Outer Membrane Vesicles (OMV)-based and Proteomics-driven Antigen Selection Identifies Novel Factors Contributing to Bordetella pertussis Adhesion to Epithelial Cells.

Despite high vaccination coverage world-wide, whooping cough, a highly contagious disease caused by Bordetella pertussis, is recently increasing in occurrence suggesting that novel vaccine formulations targeted at the prevention of colonization and transmission should be investigated. To identify new candidates for inclusion in the acellular formulation, we used spontaneously released outer membrane vesicles (OMV)1 as a potential source of key adhesins. The enrichment of Bvg+ OMV with adhesins and the ability of anti-OMV serum to inhibit the adhesion of B. pertussis to lung epithelial cells in vitro were demonstrated. We employed a proteomic approach to identify the differentially expressed proteins in OMV purified from bacteria in the Bvg+ and Bvg- virulence phases, thus comparing the outer membrane protein pattern of this pathogen in its virulent or avirulent state. Six of the most abundant outer membrane proteins were selected as candidates to be evaluated for their adhesive properties and vaccine potential. We generated E. coli strains singularly expressing the selected proteins and assessed their ability to adhere to lung epithelial cells in vitro Four out of the selected proteins conferred adhesive ability to E. coli Three of the candidates were specifically detected by anti-OMV mouse serum suggesting that these proteins are immunogenic antigens able to elicit an antibody response when displayed on the OMV. Anti-OMV serum was able to inhibit only BrkA-expressing E. coli adhesion to lung epithelial cells. Finally, stand-alone immunization of mice with recombinant BrkA resulted in significant protection against infection of the lower respiratory tract after challenge with B. pertussis Taken together, these data support the inclusion of BrkA and possibly further adhesins to the current acellular pertussis vaccines to improve the impact of vaccination on the bacterial clearance.

A novel high-throughput assay to quantify the vaccine-induced inhibition of Bordetella pertussis adhesion to airway epithelia.

BACKGROUND: Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis. Despite high vaccination coverage whooping cough is currently re-emerging in many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization. RESULTS: We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium. CONCLUSIONS: We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.

The potential of adjuvants to improve immune responses against TdaP vaccines: A preclinical evaluation of MF59 and monophosphoryl lipid A.

The successful approach of combining diphtheria, tetanus and pertussis antigens into a single vaccine has become a cornerstone of immunization programs. Yet, even if vaccination coverage is high, a resurgence of pertussis has been reported in many countries suggesting current vaccines may not provide adequate protection. To induce better tailored and more durable immune responses against pertussis vaccines different approaches have been proposed, including the use of novel adjuvants. Licensed aP vaccines contain aluminum salts, which mainly stimulate humoral immune responses and might not be ideal for protecting against Bordetella pertussis infection. Adjuvants inducing more balanced T-helper profiles or even Th1-prone responses might be more adequate. In this study, two adjuvants already approved for human use have been tested: MF59 emulsion and the combination of aluminum hydroxide with the Toll-Like Receptor 4 agonist MPLA. Adjuvanticity was evaluated in a mouse model using a TdaP vaccine containing three B. pertussis antigens: genetically detoxified pertussis toxin (PT-9K/129G), filamentous hemagglutinin (FHA) and pertactin (PRN) The physico-chemical compatibility of TdaP antigens with the proposed adjuvants, together with a quicker onset and changed quality of the antibody responses, fully supports the replacement of aluminum salts with a new adjuvant to enhance aP vaccines immunogenicity.

Lipoprotein CD0873 is a novel adhesin of Clostridium difficile.

Clostridium difficile is a cause of antibiotic-associated diarrhea and colitis, a healthcare-associated intestinal disease. Colonization of the gut is a critical step in the course of infection. The C. difficile lipoprotein CD0873 was identified as a putative adhesin through a bioinformatics approach. Surface exposure of CD0873 was confirmed and a CD0873 mutant was generated. The CD0873 mutant showed a significant reduction in adherence to Caco-2 cells and wild-type bacteria preincubated with anti-CD0873 antibodies showed significantly decreased adherence to Caco-2 cells. In addition, we demonstrated that purified recombinant CD0873 protein alone associates with Caco-2 cells. This is the first definitive identification of a C. difficile adhesin, which now allows work to devise improved measures for preventing and treating disease.

Neisseria gonorrhoeae PIII has a role on NG1873 outer membrane localization and is involved in bacterial adhesion to human cervical and urethral epithelial cells.

