Structure of the o-repeating unit and the lipid a isolated from fusobacterium nucleatum atcc 51191

Session: 
S10.1 Lipopolysaccharides
Code: 
OL10.1.1
Location (hall): 
Glucose
Start/end time: 
Thursday, July 4, 2019 - 17:15 to 17:30
Pilar
Garcia-Vello

Pilar Garcia-Vello1, Dimitra  Lamprinaki2, Flaviana  Di Lorenzo1, Antonio  Molinaro1, Nathalie Juge2, Cristina De Castro3

1Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy, 2Gut Microbes & Health Institute Strategic Programme, Quadram Institute Bioscience, Norwich Research Park, Norwich, UK, 3Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy

INTRODUCTION:
Fusobacterium nucleatum is a common member of the oral microbiota [1]. However, this symbiont has been found to cause opportunistic infections such as periodontal diseases and has been implicated in adverse pregnancy outcomes such as preeclampsia, gastrointestinal disorders such as colorectal cancer or appendicitis, as well as cardiovascular diseases, rheumatoid arthritis, respiratory tract infections, Lemierre’s syndrome and Alzheimer’s disease [2]. F. nucleatum has virulence mechanisms that lead to infections outside the mouth [3]. 

As a Gram-negative bacterium, F. nucleatum has an outer membrane layer protecting the bacterium in different environments and its external face is composed mainly by lipopolysaccharides (LPS) [4]. LPS are microbe associated molecular pattern (MAMP) molecules that play a crucial role in the interaction of the host with pathogens but also commensal bacteria as they can be recognized by the innate immunity response activating the pattern recognition receptors (PRR) [5]. The dual commensal-pathogen behaviour of F. nucleatum makes the determination of the LPS structure of this bacterium especially interesting.

METHODS:
The structure of O-antigen and lipid A from F. nucleatum ssp. animalis (ATCC 51191) was characterized using a combination of Gas Chromatography - Mass Spectrum (GC-MS) derivatization, Matrix-Assisted Laser Desorption/Ionization (MALDI) and Nuclear Magnetic Resonance (NMR) analytical techniques.

RESULTS:
Analysis of the NMR disclosed the presence of a repeating unit composed by three monosaccharides, apparently in contrast with the presence in the proton spectrum of about six signals in the anomeric region with no stoichiometric proportions. Analysis of the connectivity by combining COSY, TOCSY and NOESY spectra established that three different sugars were present: β-GlcpNAcA, β-GlcpNAc3NAlaA and α-FucpNAc4NAc. Importantly, the amino function at position 4 of α-FucpNAc4NAc is acetylated in a not stoichiometric fashion: depending on the presence of the acetyl, the chemical shifts of all signals change, giving a spectrum with six anomeric signals. Also, the Lipid A was analysed by MALDI. 

CONCLUSIONS:
The LPS structure of F. nucleatum ATCC 51191 displays great complexity due to the high level of amino functions, the partial acetylation of one of them and the presence of an amino acid, Ala. More research is necessary to understand the role that this structure plays in the interaction of this bacterium with host in the context of health and disease.

References: 
  1. Avila M, Ojcius DM, Yilmaz O. The oral microbiota: living with a permanent guest. DNA Cell Biol. 2009;28(8):405-11. 
  2. Han YW. Fusobacterium nucleatum: a commensal-turned pathogen. Curr Opin Microbiol. 2015;23:141-7.
  3. Bachrach G, Ianculovici C, Naor R, Weiss EI. Fluorescence based measurements of Fusobacterium nucleatum coaggregation and of fusobacterial attachment to mammalian cells. FEMS Microbiol Lett. 2005;248(2):235-40.
  4. Okahashi N, Koga T, Nishihara T, Fujiwara T, Hamada S. Immunobiological Properties of Lipopolysaccharides Isolated from Fusobacterium nucleatum and F. necrophorum. J Gen Microbiol. 1988;134(6):1707-15.
  5. Molinaro A., Holst O., Di Lorenzo F., Callaghan M., Nurisso A., D'Errico G., et al. Chemistry of lipid A: at the heart of innate immunity. Chemistry. 2015;21(2):500-19.

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