material and methods supp-jan14 - nature research · 2016-03-29 · pgcc4 slam1ntd pgcc4 containing...

Title: Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria

Authors: Yogesh Hooda, Christine Chieh-‐Lin Lai, Andrew Judd, Carolyn M. Buckwalter, Hyejin Esther Shin, Scott D. Gray Owen, and Trevor F. Moraes

To whom correspondence should be addressed E-mail: [email protected]

This PDF file includes: Supplementary Figures 1-12 Supplementary Table 1: Strains, plasmids and antibodies used in this study.

SUPPLEMENTARY INFORMATIONARTICLE NUMBER: 16009 | DOI: 10.1038/NMICROBIOL.2016.9

NATURE MICROBIOLOGY | www.nature.com/naturemicrobiology 1

DOI: 10.1038/NMICROBIOL.2016.9 SUPPLEMENTARY INFORMATION


~4000 Tn N. meningitidis mutants(40 plates)

Whole cell solid phase binding assay of mutants probed with α TbpB antibodies

Mutants Gene name Predicted ORF

2E1 nmb1037 Putative glutamate-cysteine ligase

5H3

24C10

28D2

3411F

nmb0313

nmb0173

nmb0617

nmb0690

TPR-containingprotein

LysR family trans-criptional regulatorTranscription termin-nation factor Rho

Amidophospho-ribosyltransferase

Kan

Kan

Kan

Sheared genomic DNA withkanamycin cassette inserted by Tn5

N. meningitidis colonies withTn insertions

a

b

c

d

e

α TbpB

WT Δtbpb

2E15H3

PFA

SDS

Emp

Compl

α TbpBPFA

SDS



WTΔnmb0313



Slam1

Slam2

Ntd

Ntd

DUF560

DUF560

SP

SP

1 31 203 488

1 19 207 505

29% id 24% id

a

bN. meningitidis Slam2HpuAHpuB

N. gonorrhoeae 80%78%

N. lactamica 98%85%

N. polysaccharea 98%81%

N. cinerea 58%52%

55%51%

N. mucosa 54%51%

N. weaveri 39%41%

N. shayeganii 37%29%

96%

97%

95%

78%

78%

78%

57%

48%



Inner membrane

Outer membrane

Fractions: 1 2 3 4 5 6 7 8 9 10 11 12

a

b

α Slam1

α TbpB

α LepB

α OmpA

100

101

102

103

104

0

100

200

300

400

PgaBPgaB + Slam1PgaB + Slam2Co

unts

PE Fluorescence

α PgaB



Intact cells Spheroplasts

Intact E. coli cells

Spheroplast E. coli cells

a

c

n.t.GST-C-lobe

α TbpB α GST

α TbpB α GST

Coun

ts

α GSTα TbpB

Mea

n Fl

uore

scen

ce

I

nten

sity

(MFI

)

b

Intact cells Spheroplasts

Mea

n Fl

uore

scen

ce

I

nten

sity

(MFI

)

PE Fluorescence PE FluorescencePE FluorescencePE Fluorescence

Coun

ts

Coun

ts

Coun

ts

GST-C-lo

be

α TbpB

Mea

n Fl

uore

scen

ce

I

nten

sity

(MFI

)

α GST

Mea

n Fl

uore

scen

ce

I

nten

sity

(MFI

)

n.t.

GST-C-lo

ben.t.

GST-C-lo

ben.t.

GST-C-lo

ben.t.



