Staphylococcus aureus Protein A (spa) Typing

Staphylococcus aureus Protein A (spa) Typing

Staphylococcus aureus Protein A
(spa) Typing
Senior scientist Henrik Hasman, National Food Institute-DTU
Henrik Hasman
[email protected]
 +45 35 88 63 47
Typing method for S. aureus
•
Gold standard - Phage typing and PFGE
– Phage and Band based
– Labourious: 2-6 days
– Analysis is subjective and can give non-typable results
– Communication of data is problematic
•
DNA sequence-based methods
– Standardized protocol: 1-2 days
– Analysis is unambiguous
– Sequence data can easily be transferred
– Eg. MLST, coa, spa successfully used for S. aureus
• MLST is not suitable for routine surveillance of MRSA
– High cost and access to a high-throughput DNA sequencing
facility
The discriminative power of PFGE is generally higher than most DNA-based methods
Protein A (spa) in S. aureus
•
•
2150bp
Surface protein
– Virulence factor
• Binds to IgG via Fc-binding domain
– Reduce phagocytosis
– X-region-variable number of repeats
– Highly polymorphic
» Deletion and duplication of repeats, and point mutations
What is a repeat?
•
•
•
Multiple copies of the same nucleotide sequence
– Tandem vs. interspersed
– Tandem:
-------------------- HENRIKHeNRIKHEnRIKHeNRIK--------------– Interspersed
Region X
spontaneous point mutations, loss and/or gain of repeats
Variations in the number of repeats
DNA polymerase slippage and various recombination events
Variations in individual repeats are a result of mutagenesis
298 repeats known today (April 19. 2009)
21-30nt (encode triplet codon)
Repeats are assigned an numerical code (in Ridom/Bionummerics)
spa type is deduced from the sequence and order of specific repeats
Spa-typing of S. aureus
PCR and sequencing
RCAMCAAAA
TAYATGTCGT
SL r04
r20
r17
r20
r34 SR
24 nt repeats (21 to 30 nt)
GAGGAAGACAATAACAAGCCTGGT
AAAGAAGACAACAACAAACCTGGC
r04r20r17r20r34 = t2906
r04
r20
The SPA principle
How to translate sequence into SPA type?
•
Manually (NOT recomended)
– Identify SL and SR
– Identify repeats
– Compare to list of all known repeat sequences
•
Computer-based assignment
– At least two software solutions exists (Ridom and Bionumerics)
– Both are quiet expensive (3000+ Euros), but you might already have
Bionumerics installed. Then the spa module is a free add-on.
•
Or you might find a collaborating institute or hospital who has one of
these programs installed. Otherwise contact your CRL (me) for help.
Bionumerics v4.61
Examples of related SPA types
Genetic
events
t034 r08r16r02r25r02r25r34
r24r25
t571 r08r16r02r25r02r25r34
r25
t011 r08r16r02r25
r34
r24r25
t108 r08r16r02r25
r24r25
t1255 r08r16
r34
r24r25
t1793 r08r16r02r25r02r25r34r24r24r25
t2876 r08r16r02r25r02r25r
24r25
t1333 r15r12r16r34
t899 r07r16
1
2
1
2
1
1
r02r25r17r24
>8
r23r02r34
>5
M
L
S
T
Examples of pittfalls in relating SPA types
t528 r04
6,72,12,43,49,67,59
ST151 CC151
t524 r04r17
18,1,1,1,1,5,3
ST71
t529 r04r34
6,72,12,43,49,67,59
ST151 CC151
CC97
SPA typing vs MLST typing
•
SPA typing has in general higher discriminative power than MLST
and lower discriminative power than PFGE!
-
This means that many SPA types can belong to the same MLST
type (also called Sequence Type – ST).
-
Furthermore, closely related Sequence Types can be organized
into so-called Clonal Complexes (CC’s).
t011
t034
t108
t571
t1255
t1793
t2876
ST398
ST804
ST1067
ST752
ST1232
ST621
CC398
SPA typing vs MLST typing
•
In far the most cases, a SPA type will always belong to a certain
Sequence Type.
•
However, a few deviations from this rule exists! The SPA type
t899 has been shown to belong to both ST9 and ST398.
QUESTIONS???
AFTER THE BREAK
The magical world of MLST typing….
