supplementary information evolutionary inactivation of a

1

Supplementary information

Evolutionary inactivation of a sialidase in group B Streptococcus

Masaya Yamaguchia, b, *, Yujiro Hirosea, c, Masanobu Nakataa, Satoshi Uchiyamab, Yuka

Yamaguchib, Kana Gotoa, Tomoko Sumitomoa, Amanda L. Lewise, Shigetada

Kawabataa, Victor Nizetb, d, f

aDepartment of Oral and Molecular Microbiology, Osaka University Graduate School

of Dentistry, Suita, Osaka, Japan; cDepartment of Urology, Nagoya University Graduate

School of Medicine, Nagoya, Aichi, Japan; bDepartment of Pediatrics, dSkaggs School

of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla,

CA, USA; eDepartment of Molecular Microbiology and Ob/Gyn, Washington

University School of Medicine, St. Louis, Missouri, USA; fRady Children’s Hospital,

San Diego, CA, USA.

*Address correspondence to Masaya Yamaguchi, [email protected]

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Supplementary Figure legends

Figure S1. Maximum likelihood tree of nanA and nonA genes. Bootstrap values are shown

near the nodes. Strains with identical DNA sequences are listed on the same branch. The scale

bar indicates nucleotide substitutions per site.

Figure S2. Schematic illustration of domain structures in streptococcal NanA, NanB, and

NanC proteins. Strains with identical sequences are listed on the same branch. S. iniae NanA, S.

pneumoniae NanB and NanC lack an LPXTG motif.

Figure S3. Sialidase activities of bacterial cells and culture supernatants. A. The sialidase

activities of GBS A909 and streptococcal type strains. B. The sialidase activities of GBS

clinical strains and S. pneumoniae D39. C. The sialidase activities of clinically isolated S. iniae

strains. The strains were isolates from brain of a diseased hybrid-stripedbass or tilapia at the

Kent SeaTech aquaculture facility in Mecca, CA. After 2 h incubation at 37°C, fluorescence of

degraded sialidase substrate was measured with excitation and emission wavelengths of 350 and

460 nm, respectively. Data are presented as the mean of triplicate or sextuplet samples. S.E.

3

values are represented by vertical lines. The limit of sensitivity of the assay is 0.3 mU/mL.

Figure S4. Maximum likelihood tree of nanA, nanB, nanC, and nonA genes. Bootstrap

values are shown near nodes. The scale bar indicates nucleotide substitutions per site.

Figure S5. Bayesian phylogenetic tree of nanA in S. pneumoniae. Percentage of posterior

probabilities is shown near the nodes. The scale bar indicates nucleotide substitutions per site.

FEL and FUBAR analyses were performed on nanA multiple alignment data and this tree.

Figure S6. Bayesian phylogenetic tree of nonA in S. agalactiae. Percentage of posterior


FEL and FUBAR analyses were performed on nonA multiple alignment data and this tree.

Figure S7. Bayesian phylogenetic tree of nanB in S. pneumoniae. Percentage of posterior


FEL and FUBAR analyses were performed on nanB multiple alignment data and this tree.

4

Figure S8. Bayesian phylogenetic tree of nanC in S. pneumoniae. Percentage of posterior


FEL and FUBAR analyses were performed on nanC multiple alignment data and this tree.

Figure S9. Bayesian phylogenetic tree of bgaA in S. pneumoniae. Percentage of posterior


FEL and FUBAR analyses were performed on bgaA multiple alignment data and this tree.

Figure S10. Bayesian phylogenetic tree of strH in S. pneumoniae. Percentage of posterior


FEL and FUBAR analyses were performed on strH multiple alignment data and this tree.

Figure S11. Expression of nonA in GBS A909. The graph shows fold transcript level of cylE

and nonA in GBS A909 wild type (filled bars) and !nonA mutant strain (open bars). RNA

extractions were performed from mid-log phase cultures. The level of DNA gyrase subunit A

gene (gyrA) transcription was used as an internal standard. The data represent the mean values

5

of triplicate samples. S.E. values are represented by vertical lines.

