Outer membrane protein P4 is not required for virulence in the human challenge model of Haemophilus ducreyi infection

BMC Microbiology

Margaret E Bauer³  Barry P Katz¹  Kate R Fortney³  Beth W Zwickl²  Diane M Janowicz² 

[1] Department of Biostatistics, Indiana University School of Medicine, 635 Barnhill Drive Room MS-218, Indianapolis, IN 46202, USA;Department of Medicine, Indiana University School of Medicine, 545 Barnhill Drive Room EH-435, Indianapolis, IN 46202, USA;Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive Room MS-218, Indianapolis, IN 46202, USA

关键词: Human;    Lipoprotein;    Chancroid;    GUD;    H. ducreyi;   

Others  :  821341
DOI  :  10.1186/1471-2180-14-166

 received in 2014-02-14, accepted in 2014-06-04,  发布年份 2014

【 摘 要 】

Background

Bacterial lipoproteins often play important roles in pathogenesis and can stimulate protective immune responses. Such lipoproteins are viable vaccine candidates. Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, expresses a number of lipoproteins during human infection. One such lipoprotein, OmpP4, is homologous to the outer membrane lipoprotein e (P4) of H. influenzae. In H. influenzae, e (P4) stimulates production of bactericidal and protective antibodies and contributes to pathogenesis by facilitating acquisition of the essential nutrients heme and nicotinamide adenine dinucleotide (NAD). Here, we tested the hypothesis that, like its homolog, H. ducreyi OmpP4 contributes to virulence and stimulates production of bactericidal antibodies.

Results

We determined that OmpP4 is broadly conserved among clinical isolates of H. ducreyi. We next constructed and characterized an isogenic ompP4 mutant, designated 35000HPompP4, in H. ducreyi strain 35000HP. To test whether OmpP4 was necessary for virulence in humans, eight healthy adults were experimentally infected. Each subject was inoculated with a fixed dose of 35000HP on one arm and three doses of 35000HPompP4 on the other arm. The overall parent and mutant pustule formation rates were 52.4% and 47.6%, respectively (P = 0.74). These results indicate that expression of OmpP4 in not necessary for H. ducreyi to initiate disease or progress to pustule formation in humans. Hyperimmune mouse serum raised against purified, recombinant OmpP4 did not promote bactericidal killing of 35000HP or phagocytosis by J774A.1 mouse macrophages in serum bactericidal and phagocytosis assays, respectively.

Conclusions

Our data suggest that, unlike e (P4), H. ducreyi OmpP4 is not a suitable vaccine candidate. OmpP4 may be dispensable for virulence because of redundant mechanisms in H. ducreyi for heme acquisition and NAD utilization.

