 & 42 >M protein type 1 S. pyogenes 484aa MAKNNTNRHYSLRKLKTGTASVAVALTVLGAGFANQTEVKA/NGDGNPREVIED LAANNPAIQNIRLRYENKDLKARLENAMEVAGRDFKRAEELEKAKQALEDQRKD LETKLKELQQDYDLAKESTSWDRQRLEKELEEKKEALELAIDQASRDYHRATAL EKELEEKKKALELAIDQASQDYNRANVLEKELETITREQEINRNLLGNAKLELD QLSSEKEQLTIEKAKLEEEKQISDASRQSLRRDLDASREAKKQVEKDLANLTAE LDKVKEDKQISDASRQGLRRDLDASREAKKQVEKDLANLTAELDKVKEEKQISD ASRQGLRRDLDASREAKKQVEKALEEANSKLAALEKLNKELEESKKLTEKEKAE LQAKLEAEAKALKEQLAKQAEELAKLRAGKASDSQTPDTKPGNKAVPGKGQAPQ AGTKPNQNKAPMKETKRQLPSTGETANPFFTAAALTVMATAGVAAVVKRKEEN Economou, TIM 1999 C-terminal LPXTG signal: cell wall attachment M-protein C-terminal Anchor to Cell Surface: role of LPXTG signal >NP_ M protein type 1 S. pyogenes, 484 aa MAKNNTNRHYSLRKLKTGTASVAVALTVLGAGFANQTEVKA/NGDGNPREVIED LAANNPAIQNIRLRYENKDLKARLENAMEVAGRDFKRAEELEKAKQALEDQRKD LETKLKELQQDYDLAKESTSWDRQRLEKELEEKKEALELAIDQASRDYHRATAL EKELEEKKKALELAIDQASQDYNRANVLEKELETITREQEINRNLLGNAKLELD QLSSEKEQLTIEKAKLEEEKQISDASRQSLRRDLDASREAKKQVEKDLANLTAE LDKVKEDKQISDASRQGLRRDLDASREAKKQVEKDLANLTAELDKVKEEKQISD ASRQGLRRDLDASREAKKQVEKALEEANSKLAALEKLNKELEESKKLTEKEKAE LQAKLEAEAKALKEQLAKQAEELAKLRAGKASDSQTPDTKPGNKAVPGKGQAPQ AGTKPNQNKAPMKETKRQLPSTGETANPFFTAAALTVMATAGVAAVVKRKEEN LPXTG signal Hydrophobic aa form a lipid anchor Navarre &Schneewind, Microbiol. Molec. Biol. Rev (1999) 63:

5 M protein 60 nm emm gene sequence typing DNA sequence from the 5 end (of coding strand) N-terminus NH3-5 3 emm gene Transcription > translation M-protein COOH JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1996, p Vol. 34, No. 4 Sequencing emm-specific PCR Products for Routine and Accurate Typing of Group A Streptococci SUMMARY BERNARD BEALL, RICHARD FACKLAM, and TERRY THOMPSON 1. emm gene sequencing is an alternative to M-serotyping of GAS 2. Members of the same M-serotype almost always belonged to the same emm-sequence type 3. Six more emm genes were sequenced (increasing the emm sequence-type database) 4. Could now sequence-type the M-nontypeable strains. Variable region at N-terminus conserved C-terminus anchored in cell wall 5. Successfully used sequence typing to track an outbreak of GAS infections in a hospital (single-source). emm gene sequence typing. - CDC definition of an emm type P1 emm gene Old Definition: Greater than or equal to 95% nt identity over the first 160 bases of sequence (using primer 1) with that of a reference emm gene sequence. NEW DEFINITION: New types share less than 92% sequence identity over the first 90 bases encoding the deduced processed M protein of the type reference strain emm gene sequence typing 5 end of gene: TGAGGGTTTTTCCTAAAAAATGATAACATAAGGAGCATAAAA ATG GCT AGA GAA M A R E AAT ACC AAT AAG CAT TAT TGG CTT AGA AAA TTA AAA AAA GGC ACT G... N T N K H Y W L R K L K K G T... *Primer EMM1 (forward) sequence is underlined. Start codon of M protein is in bold. Forward PCR primer EMM1 is within the region of the gene corresponding to the N-terminal Signal Sequence 5

emm gene sequence typing Consensus DNA primer sites relative to M protein sequence of emm gene 3 end of the gene:...1240 1250 1260 1270 1280.

