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Function of neisserial outer membrane phospholipase a in autolysis and assessment of its vaccine potential.

ABSTRACT: Outer membrane phospholipase A (OMPLA) is an outer membrane-localized enzyme, present in many gram-negative bacterial species. It is implicated in the virulence of several pathogens. Here, we investigated the presence, function, and vaccine potential of OMPLA in the human pathogen Neisseria meningitidis. Immunoblot analysis showed the presence of OMPLA in 28 out of 33 meningococcal strains investigated. The OMPLA-negative strains all contained a pldA gene, but these alleles contained premature stop codons. All six Neisseria gonorrhoeae strains tested, but only two out of seven commensal neisserial strains investigated, expressed OMPLA, showing that OMPLA is expressed by, but not limited to, many pathogenic neisserial strains. The function of OMPLA was investigated by assessing the phenotypes of isogenic strains, expressing no OMPLA, expressing wild-type levels of OMPLA, or overexpressing OMPLA. OMPLA exhibited phospholipase activity against endogenous phospholipids. Furthermore, OMPLA was characterized as an autolysin that acted under specific conditions, such as prolonged growth of the bacteria. The vaccine potential of the protein was investigated by immunizing mice with in vitro refolded, recombinant OMPLA. High levels of antibody titers were obtained, but the murine sera were neither bactericidal nor protective. Also, convalescent patients and vaccinee sera did not contain detectable levels of anti-OMPLA antibodies, indicating that OMPLA may not be sufficiently immunogenic to be included in a meningococcal vaccine.

SUBMITTER: Bos MP

PROVIDER: S-EPMC1087465 | biostudies-literature | 2005 Apr

REPOSITORIES: biostudies-literature

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Function of neisserial outer membrane phospholipase a in autolysis and assessment of its vaccine potential.

Bos Martine P MP Tefsen Boris B Voet Pierre P Weynants Vincent V van Putten Jos P M JP Tommassen Jan J

Infection and immunity 20050401 4

Outer membrane phospholipase A (OMPLA) is an outer membrane-localized enzyme, present in many gram-negative bacterial species. It is implicated in the virulence of several pathogens. Here, we investigated the presence, function, and vaccine potential of OMPLA in the human pathogen Neisseria meningitidis. Immunoblot analysis showed the presence of OMPLA in 28 out of 33 meningococcal strains investigated. The OMPLA-negative strains all contained a pldA gene, but these alleles contained premature s ...[more]

PMID: 15784566

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Project description:Outer membrane vesicles (OMVs) are spontaneously released by many gram-negative bacteria during their growth and constitute an important virulence factor for bacteria, helping them to survive through harsh environmental conditions. Native OMVs, naturally-released from bacteria, are produced at a level too low for vaccine manufacturing, requiring chemical treatment (detergent-extracted) or genetic manipulation, resulting in generalized modules for membrane antigens (GMMAs). Over the years, the nature and properties of OMVs have made them a viable platform for vaccine development. There are a few licensed OMV vaccines mainly for the prevention of meningitis caused by Neisseria meningitidis serogroup B (MenB) and Haemophilus influenzae type b (Hib). There are several candidates in clinical development against other gram-negative organisms from which the OMVs are derived, but also against heterologous targets in which the OMVs are used as carriers (e.g. coronavirus disease 2019 [COVID-19]). The use of OMVs for targets other than those from which they are derived is a major advancement in OMV technology, improving its versatility by being able to deliver protein or polysaccharide antigens. Other advances include the range of genetic modifications that can be made to improve their safety, reduce reactogenicity, and increase immunogenicity and protective efficacy. However, significant challenges remain, such as identification of general tools for high-content surface expression of heterologous proteins on the OMV surface. Here, we outline the progress of OMV vaccines to date, particularly discussing licensed OMV-based vaccines and candidates in clinical development. Recent trends in preclinical research are described, mainly focused on genetic manipulation and chemical conjugation for the use of OMVs as carriers for heterologous protein and polysaccharide antigens. Remaining challenges with the use of OMVs and directions for future research are also discussed.

Project description:In the past decade, researchers have proposed that the pldA gene for outer membrane phospholipase A (OMPLA) is important for bacterial colonization of the human gastric ventricle. Several conserved Helicobacter pylori genes have distinct genotypes in different parts of the world, biogeographic patterns that can be analyzed through phylogenetic trees. The current study will shed light on the importance of the pldA gene in H. pylori. In silico sequence analysis will be used to investigate whether the bacteria are in the process of preserving, optimizing, or rejecting the pldA gene. The pldA gene will be phylogenetically compared to other housekeeping (HK) genes, and a possible origin via horizontal gene transfer (HGT) will be evaluated through both intra- and inter-species evolutionary analyses.In this study, pldA gene sequences were phylogenetically analyzed and compared with a large reference set of concatenated HK gene sequences. A total of 246 pldA nucleotide sequences were used; 207 were from Norwegian isolates, 20 were from Korean isolates, and 19 were from the NCBI database. Best-fit evolutionary models were determined with MEGA5 ModelTest for the pldA (K80 + I + G) and HK (GTR + I + G) sequences, and maximum likelihood trees were constructed. Both HK and pldA genes showed biogeographic clustering. Horizontal gene transfer was inferred based on significantly different GC contents, the codon adaptation index, and a phylogenetic conflict between a tree of OMPLA protein sequences representing 171 species and a tree of the AtpA HK protein for 169 species. Although a vast majority of the residues in OMPLA were predicted to be under purifying selection, sites undergoing positive selection were also found.Our findings indicate that the pldA gene could have been more recently acquired than seven of the HK genes found in H. pylori. However, the common biogeographic patterns of both the HK and pldA sequences indicated that the transfer occurred long ago. Our results indicate that the bacterium is preserving the function of OMPLA, although some sites are still being evolutionarily optimized.

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Function of neisserial outer membrane phospholipase a in autolysis and assessment of its vaccine potential.

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Function of neisserial outer membrane phospholipase a in autolysis and assessment of its vaccine potential.

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