Supporting InformationCywes-Bentley et al. 10.1073/pnas.1303573110SI Text 1Underlying the barrier to isolation of poly-N-acetylglucosamine(PNAG) from most organisms producing wild-type levels of thisantigen are the findings that unlike bacterial capsular poly-saccharides that can be readily isolated from in vitro culturesupernates and microbial cell surfaces, almost all of the PNAG istightly bound to the cell surface and thus must be extracted forpurification (1–3). Analysis of levels of in vitro PNAG pro-duction by wild-type bacterial strains with a single copy ofthe intercellular adhesion (ica)/polyglucosamine (pga) locus in-dicates only 1–2 femtograms per cell are present (3, 4), a level ofsurface polysaccharide comparable to that reported for otherorganisms (5–7). This amount of surface carbohydrate is, none-theless, sufficient to be a target for protective antibody (8, 9).Thus, even in cultures that can grow to 109 cfu/mL, only ∼1–2μg/mL is available for isolation, and the actual yields are mark-edly reduced by the difficulty in extracting PNAG from the cellsurface, the insolubility of PNAG at neutral pH, which leads tosignificant losses during purification (4, 10), and additional lossesoccurring during the complex purification procedures needed toeliminate tightly associated contaminating bacterial antigens (1,4, 10). PNAG has only been isolated from cellular extracts ofnonrecombinant isolates of Staphylococcus epidermidis that werechosen due their natural production of unusually high amountsof antigen and could only be purified when these cells weregrown under conditions that markedly stimulate polysaccharidesynthesis (1, 11). In all other instances, PNAG has only beenisolated from recombinant bacteria when an altered promoter inthe biosynthetic chromosomal genetic locus is present that re-sults in strikingly enhanced transcription and PNAG synthesis(12) or the biosynthetic genes are cloned and overexpressed ona plasmid (4, 13–15). No other conditions for isolating PNAGfrommicrobial cultures have been described. These findings frommultiple investigators indicate that PNAG is produced at very lowlevels by most organisms. Therefore, when coupled with the dif-ficulties in purifying this material in sufficient quantities forchemical analysis in the absence of the overexpression of knownbiosynthetic genes, an explanation emerges as to why PNAG hasnot previously been found to be produced by diverse microbialspecies, particularly those lacking ica/pga homologous genes.

SI Text 2There is some concern that functional antibody to PNAG willdisrupt the normal PNAG-producing microbiota present in thegastrointestinal (GI) or female genital tract and on the skin,a well-known consequence of antibiotic treatments (16). Thoughhuman tests of PNAG-specific antibodies and vaccines shouldundoubtedly include careful evaluations to ensure acceptablelevels of safety, the concern about negative consequences fromdisruption of the indigenous microbiota can be allayed by severalobservations. Antibodies to antigens on typical commensal or-ganisms dominate our natural immune responses, and the effectof the indigenous microbiota on development and shaping of theimmune system is well established (17). Natural antibodies tocommensal microbial antigens are very important in protectionagainst infection, because loss of serum IgG, such as occurs incommon variable immunodeficiency (CVI) (18) greatly increasesthe occurrence of infections by commensal organisms. CVI ismost successfully treated by giving i.v. normal human IgG (19).Vaccination against PNAG would only increase the levels ofantibody to the normal microbiota by a few tenths of a percent,unlikely to be of significant consequence in the presence of all

