Mechanisms of colonization and dissemination of a bacterial pathogen in a human population, the example of Neisseria meningitidis – PATHONEM
Aim 1: Identification of N. meningitidis factors required for colonization of the air-liquid interface model of upper respiratory tract cell culture.
The use of an air-liquid culture model of human cells will allow to better understand the interaction between Neisseria meningitidis and human nasopharyngeal and nasal epithelium. It will also focus on the interaction between Neisseria meningitidis and mucus. This model will also be useful to study meningococcal colonization in the context of the nasal microbiota by co-infecting the monolayers with bacteria usually encounter in the nasopharynx. A high throughput screening of meningococcal genes will be used to uncover the bacterial attributes involved in colonization of the air-liquid culture model.
Aim 2: Identify the cellular adhesion epitopes responsible for Neisseria meningitidis interaction with human epithelial cells.
As mentioned above bacteria express numerous adhesins, however in capsulated meningococci type IV pili are first to allow bacterial adhesion. As previously mentioned pili receptors have been identified on endothelial cells but these receptors are not involved in the bacterial interaction on epithelial cells. Our preliminary data, suggest that type IV pili recognize glycan motifs, we hypothesize that the pili interact via the same glucidic motifs on various cellular receptors of different cell types. In this aim we will focus on the finding of the glycosylated type IV pili receptor(s) present on upper respiratory tract epithelial cells.
Interaction of N. meningitidis with cells in the air-liquid interface model.
We will use an air-liquid interface culture model with primary human nasal epithelial cells. The air-liquid interface culture model allows the full differentiation of nasal epithelial cells in mucus producing goblet cells and ciliated cells. We will address the behaviour of meningococci in the mucus and the impact of meningococcal colonization on the differentiated epithelium.
High throughput screening for genes involved in survival during colonization of air-interface culture.
Combined to a relevant colonization model, high-throughput insertion tracking by deep sequencing will permit exhaustive identification of N. meningitidis genes involved in colonization of the mucus layer.
Set up of a co-infection model with bacterial commensal species.
We will infect the epithelial air-liquide interface model with defined bacterial species usually encounter in nasopharynx (like Streptococci sp, Haemophilus influenzae and non pathogenic Neisseriaceae) and perform high throughput screening to find genes involved in colonization of epithelium already infected with other commensal species.
Unravel the role of carbohydrate during type IV pili dependent adhesion.
We will search for the specific composition of the glycan moiety required for N. meningitidis adhesion using human glycan array developed by the “Consortium for Functional Glycomics” (http://www.functionalglycomics.org/). We will then focus on glycosyltransferase related to the identified sugar and inhibit their expression. In parallel we will deglycosilate cells in vitro and test adherence of bacteria or that of recombinant pilin.
Determine which receptor carries the adhesion epitope.
In parallel to Task 4 we will use a second strategy to find human receptors interacting with type IV pili without concern for the carbohydrate moiety
Neisseria meningitidis is a major disease burden for human beings. N. meningitidis is a commensal of the nasopharynx from where it invades the bloodstream where they are responsible for septicemia with or without a severe sepsis and/or meningitis, after crossing of the blood-brain-barrier.
The primary focus of this project is to conduct a comprehensive study of the mechanisms by which a commensal and pathogen bacterium, Neisseria meningitidis, colonize the naso-pharyngeal epithelium and disseminate from person to person, with the ultimate objective to find new broad range vaccine target and therapeutic strategies against Neisseria meningitidis. This involves elucidation of the intricate network of dynamic interactions and molecular strategies selected by the meningococcus to establish itself within its niche. Our ambition is also to develop strategies to study other airborne pathogens.
Until now, most strategies described for the study of bacteria colonization of the human nasopharynx used non specific model of carcinoma derived cell lines which have lost or partly lost their primary phenotypes. This proposal focuses on the development and the use of dedicated in vitro models to study colonization of differentiated human epithelium and dissemination through aerosols. Expected results will improve our knowledge of bacteria dissemination and nasal colonization and will allow the development of new therapeutic strategies to limit bacterial spreading.
POSTER : 20th International Pathogenic Neisseria Conference (IPNC 2016)”, 10/2016, Manchester, England
POSTER : YOUNG RESEARCHERS IN LIFE SCIENCES
Institut Pasteur, 18-20 mai 2016
Many bacterial pathogens initially colonize as commensal the nasopharynx and the upper respiratory tract (e.g., Staphylococcus aureus, Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumonia and Bordetella pertussis). These bacteria spread from person to person by direct contact or via aerosol. The understanding of events leading to colonization and dissemination between individuals is key to limit the spreading of these bacteria and identify new therapeutic strategies. Neisseria meningitidis (meningococcus) is a paradigm of commensal bacteria of the nasopharynx that may become a dreadful pathogen. This is an obligate human Gram-negative bacterium that normally resides on the surface of the nasopharyngeal mucosa of ~10% of the human population without affecting the host. The meningococcus spread from person to person by direct contact using aerosol transmission. Pathology is initiated when meningococci translocate from the nasopharynx into the bloodstream. Once in the blood flow, a tight interaction of this bacterium with human endothelia enables bacterial access to secondary anatomical sites (meninges, skin, etc), which promotes much of the morbidity and mortality associated with infections.
Understanding meningococcal colonization within the upper respiratory tract has been hampered by the lack of relevant models. Primary human nasal epithelial cells are now available and air-liquid interface protocols already developed to obtain fully differentiated nasal epithelium containing mucus producing goblet cells and ciliated cells. Using in vitro models, this proposal aims at identifying some of the bacterial components used by a bacterial pathogen to colonize its host and disseminate in a population. Our results will bring new insights into the colonization of the nasopharyngeal epithelium by Neisseria meningitidis with the objective to identify new potent therapeutic targets aiming at preventing the colonization and/or dissemination of the bacteria.
To achieve this objective, this project will be organized in three specific aims:
Aim 1: Identification of N. meningitidis factors required for colonization of the air-liquid interface model of upper respiratory tract cell culture. We will study the interaction between N. meningitidis and the mucosal nasopharynx using a model of differentiated epithelial cells grown in air-liquid interface condition. Then using a high throughput screening of meningococcal genes we will uncover new virulence attributes required by bacteria for colonization.
Aim 2: Identification of the epithelial host cells receptors for type IV pili. Preliminary data suggest that type IV pili are lectins that interact with identical glycan motifs which are expressed by various receptors on different cell types. We will therefore search for the specific composition of the glycan moiety required for N. meningitidis adhesion using human glycan array. In parallel we will use a BiraA proximity-dependent labeling strategy to find human adhesion receptors that possess the glycan of interest.
Aim 3: Explore mechanisms responsible for bacterial dissemination. We will set up an in vitro model to study the spreading of bacteria by aerosolization. Our aim is to produce a device that combines either air-liquid interface cell cultures or liquid culture and a sterile airflow system to aerosolize bacteria and allow flight of particles. This will allow us to determine which meningococcal factors are specifically required for dissemination from person to person.
Project coordination
Mathieu COUREUIL (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
INSERM INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
Help of the ANR 252,148 euros
Beginning and duration of the scientific project:
December 2015
- 36 Months
