1

Supplementary Information for

Bacterial outer membrane vesicles engineered with lipidated antigens as

a platform for Staphylococcus aureus vaccine

Carmela Irene1+, Laura Fantappiè2+, Elena Caproni,1 Francesca Zerbini1a, Andrea

Anesi1b, Michele Tomasi1, Ilaria Zanella1, Simone Stupia1, Stefano Prete2, Silvia Valensin2,

Enrico König1, Luca Frattini1, Assunta Gagliardi1, Samine J. Isaac1, Alberto Grandi2,

Graziano Guella1 and Guido Grandi1*

Guido Grandi

Email: guido.grandi@unitn.it This PDF file includes:

Supplementary text References Figures S1 to S3 Tables S1 to S2

www.pnas.org/cgi/doi/10.1073/pnas.1905112116

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Supplementary Information Text

Material and Methods

Bacterial strains and culture conditions

HK100 and BL21(DE3) E. coli strains were routinely grown in LB broth at 37°C and used

for cloning and expression experiments, respectively. Staphylococcus Newman strain was

grown in Tryptic soy medium under aerobic conditions and CFUs were estimated by

plating bacteria on Tryptic soy agar plates. Stock preparations of E. coli strains in LB +15%

glycerol and S. aureus in TSB+15% glycerol were stored at -80°C. Each bacterial

manipulation was started using an overnight culture from a frozen stock. When required

ampicillin was added to final concentration of 100 µg/ml.

Plasmids and strains construction and OMVs preparation

BL21(DE3)ΔompA strain was generated as previously described (1). BL21

(DE3)ΔompAΔmsbBΔpagP strain was generated using the CRISPR/Cas genome editing

strategy (2). Briefly, the BL21(DE3)ΔompA strain was transformed with pCasRed plasmid

encoding the Cas9 endonuclease, the tracrRNA and the λRed cassette. Subsequently,

BL21(DE3)ΔompA (pCasRed) was co-transformed with a mutagenic double-stranded

donor DNA (ds-dDNA, Table S2) and the pCRISPR-SacB-gDNA plasmid, which carries

a synthetic DNA fragment (Table S2) coding for a gene specific RNA guide necessary to

direct the Cas9 to the target site. The ds-donor DNA was designed to cause a whole gene

deletion. Positive mutants were identified by colony PCR with gene specific primers (Table

S2).

To express the five S. aureus antigens HlaH35L, LukE, FhuD2, Csa1A and SpAKKAA the

coding sequences were chemically synthetized (Genart-Invitrogen) and PCR amplified

using the primers reported in Table S2. The PCR products were inserted into plasmids pET-

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OmpA or pET-Lpp plasmid, two pET21 derivatives carrying the sequence encoding the

leader peptide for secretion of E. coli OmpA or Lpp, respectively, using the polymerase

incomplete primer extension (PIPE) cloning method (3). Plasmids were linearized using

the primers couples: omprev/nohisflag and Lpp-R-plasmid/nohisflag (Table S2). The

correctness of the cloning was verified by sequence analysis. pET plasmid derivatives

containing the genes of interest were transformed into BL21(DE3)ΔompA and BL21

(DE3)ΔompAΔmsbBΔpagP strains. Recombinant clones were grown in 200 ml LB medium

(starting OD600=0.05) and, when the cultures had reached an OD600 value of 0.5,

recombinant protein expression was induced by addition of 0.1 mM IPTG.

Purification of recombinant S. aureus antigens and limited proteolysis of LukE,

FhuD2 and Csa1A

Recombinant S. aureus antigens were purified using the TEV protease purification strategy

