A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates.

Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria.

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms.

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (>95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.

Evaluation of a novel therapeutic approach to treating severe pneumococcal infection using a mouse model.

P4, a 28-amino-acid peptide, is a eukaryotic cellular activator that enhances specific in vitro opsonophagocytic killing of multiple bacterial pathogens. In a previous study, we successfully recreated this phenomenon in mice in vivo by using a two-dose regimen of P4 and pathogen-specific antibodies, which significantly reduced moribundity in mice. For the present study, we hypothesized that the inclusion of a low-dose antibiotic would make it possible to treat the infected mice with a single dose containing a mixture of P4 and a pathogen-specific antibody. A single dose consisting of P4, intravenous immunoglobulin (IVIG), and ceftriaxone effectively reduced moribundity compared to that of untreated controls (n = 10) by 75% (P < 0.05) and rescued all (10 of 10) infected animals (P < 0.05). If rescued animals were reinfected with Streptococcus pneumoniae and treated with a single dose containing P4, IVIG, and ceftriaxone, they could be rerescued. This observation of the repeated successful use of P4 combination therapy demonstrates a low risk of tolerance development. Additionally, we examined the polymorphonuclear leukocytes (PMN) derived from infected mice and observed that P4 enhanced in vitro opsonophagocytic killing (by >80% over the control level; P < 0.05). This finding supports our hypothesis that PMN are activated by P4 during opsonophagocytosis and the recovery of mice from pneumococcal infection. P4 peptide-based combination therapy may offer an alternative and rapid immunotherapy to treat fulminant pneumococcal infection.

A 28-aa pneumococcal surface adhesin A-derived peptide, P4, augments passive immunotherapy and rescues mice from fatal pneumococcal infection.

BACKGROUND: P4, a 28-aa peptide derived from pneumococcal surface adhesin A, is a multilineage cell activator in vitro. We hypothesized that P4-mediated activation of phagocytic cells could rapidly and substantially increase opsonophagocytosis of bacteria, which could be translated in vivo to reduced mouse morbidity from fatal pneumococcal infection. METHODS: Reference in vitro opsonophagocytic killing and uptake assays were used with suitable effector cells and pathogen-specific antibodies. P4 peptide solution was added at the preopsonization stage. ND4-SW mice were infected intranasally with Streptococcus pneumoniae serotype 3 (WU2). At 72 and 96 h, infected mice received intraperitoneal or intravenous injection of gamma globulin, followed by an injection of P4. RESULTS: P4 treatment enhanced in vitro opsonophagocytosis of bacterial pathogens by many fold, and this effect was dependent on complement, P4, and antibody concentrations. Treatment of highly virulent WU2-infected mice with the combination of P4 and serotype-specific antiserum resulted in 100% remission of bacteremia and rescued 80% of the animals (P < .05). CONCLUSION: P4 peptide in combination with pathogen-specific antibodies and complement enhances specific opsonophagocytosis and rescues mice from life-threatening pneumococcal infection. P4 peptide provides a fresh direction for therapeutic intervention through augmented passive immunotherapy.

A real-time polymerase chain reaction for the detection of Streptococcus pneumoniae in blood using a mouse model: a potential new "gold standard".

Better diagnostics for pneumococcal disease are urgently needed. In a murine model, real-time polymerase chain reaction was superior to conventional culture in detecting pneumococcus in blood, particularly in early disease and after antibiotic administration, and could distinguish between commensalism and infection.