of the rest of the natural and protective antibodies to commensalmicrobial antigens. The phase I evaluation of mAb F598 in hu-mans revealed no signs or symptoms associated with disruption ofthe indigenous microbiota (20). Approximately 5% of healthyhumans do develop natural opsonic/protective antibody to PNAG(21), indicating a relative lack of a negative effect of these anti-bodies on overall health in these individuals. Of note, a recentstudy (22) showed experimental GI infections in mice generateT-cell immune responses to the commensal microbiota that wasof the same magnitude as it was to pathogenic microbes, leadingthe authors to conclude that adaptive immune responses againstthe normal commensal flora are an integral component of mu-cosal immunity Additionally, we have vaccinated over a dozenrabbits and three goats that harbor PNAG-producing indigenousmicrobes with purified PNAG or with deacetylated PNAG or9GlcNH2 conjugated to various carrier proteins, and they allsurvived from 12 to 36 mo under frequent veterinary care andobservation without displaying any noticeable signs of distress orillness. A final consideration indicating immunity to PNAG willnot disrupt normal commensal organisms in a meaningful way isthat in the absence of stimuli promoting recruitment of comple-ment and inflammatory/phagocytic cells to sites where these or-ganisms reside, there is likely to be little pathologic consequencesfrom the presence of low levels of functional antibody to PNAG inthe GI lumen, the female genital tract, or on the skin. In con-clusion, the expression of PNAG among diverse pathogens andthe ability to demonstrate in vitro killing and in vivo protection byantibody to PNAG opens the door to use immunization strategiesthat can target many of the major microbial pathogens of humansand economically important animals.

SI Text 3Image Analysis for IFA of Sporozoites. Image analysis of the spo-rozoite IFA was carried out by placing the IFA slides on anOlympusAX80 Provis fluorescent microscope with a 40× objective,exposure time of 1/3.5 s, an isotropic signal of 400, and at1,360×1,024 resolution. The well was first scanned, then a repre-sentative photograph was taken. Images were then analyzed usingImage-Pro Plus (MediaCybernetics) using a software macro de-signed such that sporozoites were identified based on size anddimensions ignoring luminous artifacts. Sporozoites were manuallyselected until all sporozoites were analyzed (a minimum of three).The luminosity for the control sera was calculated by averaging theindividual luminosity of each parasite; maintaining a coefficient ofvariation of less than 20%. The cutoff for positivity was set at themean +2 SD of the negative control. The results from three sep-arate experiments of the individual mean fluorescent intensitiesobtained from the enzyme and periodate-treated specimens werestatistically analyzed comparing the mean ± the SD of the dif-ferences from the cutoff for positivity for the normal and anti-PNAG immune sera.

Electron Microscopy. Single antibody labeling. Bacteria were grownfor 24–48 h on chocolate agar plates at 37 oC in 5%CO2, and cellsharvested into PBS with 2% (vol/vol) BSA and 2% (vol/vol)glutaraldehyde and held overnight at 4 oC. Samples were labeledwith 15 μg MAb F598 or MAb F429/mL for 2 hr at room tem-perature (RT). Bacterial suspensions were placed onto parafilm,and then carbon coated grids made hydrophilic by a 30 s expo-sure to a glow discharge inverted onto the cells for 10 min at RT.Grids were placed in 20 μL of ice-cold 100% methanol for 1 minat RT, then inverted onto 20 μL droplets of PBS/BSA for 10 min

Cywes-Bentley et al. www.pnas.org/cgi/content/short/1303573110 1 of 9

at RT. Grids were next washed with 20 μL of PBS three timesover a 10-min period and then incubated on 20 μL of protein Agold 15 nm (1:60 dilution in 1% BSA/PBS) for 30 min at RT.Grids were washed with 20 μL of PBS twice over a 5-min period,followed by washing with 20 μL of water four times over a 10-minperiod. Grids were blotted and stored at RT. Samples wereviewed on a JEOL 1200EX–80-kV transmission electron mi-croscope and images recorded with an AMT 2k charge-coupleddevice camera.Dual antibody labeling. Samples were treated as described for singleantibody labeling. Once labeled with either mAb F598 or mAbF429 and 15 nm protein A gold, samples were incubated with 1%glutaraldehyde in 0.15 M glycine/PBS buffer for 10 min to cross-link mAb F598 or mAb F429 and protein A gold and block

binding of additional protein A. Grids were further incubated ina 1:100 dilution of a mouse mAb to serotype A N. meningitidiskindly provided by Dr. Peter Rice for 30 min at RT. Next, gridswere washed with 20 μL of PBS three times over a 10-min periodand then incubated with 20 μL of rabbit antibody to mouse IgGat a 1:100 dilution in 1% BSA/PBS for 20 min at RT. Grids werethen washed with 20 μL of PBS three times over a 10-min periodand incubated with 20 μL of protein A linked to 10 nm goldparticles (1:80 dilution in 1% BSA/PBS) for 20 min at RT. Gridswere washed with 20 μL of PBS twice over a 5-min period, fol-lowed by washing with 20 μL of water four times over a 10-minperiod. Grids were blotted and stored in holders at RT. Sampleswere viewed as described above.

