Supplemental figure captions legend

Suppl. FIG. 1. Flowchart of CesT variant library generation and functional screen.

Suppl. FIG. 2. Graphical summary of CesT variant library screening data. 1092 isolates were

screened in modified Bradford assay (left) and classified according to comparisons with control

strains. From the ‘reduced secretion’ isolates expressing normal levels of CesT protein (680

isolates), 100 were randomly selected for follow up SDS-PAGE analyses and DNA sequencing.

Mapped mutations of interest were followed up with detailed experiments and are shown in

Figures 2, 4 and 5 in the manuscript.

Suppl. FIG. 3. Structural models depicting locations of specific amino acid within the EHEC

CesT structure. In the case of serine 63, in EPEC it is a proline (denoted by an asterisk). The

black arrowheads point to the amino acid that is also highlighted in yellow.

Suppl. FIG. 4. Verification of secretion profiles and protein expression patterns for

chromosomal integration mutant cesT::cesT(S147A). Tir and CesT protein expression levels

were unchanged whereas total effector secretion was reduced (right most lane, left panel).

Suppl. FIG. 5. Constraint based alignment (COBALT) of three multicargo chaperones from

Salmonella (InvB), Shigella (Spa15) and EPEC (CesT). The asterisks denote amino acids that are

identical in all three chaperones.

Error prone (EP)-PCR using EPEC genomic DNA to generate mutated cesT alleles

Digest and clone EP-PCR fragments, clone into pRK415 plasmid

Add ligated DNA to DH5a, select for transformants

Patch (Array) CesT variant library transformants into 96 well plate pattern on plates

Subculture transformants in liquid LB in 96 well plates

Perform 96 well triparental mating to mobilize plasmids into ∆sep∆cesT

Streak purify recipient strains on selective solid agar keeping track of well number

Select for recipient ∆sep∆cesT strains with streptomycin and tetracycline in new 96 well plate

Patch (Array) ∆sep∆cesT/ pCesT variants in a 96 well plate (liquid LB)

Frozen Archive (A)

DH5α

Frozen Archive (B)

Grow strains under secretion inducing conditions

Collect supernatants for modfied Bradford assay

Identify clones of interest from Bradford results

Go back to Frozen Archive (A) , find corresponding strain and isolate plasmid DNA for sequencing

Verify Bradford data with traditional secretion assay and secreted protein profiles in SDS-PAGE with Frozen Archive (B)

Follow up experiments or mobilization of plasmids into ∆cesT for infection assays

Supplementary Figure 1. Flowchart of CesT variant library generation and functional screen

739

353 reducedsecretion

no change

n=1092 n=100

Supplementary Figure 2. Graphical summary of CesT variant library screening data. 1092 isolates were screened in modified Bradford assay (left) and classified according to comparisons with control strains. From the ‘reduced secretion’ isolates expressing normal levels of CesT protein (680 isolates), 100 were randomly selected for follow up SDS-PAGE analyses and DNA sequencing. Mapped mutations of interest were followed up with detailed experiments and are shown in Figures 2, 4 and 5 in the manuscript.

34

4

20

42 frameshift

stop codon

multiple mutations

single mutations

E68

E35

E107

G17

H128

K108

N26

N73

N129

S63*

S119

S146

V126

Supplementary Fig. 3. Structural models depicting locations of specific amino acid within the EHEC CesT structure. In the case of serine 63, in EPEC it is a proline (denoted by an asterisk). The black arrowheads point to the amino acid that is also highlighted in yellow.

α-Tir

α-CesT

α-RNApol

Total Secreted Proteins Coomassie Stain

Immunoblot: whole cell lysates

Supplementary Figure 4. Verification of secretion profiles and protein expression patterns for chromosomal integration mutant cesT::cesT(S147A). Tir and CesT protein expression levels were unchanged whereas total effector secretion was reduced (right most lane, left panel).

Tir

EspB/D

EspA

EspC

NleA

Supplementary Figure 5. Constraint based alignment (COBALT) of three multicargo chaperones from Salmonella (InvB), Shigella (Spa15) and EPEC (CesT). The asterisks denote amino acids that are identical in all three chaperones.

InvB

Spa15 CesT

InvB

Spa15

CesT

InvB

Spa15 CesT