6C

Discovery of Streptococcus pneumoniae serogroup 6 variants

with bi-specific WciNα

MB Oliver1, MPG van der Linden2, SA Küntzel2, J Saad1, MH Nahm1,3 1Department of Microbiology and 3Department of Pathology, Univ. of Alabama at Birmingham, USA, 2German National Reference

Center for Streptococci Department of Medical Microbiology, University Hospital RWTH Aachen, Germany

Mark van der Linden

University Hospital RWTH Aachen

Institute of Medical Microbiology

National Reference Center for Streptococci

Pauwelsstrasse 30, 52074 Aachen, Germany

Tel: +49 241 808 9946

Fax: +49 241 808 2483

Email: mlinden@ukaachen.de

Background: Streptococcus pneumoniae (pneumococcus) is a persistent,

opportunistic commensal of the human nasopharynx and is the leading cause of

community-acquired pneumonia. It expresses an anti-phagocytic capsular

polysaccharide (PS). Genetic variation of the capsular polysaccharide synthesis

(cps) locus is the molecular basis for structural and antigenic heterogeneity of the

capsule types (serotypes). Serogroup 6 has 4 known members (6A, 6B, 6C, and 6D)

with distinct serological properties, homologous cps loci and structurally similar PSs.

cps of serotypes 6A and 6B have wciNα, encoding α-1,3-galactosyl-transferase

whereas serotypes 6C and 6D have wciNβ encoding α-1,3-glucosyl-transferase.

Methods and results: Recently discovered in Germany were two atypical serogroup

6 isolates named 6X11 and 6X12. Using serogroup 6-specific mouse monoclonal

antibodies, 6X11 was found to have serologic properties of both 6B and 6D, whereas

6X12 has 6A and 6C. Studies of their capsular PSs with NMR methods revealed that

6X12 PS had two different repeating units; about 75% was that of 6A PS and about

25% was that of 6C PS. Similarly, 6X11 PS was found to contain two different

repeating units; about 40% of 6B and 60% of 6D. Genetic studies of the two strains

revealed mutations in their wciNα. 6X12 had one mutation (A150T) and 6X11 had

two mutations (D38N and A150T). Using site-directed mutagenesis, A150T mutation

but not D38N mutation was found to make a 6A strain acquire hybrid serologic and

chemical profiles like 6X12. The hybrid serotypes represented by 6X12 and 6X11

strains were named serotypes 6E and 6F.

Conclusions: Single amino acid changes in cps genes encoding

glycosyltransferases can alter substrate specificities, permit biosynthesis of

heterogeneous capsule repeating units, and result in new hybrid capsule types that

may differ in their interaction with the host immune system.

WciNβ

6A 2)-α-D-Galp-(13)-α-D-Glcp-(13)-α-L-Rhap-(13)-D-Rib-ol-(5P

6B 2)-α-D-Galp-(13)-α-D-Glcp-(13)-α-L-Rhap-(14)-D-Rib-ol-(5P

6C 2)-α-D-Glc’p-(13)-α-D-Glcp-(13)-α-L-Rhap-(13)-D-Rib-ol-(5P

6D 2)-α-D-Glc’p-(13)-α-D-Glcp-(13)-α-L-Rhap-(14)-D-Rib-ol-(5P

WciNα WchA WciP WciO

ABSTRACT ABSTRACT

CONCLUSIONS

• 6X12 and 6X11 capsular PSs are structural hybrids with two different

repeating units:

• 6X12 has a ratio of ~75:25 6A:6C PS

• 6X11 has a ratio of ~40:60 6B:6D PS

• 6X12 WciNα has one mutation (A150T) and 6X11 WciNα has two mutations

(D38N and A150T)

• WciNα codon 150, not 38, mediates substrate specificity

• Thr150 is bi-specific for two different donor sugar substrates (UDP-Gal and

UDP-Glc)

• WciNα residues 149-151 are ligand binding residues

• Alignment of this peptide region with protein family 01501 members revealed a

conserved “FVNXGV” motif, with X being Ala, Thr or Ser. Studies of N.

meningitidis LgtC and human glycogenin-1 revealed the N of this motif is

involved coordinating the “C1” of the donor sugar (Persson et al, 2001).

