A Listeria monocytogenes ST2 clone lacking chitinase ChiB from an outbreak of non-invasive gastroenteritis

Abstract

An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.

Keywords: Molecular surveillance; chitin; core genome MLST; invasion; listeriosis.

Figure 1.

Confirmation of the outbreak cluster by genome sequencing. (a) Minimum spanning tree calculated using cgMLST data (based on Ruppitsch’s cgMLST scheme) of gastroenteritis outbreak isolates (brown), causative food isolates (blue) as well as selected isolates from epidemiologically unrelated invasive listeriosis cases (red). Please note that distances between nodes in minimal spanning trees cannot be summed up to calculate the number of different alleles of more distantly related isolates. Circle size corresponds to isolate number and CTs are indicated. Isolate pairs with allele differences ≤10 are considered to be epidemiologically linked [24]. (b) Neighbor-joining tree illustrating phylogenetic relatedness of the same set of isolates after mapping to the genome of the L. monocytogenes serotype 4b strain CLIP 80459 [28] as the reference and SNP filtering. Colour coding of isolates is the same as in panel A. Please see the online version of the article for a colored figure.

Figure 2.

Invasion, intracellular multiplication and cell-to-cell spread of clinically invasive and gastroenteritis isolates. (a) Invasion assays. Isolates 15-01121 (gastroenteritis) and 15-01429 (invasive) were grown in BHI broth to mid-logarithmic growth phase and used to infect monolayers of HeLa, Hep-G2 and Caco-2 cells. Strains EGD-e and LMS2 (ΔdivIVA) were included to control the experiment. The experiment was performed in triplicate, EGD-e values were set to 100%, average values are shown and standard deviations are indicated. Significant differences are indicated by asterisks (t-test, P < .01). (b) Intracellular multiplication of the same set of strains as in panel A in J774 mouse macrophages. Average values and standard deviations are shown, which were calculated from an experiment performed in triplicate. Significant differences are indicated by asterisks (t-test, P < .01). (c) Plaque formation assay to monitor the ability to spread from cell to cell in 3T3 mouse fibroblasts. The same set of strains as in panel A was tested.

Figure 3.

Chitinase activity of selected gastroenteritis outbreak isolates. (a) Schematic illustration showing the lmo0105 (chiB) locus of L. monocytogenes EGD-e and the position of the premature stop codon in the gastroenteritis outbreak isolates (upper part). Domain organization of the ChiB protein according to Paspaliari et al. [60] (bottom part). The premature stop codon is indicated by a star. GH18 – family 18 glycoside hydrolases domain, FnIII – fibronectin type III-like domain, CBD – carbohydrate binding domain. (b) Multiple sequence alignment of the chiB gene sequences (partial) of all L. monocytogenes isolates sequenced in this study. The 315th codon of chiB (CGA, encoding arginine) is changed into the TGA stop codon in all gastroenteritis outbreak isolates and in all food isolates. (c) Chitinase activity of invasive isolates, gastroenteritis outbreak isolates and two selected food isolates on chitin agar plates after 4 days of incubation at 30°C. Halo formation indicates chitin hydrolysis. EGD-e was included as positive control. Please see the online version of the article for a colored figure.

Figure 4.

Restoration of ChiB production in the 15-01121 gastroenteritis isolate. (a) cgMLST analysis of the gastroenteritis isolate 15-01121, the invasive isolate 15-01429 and isolate 15-01121 after correction of chiB. (b) Chitinase production of strains 15-01429, 15-01121 and LMSB1 (15-01121 chiB⁺) on chitin containing BHI agar plates. (c) Western blot showing presence of ChiB in culture supernatants of the same set of strains as in panel A. Please see the online version of the article for a colored figure.

Figure 5.

Effect of chiB correction on virulence. (a) Invasion of the gastroenteritis isolate 15-01121, the invasive isolate 15-01429 and the gastroenteritis isolate with the corrected chiB gene 15-01121-chiB⁺ into different cell types. L. monocytogenes strain EGD-e was included as control. EGD-e values were set to 100%, average values are shown and standard deviations are indicated. Significant differences compared to 15-01121 are indicated by asterisks (t-test, P < .01). (b) Intracellular growth of the same set of strains in J774 mouse macrophages. L. monocytogenes strains EGD-e and LMS2 (ΔdivIVA) were included as controls here. All experiments were performed as triplicates and average values and standard deviations are shown. Significant differences are indicated by asterisks (t-test, P < .01). (c–d) Mouse infection experiment. C57BL/6 mice (n = 5) were infected via their tail vene with 5 × 10⁴ bacteria and the bacterial load in liver (c) and spleen (d) was determined on day 3 post infection. Data are represented as box plot and differences statistically significant according to the Brown–Forsythe test are indicated with asterisks.