6

Table S1. Homology between NanA, NanB, and NanC of S. pneumoniae D39 and NonA of

GBS A909

NanA NonA NanB NanC

NanA - 49/58/75/1 63/25/43/17 53/27/44/16

NonA 70/58/75/1 - 62/28/45/16 60/27/44/15

NanB 88/25/43/17 62/28/45/16 - 94/51/69/0

NanC 70/27/44/16 57/27/44/15 88/51/69/0 -

Cover/Indentities/Positives/Gaps (%)

7

Table S2. Bacterial strains and the accession number of the genome sequences used for phylogenetic tree Bacteria Strain Accession number Streptococcus pneumoniae D39 CP000410.1 JJA CP000919.1 CGSP14 CP001033.1 Hungary19A-6 CP000936.1 ST556 CP003357.1 Taiwan19F-14 CP000921.1 SPNA45 CP000936.1 AP200 CP002121.1 G54 CP001015.1 gamPNI0373 CP001845.1 TCH8431/19A CP001993.1 ATCC700669 FM211187.1 P1031 CP000920.1 70585 CP000918.1 TIGR4 AE005672.3 670-6B CP002176.1 NT_110_58 CP007593.1 Streptococcus agalactiae A909 CP000114.1 ILRI112 HF952106.1 GBS2-NM CP007571.1 GBS6 CP007572.1 GBS1-NY CP007570.1 SA20-06 CP003919.1 138spar CP007565.1 138P CP007482.1 2-22 FO393392.1 GD201008-001 CP003810.1 NEM316 AL766854.1 NGBS572 CP007632.1 2603V/R NC_004116.1 ILRI005 HF952105.1 CNCTC 10/84 CP006910.1 09mas018883 HF952104.1 NGBS061 CP007631.2 COH1 HG939456.1 Streptococcus iniae ISNO CP007587.1 ISET0901 CP007586.1 YSFST01-82 CP010783.1 SF1 CP005941.1 Streptococcus intermedius B196 CP003857.1 C270 CP003858.1 JTH08 AP010969.1 Streptococcus mitis B6 FN568063.1 KCOM 1350 CP012646.1 Streptococcus oralis Uo5 FR720602.1 Streptococcus pseudopneumoniae IS7493 CP002925.1 Streptococcus sp. VT 162 CP007628.2 Erysipelothrix rhusiopathiae SY1027 AP012027.1 Fujisawa CP005079.1

8

Table S3. The frameshift mutation in GBS nonA genes.

GBS strain DNA sequence (5’–3’)

A909 T GAT TGG GGA AAC ATA GGA ATG GTT ATT CGC CGT AGT GA

SA20-06 T GAT TGG GGG AAA CAT AGG AAT GGT TAT TCG CCG TAG TGA

GX026 T GAT TGG GGG AAA CAT AGG AAT GGT TAT TCG CCG TAG TGA

2-22 T GAT TGG GGG AAA CAT AGG AAT GGT TAT TCG CCG TAG TGA

138spar T GAT TGG GGG AAA CAT AGG AAT GGT TAT TCG CCG TAG TGA

138P T GAT TGG GGG AAA CAT AGG AAT GGT TAT TCG CCG TAG TGA

Bold and underlined character is the inserted nucleotide. Box shows stop codons generated by

the framshift.

9

Table S4. Evolutionary analyses of nanA, nonA, nanB, nanC, bgaA, and strH genes

Gene Species Number

of Strains dN/dS

Codons evolving under

positive selection

Codons evolving under

negative selection

nanA S. pneumoniae 16 0.29 0.940% (7/745) 15.302% (114/745)

nonA S. agalactiae 16 0.35 0.885% (4/452) 1.770% (8/452)

nanB S. pneumoniae 16 0.35 0.441% (2/454) 0.661% (3/454)

nanC S. pneumoniae 6 0.29 0% (0/740) 2.703% (20/740)

bgaA S. pneumoniae 14 0.29 0.806% (18/2233) 4.881% (109/2233)

strH S. pneumoniae 17 0.50 0.758% (10/1319) 0.607% (8/1319)

Evolutionary analysis was performed using Baysian inference of aligned nanA, nonA, nanB,

nanC, bgaA, and strH sequences from S. pneumoniae or S. agalactiae, with FUBAR in the

HyPhy software package. The dN/dS means ratio of non-synonymous changes to synonymous

changes in overall analyzed genes. Individual codons with a statistically significant signature

were also calculated and are expressed as a percentage of the total number of codons used in the

analysis.