Multi Locus Sequence Typing
(MLST)
Senior scientist Henrik Hasman, National Food Institute-DTU
Henrik Hasman
[email protected]
 +45 35 88 63 47
CC398
S. aureus isolates can in most cases be allocated
into Clonal Complexes based on their Sequence type
(at least human isolates…).
MLST….S(equence) T(ype)….C(lonal) C(omplex)????
•
MLST is performed to obtain the Sequence Type (ST)
•
The ST can often be associated with certain CC’s
•
If not, they are called “Singletons”
•
S. aureus has 10-15 major (human) CC’s
•
Each CC has a ST as founder and many ST’s as members
•
MLST is performed by sequencing 7 “household genes”
•
These are selected based on their “molecular clock”
•
MLST can not substitute PFGE as they have different
molecular clocks.
Genes which are sequenced in the MLST
• arc (Carbamate kinase)
• aro (Shikimate dehydrogenase)
• glp (Glycerol kinase)
• gmk (Guanylate kinase)
• pta (Phosphate acetyltransferase)
• tpi (Triosephosphate isomerase)
• yqi (Acetyle coenzyme A acetyltransferase)
The MLST principle
arcC
Primer 1797 (100.0%)
Primer 1798 (100.0%)
Primer 1809 (100.0%)
Primer 1810 (100.0%)
S. aureus MRSA252 BX571856
2902619 bp
yqiL
Primer 1805 (100.0%)
Primer 1806 (100.0%)
Primer 1807 (95.7%)
Primer 1808 (100.0%)
aroE
Primer 1799 (100.0%)
Primer 1803 (95.0%)
Primer 1800 (95.7%)
Primer 1804 (100.0%)
Primer 1801 (100.0%)
Primer 1802 (95.7%)
gmk
glpF
pta
tpi
Primers and PCR conditions
http://saureus.mlst.net/misc/info.asp
The MLST principle
MLST allele sizes:
Gene
arcC:
aroE:
glpF:
gmk:
pta:
tpi:
yqiL:
Size
456 bp
456 bp
465 bp
429 bp
474 bp
402 bp
516 bp
How many different alleles?
•
•
•
•
•
•
•
•
Gene
arcC:
aroE:
glpF:
gmk:
pta:
tpi:
yqiL:
Number of Alleles
109
151
118
84
112
117
108
The MLST principle
A
A
A
A
T
C
A
A
T
C
A
A
T
C
T
C
G
C
A
G
C
A
T
T
T
T
T
T
A
A
C
T
A
A
C
T
G
T
G
A
A
A
A
A
A
T
G
A
A
T
A
T
G
A
A
T
A
G
T
G
A
T
A
G
A
A
C
G
A
T
A
G
A
A
C
T
G
T
G
T
A
T
A
G
G
C
A
C
A
A
G
C
A
C
A
A
T
C
G
T
C
G
T
T
A
C
A
C
G
T
A
C
A
C
G
T
G
T
G
T
3833
0
127 128
129 130 131 132
133 134
135 136 137
T
138
139 140
141 142 143 144
145 146
147 148 149
150 151 152 153 154 155
G
T
156
157
158 159 160 161 162 163 164 165
166 167
168 169
G
170
171 172 173 174 175 176
177 178 179
T
180
181 182 183 184 185 186
187 188
T
189
Fragment
MRSA- 9B1797_179
8- 1797
The MLST principle
The MLST principle
Delete/trim
Fragment with required length!
MLST Databases
http://saureus.mlst.net/
MLST Databases
MLST Databases
MLST Databases
MLST Databases
MLST Databases
MLST profile:
Gene
arcC:
aroE:
glpF:
gmk:
pta:
tpi:
yqiL:
Allelle
108
13
1
1
12
11
13
Finding the Sequence Type (ST):
Finding the Sequence Type (ST):
Finding the Sequence Type (ST):
MLST – the fast version….
CLCbio DNA workbench + MLST module
s
o
r
u
E
0
0
0
3
0
0
5
1
Results
:
s
t
s
o
C
Drag’n Drop sequences
http://eburst.mlst.net/
Requires Java running on the computer
aroE
arcC
CC398
CC8
CC5
Known members of Clonal Complex 398
•
•
•
•
•
•
•
•
•
•
•
•
ST398 (founder)
ST804
ST1067
ST752
ST1232
ST621
ST753
ST291
ST813
ST1066
ST1277
ST1112
Thank you for your attention
Questions and remarks?