a b

dinput

- +Slam1

- +pre-im

mune

- +-+ - + - + GST

GST-C-lobe

GST

-GST-C-lobe

-GST

48-

35-

63-kDa

input pulldown

c

PFA

SDS

- +GST-C

-lobe

- +PK

TbpB

TbpB

GST-C-lo

be

-TbpB-GST-C-lobe

TbpB

TbpB

- +

GST

- +GST-C

-lobe

GST

-GST-C-lobe

-GST*

PK

-

kDa kDa

63-

48-

35-

75-

63-

48-

35-

75-



TamA KO TamB KOWT

TbpB

TbpB + Slam1

Coun

ts

Coun

ts

Coun

ts

PE Fluorescence PE FluorescencePE Fluorescence

TbpB TbpB TbpB



a b

c

N. meningitid

is B16B6 W

T

TbpB

purified TbpB

N. meningitid

is B16B6 Δtbpb

N. meningitid

is B16B6 W

T

N. meningitid

is B16B6 Δnmb0313

HpuA

E. coli C43 W

T

purified H

puA

E. coli C43 + H

puA

Slam1

TbpB

HpuA

lipidated HpuA

Slam1

PgaB

E. coli E11 W

T

E. coli E11 ΔpgaB

d

kDa kDa

kDa kDa

63- 63-75-

48-

35-

25-

75-

48-

35-

25-

75-

25-

63-48-

35-

PgaB75-

25-

63-48-

35-



Figure 1b (blue box) and Figure 2b (red box)

α TbpB α LbpB α FbpA α FbpAα fHbp

α TbpB α LbpB α fHbp α HpuA

Figure 3a-d

Figure 4c

α TbpB α GSTα BamA α BamA α TbpB α GSTα Slam1 α Slam1

GST IP Slam1 IP

B16B6 B16B6 B16B6 MC58 MC58

75

63

48

35

35

2520

35

2520

75

63

48

35

10075

63

48

35

100

75

63

48

35

100

75

63

48

35

100

75

63

48

35

25

75

63

48

35

25

75

63

48

35

100 75

63

48

35

100

75

63

48

35

100

75

63

48

35

100

75

63

48

35

75

63

48

35

75

63

48

35

75

63

48

35

75

63

48

35



75

63

48

35

100

Supplementary Figure 1b

α TbpB α TbpB α His

Supplementary Figure 1c

α TbpB

α TbpB α LepB α OmpA

Supplementary Figure 2c

α TbpB

α LepB

α GSTα TbpB

α GST

α TbpB α OmpA α Slam1

Supplementary Figure 1d

Supplementary Figure 8c Supplementary Figure 8d

N. meningitidis E. coli

75

63

48

35

10075

63

48

35

100

75

63

48

35

100

35

2520

17

11

75

63

48

35

100

75

63

48

35

75

63

48

3525

7563483525

756348

35

25

7563

48

35

25

75

63

48

35

25

705535

25

100

75

63

48

35

75

63

48

35

25

75

63

48

35

25

100

Supplementary Figure 6a

Supplementary Figure 8b



Supplementary Table 1: Strains, plasmids and antibodies used in this study. Strain or plasmid Description Reference Strains: N. meningitidis B16B6 [1] N. meningitidis B16B6 nmb0313/tn5

EZTn5-kanamycin insertion in nmb0313 This study

N. meningitidis B16B6 Δnmb0313

Δnmb0313::kan This study

N. meningitidis B16B6 ΔtbpB ΔtbpB::kan [2]

N. meningitidis B16B6 ΔlbpB ΔlbpB::kan [3]

N. meningitidis MC58 [4] N. meningitidis MC58 Δnmb0313

Δnmb0313::kan This study

E. coli MM294 F-, glnX44(AS), LAM-, rfbC1, endA1, spoT1, thiE1, hsdR17, creC510 Yale CGSC #6315 E. coli C43 F– ompT gal dcm hsdSB(rB- mB-)(DE3) [5] E. coli BW25113 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, rph-1, Δ(rhaD-rhaB)568, hsdR514 [6] E. coli JW4179-1 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, rph-1, Δ(rhaD-rhaB)568,ΔytfM769::kan, hsdR514 [6] E. coli JW4180-1 F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, rph-1, Δ(rhaD-rhaB)568,ΔytfN770::kan, hsdR514 [6] E. coli E11 K1 capsule type, clinical isolate [7] E. coli E11 ΔpgaB ΔpgaB [7] Plasmids: pUC19 Slam1-Kan pUC19 containing the 500bp upstream and downstream regions of slam1 flanking kan2 from the

EZ::Tn5 transposon kit This study

pGCC4 Neisseria complementation vector [8] pGCC4 Slam1 pGCC4 containing full length Slam1 This study

pGCC4 Slam1barrel pGCC4 containing Slam1 barrel domain This study

pGCC4 Slam1Ntd pGCC4 containing Slam1 N-terminal domain This study pGCC4 TbpB pGCC4 containing full length TbpB This study

pGCC4 GST-C-lobe pGCC4 plasmid with GST inserted after the anchoring peptide, replacing the TbpB N-lobe and in frame with the TbpB C-lobe