10

Table S5. Bacterial strains used in sialidase activity assay Bacteria Strain Remarks Streptococcus pneumoniae D39 Serotype 2 Streptococcus agalactiae A909 Serotype Ia M709 Serotype Ib GBS2-NM Serotype II GBS6 Serotype III GBS1-NY Serotype V SA20-06 Serotype VI Streptococcus mitis ATCC 49456/NCTC 12261/SK142 Type strain Streptococcus oralis ATCC 35037/NCTC 11427 Type strain Streptococcus pseudopneumoniae ATCC BAA-960/CCUG 49455/SK1069 Type strain Streptococcus intermedius ATCC 27335/SK54 Type strain Streptococcus iniae K122 Clinical strains K139 Clinical strains K277 Clinical strains K288 Clinical strains K375 Clinical strains K436 Clinical strains K540 Clinical strains 94-426 Clinical strains

11

Table S6. Real-time RT-PCR Primers used in this study

Primers Sequence (5’ to 3’)

A909nonAF GTGATTGGGGAAACATAGGAATGG

A909nonAR CGATTTGGGTATATTCTTTTTCAG

A909cylEF ATTGAAAATCCCTTGGAAGAGGAGACAG

A909cylER GTTAGGCTAGGGTGAGCCCTCG

A909gyrAF CCACATGGTGATTCATCTATTTAC

A909gyrAR CGCTGGTAAAACAAGAGGTTCACG

Supplementary Figure 1. Yamaguchi et al.

S. pneumoniae

S. agalactiae

0.3

S. pneumoniae gamPNI0373

S. agalactiae ILRI005

S. intermedius JTH08

S. agalactiae SA20-06

S. intermedius C270

S. agalactiae strain COH1


S. pneumoniae TCH8431/19A; ATCC 700669

S. agalactiae strain GBS6; GBS2-NM; GBS1-NY

Clostridium perfringens strain JP838

S. pneumoniae TIGR4

S. pneumoniae 670-6B

S. pneumoniae 70585

S. mitis strain KCOM 1350

S. mitis B6

S. pneumoniae G54


S. agalactiae A909; strain SG-M1; HN016; GD201008-001

S. intermedius B196

Erysipelothrix rhusiopathiae SY1027; str. Fujisawa

S. agalactiae strain 138spar; 138P; 2-22; strain GX026

S. pneumoniae ST556; Taiwan19F-14

S. agalactiae CNCTC 10/84

S. iniae strain YSFST01-82

S. pneumoniae D39; R6; OXC141

S. pneumoniae JJA

S. pneumoniae AP200

S. sp VT 162

S. pneumoniae Hungary19A-6

S. agalactiae strain H002; NGBS572; 2603V/R; NEM316

S. iniae strain ISNO; ISET0901; SF1

S. agalactiae strain GBS85147

S. pneumoniae CGSP14

S. pneumoniae SPNA45

Virgibacillus sp. SK37

Clostridium perfringens str. 13

S. agalactiae strain SS1; NGBS061; 09mas018883

S. pneumoniae strain NT_110_58

S. pseudopneumoniae IS7493

Clostridium perfringens ATCC 13124

S. pneumoniae P1031

Clostridium perfringens strain FORC_003

S. oralis Uo535

99.391

92

97

100

100

100

99.7

99.7

99

99.7

99.7

100

100

38.7

100


SPN D39

NanB, 697 A.A.

SPN D39

NanC, 740 A.A.

S. oralis Uo5

NanA, 1130 A.A.

S. intermedius B196

NanA, 920 A.A.

S. iniae YSFST01-82

NanA, 902 A.A.

S. mitis KCOM 1350

NanA, 1292 A.A.