BACKGROUND: Protein PIII is one of the major outer membrane proteins of Neisseria gonorrhoeae, 95% identical to RmpM (reduction modifiable protein M) or class 4 protein of Neisseria meningitidis. RmpM is known to be a membrane protein associated by non-covalent bonds to the peptidoglycan layer and interacting with PorA/PorB porin complexes resulting in the stabilization of the bacterial membrane. The C-terminal domain of PIII (and RmpM) is highly homologous to members of the OmpA family, known to have a role in adhesion/invasion in many bacterial species. The contribution of PIII in the membrane architecture and its role in the interaction with epithelial cells has never been investigated. RESULTS: We generated a ΔpIII knock-out mutant strain and evaluated the effects of the loss of PIII expression on bacterial morphology and on outer membrane composition. Deletion of the pIII gene does not cause any alteration in bacterial morphology or sensitivity to detergents. Moreover, the expression profile of the main membrane proteins remains the same for the wild-type and knock-out strains, with the exception of the NG1873 which is not exported to the outer membrane and accumulates in the inner membrane in the ΔpIII knock-out mutant strain.We also show that purified PIII protein is able to bind human cervical and urethral cells and that the ΔpIII knock-out mutant strain has a lower ability to adhere to human cervical and urethral cells. CONCLUSION: Here we demonstrated that the PIII protein does not play a key structural role in the membrane organization of gonococcus and does not induce major effects on the expression of the main outer membrane proteins. However, in the PIII knock-out strain, the NG1873 protein is not localized in the outer membrane as it is in the wild-type strain suggesting a possible interaction of PIII with NG1873. The evidence that PIII binds to human epithelial cells derived from the female and male genital tract highlights a possible role of PIII in the virulence of gonococcus and suggests that the structural homology to OmpA is conserved also at functional level.

Protective efficacy induced by recombinant Clostridium difficile toxin fragments.

Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.

Molecular analysis of two novel Neisseria gonorrhoeae virulent components: the macrophage infectivity potentiator and the outer membrane protein A.

Molecular analyses of mip and ompA genes were performed on 20 Neisseria gonorrhoeae isolates. The genes were present with a high degree of conservation in all strains. Sera from patients with urethritis or disseminated gonococcal infections were able to recognize the purified Neisseria gonorrhoeae macrophage infectivity potentiator (Ng-MIP) and Neisseria gonorrhoeae outer membrane protein A (Ng-OmpA).

Identification of a new OmpA-like protein in Neisseria gonorrhoeae involved in the binding to human epithelial cells and in vivo colonization.

Outer membrane protein As (OmpAs) are highly conserved proteins within the Enterobacteriaceae family. OmpA contributes to the maintenance of structural membrane integrity and invasion into mammalian cells. In Escherichia coli K1 OmpA also contributes to serum resistance and is involved in the virulence of the bacterium. Here we describe the identification of an OmpA-like protein in Neisseria gonorrhoeae (Ng-OmpA). We show that the gonococcal OmpA-like protein, similarly to E. coli OmpA, plays a significant role in the adhesion and invasion into human cervical carcinoma and endometrial cells and is required for entry into macrophages and intracellular survival. Furthermore, the isogenic knockout ompA mutant demonstrates reduced recovery in a mouse model of infection when compared with the wild-type strain, suggesting that Ng-OmpA plays an important role in the in vivo colonization. All together, these data suggest that the newly identified surface exposed protein Ng-OmpA represents a novel virulence factor of gonococcus.

Ng-MIP, a surface-exposed lipoprotein of Neisseria gonorrhoeae, has a peptidyl-prolyl cis/trans isomerase (PPIase) activity and is involved in persistence in macrophages.

Macrophage infectivity potentiators (MIPs) are a family of surface-exposed virulence factors of intracellular microorganisms such as Legionella, Chlamydia and Trypanosoma. These proteins display peptidyl-prolyl cis/trans isomerase (PPIase) activity that is inhibited by immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization in Neisseria gonorrhoeae of Ng-MIP, a surface-exposed lipoprotein with high homology to MIPs. The protein is an homodimer with rapamycin-inhibited PPIase activity confirming that it is a functional member of the MIP family. A knock-out strain, generated by deletion of the mip gene in N. gonorrhoeae F62 strain, was evaluated for its role in infection of mouse and human macrophages. We show that Ng-MIP promotes the intracellular survival of N. gonorrhoeae in macrophages, highlighting a possible role of this protein in promoting the persistence of gonococcal infection.