• We propose to rename 6X12 and 6X11 serotypes 6F and 6G, respectively.

DXD

103-105

D38 A150

WciNα Residue Substrate for Residue 2

Serotype 38 150 UDP-Gal UDP-Glc

6A/6B Asp Ala 100 % 0 %

6X12 (6F) Asp Thr 75 % 25 %

6X11 (6G) Asn Thr 40 % 60 %

A

B

Figure 8. A: Proposed models of 6X12 and 6X11 capsular PS RUs. B:

Predicted secondary structure of WciNα using PHYRE2 modeling program. The

enzyme’s 22 ligand binding residues are shown in red, while residues 38 and

150 are shown in yellow. The DXD motif is commonly found in type A glycosyl-

transferases.

Figure 1. Flow cytometric histograms of various

pneumococcal strains stained with serogroup-6 specific anti-

capsule mAbs.

TIGR6A

TIGR6B

TIGR6C

TIGR6D

6X11

6X12

Isolates shared serologic properties of serogroup 6

TIGR6A

TIGR6B

TIGR6C

TIGR6D

6X11

6X12

Hyp6BM810

110

210

310

40

50

100

1

0

50

100

Hyp6AM3

1 101

102

103

104

Hyp6AG1

1 101

102

103

104

Hyp6BM1

101

102

103

104

1

Hyp6DM5

101

102

103

104

1

0

50

100

0

50

100

0

50

100

0

50

100

6X12 and 6X11 PSs were unique

Figure 2. 1H-NMR spectra of the anomeric

region for 6X12, 6X11 and serogroup 6 PSs.

Vertical line drawn as a reference point for

comparison.

Rha Glc

Glc

RhaGal

Glc’

Chemical shift (ppm)

5.05.6 5.45.8 5.2

ATCC 6B

MNZ21 6D

6X11

SSI 6A

SSI 6C

6X12

C

6X12 and 6X11 express two different capsular PS repeating units

Figure 3. Overlay of 1H-13C HMQC spectra of PSs representing 6A and 6X12

(A) or 6B and 6X11 (B). For 6X12 and 6X11, new glucose (Glc’) signals

appear indicating their PSs are a mixture of two different repeating units (RU).

C: Proposed structural PS RU models of 6X12 and 6X11.

A

B

70

80

3.63.84.04.25.05.25.45.6

gal.h1

glc’.h1

6096

98

100

102

13C

(p

pm

)

13C

(p

pm

)

6A

6X12

6A

6X12

glc.h1

rha.h1

rha.h2 rib.h5

gal.h5

gal.h2

gal.h3

rib.h4glc’.h5

gal.h4

rib.h1

gal.h6

glc.h6glc’.h6

glc.h4 glc.h2

rha.h4

glc’.h4

glc.h3rib.h3

rha.h3glc’.h2

glc’.h3

rib.h2 glc.h5

rha.h5

3.63.84.04.25.05.25.45.6

96

98

100

102

70

80

60

1H (ppm)1H (ppm)

13C

(p

pm

)

13C

(pp

m)

6B

6X11

6B

6X11

glc’.h1

gal.h1

glc.h1

rha.h1rib.h5

rha.h2

gal.h5

gal.h2

rib.h4glc.h3

glc’.h2rha.h3

gal.h4

gal.h3

glc’.h5 glc.h5

Trisgal.h6

glc.h6glc’.h6

rib.h1

rha.h5glc.h4 glc.h2

rha.h4

glc’.h4rib.h3

glc’.h3

rib.h2

6X12 6A 2)-α-D-Galp-(13)-α-D-Glcp-(13)-α-L-Rhap-(13)-D-Rib-ol-(5P 75 %

6C 2)-α-D-Glc’p-(13)-α-D-Glcp-(13)-α-L-Rhap-(13)-D-Rib-ol-(5P 25 %

6X11 6B 2)-α-D-Galp-(13)-α-D-Glcp-(13)-α-L-Rhap-(14)-D-Rib-ol-(5P 40 %

6D 2)-α-D-Glc’p-(13)-α-D-Glcp-(13)-α-L-Rhap-(14)-D-Rib-ol-(5P 60 %

INTRODUCTION

Two aberrant Serogroup 6 isolates found

Among isolates from invasive pneumococcal disease in Germany, two isolates were found that clearly belonged to serogroup 6,

but could not be assigned any of the serotypes 6A, B, C or D. The pneumococci were isolated from blood from two German

males aged 70 and 73 years, in 2006 and 2011 respectively. Information on diagnosis was not available.

