The pneumococcal Pilus-1 enhances attachment to epithelial cells in the respiratory tract and subsequent invasion. Pilus-1 expression is bi-stable and positively regulated by the RlrA transcriptional regulator. To delineate the role of pilus-1 in Experimental Otitis Media (EOM), we evaluated colonization and disease due to a Streptococcus pneumoniae (SP) wild type strain (Taiwan19F-14 wt) and its otherwise isogenic pilus-1 and pilus-2 deficient mutant (Taiwan19F-14 ΔPI-1/PI-2-) as well as potential for a chimeric protein (RrgB321) vaccine candidate for prevention of middle ear (ME) disease. Methods Chinchillas were challenged intranasally with either Taiwan19F-14 wt or Taiwan19F-14PI-1/PI-2 deficient mutant. ME status was assessed and direct cultures performed. New cohorts of animals were immunized with RrgB321 or alum. Intranasal challenge with Taiwan19F-14 wt [erythromycin susceptible E(S)] was performed. Subsequently, a second cohort of animals was immunized and challenged with either Taiwan19F-14 wt or a Pilus-1 over-expressing mutant [Taiwan19F-14+pMU1328_Pc-rlrA mutant; E resistant (R)] strain. Pilus-1 expression was analyzed in SP isolated from nasopharynx (NP) and ME fluids by flow cytometry. Results Culture positive EOM developed following challenge with either wild type SP (Taiwan19F-14) or its pilus-1 deficient mutant. Culture positive EOM developed following challenge with wild type in both RrgB321 immunized and control animals. Pilus-1 expression in ME fluids was significantly higher in controls compared to immunized chinchillas. In second cohort of immunized and control animals challenged with the over-expressing Pilus-1 mutant, delayed development of EOM in the immunized animals was observed. Pneumococci recovered from ME fluid of immunized animals were no longer E(R) signifying the loss of the pMU1328_Pc-rlrA plasmid. Conclusion Pneumococcal pilus-1 was not essential for EOM. Regulation of Pilus-1 expression in ME fluids in the presence of anti RrgB321 antibody was essential for survival of S. pneumoniae. Pneumococci have evolved mechanisms of regulation of non-essential surface proteins to evade host defenses.
References
[1]
Moschioni M, Donati C, Muzzi A, Masignani V, Censini S, et al. (2008) Streptococcus pneumoniae contains 3 rlrA pilus variants that are clonally related. J Infect Dis 197: 888–896.
[2]
De Angelis G, Moschioni M, Muzzi A, Pezzicoli A, Censini S, et al. (2011) The Streptococcus pneumoniae pilus-1 displays a biphasic expression pattern. PLoS One 6: e21269.
[3]
Pancotto L, De Angelis G, Bizzarri E, Barocchi MA, Giudice GD, et al. (2013) Expression of the Streptococcus pneumoniae pilus-1 undergoes on and off switching during colonization in mice. Sci Rep 3: 2040.
[4]
Rosch JW, Mann B, Thornton J, Sublett J, Tuomanen E (2008) Convergence of regulatory networks on the pilus locus of Streptococcus pneumoniae. Infect Immun 76: 3187–3196.
[5]
Bagnoli F, Moschioni M, Donati C, Dimitrovska V, Ferlenghi I, et al. (2008) A second pilus type in Streptococcus pneumoniae is prevalent in emerging serotypes and mediates adhesion to host cells. J Bacteriol 190: 5480–5492.
[6]
Bouchet V, Hood DW, Li J, Brisson JR, Randle GA, et al. (2003) Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media. Proc Natl Acad Sci U S A 100: 8898–8903.
[7]
Alloing G, Martin B, Granadel C, Claverys JP (1998) Development of competence in Streptococcus pneumonaie: pheromone autoinduction and control of quorum sensing by the oligopeptide permease. Mol Microbiol 29: 75–83.
[8]
Achen MG, Davidson BE, Hillier AJ (1986) Construction of plasmid vectors for the detection of streptococcal promoters. Gene 45: 45–49.
[9]
Sabharwal V, Ram S, Figueira M, Park IH, Pelton SI (2009) Role of complement in host defense against pneumococcal otitis media. Infect Immun 77: 1121–1127.
[10]
Moschioni M, De Angelis G, Harfouche C, Bizzarri E, Filippini S, et al. (2012) Immunization with the RrgB321 fusion protein protects mice against both high and low pilus-expressing Streptococcus pneumoniae populations. Vaccine 30: 1349–1356.
[11]
Sabharwal V, Figueira M, Pelton SI, Pettigrew MM (2012) Virulence of Streptococcus pneumoniae serotype 6C in experimental otitis media. Microbes Infect 14: 712–718.
[12]
Babl FE, Pelton SI, Li Z (2002) Experimental acute otitis media due to nontypeable Haemophilus influenzae: comparison of high and low azithromycin doses with placebo. Antimicrob Agents Chemother 46: 2194–2199.
[13]
Figueira MA, Ram S, Goldstein R, Hood DW, Moxon ER, et al. (2007) Role of complement in defense of the middle ear revealed by restoring the virulence of nontypeable Haemophilus influenzae siaB mutants. Infect Immun 75: 325–333.
[14]
Croucher NJ, Finkelstein JA, Pelton SI, Mitchell PK, Lee GM, et al. (2013) Population genomics of post-vaccine changes in pneumococcal epidemiology. Nat Genet 45: 656–663.
[15]
Karasic RB, Beste DJ, To SC, Doyle WJ, Wood SW, et al. (1989) Evaluation of pilus vaccines for prevention of experimental otitis media caused by nontypable Haemophilus influenzae. Pediatr Infect Dis J 8: S62–65.
[16]
Moschioni M, De Angelis G, Melchiorre S, Masignani V, Leibovitz E, et al. (2010) Prevalence of pilus-encoding islets among acute otitis media Streptococcus pneumoniae isolates from Israel. Clin Microbiol Infect 16: 1501–1504.
[17]
Shi ZY, Enright MC, Wilkinson P, Griffiths D, Spratt BG (1998) Identification of three major clones of multiply antibiotic-resistant Streptococcus pneumoniae in Taiwanese hospitals by multilocus sequence typing. J Clin Microbiol 36: 3514–3519.