1. Mack D, et al. (1996) The intercellular adhesin involved in biofilm accumulation ofStaphylococcus epidermidis is a linear beta-1,6-linked glucosaminoglycan: Purificationand structural analysis. J Bacteriol 178(1):175–183.

2. Vuong C, et al. (2004) A crucial role for exopolysaccharide modification in bacterialbiofilm formation, immune evasion, and virulence. J Biol Chem 279(52):54881–54886.

3. Cerca N, et al. (2007) Molecular basis for preferential protective efficacy of antibodiesdirected to the poorly acetylated form of staphylococcal poly-N-acetyl-β-(1–6)-glucosamine. Infect Immun 75(7):3406–3413.

4. Choi AHK, Slamti L, Avci FY, Pier GB, Maira-Litrán T (2009) The pgaABCD locus ofAcinetobacter baumannii encodes the production of poly-β-1–6-N-acetylglucosamine,which is critical for biofilm formation. J Bacteriol 191(19):5953–5963.

5. Baddour LM, et al. (1992) Staphylococcus aureus microcapsule expression attenuatesbacterial virulence in a ratmodel of experimental endocarditis. J Infect Dis 165(4):749–753.

6. WesselsMR,MosesAE,Goldberg JB,DiCesareTJ (1991)Hyaluronic acid capsule is a virulencefactor for mucoid group A streptococci. Proc Natl Acad Sci USA 88(19):8317–8321.

7. Kim JO, Weiser JN (1998) Association of intrastrain phase variation in quantityof capsular polysaccharide and teichoic acid with the virulence of Streptococcuspneumoniae. J Infect Dis 177(2):368–377.

8. Kim JO, et al. (1999) Relationship between cell surface carbohydrates and intrastrainvariation on opsonophagocytosis of Streptococcus pneumoniae. Infect Immun 67(5):2327–2333.

9. Pozzi C, et al. (2012) Opsonic and protective properties of antibodies raised to conjugatevaccines targeting six Staphylococcus aureus antigens. PLoS ONE 7(10):e46648.

10. Maira-Litrán T, Kropec A, Goldmann DA, Pier GB (2005) Comparative opsonic andprotective activities of Staphylococcus aureus conjugate vaccines containing native ordeacetylated Staphylococcal poly-N-acetyl-β-(1–6)-glucosamine. Infect Immun 73(10):6752–6762.

11. Sadovskaya I, Vinogradov E, Flahaut S, Kogan G, Jabbouri S (2005) Extracellularcarbohydrate-containing polymers of a model biofilm-producing strain, Staphylococcusepidermidis RP62A. Infect Immun 73(5):3007–3017.

12. Jefferson KK, Cramton SE, Götz F, Pier GB (2003) Identification of a 5-nucleotidesequence that controls expression of the ica locus in Staphylococcus aureus andcharacterization of the DNA-binding properties of IcaR. Mol Microbiol 48(4):889–899.

13. Wang X, Preston JF III, Romeo T (2004) The pgaABCD locus of Escherichia colipromotes the synthesis of a polysaccharide adhesin required for biofilm formation.J Bacteriol 186(9):2724–2734.

14. Joyce JG, et al. (2003) Isolation, structural characterization, and immunologicalevaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus.Carbohydr Res 338(9):903–922.

15. Maira-Litrán T, et al. (2002) Immunochemical properties of the staphylococcal poly-N-acetylglucosamine surface polysaccharide. Infect Immun 70(8):4433–4440.