(4). Briefly, the synthetic genes coding for SpaKKAA, HlaH35L, LukE, FhuD2 and Csa1A

were fused at their 5’ to the sequence coding for a His6-tag and the TEV cleavage site. The

constructs were cloned in a pET15 plasmid downstream of a T7 inducible promoter and

expressed in E. coli BL21(DE3) strain. Bacterial biomass, 5 g wet weight, was

resuspended in 50 ml buffer A (50 mM NaH2PO4, 300 mM NaCl, pH 8.0) in the presence

of a protease inhibitor (0.2 mM PMSF), sonicated thoroughly at 4°C and the total cell lysate

was finally centrifuged (15.000 x g, 30 min, 4°C). The supernatant was filtered (0.22 µm)

and applied to Ni-affinity chromatography (IMAC) using an ÄKTA purifier System (GE)

and a 5 ml HiTrap IMAC column (GE) monitoring absorbance at 280 nm. Protein binding

and column washing was performed at concentrations of 20 mM and 50 mM imidazole,

respectively. Bound proteins were eluted using a linear gradient by increasing buffer B (50

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mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0) from 10% to 100% over 6

column volumes. Pooled fractions containing the His-tagged recombinant protein was

dialysed against buffer A at 4°C and subsequently digested with TEV protease (1 mg per

100 mg protein) in the presence of 5 mM β-mercaptoethanol. TEV-digested protein pool

was applied to Ni-affinity chromatography and the flow-through, containing the untagged

recombinant protein, was subjected to a final purification step by size-exclusion

chromatography using a HiLoad 16/600 Superdex 75 pg column.

For limited proteolysis experiments, LukE, FhuD2 and Csa1A were treated with 0.16-0.8

ng of Proteinase K, in 100 l PBS for 30 minutes. The reactions were blocked adding

phenylmethhylsulfonyl fluoride (PMSF; Sigma-Aldrich). Digested fragments were

separated by SDS-PAGE and analyzed by Western blot with primary antibodies raised

either against the recombinant proteins or the engineered OMVs expressing the lipidated

proteins.

Western blot and flow cytometry analysis

Western blot analysis was performed as previously reported (1). The polyclonal antibodies

against each antigen were obtained from Genscript by immunizing rabbits with specific

synthetic peptides (Csa1A: GYRDDQFDKNDKG, FhuD2: MDDGKTVDIPKDPK,

LukE: NEFVTPDGKKSAHD, SpAKKAA: AKKLNDAQAPKADN, HlaH35L:

GTNTKDKWIDRSSE) conjugated with KLH protein. Anti-MBP (maltose binding

protein) monoclonal antibody and anti-HISTAG antibodies were purchased from New

England Biolabs and Roche, respectively. Flow cytometry analysis on E.coli strain

expressing lipidated FhuD2 was performed as previously described (5). Primary antibody

against FhuD2 was obtained from Genscript as described above.

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Triton X-114 protein separation from OMVs

OMVs (100 g of proteins) were diluted in PBS, ice cold TritonX-114 was added to 1%

final concentration and the OMV-containing solution was incubated at 4°C for 1 h under

shaking. The solution was then heated at 37°C for 10 minutes and the aqueous phase was

separated from the detergent by centrifugation at 13,000 g for 10 min. Proteins in both

phases were then precipitated by standard chloroform/methanol procedure, separated by

SDS-PAGE electrophoresis and the protein of interest visualized by Western blot.

LPS purification and Mass spectrometry analysis

LPS extraction

Bacterial cells, harvested from 1 L culture, were washed twice with ethanol, acetone and

petroleum ether, then dried at room temperature. 5 ml of 90% phenol solution was added

to the dried cells, the suspension was heated at 70°C and vigorously stirred for 30 minutes.

Samples were cooled on ice and centrifuged for 20 minutes at 6,000 rpm. The water phase

was collected and dialyzed for two days against water. DNase and RNase (10mg of each)

digestion was performed incubating for 3 hours at 37°C, followed by digestion with

Proteinase K. After overnight dialysis against water, the LPS was lyophilized in a rotatory

evaporator (Büchi Labortechnik AG, Flawil, Switzerland). Dried LPS was weighted and

dissolved in water in a 1-2% solution.

LPS acidic hydrolysis

The protocol for LPS hydrolysis was slightly modified from that of El Hamidi et al. (6).

