Listeria monocytogenes is resistant to lysozyme through the regulation, not the acquisition, of cell wall-modifying enzymes

Abstract

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood.

FIG 1

Screen to identify lysozyme-sensitive mutants in L. monocytogenes. (A) L. monocytogenes transposon mutants were replica plated from BHI (left panels) onto BHI-lysozyme plates (right panels) containing 1 mg/ml chicken egg white lysozyme (Sigma). Arrows indicate colonies defective on BHI-lysozyme plates. (B, C, D) Strains were grown in 2 ml BHI overnight while shaking at 37°C, and 30 μl was spread onto BHI agar. Filter disks containing 1 mg of lysozyme were placed onto the agar and incubated overnight at 37°C, and zones of clearance were measured. Means and standard deviations from at least three separate experiments are presented. ***, P < 0.001. The dotted line indicates the zone of inhibition of WT bacteria by lysozyme.

FIG 2

Treatment of lysozyme-sensitive strains with CRAMP and cell wall-acting antibiotics. (A) A final concentration of 10 μg/ml of purified mouse CRAMP (Anaspec) was added to mid-exponential-phase cells (at 37°C in BHI) of the L. monocytogenes strains indicated, and turbidity was monitored at 10-min intervals. Data are representative of at least three separate experiments and divided into three panels for clarity. (B, C) Thirty-microliter volumes of overnight L. monocytogenes cultures were plated onto BHI, and disks containing 700 ng of cefuroxime (B) or 500 ng of penicillin G (C) were added. The plates were incubated overnight at 37°C, and zones of inhibition were measured. The data shown are the means of at least three separate experiments, and error bars represent the standard deviations of the means. *, P < 0.05; ***, P < 0.001; ns, not statistically significantly different (determined by unpaired two-tailed t test).

FIG 3

Rli31 is highly abundant and expressed in all growth phases. (A) Twenty-microgram samples of total RNA collected in the phases of growth in BHI indicated were separated on 6% polyacrylamide. Nucleotides were transferred to a nylon membrane, probed with a ³²P-labeled TB13 primer, and imaged by Typhoon. (B) Twenty-microgram samples of total RNA collected in the mid-exponential phase in BHI at 37°C from the WT (lane 1), the Δrli31 mutant (lane 2), and the Δrli31/pAM401:rli31 mutant (lane 3) were separated on 6% polyacrylamide and stained with SYBR gold (Invitrogen). The arrow indicates Rli31, and lane L contains a molecular size ladder. (C) RNA from panel B was transferred to a nylon membrane, probed with a ³²P-labeled TB13 primer, and imaged by Typhoon.

FIG 4

Characterization of the rli31 mutant phenotype. (A, B) HPLC analysis of the muropeptide composition of WT and Δrli31 mutant L. monocytogenes. Deacetylated muropeptides are red, and O-acetylated muropeptides are blue. Muropeptide abbreviations: GlcNAc, NAG; GlcN, glucosamine; M, NAM; TriPDAPNH₂, l-alanyl-γ-d-glutamyl-amidated meso-diaminopimelic acid; TetraPDAPGlcNAc, l-alanyl-γ-d-glutamyl-amidated meso-diaminopimelyl-d-alanine; OAc, O-acetylated. (A) Samples were treated with hydrofluoric acid. (B) Samples were not treated with hydrofluoric acid to retain covalent modifications. (C) Multiple rli31 and pgdA mutants were independently passaged with increasing concentrations of lysozyme (50, 100, 200, 500, 1,000 μg/ml) in BHI broth while shaking at 37°C. The resulting strains were grown to mid-exponential phase and treated with 1 mg/ml lysozyme along with the Δrli31 mutant parent strain. Turbidity was monitored at 10-min intervals. sup., suppressor. (D) PgdA and PbpX transcripts of the strains indicated were measured by qPCR, normalized to BglA, and compared to the transcript levels in WT L. monocytogenes. Error bars represent standard deviations of the means. Statistical analyses were performed by using a two-tailed t test assuming a null hypothesis of 1. **, P < 0.01.

FIG 5

The rli31 mutant phenotype is due principally to the regulation of pgdA and pbpX. Strains were grown to an OD₆₀₀ of 0.5, and the indicated concentrations of lysozyme were added. Bacteria were plated for CFU counting at the intervals shown. The data are averages of at least three independent experiments, and error bars specify standard errors. Two-tailed t tests indicate statistically significant difference between the degU::Tn and Δrli31 degU::Tn (A), ΔpgdA and ΔpgdA rli31::Tn (B), ΔpbpX and ΔpbpX rli31::Tn (C), ΔoatA and ΔoatA rli31::Tn (D), ΔpgdA ΔoatA and ΔpgdA ΔoatA rli31::Tn (E), and ΔpgdA pbpX::Tn and ΔpgdA pbpX::Tn rli31::Tn (F) mutant strains. **, P < 0.01; *, P < 0.05; ns, no significant difference.

FIG 6

Serum kills lysozyme-sensitive L. monocytogenes. (A) CFU counts in organs of CD-1 mice infected i.v. for 48 h. Shown are combined data from two separate experiments with at least eight mice per group. A two-tailed Mann-Whitney t test was used for the statistical comparison of each group with the WT. *, P < 0.05; **, P < 0.01; ***, P < 0.0001. (B) CFU counts in organs of CD-1 mice infected i.v. for 30 min with the strains of bacteria indicated. Shown are combined data from at least two separate experiments with at least eight mice per group. A two-tailed P value is reported for the comparison of each group with the WT. *, P < 0.05; **, P < 0.01; ***, P < 0.0001. (C, D) Strains were grown to mid-exponential phase in BHI, washed with PBS, and diluted 1:100 in defibrinated sheep or horse blood (Hemostat). Bacteria were plated for CFU counting at the times indicated. Data represent means and standard errors from at least three separate experiments. (D) Blood was treated with 5 mg of bentonite (Sigma) for 30 min at 4°C immediately prior to inoculation. A two-tailed P value is reported for the comparison of the pgdA oatA pbpX mutant strain (no bentonite) with the WT. A two-tailed P value is also reported for the comparison of the values obtained with the pgdA oatA pbpX mutant strain with bentonite with those obtained without bentonite. ***, P < 0.0001.