The actual sequence of the primer is the reverse and complement of this (see table on previous page). Stop codon for M protein ORF is further downstream at 1519-1521.

6 emm gene sequence typing Consensus DNA primer sites relative to M protein sequence of emm gene 3 end of the gene: CAA TTA GCA AAA CAA GCT GAA GAA CTT GCA AAA CTA AGA GCT GGA......Q L A K Q A E E L A K L R A G... *Region of primer EMM2 is underlined. The actual sequence of the primer is the reverse and complement of this (see table on previous page). Stop codon for M protein ORF is further downstream at Reverse PCR primer EMM2 is within the region of the gene corresponding to the C-terminal C-repeats, but before the LPXTG signal sequence. >NP_ M protein type 1 S. pyogenes, 484 aa MAKNNTNRHYSLRKLKTGTASVAVALTVLGAGFANQTEVKA/NGDGNPREVIED LAANNPAIQNIRLRYENKDLKARLENAMEVAGRDFKRAEELEKAKQALEDQRKD LETKLKELQQDYDLAKESTSWDRQRLEKELEEKKEALELAIDQASRDYHRATAL EKELEEKKKALELAIDQASQDYNRANVLEKELETITREQEINRNLLGNAKLELD QLSSEKEQLTIEKAKLEEEKQISDASRQSLRRDLDASREAKKQVEKDLANLTAE LDKVKEDKQISDASRQGLRRDLDASREAKKQVEKDLANLTAELDKVKEEKQISD ASRQGLRRDLDASREAKKQVEKALEEANSKLAALEKLNKELEESKKLTEKEKAE LQAKLEAEAKALKEQLAKQAEELAKLRAGKASDSQTPDTKPGNKAVPGKGQAPQ AGTKPNQNKAPMKETKRQLPSTGETANPFFTAAALTVMATAGVAAVVKRKEEN First part of study: For 62 of the 75 isolates, the emm sequences of 1-3 strains of each M-type had!95% sequence identity over the first 160nt with corresponding emm genes in Genbank. Six other M types had emm sequences that matched emm genes of different designations. Second part - typing clinical isolates 77 recent clinical GAS isolates emm sequences obtained from 74 Results in Table

Second part - typing clinical isolates emm sequences of 69 (out of 74) matched known emm-types Those that didn t match, belonged to new emm-types Third part - typing M types previously not sequenced

7 Second part - typing clinical isolates emm sequences of 69 (out of 74) matched known emm-types Those that didn t match, belonged to new emm-types Third part - typing M types previously not sequenced Six 5 emm sequences for known M-serotypes were obtained All showed <95% nt identity to other emmtypes (Table 2) JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1996, p Vol. 34, No. 4 Sequencing emm-specific PCR Products for Routine and Accurate Typing of Group A Streptococci SUMMARY BERNARD BEALL, RICHARD FACKLAM, and TERRY THOMPSON 1. emm gene sequencing is an alternative to M-serotyping of GAS 2. Members of the same M-serotype almost always belonged to the same emm-sequence type 3. Six more emm genes were sequenced (increasing the emm sequence-type database) 4. Could now sequence-type the M-nontypeable strains. 5. Successfully used sequence typing to track an outbreak of GAS infections in a hospital (single-source). Concluding remarks emm sequence typing is the current gold standard for typing GAS isolates You need to know the definition of emm types, and the way emm types are determined. You should know the structure and significance of the M protein (and its gene). emm genes occur in group G and C streptococci, and these organisms can be typed using the same scheme as for GAS. 7

Sequencing emm-specific PCR Products for Routine and Accurate Typing of Group A Streptococci

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1996, p. 953 958 Vol. 34, No. 4 0095-1137/96/$04.00 0 Copyright 1996, American Society for Microbiology Sequencing emm-specific PCR Products for Routine and Accurate