Briefly, LPS was cleaved by acidic hydrolysis in 2% acetic acid at 100°C for 2 h under

stirring and reflux. The mixture was centrifuged at 3,000 rpm for 30 min at RT, the

recovered pellet was re-suspended in 1 ml of chloroform: methanol: water 3:2:0.25 (v/v)

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and the solution was centrifuged at 3,000 rpm for 15 min. The organic phase containing

lipid A was collected and the extraction was repeated on the aqueous phase. The pooled

organic phases were dried in a rotatory evaporator and re-suspended in 500 µl of

chloroform: methanol 4:1 (v:v).

Reverse Phase Liquid Chromatography-ElectroSpray Ionization-Mass Spectrometry

(RPLC-ESI-MS)

The chromatographic separations were conducted on a Hewlett-Packard Model 1100 Series

liquid chromatograph (Hewlett-Packard Development Company, L.P., Palo Alto, CA,

USA) coupled to a Bruker Esquire 3000-LC quadrupole ion trap-mass spectrometer

equipped with an ESI source (Bruker Optik GmbH, Ettlingen, Germany). The column was

a Zorbax Eclips XDB-C8 column (150 × 4.6 mm i.d., pore size 200 Å, particle size 3.5

µm) (Hewlett Packard, Palo Alto, CA, USA) maintained at 50°C; mobile phase A was

methanol: water 95:5 (v:v) while mobile phase B was isopropanol. The linear gradient, at

a constant flow rate of 1 ml/min started from 0% B and reached 50% B in 50 min, those

final conditions were kept for 20 min to ensure the complete elution of lipids. Starting

conditions were reached in 2 minutes and column was re-equilibrated for 13 min. Aliquots

of 10 µl of crude extract were injected. ESI was operated in negative ion mode with the

high voltage capillary set at -4000V in the range 500-2000 m/z. Other parameters: high

purity nitrogen at 35 psi, 300°C and flow rate of 7 L/min.

TLR4 reporter cell assay

HEK-blue™ mTLR4 and HEK-blue™ hTLR4 cells (Invivogen) were seeded in a flat-

bottom 96-well plate (5 x 104 cells/well) and stimulated for 16-17 hours with different

concentrations of OMVs or LPS-EK ultrapure (TLR4 agonist), as positive control (10 fold

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serial dilutions, starting from 0.01 mg/ml). Detection of SEAP activity from cell culture

supernatants was performed the following day by mixing 180 μl QUANTI-Blue™

(Invivogen) per well of a flat-bottom 96-well plate with 20 μl supernatant of stimulated

with OMVS or LPS. After 1h, OD (655 nm) was measured with a spectrophotometer.

Mice Immunization, challenge, ELISA and Hla neutralization assay

Five-week old CD1 female mice were immunized intraperitoneally (i.p.) three times every

two weeks with either 10g of recombinant proteins or different amounts of OMVs

formulated with or without 2mg/ml Alum hydroxide. 5-Combo-OMVs vaccine contained

5g of each engineered OMV (25 g total OMV dose). Sera were collected 10 days after

the last immunization.

For challenge studies, mice were immunized at day 0 and 14 (and day 21 for renal abscess

model) with a combination of 25 g of 5-Combo-OMVs (from BL21ompA strain or

BL21ompAmsbpagP) with or without 2mg/ml Alum hydroxide. For the sepsis

model of infection two weeks after the second immunization mice were i.p. challenged

with 4 x 108 colony forming unit (CFU) of S. aureus Newman strain. Mice were monitored

daily for a 7-day period. Animals health was evaluated using a 1 to 4 pain scale. A value

of 4 was given to mice with: loss of weight >15%, very rough hair coat, impaired mobility.

A score of 3 was given to mice with loss of weight of about 15% and rough hair coat, while

scores of 2 and 1 were given to mice with a loss of weight between 6% and 14% or 0% and

5%, respectively. All procedures were approved by the National Health Institution and

Ethical Committee and for human reasons animals were sacrificed at symptoms of sickness

as recommended by 3Rs rules (‘‘Refinement, Reduction, Replacement’’ policy towards

the use of animals for scientific procedures_ 99/167/EC, Council Decision of 25/1/99).

