Vibrio cholerae proteome-wide screen for immunostimulatory proteins identifies phosphatidylserine decarboxylase as a novel Toll-like receptor 4 agonist.
Recognition of conserved bacterial components provides immediate and efficient immune responses and plays a critical role in triggering antigen-specific adaptive immunity. To date, most microbial components that are detected by host innate immune system are non-proteinaceous structural components. In order to identify novel bacterial immunostimulatory proteins, we developed a new high-throughput approach called "EPSIA", Expressed Protein Screen for Immune Activators. Out of 3,882 Vibrio cholerae proteins, we identified phosphatidylserine decarboxylase (PSD) as a conserved bacterial protein capable of activating host innate immunity. PSD in concentrations as low as 100 ng/ml stimulated RAW264.7 murine macrophage cells and primary peritoneal macrophage cells to secrete TNFalpha and IL-6, respectively. PSD-induced proinflammatory response was dependent on the presence of MyD88, a known adaptor molecule for innate immune response. An enzymatically inactive PSD mutant and heat-inactivated PSD induced approximately 40% and approximately 15% of IL-6 production compared to that by native PSD, respectively. This suggests that PSD induces the production of IL-6, in part, via its enzymatic activity. Subsequent receptor screening determined TLR4 as a receptor mediating the PSD-induced proinflammatory response. Moreover, no detectable IL-6 was produced in TLR4-deficient mouse macrophages by PSD. PSD also exhibited a strong adjuvant activity against a co-administered antigen, BSA. Anti-BSA response was decreased in TLR4-deficient mice immunized with BSA in combination with PSD, further proving the role of TLR4 in PSD signaling in vivo. Taken together, these results provide evidence for the identification of V. cholerae PSD as a novel TLR4 agonist and further demonstrate the potential application of PSD as a vaccine adjuvant.
Thanawastien
Ann
A
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA.
Montor
Wagner R
WR
Labaer
Joshua
J
Mekalanos
John J
JJ
Yoon
Sang Sun
SS
D000276:Adjuvants, Immunologic / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002262:Carboxy-Lyases / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D054884:Host-Pathogen Interactions; D015850:Interleukin-6 / Q000096:biosynthesis / Q000276:immunology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D053594:Myeloid Differentiation Factor 88 / Q000276:immunology; D020543:Proteome / Q000032:analysis* / Q000276:immunology; D040901:Proteomics / Q000379:methods; D012710:Serum Albumin, Bovine / Q000276:immunology; D015398:Signal Transduction / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000819:agonists* / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000276:immunology; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics / Q000276:immunology
D000276:Adjuvants, Immunologic; D015850:Interleukin-6; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D020543:Proteome; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D012710:Serum Albumin, Bovine; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase
J Immunol. 2006 Jul 15;177(2):1272-81
16818787
Plant Physiol. 2007 Jun;144(2):904-14
17449644
J Immunol. 2002 Jul 1;169(1):10-4
12077222
Infect Immun. 2006 Jan;74(1):694-702
16369026
Cell. 2007 Sep 21;130(6):1071-82
17889651
Biochem J. 1996 Oct 1;319 ( Pt 1):33-8
8870646
J Biol Chem. 1990 Mar 5;265(7):4111-5
2406271
J Biol Chem. 1974 May 25;249(10):3079-84
4598120
Curr Biol. 2000 Sep 21;10(18):1139-42
10996797
Curr Opin Gastroenterol. 2006 Jan;22(1):8-12
16319670
Apoptosis. 2003 Mar;8(2):115-28
12766472
Biochim Biophys Acta. 1997 Sep 4;1348(1-2):236-44
9370338
J Immunol. 2006 Jul 1;177(1):322-32
16785528
J Bacteriol. 1998 Jan;180(1):100-6
9422599
Proc Natl Acad Sci U S A. 2008 Feb 26;105(8):3070-5
18287038
J Endotoxin Res. 2003;9(1):55-9
12691620
Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4364-9
18337508
Seikagaku. 1999 Jan;71(1):1-15
10067119
Methods Enzymol. 1997;280:81-8
9211308
J Biol Chem. 2006 Apr 7;281(14):9049-57
16455646
J Bacteriol. 2003 Aug;185(15):4585-92
12867469
Cold Spring Harb Symp Quant Biol. 1999;64:429-35
11232318
Curr Opin Immunol. 1997 Feb;9(1):4-9
9039775
Biochim Biophys Acta. 2002 Feb 13;1589(1):1-13
11909637
Trends Microbiol. 1999 Dec;7(12):493-9
10603485
Scand J Infect Dis. 2003;35(9):555-62
14620134
J Endotoxin Res. 2001;7(3):167-202
11581570
J Endotoxin Res. 2004;10(6):413-8
15588424
Cell Mol Life Sci. 2003 Sep;60(9):1793-804
14523544
Nature. 1988 Jul 21;334(6179):255-8
2456466
Nature. 2001 Oct 18;413(6857):732-8
11607032
Nature. 2001 Apr 26;410(6832):1099-103
11323673
Nature. 1998 Sep 17;395(6699):284-8
9751057
J Bacteriol. 2008 Aug;190(15):5493-501
18502869
Infect Immun. 2008 Mar;76(3):1282-8
18174340
Mol Microbiol. 2006 Apr;60(1):51-66
16556220
Nat Immunol. 2003 Dec;4(12):1247-53
14625549
Trends Microbiol. 2000 Oct;8(10):452-6
11044679
Proc Natl Acad Sci U S A. 2008 Jun 24;105(25):8736-41
18574146
J Immunol. 2007 Feb 1;178(3):1243-9
17237368
Int Immunol. 2005 Jan;17(1):1-14
15585605
Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9237-42
11470918
J Immunol. 2006 Jun 15;176(12):7335-45
16751377
J Exp Med. 1994 Jan 1;179(1):269-77
7505800
J Exp Med. 1997 Nov 17;186(10):1623-31
9362523
J Biol Chem. 2005 Dec 2;280(48):40032-40
16192276
J Cell Sci. 2002 Jun 15;115(Pt 12):2603-11
12045230
Science. 2005 Aug 19;309(5738):1248-51
16020693
J Virol. 2005 Oct;79(20):12893-904
16188991
J Exp Med. 1998 Dec 7;188(11):2091-7
9841923
J Immunol. 1999 Apr 1;162(7):3749-52
10201887
Immunity. 1999 Jul;11(1):115-22
10435584
Curr Top Microbiol Immunol. 2002;270:155-67
12467250
Curr Opin Microbiol. 2007 Feb;10(1):4-9
17161645
J Lipid Res. 1972 Mar;13(2):263-7
5016308