【 授权许可】

   
2014 Janowicz et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Pizza M, Scarlato V, Masignani V, Giuliani MM, Arico B, Comanducci M, Jennings GT, Baldi L, Bartolini E, Capecchi B, Galeotti CL, Luzzi E, Manetti R, Marchetti E, Mora M, Nuti S, Ratti G, Santini L, Savino S, Scarselli M, Storni E, Zuo P, Broeker M, Hundt E, Knapp B, Blair E, Mason T, Tettelin H, Hood DW, Jeffries AC, et al.: Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 2000, 287(5459):1816-1820.
• [2]Fraser CM, Casjens S, Huang WM, Sutton GG, Clayton R, Lathigra R, White O, Ketchum KA, Dodson R, Hickey EK, Gwinn M, Dougherty B, Tomb JF, Fleischmann RD, Richardson D, Peterson J, Kerlavage AR, Quackenbush J, Salzberg S, Hanson M, van Vugt R, Palmer N, Adams MD, Gocayne J, Weidman J, Utterback T, Watthey L, McDonald L, Artiach P, Bowman C, et al.: Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 1997, 390(6660):580-586.
• [3]Sigal LH, Zahradnik JM, Lavin P, Patella SJ, Bryant G, Haselby R, Hilton E, Kunkel M, Adler-Klein D, Doherty T, Evans J, Molloy PJ, Seidner AL, Sabetta JR, Simon HJ, Klempner MS, Mays J, Marks D, Malawista SE: A vaccine consisting of recombinant borrelia burgdorferi outer-surface protein a to prevent Lyme disease. Recombinant outer-surface protein a Lyme disease vaccine study consortium. N Engl J Med 1998, 339(4):216-222.
• [4]Steere AC, Sikand VK, Meurice F, Parenti DL, Fikrig E, Schoen RT, Nowakowski J, Schmid CH, Laukamp S, Buscarino C, Krause DS: Vaccination against Lyme disease with recombinant borrelia burgdorferi outer-surface lipoprotein a with adjuvant. Lyme disease vaccine study group. N Engl J Med 1998, 339(4):209-215.
• [5]Bauer ME, Townsend CA, Doster RS, Fortney KR, Zwickl BW, Katz BP, Spinola SM, Janowicz DM: A fibrinogen-binding lipoprotein contributes to the virulence of Haemophilus ducreyi in humans. J Infect Dis 2009, 199(5):684-692.
• [6]Leduc I, Richards P, Davis C, Schilling B, Elkins C: A novel lectin, DltA, is required for expression of a full serum resistance phenotype in Haemophilus ducreyi. Infect Immun 2004, 72:3418-3428.
• [7]Hiltke TJ, Campagnari AA, Spinola SM: Characterization of a novel lipoprotein expressed by Haemophilus ducreyi. Infect Immun 1996, 64:5047-5052.
• [8]Spinola SM, Griffiths GE, Bogdan JA, Menegus MA: Characterization of an 18,000 molecular-weight outer membrane protein of Haemophilus ducreyi that contains a conserved surface-exposed epitope. Infect Immun 1992, 60:385-391.
• [9]Fortney KR, Young RS, Bauer ME, Katz BP, Hood AF, Munson RS, Spinola SM: Expression of peptidoglycan-associated lipoprotein is required for virulence in the human model of Haemophilus ducreyi infection. Infect Immun 2000, 68(11):6441-6448.
• [10]Janowicz DM, Leduc I, Fortney KR, Katz BP, Elkins C, Spinola SM: A DltA mutant of Haemophilus ducreyi is partially attenuated in its ability to cause pustules in human volunteers. Infect Immun 2006, 74(2):1394-1397.
• [11]Spinola SM, Wild LM, Apicella MA, Gaspari AA, Campagnari AA: Experimental human infection with Haemophilus ducreyi. J Infect Dis 1994, 169:1146-1150.
• [12]Janowicz DM, Ofner S, Katz BP, Spinola SM: Experimental infection of human volunteers with Haemophilus ducreyi: fifteen years of clinical data and experience. J Infect Dis 2009, 199:1671-1679.
• [13]Bauer ME, Fortney KR, Harrison A, Janowicz DM, Munson RS Jr, Spinola SM: Identification of Haemophilus ducreyi genes expressed during human infection. Microbiology 2008, 154(Pt 4):1152-1160.
• [14]Green BA, Farley JE, Quinn-Dey T, Deich RA, Zlotnick GW: The e (P4) outer membrane protein of Haemophilus influenzae: biologic activity of anti-e serum and cloning and sequencing of the structural gene. Infect Immun 1991, 59(9):3191-3198.
• [15]Morton DJ, Smith A, VanWagoner TM, Seale TW, Whitby PW, Stull TL: Lipoprotein e (P4) of Haemophilus influenzae: role in heme utilization and pathogenesis. Microbes Infect 2007, 9(8):932-939.
• [16]Reidl J, Mekalanos JJ: Lipoprotein e(P4) is essential for hemin uptake by Haemophilus influenzae. J Exp Med 1996, 183(2):621-629.
• [17]Reidl J, Schlor S, Kraiss A, Schmidt-Brauns J, Kemmer G, Soleva E: NADP and NAD utilization in Haemophilus influenzae. Mol Microbiol 2000, 35(6):1573-1581.
• [18]Mason KW, Zhu D, Scheuer CA, McMichael JC, Zlotnick GW, Green BA: Reduction of nasal colonization of nontypeable Haemophilus influenzae following intranasal immunization with rLP4/rLP6/UspA2 proteins combined with aqueous formulation of RC529. Vaccine 2004, 22(25–26):3449-3456.
• [19]Hotomi M, Ikeda Y, Suzumoto M, Yamauchi K, Green BA, Zlotnick G, Billal DS, Shimada J, Fujihara K, Yamanaka N: A recombinant P4 protein of Haemophilus influenzae induces specific immune responses biologically active against nasopharyngeal colonization in mice after intranasal immunization. Vaccine 2005, 23(10):1294-1300.