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Experiments using the renal abscess and skin infection model were performed as

previously described (7). For the renal model mice were challenged i.v. 10 days after the

third immunization with 1 x 107 colony forming units (CFUs) of S. aureus Newman strain,

while in the skin infection model mice were challenged s.c. 14 days after the second

immunization with 5 x 107 CFUs of S. aureus Newman strain.

ELISA assays on mice sera collected after immunizations were performed as previously

described (1).

Hla neutralization assay was performed as previously described (7) with some minor

modifications. Briefly, serial twofold dilutions of antisera against “Empty”-OMV, rHlaH35L

and HlaH35L-OMVs were incubated with 20 ng rHla (Abcam) for 20 min at 37°C. Then,

erythrocytes derived from de-fibrinated rabbit blood were added, and incubation was

prolonged for a further 15 min at 37 °C. Incubation with water was used as a positive

control (maximal hemolysis). Plates were then centrifuged for 5 min at 1,000 × g, and the

supernatant was analyzed spectrophotometrically by an absorbance microplate reader at

540 nm.

LPS analysis by Mass Spectrometry

The LPS was purified from the four strains and the Lipid A was analysed by Reverse Phase

Liquid Chromatography-ElectroSpray Ionization-Mass Spectrometry (RPLC-ESI-MS).

The spectra obtained for the four preparations are shown in Figure 5. The total ion current

(TIC) of Lipid A from BL21(DE3)ΔompA strain showed a predominant peak (hexa-Ac,

m/z of 1717.2) corresponding to the pseudomolecular ion of monophosphoryl hexa-

acylated lipid A containing four units of 3-hydroxytetradecanoic acid (C14OH), one unit of

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both dodecanoic acid (C12) and tetradecanoic acid (C14) (Figure 1S). The peak with a

slightly lower retention time (28 min using our chromatographic setup), was identified as

the phosphorylethanolamine analogue of hexa-acylated lipid A (hexa-Ac-PE) (1717+123

Da). Finally, the other minor peaks were attributed to monophosphoryl penta-acylated

lipid A (penta-Ac, m/z 1506.6 at 16.8 min) with no C14 unit, its phosphorylethanolamine

analogue (penta-Ac-PE, m/z 1629.6 at 14.6 min) and tetra-acylated lipid A (tetra-Ac-PE,

m/z 1280.1 at 9 min), carrying three C14OH and one C12 units. The assignment of fatty acyl

units was confirmed by tandem MS. Overall, the spectrum was essentially in line with what

reported for LPS purified from wild type E. coli strains (8), even though the

phosphorylethanolamine analogues are usually not detected in bacteria grown in rich

media. A similar profile was obtained when the Lipid A from BL21(DE3)ΔompA strain

expressing lipidated FhuD2 was analysed, with the difference that the monophosphoryl

hexa-acylated lipid A appeared to be sligthly more abundant with respect to the other

species. The TICs of the Lipid A from BL21(DE3)ΔompAΔmsbBΔpagP presented

substantial differences from BL21(DE3)ΔompA Lipid A. Consistent with the inactivation

of the msbB and pagP genes, the hexa-acylated lipid A species was absent. The

predominant form was the phosphorylethanolamine penta-acylated lipid A (penta-Ac-PE,

m/z 1629.9 at 15.30 min), followed by the penta-acylated species containing no C14 unit

(penta-Ac, m/z 1506.6 at 15.82 min) and, to less extent, the tetra-acylated containing no

C14 unit (tetra-Ac, m/z 1280.1 at 8.44 min) and its phosphorylethanolamine analogue (tetra-

Ac-PE, m/z 1403.1 at 7.60 min). Finally, the spectrum of the

BL21(DE3)ΔompAΔmsbBΔpagP(pET-FhuD2) strain showed two interesting features.

First, the tetracylated monophosphoryl species (tetra-Ac) was more abundant compared to

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the Lipid A from the same strain not expressing lipidated FhuD2. Second, both penta- and

tetra-acylated phosphorylethanolamine analogues (penta-Ac-PE and tetra-Ac-PE) were

completely missing in the Lipid A preparation of this strain (Figure 5 and Figure 1S).

