Listeria monocytogenes CtaP is a multifunctional cysteine transport-associated protein required for bacterial pathogenesis

Abstract

The bacterial pathogen Listeria monocytogenes survives under a myriad of conditions in the outside environment and within the human host where infections can result in severe disease. Bacterial life within the host requires the expression of genes with roles in nutrient acquisition as well as the biosynthesis of bacterial products required to support intracellular growth. A gene product identified as the substrate-binding component of a novel oligopeptide transport system (encoded by lmo0135) was recently shown to be required for L. monocytogenes virulence. Here we demonstrate that lmo0135 encodes a multifunctional protein that is associated with cysteine transport, acid resistance, bacterial membrane integrity and adherence to host cells. The lmo0135 gene product (designated CtaP, for cysteine transport associated protein) was required for bacterial growth in the presence of low concentrations of cysteine in vitro, but was not required for bacterial replication within the host cytosol. Loss of CtaP increased membrane permeability and acid sensitivity, and reduced bacterial adherence to host cells. ctaP deletion mutants were severely attenuated following intragastric and intravenous inoculation of mice. Taken together, the data presented indicate that CtaP contributes to multiple facets of L. monocytogenes physiology, growth and survival both inside and outside of animal cells.

Fig. 1

Organization of lmo0135 region and construction of lmo0135 loss of function mutants and complement. (A) Schematic of the lmo0135 coding region and surrounding genes. The black arrow denotes the open reading frame of lmo0135, while the gray arrows indicate the flanking genes. Predicted transcriptional terminators are depicted by stem loops. The solid broken line depicts the Δlmo0135::erm deletion, and the region used for complementation is shown by the solid black line. lmo0135 gene encodes a cysteine transport associated protein, hence the ctaP designation. (B) Predicted topology of Lmo0135 (CtaP) using TMpred program for the ExPASY Proteomics website (http://www.expasy.org/tools). Numbers denote amino acid number. The gray oval represents a putative transmembrane domain. The black arrow denotes the location of a putative lipoprotein membrane attachment site with the amino acid highlighted in bold to indicate the predicted cysteine residue involved in anchoring the protein to the membrane characteristic of known lipoproteins.

Fig. 2

Lmo0135 is required for growth in broth culture. Growth of the Δlmo0135::erm mutant (■) was compared to wild type (●) and the complemented pPL2::0135 (△) strains in the rich media (A) BHI, (B) LB, or minimal media (C) HTM. Overnight cultures of each strain were grown in BHI or LB, normalized to OD_600, and diluted 1:20 in fresh media. Absorbance was measured at the indicated time points. For measurement of bacterial growth in HTM, 1 ml of each overnight culture grown in BHI was normalized to OD_600, washed in PBS and resuspended in 1 ml HTM prior to the 1:20 dilution used for the growth analyses. Data shown is representative of three separate experiments.

Fig. 3

Addition of tryptone or high levels of free cysteine can rescue the growth defect of the Δlmo0135::erm (Δctap::erm) mutant in minimal media. A) Growth of the Δlmo0135::erm (Δctap::erm) mutant (squares) was compared to wild type 10403S (circles) in HTM media +/− 10 g/L of Bacto-tryptone. Closed symbols indicate media without added tryptone, and open symbols indicate media with added tryptone. Measurement of growth of wild-type (●) and Δlmo0135::erm (Δctap::erm) mutant (■) in HTM media supplemented with either (B) 1.0 mg/ml cysteine (Cys), or (C) 1.0 mg/ml methionine (Met). One ml of an overnight culture grown in BHI was normalized to OD_600, washed in PBS and resuspended in 1 ml HTM prior to the 1:20 dilution used for the growth analyses. Similar results were obtained using 0.5 mg/ml free cysteine or methione (data not shown). Data shown is representative of three separate experiments.

Fig. 4

The ΔctaP::erm mutant is not deficient for growth inside macrophages. Murine J774 macrophage-like cells were infected with wild type (●) or an ΔctaP::erm mutant (■) at an MOI of 0.1:1. Thirty minutes post-infection, J774 infected monolayers were washed and 5 μg/ml gentamicin was added to kill extracellular bacteria. At indicated time points, coverslips containing infected J774 monolayers were removed and cells were lysed to release intracellular bacteria for enumeration of colony forming units. Data shown is representative of two independent experiments done in duplicate.

