A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system

Boneca et al. 10.1073/pnas.0609672104.

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SI Figure 6
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SI Figure 6

Fig. 6. Growth of the pgdA mutant in serum. Despite the in vitro sensitivity of the pgdA mutant to lysozyme, either strain (EGDe and the pgdA mutant) grew normally in 25% FCS independently of prior decomplementation (A). Growth in 25% serum correlated with an increase in CFU/ml (B) indicating that the pgdA mutant was not sensitive to the complement or the normal amounts of lysozyme found in the serum.

SI Figure 7

Fig. 7. Morphology of Listeria monocytogenes EGDe (A and B) and the pgdA mutant (C and D) in the absence or presence of lysozyme (10 mg/ml). Both strains had normal bacillary morphology (A and C) in BHI broth as observed by optical microscopy. Addition of lysozyme in stationary phase had no effect on the wild-type EGDe strain (B) but lead to cell rounding of the pgdA mutant (D).

SI Figure 8

Fig. 8. (A) Escherichia coli MC1061 and L. monocytogenes EGDe PG were compared for their ability to activate NF-kB in a Nod1- and Nod2-dependent manner. Transiently transfected HEK293 cells were stimulated with either native, amidase-digested, or muramidase-digested PG. Listeria PG induced NF-kB in a Nod1- and Nod2-dependent manner only after digestion into soluble muropeptides by the muramidase. (B) Native PG from strain EGDe and its isogenic pgdA mutant were used to activate NF-kB in a Nod1-and Nod2-dependent manner. Native PG from the pgdA mutant was more efficient in inducing a Nod1 and Nod2-dependent response. Taken together, our results suggested that L. monocytogenes contains in its native PG the Nod agonists but that these are not readily available to the host. (C) To further establish a possible link between N-deacetylation of PG and escape from Nod proteins, we decided to N-deacetylate a PG known to be fully acetylated using a recombinant PG N-deacetylase BC1960 from Bacillus cereus (1) and highly purified PG from Helicobacter pylori. This PG was used to activate NF-kB in a Nod1- and Nod2-dependent manner. Whereas native PG induced both Nod1 and Nod2, in vitro fully N-deacetylated native H. pylori PG failed to activate NF-kB in a Nod1 and Nod2-dependent manner. (D) Analysis of native and in vitro N-deacetylated H. pylori peptidoglycan by HPLC and MALDI-PSD. H. pylori has a classical mesoDAP-type PG. Treatment of native H. pylori PG with recombinant BC1960 PG deacetylase from B. cereus resulted in a complete N-deacetylation of H. pylori PG as assessed by HPLC and MALDI-PSD of soluble muropeptides. Peaks 1-5, and, 1* to 9* represent native monomeric and in vitro N-deactylated monomeric muropeptides of H. pylori. The nomenclature of each peak correspond to previously described nomenclature (1).

SI Figure 9

Fig. 9. Enhanced inflammatory response of the pgdA mutant. TNF-a (A) and IL-1b (B) production by RAW264.7 macrophages was enhanced by the pgdA mutant compared with the parental strain EGDe. Cytokine production was assayed by the ELISA method. The inflammatory response was also enhanced, as measured by the amount of IL-6 (see Fig. 5 A and C) and, particularly, IFN-b (see Fig. 5 B and D) production.

SI Figure 10

Fig. 10. Model of host recognition of the pgdA mutant. In the intestinal lumen, the pgdA mutant is rapidly eliminated as a result of the bacteriolytic activity of lysozyme (thick arrow) that is massively produced by Paneth cells. The few bacteria that escaped from the intestinal lumen (dashed arrow) disseminate by spreading either from cell to cell or by entry into phagocytic cells. The pgdA mutant sensitivity to lysozyme results in its enhanced destruction in phagosomes and release of cell-wall components such as muropeptides and LTA. LTA is readily available to target membrane-bound TLR2, whereas muropeptides can be delivered to the cytosol to target Nod1 through either a listeriolysin O-dependent mechanism or by endogenous transporters. Activation of the TLR2 and Nod1 pathway results in an induction of proinflammatory cytokines. Type I interferons are also induced in a TLR2-dependent fashion. Production of IFN-b induced by the pgdA mutant depends only partially on MyD88, indicating that TLR2 might activate Type I interferons by an as-yet-unknown pathway. Type I interferons and cytokines enhanced production by the pgdA mutant results in a stronger inflammatory response contributing to its impaired virulence.

1. Psylinakis E Boneca IG, Mavromatis K, Deli A, Hayhurst E, Foster SJ, Varum KM, Bouritis V (2005) J Biol Chem 280:30856-30863.

SI Text

Expression Plasmids, Transient Transfections, and NF-kB Activation Assays. The expression plasmid for FLAG-tagged hNod1 was from Gabriel Nuñez (University of Michigan Medical School, Ann Arbor, MI) and has been described (1). The expression plasmid for hNod2 was from Gilles Thomas (Fondation Jean Dausset/Centre d'Étude du Polymorphisme Humain). Briefly, HEK293T cells were transfected overnight with 1 ng of hNod1 or 1 ng of hNod2 plus 75 ng of Ig luciferase reporter plasmid. PG samples (0.1 mg/ml) were digested with 0.25 mg/ml mutanolysin or human serum amidase. At the same time, 0.3 mg of PG preparations or 10 pmol of muramyl-peptides were added to the cell culture medium, and synergistic NF-kB-dependent luciferase activation was measured after 24 h of coincubation. NF-kB-dependent luciferase assays were performed in duplicate, and data reported represent at least three independent experiments.

Infection of Murine Macrophages. For primary cultures of BMDM, the contents of murine femurs and tibias were flushed and cells grown for a week in RPMI medium 1640 supplemented with 2 mM glutamine, 10% FCS, and 6 ng/ml M-CSF. Nonadherent cells were recovered and incubated in RPMI medium 1640 supplemented with 2 mM glutamine, 10% FCS, and 6 ng/ml M-CSF for 24 h to obtain a homogenous monolayer of macrophages.

Mouse Experiments. C57/BL6J mice were purchased from Janvier (Le Genest, France) or Charles River Laboratories. TLR2^–/– and MyD88^–/– mice initially provided by S. Akira (Osaka, Japan) were further back-crossed in C57/BL6J to reach the eighth back-cross by Michel Chignard (Institut Pasteur). Nod1^–/– mice were generated under specific pathogen-free conditions in the animal facilities of the Institut Pasteur from breeding pairs that had been kindly supplied by J. Bertin (Millenium). Nod2^–/– were initially provided by J.-P. Hugot (Hôpital Robert Debré). Nod1^–/– and Nod2^–/– were further back-crossed in C57/BL6J to reach the eighth back-cross by Jérôme Viala (Institut Pasteur). iFABP-hEcad transgenic mice were generated at the Institut Pasteur and were described (2). Animals were housed in polycarbonate cages in isolators and fed a commercial pellet diet with water ad libitum. Animal handling and experimentation was performed in accordance with institutional guidelines and current French legislation (Law No. 87-848) and European Regulations on Animal Welfare and with Public Health Service recommendations.

1. Inohara N, Koseki T, del Peso L, Hu Y, Yee C, Chen S, Carrio R, Merino J, Liu D, Ni J, Nunez G (1999) J Biol Chem 274:14560-14567.

2. Lecuit M, Vandormael-Pournin S, Lefort J, Huerre M, Gounon P, Dupuy C, Babinet C, Cossart P (2001) Science 292:1722-1725.

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