Aminosugars but not sialic acids sustain growth of <i>C. canimorsus.</i>

<p>Viable counts after challenge with 2×10⁶ wt <i>Cc5</i> (black) or <i>siaC</i> (grey) grown for 24 h with J774.1 in cRPMI (control) or in the same condition with the addition of Neu5Ac, Neu5Ac- CMP, 12.5 ng/ml enzyme SiaC_FL, SiaC_Y488C or NanH from <i>C. perfringens</i> (A) or with the addition of GalNAc, GlcNAc or LacNAc (B) or with the addition of mannose, galactose, glucose or sialyl-lactose (C). Mean values from 3 or more experiments and s.d. are shown including statistical difference between wt <i>Cc5</i> and <i>siaC</i> with * p<0.05, ** p<0.01 and *** p<0.001 for each pair of columns (2-tailed unpaired Student's t test). The grey dotted line indicates the bacterial number inoculated.</p

<i>C. canimorsus</i> desialylates macrophage and epithelial cell surfaces.

<p>(A) The targets of the lectins used in this study are schematically represented (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000164#ppat.1000164-Varki2" target="_blank">[44]</a>). Surface carbohydrates of J774.1 macrophages (B) or HeLa epithelial cells (C) were analyzed by lectin binding after 2 h of infection with 4×10⁷ wt (<i>Cc5</i>) or <i>siaC</i> bacteria. Cells were fixed with paraformaldehyde and incubated for 1 h with lectin SNA, which recognizes terminal sialic acids (2- 6 or 2- 3) linked to Gal or to GalNAc or PNA that binds to the disaccharide Gal 1–3 GalNAc only after removal of terminal sialic acids. SiaC was added to cells alone or with <i>siaC</i> bacteria at 100 ng/ml. Biotinylated lectins were visualized by FITC conjugated streptavidin.</p

Identification of the Tn integration site and analysis of mRNA present in wt <i>C. canimorsus</i> 5.

<p>(A) Amino acid sequence of the <i>C. canimorsus</i> sialidase showing the signal peptide (italics) and the BNR/asp repeats (Ser/Thr-X-Asp-X-Gly-X-Thr-Trp/Phe) of bacterial sialidases (boxed). Domain predictions were analyzed by InterProScan <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000164#ppat.1000164-Quevillon1" target="_blank">[42]</a>. The residues conserved in sialidases at the C-terminus are underlined and the tyrosine 488 is bold <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000164#ppat.1000164-Roggentin1" target="_blank">[43]</a>. The Tn<i>4351</i> integration site in SiaC at amino acid 77 is indicated, boxed in grey and bold. (B) Genetic locus of the sialidase gene (<i>siaC</i>) including its upstream genes, <i>gntR</i>-like gene (CAPCA_MM1) and putative N-acyl-glucosamine epimerase encoding gene (CAPCA_MM2); and downstream coding sequence (CAPCA_MM3). (C) Reverse transcription performed on total RNA with specific primers (5129 or 5132) followed by PCR to identify transcripts present in wt <i>Cc5</i> (cDNA). PCR reactions were also performed using genomic DNA (gDNA) as template instead of cDNA as a positive control. As a control, reverse transcription was performed without reverse transcriptase in a parallel assay and used as template for the subsequent PCR reaction (-RT).</p

Surface localized sialidase is required for growth.

<p>(A) Sialidase activity of intact bacteria, measured with the substrate MUAN as the mean of triplicate measurements and s.d. of a representative experiment. (B) Viable counts after challenge with 2×10⁶<i>Cc5</i> (black), <i>siaC</i> (light grey) or <i>siaC</i> complemented with plasmids containing <i>siaC</i>_FL, <i>siaC</i>_Δ1–21 and <i>siaC</i>_Y488C after 24 h in presence of J774.1 with the grey dotted line indicating the bacterial number inoculated. Sialidase was detected by immunoblotting with a polyclonal antibody against SiaC in total cells (TC). (C) Outer membrane protein fractions (OMP), cell free supernatants (SN) of the J774.1 cultures shown in (B) including as control TC of <i>Cc5</i> were analyzed by immunoblotting for the presence of SiaC. (D) Surface localization of SiaC was tested by immunofluorescence on paraformaldehyde fixed but not permeabilized bacteria using anti-SiaC followed by anti- rabbit IgG conjugated to FITC.</p

The sialidase mutant is hypo-virulent in a tissue cage mouse infection model.

<p>Tissue cages were implanted in C57BL/6 mice and infected with 10⁷<i>Cc5</i> wt and <i>siaC</i> bacteria (n = 5) singly (A) or in competition (B). Bacteria were counted in tissue cage fluid (TCF) during 27 days (<i>Cc5 = </i>black circles; <i>siaC</i> = open circles). Individual values are shown; horizontal lines indicate the median value of each group. The black dotted line is the detection limit of 20 bacteria per ml TCF. (A) Cfu numbers between groups were significantly different on days 2, 5 and 9 with p<0.01 and on days 14 and 27 with p<0.05 (Mann Whitney test). (B) 10⁷ cfu <i>Cc5</i> and erythromycin resistant <i>siaC</i> were inoculated at a 1∶1 ratio. Bacterial numbers were analyzed for 27 days (n = 5). Viable counts between wt and <i>siaC</i> were significantly different on day 2, 5 and 9 with p<0.01 and on day 14 with p<0.05. (C) <i>Ex vivo</i> isolated leukocytes were resuspended in serum free RPMI and inoculated at a moi of 20 (2×10⁶ bacteria) or 0.2 (2×10⁴ bacteria) indicated with grey dotted lines and bacterial viable count was monitored after 24 h. Values represent the mean using TCF cells from 3 uninfected mice. TCF leukocytes consist of 68% +/− 4.8% polymorphonuclear neutrophils (PMNs), 18% +/− 3.2% monocytes and 9.1% +/− 3.7% macrophages. Wt and <i>siaC</i> numbers were significantly different with p<0.05 (*) and p<0.001 (**) using 2-tailed unpaired student's t test. (D) <i>In vitro</i>, <i>Cc5</i> and <i>siaC</i> were tested in heart infusion broth with FBS inoculated at a 1∶1 ratio with approximately 100 bacteria total and bacterial growth was monitored for 2, 6, 10 and 24 h. (E) Viable counts after challenge with 2×10⁶ (grey dotted line) <i>Cc5</i> (black) or <i>siaC</i> (grey) grown for 24 h with J774.1 in cRPMI singly (control) or at a 1∶1 ratio (cross-feeding).</p

Growth of <i>C. canimorsus 5</i> is dependent on cell contact.

<p>(A) Viable counts of 2×10⁶<i>Cc5</i> after 24 h in presence of J774.1 macrophages in RPMI supplemented with 10% FBS (moi = 20) (black) or in RPMI with FBS without cells (grey) and in a transwell system preventing physical contact between bacteria and macrophages in RPMI with FBS (white). (B) Viable counts of <i>Cc5</i> and Tn mutant after 24 h culture with macrophages in RPMI and FBS (black), with macrophages in RPMI and FBS in addition of cytochalasin D (grey), with HeLa cells (light grey) and MDCK cells in DMEM and FBS (white). The grey dotted line represents the bacterial number inoculated. The difference is statistically significant between <i>Cc5</i> and Tn mutant (2-tailed unpaired Student's t test p<0.05) in 3 or more experiments. (C) Growth curve of wt <i>Cc5</i> (triangles) and Tn mutant (squares) in heart infusion broth (HIB) supplemented with 10% FBS, represented as the mean of 3 or more experiments with the error bars showing the s.d.</p