Considering that the tetra-acylated monophoryl lipid A is known to have a poor TLR4

agonistic activity and that phosphorylethanolamine modification slightly enhances the

TLR4 agonistic activity of Lipid A, these data offer an explanation of the reduced TLR4

stimulation of the OMVsmsbBpagP decorated with the lipidated antigens (see also

Discussion).

References

1. Fantappiè L, de Santis M, Chiarot E, Carboni F, Bensi G, Jousson O, Margarit I,

Grandi G. Antibody-mediated immunity induced by engineered Escherichia coli

OMVs carrying heterologous antigens in their lumen. J Extracell vesicles (2014)

3:24015. doi:10.3402/jev.v3.24015

2. Zerbini F, Zanella I, Fraccascia D, König E, Irene C, Frattini LF, Tomasi M, Fantappiè

L, Ganfini L, Caproni E, Parri M, Grandi A and Grandi G. Large scale validation of

an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli.

Microb Cell Fact (2017) 16:68. doi:10.1186/s12934-017-0681-1

3. Klock HE, Lesley SA. The Polymerase Incomplete Primer Extension (PIPE) method

applied to high-throughput cloning and site-directed mutagenesis. Methods Mol Biol

Clift Nj (2009) 498:91–103. doi: 10.1007/978-1-59745-196-3_6

4. Cesaratto F, Burrone OR, Petris G. Tobacco Etch Virus protease: A shortcut across

biotechnologies. J. Biotechnol. (2016) 231, 239–249

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5. Fantappie L, Irene C, Santis M De, Armini A, Gagliardi A, Tomasi M, Parri M, Cafardi

V, Bonomi S, Ganfini L, Zerbini F, Zanella I, Carnemolla C, Bini L, Grandi A, Grandi

G. Some Gram-negative lipoproteins keep their surface topology when transplanted

from one species to another and deliver foreign polypeptides to the bacterial surface.

Mol Cell Proteomics (2017) 1348–1364. doi:10.1074/mcp.M116.065094

6. El Hamidi A, Tirsoaga A, Novikov A, Hussein A, Caroff M. Microextraction of

bacterial lipid A: easy and rapid method for mass spectrometric characterization. J

Lipid Res (2005) 46:1773–1778. doi:10.1194/jlr.D500014-JLR200

7. Bagnoli F, Fontana MR, Soldaini E, Mishra RPN, Fiaschi L, Cartocci E, Nardi-Dei V,

Ruggiero P, Nosari S, De Falco MG, Lofano G, Marchi S, Galletti B, Mariotti P,

Bacconi M, Torre A, Maccari S, Scarselli M, Rinaudo CD, Inoshima N, Savino S, Mori

E, Rossi-Paccani S, Baudner B, Pallaoro M, Swennen E, Petracca R, Brettoni C,

Liberatori S, Norais N, Monaci E, Bubeck Wardenburg J, Schneewind O, O'Hagan DT,

Valiante NM, Bensi G, Bertholet S, De Gregorio E, Rappuoli R, Grandi G. Vaccine

composition formulated with a novel TLR7-dependent adjuvant induces high and broad

protection against Staphylococcus aureus. Proc Natl Acad Sci (2015) 112:201424924.

doi:10.1073/pnas.1424924112

8. Raetz CRH, Whitfield C. Lipopolysaccharide Endotoxins. Annu Rev Biochem (2002)

71:635–700. doi:10.1146/annurev.biochem.71.110601.135414

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Fig. S1.

A. Structures of the different Lipid A chemical species.

B. Retention times, measured m/z values (standard deviation in ppm), calculated m/z

values on the basis of the corresponding molecular formula of the structures in

panel A.

RT min Accurate massExact mass

monosotopicMolecular Formula Structure

28,44 1716.2262 (11ppm) 1716.2457 C94H176N2O22P- Hexa-Ac (1)

27,60 1839.2326 (12 ppm) 1839.2543 C96H182N3O25P2- Hexa-Ac-PE (4)

15,82 1506.0141 (22ppm) 1506.0474 C80H150N2O21P- Penta-Ac (2)

15,301629.0231 (20 ppm) 1629.0559 C82H156N3O24P2- Penta-Ac-PE (5)

8,44 1279.8264 (21) 1279.8541 C66H124N2O19P- Tetra-Ac (3)

7,60 1402.8309 (22) 1402.8626 C68H130N3O22P2- Tetra-Ac-PE (6)

A

B

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Fig. S2.

