LPXTG protein InlJ, a newly identified internalin involved in Listeria monocytogenes virulence - PubMed (original) (raw)
LPXTG protein InlJ, a newly identified internalin involved in Listeria monocytogenes virulence
Christophe Sabet et al. Infect Immun. 2005 Oct.
Abstract
Listeria monocytogenes expresses surface proteins covalently anchored to the peptidoglycan by sortase enzymes. Inactivation of srtA attenuates Listeria virulence in mice (H. Bierne, S. K. Mazmanian, M. Trost, M. G. Pucciarelli, G. Liu, P. Dehoux, L. Jansch, F. Garcia-del Portillo, O. Schneewind, and P. Cossart, Mol. Microbiol. 43:869-881, 2002). We show here that an srtA mutant is more attenuated than an internalin mutant in orally infected guinea pigs and transgenic mice expressing human E-cadherin (hEcad mice), indicating the involvement of other SrtA substrates, LPXTG proteins, in food-borne listeriosis. Data recently generated with a listerial DNA macroarray identified two LPXTG protein-encoding genes present in the genomes of L. monocytogenes strains and absent from all other Listeria species, inlI (lmo0333) and inlJ (lmo2821). They also revealed two other LPXTG protein-encoding genes, ORF29 and ORF2568, present only in a subclass of L. monocytogenes serovars, including the epidemic serovar 4b. We report here that an inlJ deletion mutant, in contrast to inlI and ORF29 mutants, is significantly attenuated in virulence after intravenous infection of mice or oral inoculation of hEcad mice. Interestingly, a DeltaORF2568 strain showed a slight increase in virulence. inlJ encodes a leucine-rich repeat (LRR) protein that is structurally related to the listerial invasion factor internalin. However, the consensus sequence of the InlJ LRR defines a novel subfamily of cysteine-containing LRRs in bacteria. In conclusion, this postgenomic approach identified InlJ as a new virulence factor among the proteins belonging to the internalin family in L. monocytogenes.
Figures
FIG. 1.
srtA is involved in L. monocytogenes oral infections in guinea pigs. The wild-type EGDe strain (wt) and isogenic Δ_srtA_, Δ_inlA_, and Δ_srtB_ strains were orally inoculated into guinea pigs, as described in Materials and Methods. Animals were euthanized 96 h after infection, and organs were recovered, homonogenized, and plated. The numbers of bacteria able to colonize the intestine, mesenteric lymph nodes (M.L.N), liver, and spleen are expressed in log10 CFU. In each experiment four animal were used for each bacterial strain. The results are representative of two independent experiments. An asterisk indicates a significant difference (P < 0.05) between a mutant strain and the wild-type strain.
FIG. 2.
InlJ LRR defines a new family of bacterial cysteine-containing LRRs. (A) Amino acid sequence of the inlJ gene product. The signal peptide is indicated by boldface letters. The 15 LRRs are aligned, with leucines, isoleucines, and valines indicated by a red background and cysteines indicated by a blue background. The other conserved residues are indicated by colored letters. The immunoglobulin (Ig)-like domain is indicated by a gray background. The four MucBP repeats are aligned with conserved residues in boldface letters. The LPXTG motif in the C-terminal sorting signal is underlined, and the hydrophobic residues are indicated by italics. (B) Comparison of InlA and InlJ LRR consensus sequences. Residue positions are numbered as described by Schubert et al. (58). The positions of the expected β strand and 310-helix are indicated. Conserved residues are indicated by colored letters (hydrophobic core, red; cysteines, blue). (C) Alignment of LRR consensus sequences of InlJ and proteins from different species. (Top) Alignment of InlJ LRR with LRRs of bacterial or viral proteins. (Bottom) Alignment of InlA and InlJ LRR consensus sequences with LRRs of RNase inhibitor (RI) and cysteine-containing (CC) types from eukaryotes. Conserved residues are indicated by colored letters (leucines, valines, and isoleucines, red; cysteines, dark blue; threonine, light blue; asparagine, green; aspartic acid, orange). O, hydrophobic residues; bCC, bacterial cysteine containing.
FIG. 3.
Expression of inlI and inlJ. (A) RT-PCR for total RNAs from the end of the exponential phase (OD600, 0.8) for cultures in BHI medium at 37°C of L. monocytogenes wild-type strain. Serial dilutions of the RNA templates were used. The gyrA and inlA genes were used as controls of known expressed genes. (B) RT-PCR for RNAs from L. monocyogenes wild-type and Δ_inlI_ or Δ_inlJ_ strains. Amplification of the iap gene was used to control RNA amounts. wt, wild type.
FIG. 4.
inlJ is involved in L. monocytogenes virulence. (A) Mice were inoculated intravenously with 104 CFU of the wild-type EGDe strain or Δ_inlI_ or Δ_inlJ_ strain. Bacterial growth was monitored in the liver and the spleen at 24 h, 48 h, and 72 h. The results are the means of two independent experiments. (B) hEcad mice were orally infected with 109 CFU of the wild-type strain or the Δ_inlI_, Δ_inlJ_, Δ_inlA_, or Δ_srtA_ strain. The numbers of bacteria able to colonize the intestine, mesenteric lymph nodes (M.L.N), liver, and spleen were determined at 72 h. The data are expressed in log10 CFU (see Materials and Methods). In each experiment four animals were used for each bacterial strain. The results are the means of two (Δ_inlI_ strain) or three (wild-type, Δ_inlJ_, Δ_inlA_, and Δ_srtA_ strains) independent experiments. An asterisk indicates a significant difference (P < 0.05) between a mutant strain and the wild-type strain. wt, wild type.
FIG. 5.
(A) Schematic representation of ORF29 and ORF2568 LPXTG proteins specific for serovar 4b. The signal peptide (SP), repeat region, MucBP domains, and C-terminal sorting signal (solid box) are indicated. (B) Effect of ORF29 and ORF2568 inactivation on L. monocytogenes serovar 4b virulence. The wild-type L. monocytogenes serovar 4b strain or ΔORF29 and ΔORF2568 isogenic mutants were orally inoculated into hEcad mice (109 CFU). The numbers of bacteria able to colonize the intestine, mesenteric lymph nodes (M.L.N), liver, and spleen were determined at 72 h and expressed in log10 CFU (see Materials and Methods). In each experiment four animals were used for each bacterial strain. The results are the means of two (ΔORF29 strain) or three (wild-type and ΔORF2568 strains) independent experiments. An asterisk indicates a significant difference (P < 0.05) between a mutant strain and the wild-type strain. wt, wild type. (C) Genetic organization of the ORF2568 region in L. monocytogenes serovar 4b strain CLIP80459 and in L. monocytogenes serovar 1/2a strain EGDe. Open reading frames are indicated according to the genome nomenclature described previously (ORF [14] and lmo [28]). Open reading frames present only in L. monocytogenes serovar 4b are indicated by solid arrows. Other open reading frames are indicated by open arrows. The ovals indicate putative transcription terminators.
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