SPN CGSP14

NanA, 997 A.A.

LamG domain Sialidase domain LPXTG motif




LamG domain Sialidase domain



0

1

2

3

4

5

GBS A909

S. mitis

ATCC 49

456

S. oral

is ATCC 35

037

S. pse

udop

neum

oniae

ATCC BAA-96

0 S. in

termed

ius

ATCC 2733

5

0

1

2

3

4

5 S

ialid

ase

activ

itry

(mU

/mL)

Sia

lidas

e ac

tivitr

y (m

U/m

L)

Bacterial cell Supernatant

GBS A909

S. mitis

ATCC 49

456

S. oral

is ATCC 35

037

S. pse

udop

neum

oniae

ATCC BAA-96

0 S. in

termed

ius

ATCC 2733

5

0

2

4

6

8

M709 (

Ib)

DK23 (II

)

COH1 (III)

CNCTC 10/84

(V)

NT6 (VI)

S. pne

umon

iae D

39

0

2

4

6

8

Sia

lidas

e ac

tivitr

y (m

U/m

L)

Sia

lidas

e ac

tivitr

y (m

U/m

L)Bacterial cell Supernatant

A

B


C

A909 (

Ia)

M709 (

Ib)

DK23 (II

)

COH1 (III)

CNCTC 10/84

(V)

NT6 (VI)

S. pne

umon

iae D

39

A909 (

Ia)

GBS (Serotype) GBS (Serotype)

K122

K139

K540

K436

K375

K288

K277

S. pne

umon

iae D

39

94-42

6

S. iniae

K122

K139

K540

K436

K375

K288

K277

S. pne

umon

iae D

39

94-42

6

S. iniae

0

1

2

3

4

Sia

lidas

e ac

tivitr

y (m

U/m

L)

0

1

2

3

4

Sia

lidas

e ac

tivitr

y (m

U/m

L)