Fig. 5

CtaP aids in adhesion and/or invasion in non-professional phagocytic cells. (A) Measurement of intracellular growth and cell-to-cell spread in murine L2 fibroblasts. Monolayers of L2 fibroblasts were infected with either wild type, the ΔctaP::erm mutant or complemented strain at an MOI of 10:1. One hour post-infection, monolayers were washed in PBS and 10 μg/ml of gentamicin was added. Plaques were visualized 3 days post-infection. Numbers of plaques (+/− SEM) are indicated below the figure. Data shown is representative of three independent experiments done in duplicate. (B) Bacterial intracellular growth and (C) adhesion in PtK2 epithelial cells. Monolayers of PtK2 cells were infected with either wild type (●) or the ΔctaP::erm mutant (■) at an MOI of 100:1. One hour post-infection, monolayers were washed in PBS and 10 μg/ml of gentamicin was added. At indicated time points, coverslips containing infected PtK2 monolayers were removed and cells were lysed to release intracellular bacteria for enumeration of colony forming units. Data shown is representative of three independent experiments done in duplicate.

Fig. 6

CtaP functions as an adhesin. His-purified CtaP and BSA were covalently coupled to red fluorescent latex beads. CtaP- or BSA-coupled beads were incubated with a monolayer of PtK2 cells at a ratio of 1000:1 for 1 hour at 37°C. Cells were washed 3X in PBS and bound beads were visualized by microscopy. Fifteen different fields were viewed for each sample and the number of bound beads per field was quantified. The average number of beads per microscopic field (an area larger than the images shown) with standard deviation (SD) is indicated below panel images. Data shown is representative of two independent experiments.

Fig. 7

CtaP is required for virulence in mice. A) Swiss Webster mice were intravenously infected with 2 × 10⁴ CFUs and B) C57BL/6 mice were orally gavaged with approximately 1 × 10⁸ CFUs of wild type (●), ΔctaP::erm mutant (□) or complemented mutant strain (■). At indicated time points post-infection, specified organs were harvested and homogenized. Homogenized organs were serially diluted and plated for enumeration of bacterial CFUs. Each data point represents one mouse, and the solid lines denote the median for the data points in each group. Data was obtained from at least two independent experiments. Dashed lines indicate the lowest detection level and X's are representative of mice where the number of bacteria recovered were below the detection level. Asterisks indicate statistical significance of * p<0.05, **p<0.001, and ***p<0.0001 using a two-tailed unpaired student t-test (GraphPad V.5.0A) between the wild type and ΔctaP::erm mutant or the wild type and complemented strains. No statistical difference was observed between wild type and complemented strain in mice infected intravenously.

Fig. 8

CtaP contributes to acid resistance. Growth of the ΔctaP::erm mutant (■) was compared to wild type (●) and the complemented mutant (□) under in vitro acid stress (A) BHI pH 5.5 and salt stress(B) BHI % NaCl conditions. Overnight cultures of each strain were grown in BHI, normalized to OD_600, and diluted 1:20 in fresh media. Absorbance was measured at the indicated time points. Solid lines indicate growth measured in normal BHI broth and dashed lines denote growth measured in the same batch of BHI broth + stress conditions. Data shown is representative of three independent experiments.

Fig. 9

CtaP influences surface hydrophobicity and membrane integrity. A) Surface hydrophobicity changes were analyzed by Congo Red (CR) staining of wild type, ΔctaP::erm mutant, and complemented mutant strains. Each strain was streaked onto BHI agar plates containing 100 μg/ml of CR and plates were incubated at 37°C for 72 hours. B) Membrane integrity alterations were assessed by using the LIVE/DEAD BacLight Bacterial Viability Kit. Strains were grown to OD₆₀₀ of 0.6 and cells were stained accordingly to the manufacture's protocol before visualization by microscopy. A total of ten different fields were viewed for each strain and the number of green, red, or mixed colored bacterial cells were counted for each field. Data shown for both panels A and B are representative of three independent experiments.

Fig. 10

Model of the multifunctional CtaP protein and its contribution to L. monocytogenes pathogenesis. CtaP functions as a nutrient transporter involved in the uptake of free cysteine. The loss of CtaP-associated cysteine transport does not affect bacterial growth in the cytosol of infected cells (where available peptides presumably serve as sources of cysteine) but may limit extracellular proliferation in other body sites where peptide and cysteine concentrations are low. CtaP contributes to bacterial membrane integrity, acid resistance, and adherence to host cells. The increased sensitivity of the ΔctaP::erm mutant to acid stress, alterations in membrane integrity, and reduced adherence to host cells would serve to decrease bacterial survival during transit through the stomach and small intestine, thereby reducing the numbers of bacteria capable of crossing the intestinal epithelial cell barrier and entering the bloodstream. Loss of CtaP-associated membrane integrity may result in increased killing of L. monocytogenes by PMNs within the liver, spleen, and possibly within other infected organs and tissues.