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Supplementary Material Contents Immune modulating proteins identified from exosomal samples.....................................................................2 Figure S1: Overlap between exosomal and soluble proteomes.................................................................................... 4 Bacterial strains:..............................................................................................................................................4 Figure S2: Variability between subjects of effects of exosomes on BL21-lux growth.................................................... 5 Figure S3: Early effects of exosomes on growth of BL21 E. coli .................................................................................... 5 Figure S4: Exosomal Lysis............................................................................................................................................ 6 Figure S5: Effect of pH on exosomal action.................................................................................................................. 7 Figure S6: Effect of exosomes on growth of UPEC (pH = 6.5) suspended in exosome-depleted urine supernatant ....... 8 Effective exosomal concentration....................................................................................................................8 Figure S7: Sample constitution for luminometry experiments..................................................................................... 8 Figure S8: Determining effective concentration .......................................................................................................... 9 Figure S9: Independent confirmation of immune proteins by Western blot .............................................................. 10 Figure S10: Effect of serial washes on exosomal action against E coli ........................................................................ 11 Supplementary methods ...............................................................................................................................12 1. Protein precipitation for mass spectrometry ......................................................................................................... 12 2. Espresso protein type-I error estimating algorithm ............................................................................................... 12 3. Antibodies ............................................................................................................................................................ 13 Supplementary references.............................................................................................................................14 Supplementary Table S1. Proteins identified from human urinary exosomal samples by mass spectrometry.................................................................................................................................................17 1 Immune modulating proteins identified from exosomal samples (see Table 1) Aminopeptidase-N (ANPEP) serves as an E. coli fimbrial receptor,1 and functions as a viral receptor.2 Transferrin similarly sequesters iron and destabilises bacterial membranes,3 though not at as acidic a pH as lactoferrin.4 LGALS3BP is present in breast milk and protects neonates from diarrhoeal illness, may protect against viral invasion, and is implicated in NK cell-mediated immunity.5 TRIM5 is involved in viral sensing and restricts retroviral infection.6, 7 CARD9 is indispensible for fungal innate and acquired fungal immunity, and its deficiency results in mucosal and systemic candidiasis.8 IFIT5 is highly conserved across species and reduces viral replication and mediates host antiviral defense,9 likely through direct binding of viral RNA.10 PIGR is a key mediator of mucosal immunity. Synthesised in epithelia, it binds basolateral IgA, is transcytosed to the apical surface where it is either expressed with bound IgA, or cleaved as "secretory component" which served to mediate IgA action in host defense.11 Neprilysin (also known as CD10 or CALLA) is an endopeptidase expressed on neutrophils and lymphocytes, and is also highly expressed in kidney. It regulates levels of inflammatory peptides induced during infection with E. coli.12 The homologue in Manduca sexta (Q86RS4) is implicated in the immune response to bacterial infection.13 Keratin 10 is a cytoskeletal protein that is widely expressed. In epithelia, Staphylococcus aureus adheres to the cell surface by binding to surface-expressed keratin 10 through the S aureus clumping factor B (ClfB).14 Calprotectin (S100A8/A9) is a heterotetramer consisting of S100A8 and S100A9. It inhibits growth and kills gram positive and gram-negative bacteria, and Candida albicans.15, 16 