Two Permeases Associated with the Multifunctional CtaP Cysteine Transport System in Listeria monocytogenes Play Distinct Roles in Pathogenesis

Abstract

The soil-dwelling bacterium Listeria monocytogenes survives a multitude of conditions when residing in the outside environment and as a pathogen within host cells. Key to survival within the infected mammalian host is the expression of bacterial gene products necessary for nutrient acquisition. Similar to many bacteria, L. monocytogenes uses peptide import to acquire amino acids. Peptide transport systems play an important role in nutrient uptake as well as in additional functions that include bacterial quorum sensing and signal transduction, recycling of peptidoglycan fragments, adherence to eukaryotic cells, and alterations in antibiotic susceptibility. It has been previously described that CtaP, encoded by lmo0135, is a multifunctional protein associated with activities that include cysteine transport, resistance to acid, membrane integrity, and bacterial adherence to host cells. ctaP is located next to two genes predicted to encode membrane-bound permeases lmo0136 and lmo0137, termed CtpP1 and CtpP2, respectively. Here, we show that CtpP1 and CtpP2 are required for bacterial growth in the presence of low concentrations of cysteine and for virulence in mouse infection models. Taken together, the data identify distinct nonoverlapping roles for two related permeases that are important for the growth and survival of L. monocytogenes within host cells. IMPORTANCE Bacterial peptide transport systems are important for nutrient uptake and may additionally function in a variety of other roles, including bacterial communication, signal transduction, and bacterial adherence to eukaryotic cells. Peptide transport systems often consist of a substrate-binding protein associated with a membrane-spanning permease. The environmental bacterial pathogen Listeria monocytogenes uses the substrate-binding protein CtaP not only for cysteine transport but also for resistance to acid, maintenance of membrane integrity, and bacterial adherence to host cells. In this study, we demonstrate complementary yet distinct functional roles for two membrane permeases, CtpP1 and CtpP2, that are encoded by genes linked to ctaP and that contribute to bacterial growth, invasion, and pathogenicity.

Keywords: ABC transporters; CtaP; PrfA; bacterial adhesion; cysteine transport; virulence; virulence factors.

FIG 1

Location and topology of the CtaP permeases. (A) Organization of the CtaP permeases. Dashed lines indicate locations of the specific deletion mutations constructed. (B) Predicted topology of Lmo0136 and Lmo0137. ^, predicted ATP binding loop; *, predicted to interact with the substrate-binding protein CtaP.

FIG 2

ctpP1 and ctpP2 exhibit different growth patterns in broth culture. (A) Assessment of growth of the ctaP transporter, permease mutants, and complements in BHI broth culture media. Overnight cultures of each strain grown shaking in BHI at 37°C were diluted 1:20 in fresh BHI media, and the OD₆₀₀ was measured at the indicated time points. (B and C) Assessment of growth of the ctaP transporter, permease mutants, and complements in defined culture media. Cultures were grown overnight shaking in BHI at 37°C. Strains were normalized by the OD₆₀₀, pelleted, and washed 2 times in PBS before resuspension in PBS to a final volume equal to the normalized culture volume. Bacterial suspensions were diluted 1:20 in HTM media containing the indicated concentrations of free cysteine. Growth was measured as the OD₆₀₀ at indicated time points.

FIG 3

Mutations in the ctaP gene of the ΔctpP1 ΔctpP2 double deletion mutant shown in the L. monocytogenes reference strain 10403S. CtaP protein-coding region with predicted protein domains. Arrows indicate amino acid changes identified in CtaP’s substrate-binding region due to single-nucleotide polymorphisms at residues T341 and K473.

FIG 4

ctpP1 and ctpP2 contribute to host cell invasion, based here on measurement of cell-to-cell spread in murine L2 fibroblasts infected with an MOI of 10:1. Plaques were counted 3 days postinfection, and the number of plaques (± the standard error of the mean) were plotted. Data shown are representative of three independent experiments. Statistical significance is indicated with letters b or c comparing WT versus the indicated mutant strains (Mann-Whitney two-tailed nonparametric test for comparison): a, nonsignificantly different from WT; b, P < 0.0001; c, P < 0.005.

FIG 5

CtpP1 and CtpP2 contribute to virulence in vivo. (A) Eight- to 10-week-old female Swiss Webster mice were intravenously infected with 2 × 10⁴ CFU of the wild type, ΔctaP::erm, or permease mutant strains. Target organs were harvested 72 h postinfection for the determination of bacterial burdens. (B) Intragastric inoculation of 8- to 10-week-old C57BL/6 mice with 1 × 10⁸ CFU of either L. monocytogenes inlA^m, inlA^m ΔctaP::erm, or inlA^m containing the various permease deletions. Statistical significance is indicated for WT versus indicated mutant strain comparisons, using Mann-Whitney two-tailed nonparametric test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.