Campylobacter jejuni Virulence Factors Identified by Modulating Their Synthesis on Ribosomes With Altered rRNA Methylation

Campylobacter jejuni is a major cause of food poisoning worldwide, and remains the main infective agent in gastroenteritis and related intestinal disorders in Europe and the USA. As with all bacterial infections, the stages of adhesion to host tissue, survival in the host and eliciting disease all require the synthesis of proteinaceous virulence factors on the ribosomes of the pathogen. Here, we describe how C. jejuni virulence is attenuated by altering the methylation of its ribosomes to disrupt the composition of its proteome, and how this in turn provides a means of identifying factors that are essential for infection and pathogenesis. Specifically, inactivation of the C. jejuni Cj0588/TlyA methyltransferase prevents methylation of nucleotide C1920 in the 23S rRNA of its ribosomes and reduces the pathogen’s ability to form biofilms, to attach, invade and survive in host cells, and to provoke the innate immune response. Mass spectrometric analyses of C. jejuni TlyA-minus strains revealed an array of subtle changes in the proteome composition. These included reduced amounts of the cytolethal distending toxin (CdtC) and the MlaEFD proteins connected with outer membrane vesicle (OMV) production. Inactivation of the cdtC and mlaEFD genes confirmed the importance of their encoded proteins in establishing infection. Collectively, the data identify a subset of genes required for the onset of human campylobacteriosis, and serve as a proof of principle for use of this approach in detecting proteins involved in bacterial pathogenesis.

Enhancement of membrane vesicle production by disrupting the degP gene in Meiothermus ruber H328

The phenomenon of membrane vesicle (MV) production is known to be common to all bacterial cells. Although MVs are expected to be employed in a variety of applications, improving MV productivity is essential for applications. Since the deletion of the degP gene, a periplasmic dual-function protease and chaperone, in Escherichia coli has successfully improved MV production capacity, we tried to enhance MV productivity in the thermophilic M. ruber H328 by deleting the degP gene. One gene (mrH_0331) was selected for degP gene from the H328 genome and we constructed the mutant strain ∆degP by deleting the degP gene of the H328 strain that was replaced with the htk gene showing thermophilic kanamaycin resistance by homologous recombination. The mutant strain ∆degP exhibited smooth growth but a lower level of turbidity at 60 °C although there was no difference in growth at 55 °C between the wild strain and the mutant strain. Finally, we have confirmed that incubation at 60 °C increases MV in the mutant strain ∆degP strain about fivefold by using two fluorescent dyes, DiI and FM4-64, which is followed by TEM analysis. The deletion of the degP gene presumably causes an increase in denatured proteins at 60 °C, leading to enhanced MV production. Meanwhile, the S-layer protein included in the outer membrane of the H328 strain increased in the MV fraction prepared from the mutant cells incubated at 60 °C. This indicates that this method is effective for MV production and that degP deletion enhances it in strain H328.

Multi-Antigen Outer Membrane Vesicle Engineering to Develop Polyvalent Vaccines: The Staphylococcus aureus Case

Modification of surface antigens and differential expression of virulence factors are frequent strategies pathogens adopt to escape the host immune system. These escape mechanisms make pathogens a “moving target” for our immune system and represent a challenge for the development of vaccines, which require more than one antigen to be efficacious. Therefore, the availability of strategies, which simplify vaccine design, is highly desirable. Bacterial Outer Membrane Vesicles (OMVs) are a promising vaccine platform for their built-in adjuvanticity, ease of purification and flexibility to be engineered with foreign proteins. However, data on if and how OMVs can be engineered with multiple antigens is limited. In this work, we report a multi-antigen expression strategy based on the co-expression of two chimeras, each constituted by head-to-tail fusions of immunogenic proteins, in the same OMV-producing strain. We tested the strategy to develop a vaccine against Staphylococcus aureus, a Gram-positive human pathogen responsible for a large number of community and hospital-acquired diseases. Here we describe an OMV-based vaccine in which four S. aureus virulent factors, ClfAY338A, LukE, SpAKKAA and HlaH35L have been co-expressed in the same OMVs (CLSH-OMVsΔ60). The vaccine elicited antigen-specific antibodies with functional activity, as judged by their capacity to promote opsonophagocytosis and to inhibit Hla-mediated hemolysis, LukED-mediated leukocyte killing, and ClfA-mediated S. aureus binding to fibrinogen. Mice vaccinated with CLSH-OMVsΔ60 were robustly protected from S. aureus challenge in the skin, sepsis and kidney abscess models. This study not only describes a generalized approach to develop easy-to-produce and inexpensive multi-component vaccines, but also proposes a new tetravalent vaccine candidate ready to move to development.

