Naturally occurring InlB variants that support intragastric Listeria monocytogenes infection in mice

Abstract

Listeria monocytogenes is a causative agent of foodborne infection in humans and animals. The virulence factor InlB interacts with mammalian receptor c-Met via its internalin domain to provide L. monocytogenes invasion in non-professional phagocytes. Naturally occurring InlB internalin domain variants form four subclusters on the maximal likelihood tree. Four variants belonging to distinct subclusters were cloned into the vector carrying 3΄ and 5΄-flanking sequences to restore full length inlB and expressed in the L. monocytogenes strain EGDeΔinlB. The substitutions Val132Ile, Thr117Ala and Ile138Leu, Thr251Met/Ser were specific for variants 13, 14 and 1, respectively, the variant 9 carried Ser73Asn, Ile91Val, Leu164Pro, Met251Ser/Thr substitutions. All InlB variants improved invasion of the parental strain in murine colon carcinoma C26 cells with 4.6-fold difference between the most and least effective variants (variants 14 and 13, respectively, P < 0.05). Bacterial loads in livers of intragastrically infected mice were 258, 149 and 92 times higher for variant 14, 13 and 1 carrying strains, respectively, than for EGDeΔinlB (P < 0.01). In contrast, the variant 9 did not noticeably improve infection comparatively to the parental strain. Overall, obtained results demonstrated that naturally occurred InlB internalin domain variants differed in their ability to support intragastric infection in mice.

Route of Injection Affects the Impact of InlB Internalin Domain Variants on Severity ofListeria monocytogenesInfection in Mice

“…Previously, we have demonstrated that the idInlBs differed in their ability to support intragastric (i.g.) infection providing different bacterial loads in the liver but not in the spleen [ 25 ]. The idInlB14 gave rise to the highest loads in the liver 3 days after i.g.…”

“…Previously, we suggested that the sequence of the invasion factor InlB might be critical for L. monocytogenes spreading among small rodents in the pristine environment [ 21 , 25 ]. Indeed, InlB is a key invasion factor responsible for the intestine barrier crossing in mice [ 19 ].…”

“…Recombinant strain construction was described previously [ 25 ]. Shortly, the idInlB encoding DNA fragments obtained on lysates of L. monocytogenes strains VIMHA004, VIMHA015, and VIMHA034 ( Table 1 ) with primers InlB3: 5′-CAAGCG GGATCC ATCACCGTGCCAACGCC and InlB4: 5′-TATCCG GGATCC TGCTTCTACTTTTG were cloned into the derivative of the pTRKH2 vector [ 29 ] carrying the inlB gene, lacking the internalin domain encoding part.…”

“…Bacteria were prepared from the mid-exponential culture as it was described above. The “gentamycin assay” was performed as described previously [ 25 ]. Briefly, bacteria resuspended in DMEM were added with MOI 100 : 1 (bacteria to cell) to cells, incubated 1 h at 37°C in 5% CO 2 atmosphere, and washed with PBS, and then the fresh medium supplemented with 100 μ g ml −1 gentamycin (Fluka) was added.…”

“…To evaluate an impact of naturally occurring InlB variants on L. monocytogenes virulence, we used a set of previously described isogenic strains that carried distinct InlB internalin domains (idInlBs) [ 25 ]. Virulence of the recombinant strains was compared in mouse models.…”