Figure 5. Comparison of German strains’ cps loci to canonical 6A and

6C cps (GenBank Acc. # CR931638 and EF538714 ). 6X12 and 6X11

were respectively 98.9% and 99.9% identical to 6A. The wciN and

wciP alleles are indicated by α and β. The dashed or dark lines

respectively indicate mutations at codons 38 or 150 of WciNα.

Hypothesis:

The two mutations, A150T and D38N, may broaden

WciNα’s specificity from UDP-Gal only to UDP-Gal

and UDP-Glc, and be responsible for the observed

serologic and biochemical changes in 6X12 and 6X11.

German strains have mutated WciNα

6X11 βα

6A α α

6C β α

6X12 α α glf-like

6X11

6X12

6A

Figure 6. Schematic for creation of isogenic mutant strains.

Allelic exchange is described by dashed lines. Primer names

and binding sites shown as black dots. Mutations are

indicated by symbols *, A150T; #, D38N; $, A150S.

Creation of WciNα mutants

Hyp6AG4 bound (MFI)

5.05.6 5.45.8 5.2

Chemical shift (ppm)

MBO172 (A150T)

MBO177 (A150S)

SSI 6A

SSI 6C

6X12

MBO182 (A150T, D38N)

MBO184 (D38N)

10001001011

Hyp6D

M5 b

ound (M

FI)

RhaGlcGal

Glc’

TIGR6C

TIGR6A

MBO184

MBO1726X12

MBO182

MBO177

10

100

1000

WciNα A150T and A150S, but not D38N, alter immunologic and chemical properties

Figure 7. A: Pneumococcal strains were stained with 6A (Hyp6AG4) and 6C (Hyp6DM5) specific mAbs, fluorescence

was measured with a flow cytometer, and the mean fluorescence intensity (MFI) was plotted on both axes. *The

amount of Hyp6AG4 bound to MBO182 was artificially reduced by 20% to provide better visual separation between

MBO182 and MBO177. B: 1H-NMR spectra of the anomeric region for purified mutant capsular PSs.

A B

*

METHODS AND RESULTS

Serogroup 6 is well characterized. Its members (serotypes 6A-6D) have highly homologous cps loci and structurally similar

capsular PS repeating units (RUs). Rha, rhamnose; Rib-ol, phosphoribitol; Gal, galactose; Glc, glucose; Glc’, second Glc

residue in 6C and 6D PS RUs. cps of serotypes 6A/6B have wciNα, encoding α-1,3-galactosyl-transferase whereas serotypes

6C/6D have wciNβ encoding α-1,3-glucosyl-transferase.

The goal of this study was to characterize the two recently discovered serogroup 6 variants.

Figure 4. Antibody bound to ELISA plates (y-axis)

against at various PS concentrations (x-axis).

ELISA plates were coated with a 6C-specific mAb

and detection antibodies were either a 6C/6D

specific mAb (Panel A) or a 6A-specific mAb

(Panel B).

0,0

1,0

2,0

3,0

4,0

None 0.03 0.3 3 30 300 3000

OD

40

5 n

m

6A + 6C mix.

6C 6X12

6D

6A, 6B

0,0

1,0

2,0

3,0

4,0

None 0.03 0.3 3 30 300 3000

OD

40

5 n

m

PS (ng/mL)

6X12

6A, 6B, 6C, 6D 6A + 6C mix.

8th International Symposium on

Pneumococci and Pneumococcal

Diseases - ISPPD-9

Hyderabad, India

March 9-13, 2014

Poster No: OP-213

Isolate Year Patient Serotype MLST aroE gdh gki recP spi xpt ddl Factor sera wciP wciN INDEL

6X11 2006 male, 73y. 6B or 6D 176 7 13 8 6 10 6 14 6c +, 6d + wciPβ wciNα no

6X12 2011 male, 70y. 6A or 6C 681 2 5 9 1 6 19 14 6b +, 6d + wciPα wciNα no