Bacterial cell Supernatant

0.4

S. pneumoniae 670-6B

Clostridium perfringens ATCC 13124

nanC NT_110_58

nanB G54

S. oralis Uo5

nanC ST556; Taiwan19F-14

S. pneumoniae D39; R6; OXC141

nanC 70585

S. pneumoniae Hungary19A-6

Virgibacillus sp. SK37

nanB 670-6B


nanC ATCC700669

nanB TIGR4

S. pseudopneumoniae IS7493

S. agalactiae strain H002; NGBS572; 2603V/R; NEM316

nanB P1031

S. pneumoniae G54

nanC 670-6B

S. agalactiae strain GBS85147

S. agalactiae strain COH1


S. pneumoniae JJA


S. mitis B6

nanC gamPNI0373

S. agalactiae strain GBS6; GBS2-NM; GBS1-NY

S. pneumoniae CGSP14

nanC R6; D39

S. agalactiae SA20-06

S. agalactiae A909; strain SG-M1; HN016; GD201008-001

S. mitis strain KCOM 1350

nanB AP200

nanB THC8431/19A

S. pneumoniae SPNA45

nanC AP200

nanC OXC141

S. pneumoniae TIGR4

nanB Hungary19A-6

Erysipelothrix rhusiopathiae SY1027; str. Fujisawa

nanC TIGR4; Hungary19A-6; G54

nanB gamPNI0373

nanC THC8431/19A

S. agalactiae strain 138spar; 138P; 2-22; strain GX026

nanC CGSP14

S. intermedius C270

nanB R6; D39

Clostridium perfringens str. 13

nanB ATCC 700669

nanC SPNA45

nanB ST556; Taiwan19F-14

nanB JJA

S. pneumoniae TCH8431/19A; ATCC 700669

nanC JJA

S. intermedius JTH08

S. pneumoniae AP200

nanB 70585

S. iniae strain ISNO; ISET0901; SF1

S. pneumoniae strain NT_110_58


S. pneumoniae 70585

S. intermedius B196

S. sp. VT 162

nanB CGSP14

S. agalactiae strain SS1; NGBS061; 09mas018883

nanC P1031nanB SPNA45

S. pneumoniae P1031

S. pneumoniae gamPNI0373

nanB OXC141

Clostridium perfringens strain FORC_003

S. iniae strain YSFST01-82

nanB NT_110_58

S. agalactiae CNCTC 10/84

S. pneumoniae ST556; Taiwan19F-14

97

100

97

86

100

100

100

41

91

81

100

100

98.3

15

100

100

64

98

95

100

67

S. pneumoniae nanA

S. agalactiae nonA

S. pneumoniae nanB or nanC



0.02

nanA_SPN_Hungary19A_6

nanA_SPN_SPNA45

nanA_SPN_P1031

nanA_SPN_Taiwan19F_14

nanA_SPN_G54

nanA_SPN_CGSP14

nanA_SPN_70585

nanA_SPN_TCH8431_19A

nanA_SPN_ATCC_700669

nanA_SPN_670_6B

nanA_SPN_TIGR4

nanA_SPN_gamPNI0373

nanA_SPN_AP200

nanA_SPN_JJA

nanA_SPN_ST556

nanA_SPN_D39

6 8

100

6 6

5 2

100

9 7

9 3

8 8100

9 8

100


6.0E-4

nonA_GBS_GBS6

nonA_GBS_GBS2-NM

nonA_GBS_GBS1-NY

nonA_GBS_138spar

nonA_GBS_CNCTC_10_84

nonA_GBS_NGBS061

nonA_GBS_ILRI112

nonA_GBS_ILRI005

nonA_GBS_2603V/R

nonA_GBS_2-22

nonA_GBS_COH1

nonA_GBS_A909

nonA_GBS_138P

nonA_GBS_SA20_06

nonA_GBS_NGBS572

nonA_GBS_NEM316

9 8

100

9 55 6

100

9 2

100

7 5

9 9

8 8


6.0E-4

nanB_SPN_AP200

nanB_SPN_JJA

nanB_SPN_ATCC700669

nanB_SPN_CGSP14

nanB_SPN_P1031

nanB_SPN_TIGR4

nanB_SPN_G54

nanB_SPN_70585

nanB_SPN_Taiwan19F_14

nanB_SPN_670_6B

nanB_SPN_NT_110_58

nanB_SPN_THC8431_19A

nanB_SPN_SPNA45

nanB_SPN_D39

nanB_SPN_OXC141

nanB_SPN_gamPNI0373

5 8

100

7 3

6 0

9 9

9 3


0.0020

NanC_SPN_670_6B

NanC_SPN_TIGR4

NanC_SPN_CGSP14

NanC_SPN_JJA

NanC_SPN_ATCC700669

NanC_SPN_AP200

100

0.0040

bgaA_SPN_R6; D39

bgaA_SPN_670_6B

bgaA_SPN_G54

bgaA_SPN_TIGR4

bgaA_SPN_70585

bgaA_SPN_AP200

bgaA_SPN_P1031

bgaA_SPN_ST556; TCH8431_19A; Taiwan19F_14

bgaA_SPN_ATCC700669; CGSP14

bgaA_SPN_Hungary19A_6

bgaA_SPN_SPNA45

bgaA_SPN_JJA

bgaA_SPN_gamPNI0373

bgaA_SPN_OXC141

100

98

99

97

100

10068

100

100

100

100


0.0020

strH_SPN_TIGR4

strH_SPN_SPNA45

strH_SPN_JJA

strH_SPN_P1031

strH_SPN_AP200; G54

strH_SPN_670_6B

strH_SPN_strain_NT_110_58

strH_SPN_D39

strH_SPN_CGSP14

strH_SPN_gamPNI0373

strH_SPN_70585

strH_SPN_ST556; Taiwan19F_14

strH_SPN_Hungary19A_6

strH_SPN_R6

strH_SPN_ATCC700669

strH_SPN_TCH8431_19A

strH_SPN_OXC14192

73

72

100

100

97

100

93

100



Rel

ativ

e ex

pres

sion

0

2

4

6

8

10

12

14

cylE nonA

N.D.

WT WT∆nonA ∆nonA

supplementary information evolutionary inactivation of a

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