This study



pGCC4 GST pGCC4 plasmid with GST inserted after the anchor peptide, replacing both the TbpB N- and C-lobes

This study

pET26b E.coli expression vector Addgene, 69862-3

pET26 Slam1 pET26 containing pelB signal peptide, 7xHis and mature B16B6 Slam1 This study pET26 Slam1barrel pET26 containing pelB signal peptide, 7xHis and B16B6 Slam1 barrel domain This study

pET26 Slam2 pET26 containing pelB signal peptide, 7xHis and mature MC58 Slam2 This study

pETDuet E.coli expression vector Addgene, 71146

pETDuet Slam1 pETDUET containing pelB signal peptide and mature B16B6 Slam1 in MCS site 1 This study pETDuet TbpB pETDUET containing pelB signal peptide and mature B16B6 TbpB in MCS site 2 This study

pETDuet T5 TbpB pETDUET containing full length B16B6 TbpB in MCS site 2 with pT5-Lac promoter This study

pETDuet LolA R43L_TbpB pETDUET containing full length B16B6 TbpB in MCS site 2 and His-LolA R43L in MCS site 1 This study pETDuet LbpB pETDUET containing pelB signal peptide and mature MC58 LbpB in MCS site 2 This study

pETDuet fHbp pETDUET containing pelB signal peptide and mature MC58 fHbp in MCS site 2 This study

pETDuet HpuA pETDUET containing pelB signal peptide and mature B16B6 HpuA in MCS site 2 This study pETDuet GST-C-lobe pETDUET containing full length B16B6 TbpB with the N-lobe replaced by a GST domain, in MCS

site 2 This study

pHERD30T pBAD-based shuttle vector [9]

pHERD30T pelB Slam1 pHERD30T containing pelB signal peptide and mature Slam1, sub-cloned from pET26b This study

pCR2.1 TOPO PgaB pCR2.1 TOPO containing pgaB (1-672) [10]

Antibodies:

α-LepB Rabbit polyclonal antibodies were obtained from Dr. Jan Willem de Gier [11] α -OmpA Rabbit polyclonal antibodies were obtained from Dr. Jan Willem de Gier [12] α-Slam1 Supplementary Figure 10c, rabbit polyclonal sera This study

α-fHbp Mouse monoclonal Jar5 antibodies were obtained from Dr. D. Granoff [13]

α-HpuA Supplementary Figure 10b, rabbit polyclonal sera This study α-TbpB Supplementary Figure 10a, rabbit polyclonal sera This study



α-BamA Mouse polyclonal antibodies were obtained from Dr. N. Noinaj [14]

α-FbpA Rabbit polyclonal antibodies were obtained from from Dr. A. B. Schryvers [15]

α-PgaB Rabbit polyclonal antibodies were obtained from Dr. D. Little and Dr. L. Howell This study α-LbpB Rabbit polyclonal antibodies were obtained from Dr. M. Joanne Lemieux [16]

α-6x His Mouse anti-6x His monoclonal antibody Pierce, MA121315 α-GST Rabbit anti-GST polyclonal antibody Cell Signalling, #2622

α-GST Mouse anti-GST monoclonal antibody GenScript, A00865-100

Bio-hTf Biotinylated human transferrin Sigma, T3915-5MG

PE-Streptavidin R-Phycoerythrin-Streptavidin Cederlane, 016-110-084 PE-rabbit IgG Phycoerythrin Conjugated F(ab')2 Affinity Purified anti-rabbit IgG Rockland Immunochemical,

CA711-108-002 PE-mouse IgG Immunopure PE polyclonal goat anti-mouse IgG Pierce, PI31861

HRP-rabbit IgG Goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) Cell Signalling, #7074S

HRP-mouse IgG Goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) Pierce, PI31430

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material and methods supp-jan14 - nature research · 2016-03-29 · pgcc4 slam1ntd pgcc4 containing...

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