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

Abstract

Listeria monocytogenes is a human intracellular pathogen that is able to survive in the gastrointestinal environment and replicate in macrophages, thus bypassing the early innate immune defenses. Peptidoglycan (PG) is an essential component of the bacterial cell wall readily exposed to the host and, thus, an important target for the innate immune system. Characterization of the PG from L. monocytogenes demonstrated deacetylation of N-acetylglucosamine residues. We identified a PG N-deacetylase gene, pgdA, in L. monocytogenes genome sequence. Inactivation of pgdA revealed the key role of this PG modification in bacterial virulence because the mutant was extremely sensitive to the bacteriolytic activity of lysozyme, and growth was severely impaired after oral and i.v. inoculations. Within macrophage vacuoles, the mutant was rapidly destroyed and induced a massive IFN-beta response in a TLR2 and Nod1-dependent manner. Together, these results reveal that PG N-deacetylation is a highly efficient mechanism used by Listeria to evade innate host defenses. The presence of deacetylase genes in other pathogenic bacteria indicates that PG N-deacetylation could be a general mechanism used by bacteria to evade the host innate immune system.

Fig. 1.

Characterization of Listeria PG. (A) Each muropeptide peak highlighted by a number was purified by HPLC, desalted, and analyzed by MALDI-TOF. Structural assignment was done by muropeptide fragmentation using MALDI-postsource decay (PSD). The structure of major muropeptides is indicated by full arrows. Structures or substructures in red and black indicate fully acetylated and N-deacetylated moieties, respectively, of the different muropeptides. Peaks 1–22 correspond to monomeric muropeptides, peaks 23- 44 correspond to dimeric muropeptides, and 46 and over correspond to trimeric muropeptides. Approximately 50% of the muropeptides present a glucosamine residue instead of the canonical N-acetylglucosamine residue. (B) HPLC analysis of the muropeptide composition of Listeria WT EGDe strain and its pgdA isogenic mutant. Each muropeptide peak was purified by HPLC and analyzed by MALDI-PSD mass spectrometry. Muropeptide peaks indicated with an asterisk correspond to N-deacetylated muropeptides. N-deacetylated muropeptides characteristic of the parental strain EGDe were completely absent from the elution pattern of the pgdA mutant. GlcNAc, N-acetylglucosamine; GlcN, glucosamine; M, N-acetylmuramic acid, TriPDAP, l-alanyl-γ-d-glutamyl-mesodiaminopimelic acid; TriPDAPNH₂, l-alanyl-γ-d-glutamyl-amidated mesodiaminopimelic acid; TetraPDAP, l-alanyl-γ-d-glutamyl-mesodiaminopimelyl-d-alanine; TetraPDAPNH₂, l-alanyl-γ-d-glutamyl-amidated mesodiaminopimelyl-d-alanine.

Fig. 2.

Effect of lysozyme on growth and impaired survival in macrophages of the pgdA mutant. Strain EGDe and its isogenic pgdA mutant were grown in BHI media and incubated with lysozyme (10 μg/ml) or the human serum amidase (1 μg/ml). (A) The pgdA mutant was selectively sensitive to the action of lysozyme upon entry into stationary phase. (B) Decrease of the optical density of the pgdA mutant correlated with a loss of viability, whereas the parental strain was insensitive to lysozyme. Lysozyme induced cell rounding of the pgdA mutant (see SI Fig. 7). (C and D) RAW264.7 macrophages (C) and PEM (D) were infected with WT EGDe and its pgdA mutant. Sensitivity of the pgdA mutant to lysozyme correlated with its impaired survival in macrophages.

Fig. 3.

Impaired survival of the pgdA mutant in macrophages. (A–D) RAW264.7 cells after 8 h of infection with the parental strain EGDe (A) and the pgdA mutant (B). Impaired survival was correlated with delay in escape of the pgdA mutant (C) from phagosomes compared with the WT strain EGDe (D). (E and F) PEM after 7 h of infection with the parental strain EGDe (E) and the pgdA mutant (F). [Scale bars: 2 μm and 1 μm (Insets).] Impaired survival correlated with delay in escape from phagosomes and bacterial lysis of the pgdA mutant compared with the WT strain EGDe.

Fig. 4.

Impaired virulence of the pgdA mutant in vivo. (A and B) BALB/c and C57/BL6J mice were challenged by i.v. injection with the parental strain EGDe and its pgdA mutant with a sublethal dose (5 × 10³ CFU per mouse). After 72 h, mice were killed, and bacterial counts in the liver (A) and the spleen (B) were determined. (C–G) Transgenic human E-cadherin mice were used as model for the oral route of infection, and colonization of several organs was followed after 3, 24, 48, and 72 h after challenge. Interestingly, the pgdA mutant was particularly vulnerable to persistence in the intestinal lumen as assayed by bacterial counts per grams of feces (C). Survival was more robust in the intestine (D) and the mesenteric lymph nodes (E) where colonization was particularly impaired after 72 h. The mutant was impaired in the survival of the liver (F) and spleen (G) at all time points as in the IV model shown in A and B.

Fig. 5.

Enhanced inflammatory response of the pgdA mutant. Cytokine production such as IL-6 (A) and IFN-β (B) by RAW264.7 macrophages was enhanced by the pgdA mutant compared with the parental strain EGDe (see also SI Fig. 9). PEM of WT C57/BL6J, Nod1^−/−, Nod2^−/− TLR2^−/− and MyD88^−/− mice were infected with strains EGDe and its isogenic pgdA mutant. After 7 h of infection, the inflammatory response was enhanced as measured by the amount of IL-6 (C) and, particularly, IFN-β (D) production. The cytokine response depended mainly on TLR2 and Myd88, although their contribution varied according to the cytokine measured. IFN-β production depended almost entirely on TLR2 in a MyD88-dependent and -independent manner (D). IL-6 production depended only partially on TLR2 and Myd88 (C) and also required Nod1. Surprisingly, Nod1 seemed to function as an inhibitor of IFN-β production, because the pgdA mutant induced 3-fold more IFN-β in Nod1^−/− compared with C57/BL6J peritoneal macrophages (D). Note that the role of Nod1 is entirely restricted to the response to the pgdA mutant and does not participate in the response to the WT EGDe strain.