02. Beyond B Antigen Coverage: The Potential of the 4CMenB Vaccine for Cross-protection Against Pathogenic Neisseria Infections

Background Two human pathogenic Neisseria species exist: N. meningitidis (Nm) and N. gonorrhoeae (Ng). Although causing disparate clinical syndromes, invasive meningococcal disease (IMD) and gonorrhea, they are genetically similar and share key protein antigens. The 4CMenB vaccine, licensed against meningococcal B disease, comprises 4 antigenic components (factor H binding protein (fHbp), variant 1.1, subfamily B; Neisseria heparin binding antigen (NHBA) peptide 2; Neisserial adhesin A (NadA) variant 3; and Porin A (PorA) P1.4), and potentially protects against non-B invasive meningococcal and gonococcal strains. In this review, we summarize the similarities between these antigens and those in Nm serogroups A, C, W, X and Y and Ng. Methods Published data in humans were analyzed to conduct a narrative literature review of the potential extent of meningococcal vaccine-induced protection against non-B meningococcal strains and Ng. Techniques applied to indirectly measure this effect are based on genotype-phenotype modelling, strain coverage, bactericidal killing and direct impact on disease reduction. Results Data were identified from countries in America, Europe, Africa and Oceania. The genes encoding for fHbp and NHBA are also present in strains belonging to the five non-B serogroups, while NadA is present in several strains of serogroups C, W and Y, and PorA P1.4 mainly in serogroup W. At the genome level, Ng and Nm share up to 90% homology. Most of the outer membrane vesicle antigens, like PilQ, Omp85 (BamA), NspA, MtrE, MetQ, LbpA, PorB, FetA, OpcA and NHBA, are highly conserved in Ng. In addition, a synergistic effect might enhance immunogenicity against non-B serogroups as shown against serogroup B. Conclusion 4CMenB components are present and conserved in several Ng and Nm strains. Recent results demonstrate that 4CMenB reduces MenW disease incidence in infants and might generate cross-protection against other non-B serogroups. In addition, 4CMenB has been proven to be effective in reducing gonococcal infections in adolescents. Research on future genomic and proteomic characterizations of IMD and gonorrhea strains will provide information on the molecular basis of the underlying broad strain coverage, while informing decisions regarding prevention and immunization programmes. Disclosures Yara Ruiz Garcia, MSc, PhD, GSK group of companies (Employee) Woo-Yun Sohn, MD, GSK group of companies (Employee, Shareholder) Mariagrazia Pizza, Biological Sciences, PhD, GSK group of companies (Employee, Shareholder) Rafik Bekkat-Berkani, M.D, GSK group of companies (Employee, Shareholder)

Enhancement of Membrane Vesicle Production by Disrupting the degP Gene in Meiothermus Ruber H328

The phenomenon of membrane vesicle (MV) production is known to be common to all bacterial cells. Although MVs are expected to be employed in a variety of applications, improving MV productivity is essential for applications. Since the deletion of the degP gene, a periplasmic dual-function protease and chaperone, in Escherichia coli has successfully improved MV production capacity, we tried to enhance MV productivity in the thermophilic M. ruber H328 by deleting the degP gene. One gene (mrH_0331) was selected for degP gene from the H328 genome and we constructed the mutant strain DdegP by deleting the degP gene of the H328 strain that was replaced with the htk gene showing thermophilic kanamaycin resistance by homologous recombination. The mutant strain DdegP exhibited smooth growth but a lower level of turbidity at 60ºC although there was no difference in growth at 55ºC between the wild strain and the mutant strain. Finally, we have confirmed that incubation at 60°C increases MV in the mutant strain DdegP strain about fivefold by using two fluorescent dyes, DiI and FM4-64, which is followed by TEM analysis. The deletion of the degP gene presumably causes an increase in denatured proteins at 60°C, leading to enhanced MV production. Meanwhile, the S-layer protein included in the outer membrane of the H328 strain increased in the MV fraction prepared from the mutant cells incubated at 60°C. This indicates that this method is effective for MV production and that degP deletion enhances it in strain H328.