• [20]Green BA, Baranyi E, Reilly TJ, Smith AL, Zlotnick GW: Certain site-directed, nonenzymatically active mutants of the Haemophilus influenzae P4 lipoprotein are able to elicit bactericidal antibodies. Infect Immun 2005, 73(7):4454-4457.
• [21]Kemmer G, Reilly TJ, Schmidt-Brauns J, Zlotnik GW, Green BA, Fiske MJ, Herbert M, Kraiss A, Schlör S, Smith A, Reidl J: NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae. J Bacteriol 2001, 183(13):3974-3981.
• [22]Yamaguchi K, Yu F, Inouye M: A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell 1988, 53(3):423-432.
• [23]Tokuda H, Matsuyama S: Sorting of lipoproteins to the outer membrane in E. coli. Biochim Biophys Acta 2004, 1694(1–3):IN1-9.
• [24]Spinola SM, Peacock J, Denny FW, Smith DL, Cannon JG: Epidemiology of colonization by nontypable Haemophilus influenzae in children: a longitudinal study. J Infect Dis 1986, 154:100-109.
• [25]Bong CTH, Throm RE, Fortney KR, Katz BP, Hood AF, Elkins C, Spinola SM: A DsrA-deficient mutant of Haemophilus ducreyi is impaired in its ability to infect human volunteers. Infect Immun 2001, 69:1488-1491.
• [26]Janowicz DM, Fortney KR, Katz BP, Latimer JL, Deng K, Hansen EJ, Spinola SM: Expression of the LspA1 and LspA2 proteins by Haemophilus ducreyi is required for virulence in human volunteers. Infect Immun 2004, 72:4528-4533.
• [27]Cole LE, Toffer KL, Fulcher RA, San Mateo LR, Orndorff PE, Kawula TH: A humoral immune response confers protection against Haemophilus ducreyi infection. Infect Immun 2003, 71:6971-6977.
• [28]White CD, Leduc I, Olsen B, Jeter C, Harris C, Elkins C: Haemophilus ducreyi outer membrane determinants, including DsrA, define two clonal populations. Infect Immun 2005, 73(4):2387-2399.
• [29]Post DM, Gibson BW: Proposed second class of Haemophilus ducreyi strains show altered protein and lipooligosaccharide profiles. Proteomics 2007, 7:3131-3142.
• [30]Leduc I, Banks KE, Fortney KR, Patterson KB, Billings SD, Katz BP, Spinola SM, Elkins C: Evaluation of the repertoire of the TonB-dependent receptors of Haemophilus ducreyi for their role in virulence in humans. J Infect Dis 2008, 197:1103-1109.
• [31]Al-Tawfiq JA, Fortney KR, Katz BP, Elkins C, Spinola SM: An isogenic hemoglobin receptor-deficient mutant of Haemophilus ducreyi is attenuated in the human model of experimental infection. J Infect Dis 2000, 181:1049-1054.
• [32]Gazzaniga F, Stebbins R, Chang SZ, McPeek MA, Brenner C: Microbial NAD metabolism: lessons from comparative genomics. Microbiol Mol Biol Rev 2009, 73(3):529-541. Table of Contents
• [33]Niven DF, O'Reilly T: Significance of V-factor dependency in the taxonomy of Haemophilus species and related organisms. Int J Syst Bacteriol 1990, 40(1):1-4.
• [34]Gerlach G, Reidl J: NAD + utilization in Pasteurellaceae: simplification of a complex pathway. J Bacteriol 2006, 188(19):6719-6727.
• [35]Singh H, Schuermann JP, Reilly TJ, Calcutt MJ, Tanner JJ: Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase. J Mol Biol 2010, 404(4):639-649.
• [36]Sauer E, Merdanovic M, Mortimer AP, Bringmann G, Reidl J: PnuC and the utilization of the nicotinamide riboside analog 3-aminopyridine in Haemophilus influenzae. Antimicrob Agents Chemother 2004, 48(12):4532-4541.
• [37]Kurnasov OV, Polanuyer BM, Ananta S, Sloutsky R, Tam A, Gerdes SY, Osterman AL: Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesis. J Bacteriol 2002, 184(24):6906-6917.
• [38]Martin PR, Shea RJ, Mulks MH: Identification of a plasmid-encoded gene from Haemophilus ducreyi which confers NAD independence. J Bacteriol 2001, 183(4):1168-1174.
• [39]Munson RS Jr, Zhong H, Mungur R, Ray WC, Shea RJ, Mahairas GG, Mulks MH: Haemophilus ducreyi strain ATCC 27722 contains a genetic element with homology to the vibrio RS1 element that can replicate as a plasmid and confer NAD independence on Haemophilus influenzae. Infect Immun 2004, 72:1143-1146.
• [40]Al-Tawfiq JA, Thornton AC, Katz BP, Fortney KR, Todd KD, Hood AF, Spinola SM: Standardization of the experimental model of Haemophilus ducreyi infection in human subjects. J Infect Dis 1998, 178:1684-1687.
• [41]Menard R, Sansonetti PJ, Parsot C: Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol 1993, 175:5899-5906.
• [42]Bozue JA, Tarantino L, Munson RS Jr: Facile construction of mutations in Haemophilus ducreyi using lacz as a counter-selectable marker. FEMS Microbiol Lett 1998, 164:269-273.
• [43]Spinola SM, Bong CT, Faber AL, Fortney KR, Bennett SL, Townsend CA, Zwickl BE, Billings SD, Humphreys TL, Bauer ME, Katz BP: Differences in host susceptibility to disease progression in the human challenge model of Haemophilus ducreyi infection. Infect Immun 2003, 71(11):6658-6663.
• [44]Banks KE, Fortney KR, Baker B, Billings SD, Katz BP, Munson RS Jr, Spinola SM: The enterobacterial common antigen-like gene cluster of Haemophilus ducreyi contributes to virulence in humans. J Infect Dis 2008, 197:1531-1536.