16. Wiström J, et al. (2001) Frequency of antibiotic-associated diarrhoea in 2462antibiotic-treated hospitalized patients: A prospective study. J Antimicrob Chemother47(1):43–50.

17. Hooper LV, Littman DR, Macpherson AJ (2012) Interactions between the microbiotaand the immune system. Science 336(6086):1268–1273.

18. Cunningham-Rundles C, Maglione PJ (2012) Common variable immunodeficiency.J Allergy Clin Immunol 129(5):1425–1426.

19. Cunningham-Rundles C (2011) Key aspects for successful immunoglobulin therapyof primary immunodeficiencies. Clin Exp Immunol 164(Suppl 2):16–19.

20. Vlock D, Lee JC, Kropec-Huebner A, Pier GB (2010) Pre-clinical and initial phase Ievaluations of a fully human monoclonal antibody directed against the PNAG surfacepolysaccharide on Staphylococcus aureus. Abstracts of the 50th InterscienceConference on Antimicrobial Agents and Chemotherapy 2010 (Am Soc Microbiology,Washington, DC), G1-1654/329 (abstr).

21. Skurnik D, et al. (2012) Natural antibodies in normal human sera inhibitStaphylococcus aureus capsular polysaccharide vaccine efficacy. Clin Infect Dis205(11):1709–1718.

22. Hand TW, et al. (2012) Acute gastrointestinal infection induces long-lived microbiota-specific T cell responses. Science 337(6101):1553–1556.

Cywes-Bentley et al. www.pnas.org/cgi/content/short/1303573110 2 of 9

Fig. S1. Binding characteristics of mAbs to Pseudomonas aeruginosa alginate (F429) or PNAG (F598) and binding of polyclonal antibody to Plasmodiumfalciparum sporozoites. (A) mAb F429-AF488 to P. aeruginosa alginate binds to P. aeruginosa FRD1 cells but not to P. aeruginosa ΔalgD cells unable to makealginate, or to Staphylococcus aureus Δica unable to make PNAG or S. aureus Δica + ica with a chromosomal copy of the intact ica locus restored. mAb F598-AF488 does not bind to P. aeruginosa or to S. aureus Δica but does bind to PNAG-producing S. aureus Δica + ica. (Scale bars: 10 μM.) (B) Inhibition of binding ofmAb F598 to PNAG-coated ELISA plate by increasing concentrations of purified PNAG antigen, but no inhibition is obtained when using fungal β-glucans [mixof β-(1→3) and β-(1→6)–linked glucose monomers]. (C) ELISA binding of mAb F598 to β-(1→6)–linked N-acetylglucosamine oligosaccharides of indicated length(5, 7, 9, or 11 monomers per oligosaccharide). (D) Binding of a 1:50 dilution of normal rabbit serum or immune rabbit antibody raised to 9GlcNH2-TT toPlasmodium falciparum sporozoites (∼1 × 10 μM) in an indirect immunofluorescence assay. (Scale bars: 10 μM.) (E) Effect of treatment with chitinase (control)or the PNAG-degrading enzyme dispersin B or sodium metaperiodate on reactivity of the anti-PNAG antibody raised to synthetic β-(1→6)–linked glucosamineoligosaccharide conjugated to tetanus toxoid (9GlcNH2-TT) with P. falciparum sporozoites. A luminosity of zero indicates the positivity limit, which was 2 SDabove the mean of a negative control. Bars represent mean luminosity for three experiments, and error bars are SEM. P values by unpaired t test.