Figure S2: In vivo protective activity of COMBO-OMVs in the sepsis model of

infection. Analysis of body weight. The figure refers to the same experiment as

described in Figure 6. Mice (30 animals/group) were immunized with 2 doses of

either “Empty-OMV” (25 g/dose) (grey), or combinations of the five engineered

OMVs (black). As control, mice were also immunized with Alum (white). After 2

weeks, mice were infected intra-peritoneally with a lethal dose of S. aureus

Newman strain (4 x 108 CFUs). Mouse weight at day 1, 3 and 5 post infection was

monitored and reported as % of weight loss/gain with respect to the previous weight

measurement.

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Fig. S3.

Figure S3: (A) Assessment of FhuD2 localization by FACS analysis. Bacterial cells

from BL21ompA and BL21ompA (pET_FhuD2) strains were incubated first with anti-

FhuD2 specific antibodies and subsequently with FITC-labeled anti-rabbit secondary

antibodies. Fluorescence was measured by flow cytometry. Grey areas represent the

background fluorescence signals obtained incubating the cells with the secondary antibody

only. (B) Inhibition of Hla hemolytic activity by sera from mice immunized with

HlaH35L-OMVs. Hemolysis of rabbit erythrocytes was measured by incubating 20 ng Hla

in the presence of increasing dilutions of sera from mice immunized with either 10 g of

purified HlaH35L + Alum, or 10 g of “Empty”-OMVs, or 10 g of HlaH35L-OMVs.

Hemolytic activity is expressed as percentage of OD values over the OD values obtained

incubating erythrocytes with water (100% haemolysis). (C) In vivo protective activity of

HlaH35L-OMVs in the sepsis model of infection. – Groups of 16 mice were immunized

with 2 doses of either “Empty-OMV” (2 g/dose) (grey) or HlaH35L-OMVs (black). After

2 weeks, mice were infected intra-peritoneally with a lethal dose of S. aureus Newman

strain (4 x 108 CFUs). Mice survival 7 days after infection is reported.

0

10

20

30

40

50

60

70

80

0

20

40

60

80

100

1:80 1:160 1:320 1:640 1:1280

"Empty"-OMVs rHla HlaH35L-OMVs

BA

BL21(DE3)/ΔompA(pET FhuD2)

FITC-A

% o

f M

ax

BL21(DE3)/ΔompA(pET empty)

FITC-A

% o

f M

ax

Hem

loly

sis

inh

ibit

ion

(%

)Serum dilutions

Surv

ival

(%

)

C«Empty»-OMVs

HlaH35L-OMVs

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Table S1.

Sequence homology of FhuD2, Csa1A, LukE, SpaKKAA and HlaH35L proteins as expressed

in OMVs with the same proteins expressed in S. aureus isolates

FhuD2 Csa1A LukE SpAKKAA Hla

% protein identity

Newman strain 100% 100% 98.93% 95.88% 100%

% protein identity

2449 strains in NCBI ≥98.94% ≥ 92.03% ≥ 99.29% ≥99.44% ≥98.66%

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Table S2. Oligos used in this study

Name Sequence

Primers for gDNA cloning

g-ompA f aaacTTTAGCACCAGTGTACCAGGTGTTATCTTTg

g-ompA r aaaacAAAGATAACACCTGGTACACTGGTGCTAAA

g-msbB f aaacTCCTTTCGCCACCCGCGCTACTGGGGAGCAg

g-msbB r aaaacTGCTCCCCAGTAGCGCGGGTGGCGAAAGGA

g-pagP f aaacACAACGTTTAGAGAAAATATTGTACAAACCg

g-pagP r aaaacGGTTTGTACAATATTTTCTCTAAACGTTGT

Oligo donor

Donor ompA f ACCGTGTTATCTCGTTGGAGATATTCATGGCGTATTTTGGATGATAACGAGGCGCAAAAAGTTCTCGTCTGGTAGAAAAACCCCGCTGCTGCGGGGTTTTTTTTGCCTTTAGTAAATTGA

