Oral Presentation MedVetPATHOGENS 2018

Comparative study of natural variants of Listeria monocytogenes invasion factor InlB: biological and physical aspects (#30)

Svetlana Ermolaeva 1 , Yaroslava Chalenko 1 , Egor Kalinin 1 , Victor Marchenkov 2 , Alexey Surin 2 , Konstantin Sobyanin 1 , Elena Sysolyatina 1
  1. Gamaleya Research Center of Epidemiology and Microbiology, Moscow, MOSCOW REGION, Russia
  2. Institute of Protein Research RAS, Puschino, Moscow region, Russia

InlB promotes Listeria monocytogenes invasion in mammalian cells via interactions with c-Met receptor. InlB includes internalin domain, B-repeat, and GW domains. The internalin domain (InlB321) is sufficient to cause c-Met activation(1,2). To evaluate an input of InlB natural variability in L. monocytogenes virulence, we analyzed distribution, biological and physical properties of InlB321 natural variants.

InlB321 variability was analyzed on a laboratory collection of 65 invasive strains and compared with data available from GeneBank. Predominant InlB321 variants were cloned into the developed vector to restore full-length InlB and used to complement inlB deletion in the EGDeΔinlB strain. Virulence was assessed in cell invasion assay and mouse models  of intravenous, intraperitoneal and intragastric infection. Purified InlB321 variants were characterized by gel-filtration chromatography and Circular dichroism (CD) and tested in signaling pathway activation assays.

Four InlB321 variants were prevalent among L. monocytogenes strains while others seemed to be their derivations. Being cloned in EGDeΔinlB, all variants restored invasion in HEK239 and C26 cells. Three variants gave similar results in intravenous infection, the last (Var14) showed decreased mortality and 2log10 decreased loads in liver 6h post infection. After intraperitoneal injection, Var14 was the worst and showed 40-fold less loads in the liver comparatively to InlB321 from EGDe. In contrast, Var14 was the most active in intragastric infection.

When purified, all InlB321 variants were mostly monomeric with Var14 forming high molecular weight oligomers in small amounts. CD analysis demonstrated that Var14 provided more flexible structure comparatively to others.  The var14 differed from EGDe InlB321 by Ala117Thr and Val132Ile substitutions. Var14 caused 4- and 2-fold activation of Erk1/2 and Akt, respectively, comparatively to HGF while EGDe InlB321 was inactive in our assay.

Taken together, obtained results demonstrated that InlB321 natural variants differed in their biological and physical properties that might be important for L. monocytogenes virulence.

  1. Braun L, Nato F, Payrastre B, Mazié JC, Cossart P. The 213-amino-acid leucine-rich repeat region of the listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling. Mol Microbiol. 1999 34:10-23
  2. Banerjee M, Copp J, Vuga D, Marino M, Chapman T, van der Geer P, Ghosh P. GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation. Mol Microbiol. 2004 52:257-71