ABSTRACT
Lysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity.
IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes. However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only affects lysozyme resistance, but also endogenous cell lysis, cell wall biosynthesis, cell division and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is by a yet unknown mechanism an important determinant for cell wall integrity in L. monocytogenes.
INTRODUCTION
Gram-positive bacteria are surrounded by a complex cell wall, which is composed of a thick layer of peptidoglycan (PG) and cell wall polymers. The bacterial cell wall is important for the maintenance of cell shape, the ability of bacteria to withstand harsh environmental conditions and to prevent cell lysis (1, 2). Due to its importance, cell wall-targeting antibiotics such as β-lactam, glycopeptide and fosfomycin antibiotics are commonly used to treat bacterial infections (3, 4). These cell-wall targeting antibiotics inhibit enzymes involved in different stages of the PG biosynthesis process or sequester substrates of these enzymes (4). Moenomycin, another cell wall-targeting antibiotic, and β-lactam antibiotics, for instance, block the glycosyltransferase and transpeptidase activity of penicillin binding proteins, respectively, which are required for the polymerization and crosslinking of the glycan strands (5–7). Peptidoglycan is also the target of the cell wall hydrolase lysozyme, which is a component of animal and human secretions such as tears and mucus. Lysozyme cleaves the glycan strands of PG by hydrolysing the 1,4-β-linkage between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). This reaction leads to a loss of cell integrity and results in cell lysis (8). The intracellular human pathogen Listeria monocytogenes is intrinsically resistant to lysozyme due to modifications of its PG. The N-deacetylase PgdA deacetylates GlcNAc residues, whereas MurNAc residues are acetylated by the O-acetyltransferase OatA (9, 10). Consequently, deletion of either of these enzymes results in reduced lysozyme resistance (9, 10). One or both of these enzymes are also present in other bacterial pathogens and important for lysozyme resistance, such as PgdA in Streptococcus pneumoniae, OatA in Staphylococcus aureus and PgdA and OatA in Enterococcus faecalis (11–14). Besides enzymes that directly alter the peptidoglycan structure, a number of other factors have been shown to contribute to lysozyme resistance in diverse bacteria. For instance, the cell wall polymer wall teichoic acid and the two-component system GraRS contribute to lysozyme resistance in S. aureus (15, 16). In E. faecalis, the extracytoplasmic function sigma factor SigV is required for the upregulation of pgdA expression in the presence of lysozyme (11, 17). Recently, some additional factors have been identified, which contribute to the intrinsic lysozyme resistance of L. monocytogenes such as the predicted carboxypeptidase PbpX, the transcription factor DegU and the noncoding RNA Rli31 (18). DegU and Rli31 are involved in the regulation of pgdA and pbpX expression in L. monocytogenes (18). Furthermore, components of a predicted ABC transporter encoded by the lmo2769-6 operon in L. monocytogenes and here referred to as eslABCR for elongation, sugar& and lysozyme sensitive phenotype (Fig. 1) have been associated with lysozyme resistance (18–20). An eslB transposon insertion mutant was also shown to be more sensitive to cefuroxime and cationic antimicrobial peptides (18).
ABC transporters can either act as importers or exporters. Importers are involved in the uptake of sugars, peptides or other metabolites, which are recognized by substrate binding proteins. On the other hand, toxic compounds such as antibiotics can be exported by ABC exporters (21–23). They are usually composed of homo& or heterodimeric cytoplasmic nucleotide-binding domain (NBD) proteins, also referred to as ATP-binding cassette proteins, and homo& or heterodimeric transmembrane domain (TMD) proteins (24). In addition to NBDs and TMDs, ABC importers have an extracellular substrate binding protein (SBP) or a membrane-integrated S-component, which are important for the delivery of specific substrate molecules to the transporter or substrate binding, respectively (25–27). The esl operon encodes EslA, the NBD protein, EslB, the TMD protein forming part of the ABC transporter, EslC, a membrane protein of unknown function and EslR, a RpiR-type transcriptional regulator (Fig. 1). So far, it has not been investigated whether EslC is a component of the ABC transporter encoded in the esl operon. EslB and EslC could for instance interact with each other and form the transmembrane domain of the ABC transporter, or EslC could function independent from EslAB. Furthermore, it is not known whether the predicted ABC transporter EslABC acts as an importer or exporter and its exact cellular function has not been identified. Here, we show that the absence of EslB, one of the transmembrane components of the ABC transporter, leads to an increased lysozyme sensitivity due to an altered PG structure. In addition, deletion of eslB resulted in the production of a thinner cell wall, and thus to an increased endogenous cell lysis. Furthermore, cell division is perturbed in the absence of EslB. We hypothesize that EslB may be required for processes, which are important for the maintenance of the cell wall integrity of L. monocytogenes during stress conditions.