Donor ompA r TCAATTTACTAAAGGCAAAAAAAACCCCGCAGCAGCGGGGTTTTTCTACCAGACGAGAACTTTTTGCGCCTCGTTATCATCCAAAATACGCCATGAATATCTCCAACGAGATAACACGGT

Donor msbB f CAAGTTGCGCCGCTACACTATCACCAGATTGATTTTTGCCTTATCCGAAACTGGAAAAGCAAAAGCCTCTCGCGAGGAGAGGCCTTCGCCTGATGATAAGTTCAAGTTTGCTTCAGAATA

Donor msbB r TATTCTGAAGCAAACTTGAACTTATCATCAGGCGAAGGCCTCTCCTCGCGAGAGGCTTTTGCTTTTCCAGTTTCGGATAAGGCAAAAATCAATCTGGTGATAGTGTAGCGGCGCAACTTG

Donor pagP f TGTTAATTGTAGCTTTGCTATGCTAGTAGTAGATTTTTGATAAATGTTTTATGGTCACAAAGTTTTAGTAACTTCTTTAAAATCAATAGCTAAAATAAGTAACATCAAAAATAACGCGAC

Donor pagP

GTCGCGTTATTTTTGATGTTACTTATTTTAGCTATTGATTTTAAAGAAGTTACTAAAACTTTGTGACCATAAAACATTTATCAAAAATCTACTACTAGCATAGCAAAGCTACAATTAACA

Screening primers for the genomic loci

ompA F CGTTGTAGACTTTACATCGCCAG

17

ompA R GTCTTCTCTGAAGCAGGATCTGC

msbB F GCCAAAGAGATTGTGCCGCAGC

msbB R CGGTAGAGTAAGTACGTTGCCG

pagP F GCATCATCTTTAATCGATGCGCGG

pagP R GCTGTGTCGGTTACCAGTACACC

Primers for antigens fusion to the OmpA and Lpp Leader sequence and cloning into pET21B

Nohisflag V-f TAACATCACCATCACCATCACGATTACAAAGA

omprev GGCCTGCGCTACGGTAGCGAAA

Lpp-R-plasmid GCTGGAGCAACCTGCCAGCAGAG

OmpA-Hla f1 Accgtagcgcaggcc GCAGATTCTGATATTAATATTAAAACCGGT

Lpp-Hla-f1 ctgctggcaggttgcGCAGATTCTGATATTAATATTAAAACCGGT

Hla-r1 gtgatggtgatgttaATTTGTCATTTCTTCTTTTTCCCAATCGAT

OmpA-Sta006 f1 accgtagcgcaggccGGGAACCAAGGTGAAAAAAATAACAAAG

Lpp-FhuD2-f1 ctgctggcaggttgcGGGAACCAAGGTGAAAAAAATAACAAAG

FhuD2-r1 gtgatggtgatgttaTTTTGCAGCTTTAATTAATTTTTCTTTTAAATCTTTAC

OmpA-Sta011 f1 accgtagcgcaggccGGCATAGGTAAAGAAGCGGAAG

Lpp-CsA1-f1 Ctgctggcaggttgc GGCATAGGTAAAGAAGCGGAAG

CsA1-r1 gtgatggtgatgttaTACATCTCCGCTTTTTTTATAATCTAAGC

OmpA-SpA_DEABC f1 accgtagcgcaggccGCACAGCATGATGAAGCCAAAAAA

Lpp-SpA-f1 Ctgctggcaggttgc GCACAGCATGATGAAGCCAAAAAA

SpA-r1 gtgatggtgatgttaTTTAGGTGCCTGTGCGTCGTT

OmpA-LukE f accgtagcgcaggccAATACTAATATTGAAAATATTGGTGATGGTGC

Lpp-Luke-f1 ctgctggcaggttgcAATACTAATATTGAAAATATTGGTGATGGTGC

Luke-r1 gtgatggtgatgttaATTATGTCCTTTCACTTTAATTTCGTGTGTTTTCCA