Lysozyme C is microbicidal against gram-positive and -negative bacteria and Candida albicans, inducing lysis of gram-negative bacteria by destabilising the outer membrane and forming pores in the inner membrane.17, 18 2 MASP2 is a mannan-binding lectin associated serine protease that is important for activation of the complement pathway. Deficiency in MASP2 is permissive to pneumococcal infection.19 Polymorphisms in the MASP2 gene are associated with HTLV-1 infection.20 Mucin-1 is widely expressed in epithelia. It binds to the surfaces of Pseudomonas aeruginosa and E. coli.21, 22 Furthermore, female mucin-1 null mice develop chronic genito-urinary infections.23 Myeloperoxidase (MPO) selectively binds and kills many gram-positive and -negative bacteria,24 and also has candidicidal and antiviral properties.25, 26 Its activity is enhanced at acidic pH.27 CD14 is the principal mammalian bacterial lipopolysaccharide receptor,28, 29 binds gram positive and gram negative bacterial surfaces,30 and may also function as a viral receptor.31 S100A7 (Psoriasin) is present in urine,32 kills E. coli by pore formation, and is maximally effective at pH 6.33, 34 Superoxide dismutase (SOD1) may inhibit bacterial growth by sequestering copper and zinc, and is implicated in host defence against Schistosoma infection.35 Dermcidin adheres to bacterial cell walls and inhibits growth of gram positive and negative bacteria, and Candida albicans,36 by inhibiting bacterial RNA and protein synthesis.37 The D3 fragment of kininogen-1 (KNG-1) is potently bactericidal with activity against gram-positive and - negative bacteria.38 Protein C Receptor (PROCR) contributes to host defence against E. coli, although the mechanism is not clear.39 Fibronectin is a glycoprotein that is expressed at cell surfaces as a dimer, or secreted. It is the principle human receptor for Treponoema pallidum40 and Trichomonas vaginalis.41 PLUNC binds bacterial lipopolysaccharide, destabilises membranes and inhibits growth of Pseudomonas aeruginosa,42, 43 while overexpression of PLUNC in bronchial epithelium protects against mycoplasma infection.44 PGLYRP1 is a peptidoglycan recognition protein that demonstrates zinc-dependent killing of gram positive and gram negative bacteria.45 Lactoferrin is a cationic protein that inhibits bacterial growth by sequestering iron, and by adhering to and destabilising bacterial membranes.46, 47 3 Histatin-1 is a candidicidal protein.48 Lipocalin 1 and Lipocalin 2 are bacteriostatic proteins that inhibit bacterial growth by their ability to bind iron.49, 50 Figure S1: Overlap between exosomal and soluble proteomes Exosomal Non-exosomal 476 125 239 Of the exosomal proteome, 21% were shared with soluble urinary proteins from simultaneously prepared samples. Bacterial strains: Strain Source BL21 Commercially available, e.g. New England Biolabs, Ipswich MA, USA. Catalogue number C2530H UPEC Clinical isolate from a patient with urinary tract infection, Addenbrooke's Hospital, Cambridge. Organism characterised using the VITEK2 system version 04.02, bioMerieux Nissle Mutaflor®, sourced from Ardeypharm GmBH, Hagen, Germany UTI89 Gift of Dr Menna Clatworthy, Deparment of Medicine, University of Cambridge CFT073 ATCC®, Manassas VA, USA Catalogue number 700928™ 4 Figure S2: Variability between subjects of effects of exosomes on BL-21-lux growth Each of 4 volunteer samples, displayed in aggregate form in Figure 4, significantly inhibited growth of BL21 E. coli. Figure S3: Early effects of exosomes on growth of BL21 E. coli Addition of exosomes to BL21 (red arrow/curve) resulted in arrest of growth after approximately 30 min, with complete abolition of growth after 45 min. Uromodulin alone was not inhibitory; combination of exosomes with uromodulin after 45 min (pink arrow/curve) again abolished growth. BL21 was grown in LB medium, pH = 5.5. 5 Figure S4: Exosomal Lysis To determine whether exosomal structural integrity was required for the observed bacterial growth inhibition, exosomal preparations were divided; one part was retained intact, whereas the other was subjected to chemical lysis. Lysis was achieved with ammonium acetate and acetone precipitation as described under supplementary methods. Briefly, samples were incubated with 5 volumes 100% ammonium acetate in methanol overnight at 4°C, and centrifuged for 10 minutes at 3,000 x g. the resulting pellets were washed with 80% ammonium acetate at 4°C, and centrifuged for 10 minutes at 3,000 x g. The resulting pellets were washed with 80% acetone and centrifuged for 10 minutes at 3,000 x g. After removal of acetone, the final pellet was resuspended in 100 µl of a 250 mM sucrose solution, and equilibrated to a protein concentration matching that of the untreated exosomal aliquot. To confirm that
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