Cywes-Bentley et al. www.pnas.org/cgi/content/short/1303573110 3 of 9

Fig. S2. Immunochemical verification of reactivity of mAb F598-AF488 with PNAG expressed by multiple pathogens. Microbial cells were fixed in para-formaldehyde, then treated with either chitinase or the PNAG-degrading enzyme dispersin B dissolved in TBS (pH 6.4) at 50 μg/mL overnight at 37 °C or in 0.4 Msodium periodate (IO4) for 2 h at 37 °C. Cells were washed, placed on microscope slides, air-dried, exposed to ice-cold methanol for 1 min, and then reactedwith mAb F598 to PNAG conjugated to AF488 (green fluorescence) as well as SYTO 83 to visualize DNA (red fluorescence) and photographed using scanningconfocal laser microscopy. Chitinase-resistant but dispersin B/IO4-sensitive binding to mAb F598 is indicative of PNAG expression. (Scale bars: 10 μm; somemicrographs lack bars due to cropping but are at same magnification as other micrographs of same strain.)

Cywes-Bentley et al. www.pnas.org/cgi/content/short/1303573110 4 of 9

Fig. S3. Expression of PNAG among Streptococcus pneumoniae strains. Cells from cultures of the serotype of S. pneumoniae indicated on the left side of eachpanel in yellow, either contained within (S. pneumoniae vaccine strains) or absent (S. pneumoniae nonvaccine strains) from the current 13-valent conjugatevaccine (Prevnar-13; Pfizer) were fixed in suspension with 4% paraformaldehyde and then placed onto slides, air-dried, exposed to cold methanol for ∼1 min,and then reacted with either control mAb F429 conjugated to AF488 or mAb F598 to PNAG conjugated to AF488 (green fluorescence) as well as SYTO 83 tostain DNA (red fluorescence), and visualized and photographed using scanning confocal laser microscopy. (Scale bars: 10 μm; some micrographs lack bars due tocropping but are at same magnification as other micrographs of same strain.)

Cywes-Bentley et al. www.pnas.org/cgi/content/short/1303573110 5 of 9

Fig. S4. Specificity of bactericidal activity for PNAG antigen against Neisseria meningitidis serogroup B and Neisseria gonorrhoeae in a rabbit antiserum raisedto PNAG. Rabbit antibody to the 9GlcNH2-TT conjugate vaccine was incubated with indicated concentration of purified PNAG or alginate antigen beforeaddition to bactericidal assays. Percent killing was calculated in comparison with normal rabbit serum at same concentration with the same inhibitor. Heat-inactivated complement (HI C′) control represents the anti–9GlcNH2-TT serum heated at 56 °C for 30 min with no inhibitor added. Blue bars indicate no (0)inhibitor added to immune serum. (A) Inhibition of bactericidal killing of N. meningitidis serogroup B strains by PNAG but not alginate. (B) Inhibition ofbactericidal killing of N. gonorrhoeae strains by PNAG but not alginate. Bars represent means of quadruplicate determinations.

Fig. S5. Detailed quantification of pathology scored for eukaryotic pathogen infections of mice. (A) Pathology score for murine BALB/c T-bet−/− RAG2−/−

(TRUC) mouse colitis model using a scale of 0–3 (1) for four indicated parameters evaluated to determine total score. TRUC mice were treated from week 2 ofbirth with either mAb F598 or control IgG1 mAb F105. (B) Pathology score of 0 or 1 (full scale was available for scoring only scores of 0 or 1 achieved) of WTmice cross-fostered on TRUC females and therapeutically treated starting at 4 wk of age with mAb F598 or control IgG1 mAb F105. Each square (filled for mAbF598-treated mice, open for control IgG1-treated mice) is one mouse, and bars represent the medians. (C and D) Corneal pathology score on scale of 0–4 (2)determined by masked observer in eyes of mice challenged with indicated dose of Candida albicans and treated as indicated in the figure with either controlIgG1 mAb or mAb F598 to PNAG. P values in all graphs determined by unpaired, nonparametric t tests. NS, not significant. Each symbol is one mouse, and barsrepresent the medians.

Cywes-Bentley et al. www.pnas.org/cgi/content/short/1303573110 6 of 9

1. Garrett WS, et al. (2007) Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system. Cell 131(1):33–45.2. Zaidi TS, Zaidi T, Pier GB (2010) Role of neutrophils, MyD88-mediated neutrophil recruitment, and complement in antibody-mediated defense against Pseudomonas aeruginosa

keratitis. Invest Ophthalmol Vis Sci 51(4):2085–2093.

Fig. S6. Additional parameters of effect of treatment with antibody to PNAG on infection with Plasmodium berghei ANKA. (A) Development of cerebralmalaria (occurs by day 9 postinfection) and associated death in infected mice treated with either normal goat serum or goat serum raised to 9GlcNH2-TTconjugate vaccine. P value: log-rank test. (B and C) Development of red blood cell parasitemia in mice (different colored line for individual animal) infectedwith P. berghei ANKA treated with indicated serum. Five of eight mice treated with normal serum died before the day-9 sample could be obtained to de-termine the level of parasitemia. (B) Compared with one of eight mice given immune serum (C). (D and E) Detection of PNAG antigen in P. berghei ANKA (D) orP. berghei ANKA GFPcon 259cl2 (E). Blood smears from infected mice were permeabilized with methanol and reacted with either control mAb F429-AF488 ormAb F598-AF488 to PNAG. The GFP-positive clone was also reacted with a primary rabbit antibody to GFP followed by goat anti-rabbit IgG conjugated toAF568 to produce red fluorescence. In D, green arrowheads indicate infected red blood cells observed in the phase-contrast micrograph. (Scale bars: 10 μm.)

Fig. S7. Survival of pga/ica-positive and mutant bacterial strains for 48 h in 5% ethanol. The indicated bacterial strain with an intact ica/pga locus (wild-type),a deletion in this locus (mutant), or the mutant containing the cloned locus in trans (complemented) were grown on agar plates then recovered, washed, andsuspended at ∼106 cfu/mL in water with 5% ethanol, at which time the initial inoculum (time 0) was ascertained by diluting and plating for bacterial enu-meration. Cultures were kept at room temperature for 48 h, after which the cultures were sonicated for 15 s, then the viable cfu counts determined by serialdilution and plating. Fold change was calculated in comparison with cfu counts per milliliter at time 0. Bars represent means of triplicates, and error bars theSEM. P values determined by unpaired t tests.

Cywes-Bentley et al. www.pnas.org/cgi/content/short/1303573110 7 of 9

Table S1. Microbial species and strains used, characteristics, and source

Species Strain Characteristics Source

Staphylococcus aureus MN8 Wild-type Laboratory stockMN8 Δica ica locus deleted strain, PNAG negative Laboratory stockMN8 Δica + ica ica complemented strain, PNAG positive Laboratory stock

Streptococcus pneumoniae D39 Serotype 2 Janet YotherATCC8 Serotype 8 Janet YotherBG5-8A Serotype 6A Janet YotherBG9273 Serotype 6A Janet YotherEF3030 Serotype 19F Rachel McLoughlin519/43 Serotype 1 Rachel McLoughlinATCC 6301 Serotype 1 Rachel McLoughlin243 Serotype 4 Rick Malley/Marc Lipsitch244 Serotype 5 Rick Malley/Marc Lipsitch246 Serotype 14 Rick Malley/Marc Lipsitch248 Serotype 6B Rick Malley/Marc Lipsitch284 Serotype 18C Rick Malley/Marc Lipsitch291 Serotype 7F Rick Malley/Marc Lipsitch295 Serotype 3 Rick Malley/Marc LipsitchSPEC1 Serotype 1 BEI ResourcesOREP3 Serotype 3 BEI ResourcesOREP4 Serotype 4 BEI ResourcesSTREP5 Serotype 5 BEI ResourcesTREP6A Serotype 6A BEI ResourcesSPEC6B Serotype 6B BEI ResourcesOREP7F Serotype 7F BEI ResourcesEMC9V Serotype 9V BEI ResourcesSTREP14 Serotype 14 BEI ResourcesOREP18C Serotype 18C BEI ResourcesTREP19A Serotype 19A BEI ResourcesSPEC19F Serotype 19F BEI ResourcesEMC23F Serotype 23F BEI ResourcesDSM11865 Serotype 9V A.R.19A Serotype 19A S.I.P.457 Serotype 11A Sandra Richter156 Serotype 12F Sandra Richter975 Serotype 15A Sandra Richter212 Serotype 22F Sandra Richter255 Serotype 31 Sandra Richter731 Serotype 35B Sandra Richter

Streptococcus pyogenes 950771 Encapsulated type M3, clinical isolate Michael Wessels188 Acapsular isogenic mutant of 950771 Michael WesselsDLS003 Encapsulated type M3, clinical isolate Michael Wessels

Streptococcus dysgalactiae AMI 05 Bovine mastitis Yung-Fu ChangEnterococcus faecalis V583 Sequenced strain Johannes Huebner

T2 Serotype 2 Johannes HuebnerT5 Serotype 5 Johannes Huebner

Listeria monocytogenes 481 Serotype 1/2b Darren HigginsClostridium difficle 14372 Clinical isolate Neeraj (Neil) SuranaMycobacterium tuberculosis H37Rv Sequenced strain Eric RubinMycobacterium smegmatis MC2 155 Sequenced strain Eric RubinNeisseria meningitidis serogroup A 80084313 Lee WetzlerNeisseria meningitidis serogroup B B16B6 Lee Wetzler

M981 Peter Rice2996 Peter RiceNMB Peter RiceH44/76 Peter Rice

Neisseria gonorrhoeae 2399 Peter Rice252 Peter RiceFA1090 Sequenced strain/ATCC 700825 Peter Rice24–1 ATCC BAA-1842 Peter Rice15253 Peter Rice442089 Peter RiceDGI-37 AHU Peter Rice

Hemophilus influenzae (nontypable) 200 Clinical isolate L.O.B.

Cywes-Bentley et al. www.pnas.org/cgi/content/short/1303573110 8 of 9

Table S1. Cont.

Species Strain Characteristics Source

140 Clinical isolate L.O.B.75 Clinical isolate L.O.B.

Hemophilus ducreyi 35000HP Sequenced strain/ATCC 700724 Stanley SpinolaHelicobacter pylori G27 Sequenced strain Joanna GoldbergCampylobacter jejuni CG8421 Sequenced strain Janet LindowBacillus fragilis 9343 Sequenced strain Laurie ComstockCitrobacter rodentium ATCC 51459 Prototype isolate for transmissible

murine colonic hyperplasiaLynn Bry

Salmonella enterica serovar typhi Ty2 Sequenced strain Laboratory stockSalmonella enterica serovar typhimurium LT2 Sequenced strain Laboratory stockCandida albicans SC5314 Sequenced strain Laboratory stockAspergillus flavus BP09-1 Clinical isolate Darlene MillerFusarium solani B1-11 Clinical isolate Darlene MillerCryptococcus neoformans 24062 Arturo CasadevallPlasmodium berghei ANKA strain Causes cerebral malaria in mice D.T.G./R.B.D.

(ANKA) GFPcon259cl2, MRA-865

Causes cerebral malaria inmice; PNAG-negative

BEI Resources

Plasmodium falciparum Clinical isolate Daniel MilnerKlebsiella pneumoniae Serotype K2 Wild-type Alan Cross

Δpga pga locus deleted strain D.R.Δpga + ppga pga complemented strain D.R.

Escherichia coli Serotype K1 Wild-type Kwang Sik KimΔpga pga locus deleted strain D.R.Δpga + ppga pga complemented strain

(ppga plasmid)D.R.

Trichomonas vaginalis UR1 clone Derivative of clinical isolate UR1 R.N.F.B7RC2 T. vaginalis genome reference strain R.N.F.UH9 Clinical isolate R.N.F.

For organisms where multiple strains were tested for PNAG production but the results not depicted in the figures, the first strain listed for each organismis the one wherein the results are provided in the figure and the remaining strains were tested for PNAG production by both mAb binding and susceptibilityto degradation by dispersin B and sodium periodate.

Cywes-Bentley et al. www.pnas.org/cgi/content/short/1303573110 9 of 9