MATERIALS AND METHODS
Bacterial strains and growth conditions
All strains and plasmids used in this study are listed in Table S1. Escherichia coli strains were grown in Luria-Bertani (LB) medium and Listeria monocytogenes strains in brain heart infusion (BHI) medium at 37°C unless otherwise stated. If necessary, antibiotics and supplements were added to the medium at the following concentrations: for E. coli cultures, ampicillin (Amp) at 100 μg/ml, chloramphenicol (Cam) at 20 μg/ml and kanamycin (Kan) at 30 μg/ml, and for L. monocytogenes cultures, Cam at 10 μg/ml, erythromycin (Erm) at 5 μg/ml, Kan at 30 μg/ml, nalidixic acid (Nal) at 20 μg/ml, streptomycin (Strep) at 200 μg/ml and IPTG at 1 mM.
Strain and plasmid construction
All primers used in this study are listed in Table S2. For the markerless in-frame deletion of lmo2768 (lmrg_01927, eslB), approximately 1kb-DNA fragments up& and downstream of the eslB gene were amplified by PCR using primer pairs ANG2532/2533 and ANG2534/2535. The resulting PCR products were fused in a second PCR using primers ANG2532/2535, the product cut with BamHI and XbaI and ligated with plasmid pKSV7 that had been cut with the same enzymes. The resulting plasmid pKSV7-ΔeslB was recovered in E. coli XL1-Blue yielding strain ANG4236. The plasmid was subsequently transformed into L. monocytogenes strain 10403S and eslB deleted by allelic exchange using a previously described procedure (28). The deletion of eslB was verified by PCR. The deletion procedure was performed with two independent transformants and resulted in the construction of two independent eslB mutant strains 10403SΔeslB(1) (ANG4275) and 10403SΔeslB(2) (ANG5662). For complementation analysis, pIMK3-eslB was constructed, in which the expression of eslB can be induced by IPTG. The eslB gene was amplified using primer pair ANG2812/ANG2813, the product cut with NcoI and SalI and fused with pIMK3 that had been cut with the same enzymes. The resulting plasmid pIMK3-eslB was recovered in E. coli XL1-Blue yielding strain ANG4647. Due to difficulties in preparing electrocompetent cells of L. monocytogenes eslB mutant strains, plasmid pIMK3-eslB was first electroporated into the wildtype L. monocytogenes strain 10403S yielding strain 10403S pIMK3-eslB (ANG4678). In the second step, eslB was deleted from the genome of strain ANG4678 resulting in the construction of the first eslB complementation strain 10403SΔeslB(1) pIMK3-eslB (ANG4688, short 10403SΔeslB(1) compl.). In addition, complementation plasmid pPL3e-PeslA-eslABC was constructed. To this end, the eslABC genes including the upstream promoter region were amplified by PCR using primers ANG3349/ANG3350. The resulting PCR product was cut with SalI and BamHI and fused with plasmid pPL3e that had been cut with the same enzymes. Plasmid pPL3e-PeslA-eslABC was recovered in E. coli XL1-Blue yielding strain ANG5660. Next, plasmid pPL3e-PeslA-eslABC was transformed into E. coli SM10 yielding strain ANG5661. Lastly, SM10 pPL3e-PeslA-eslABC was used as a donor strain to transfer plasmid pPL3e-PeslA-eslABC by conjugation into L. monocytogenes strain 10403SΔeslB(2) (ANG5662) using a previously described method (29). This resulted in the construction of the second eslB complementation strain 10403SΔeslB(2) pPL3e-PeslA-eslABC (ANG5663, short 10403SΔeslB(2) compl.). For the markerless in-frame deletion of lmo2769 (lmrg_01926, eslA), and lmo2767 (lmrg_01928, eslC), approximately 1kb-DNA fragments up& and downstream of the corresponding gene were amplified by PCR using primer pairs LMS160/161 and LMS159/162 (eslA), and LMS155/158 and LMS156/157 (eslC). The resulting PCR products were fused in a second PCR using primers LMS159/160 (eslA) and LMS155/156 (eslC). The products were cut with BamHI and EcoRI (eslA) and BamHI and KpnI (eslC) and ligated with plasmid pKSV7 that had been cut with the same enzymes. The resulting plasmids pKSV7-ΔeslA and pKSV7-ΔeslC were recovered in E. coli XL1-Blue yielding strains EJR54 and EJR43, respectively. The plasmids were subsequently transformed into L. monocytogenes strain 10403S and eslA and eslC deleted by allelic exchange yielding strains 10403SΔeslA (LJR33) and 10403SΔeslC (LJR7). Plasmid pPL3e-PeslA-eslABC was transferred into LJR33 and LJR7 via conjugation using strain SM10 pPL3e-PeslA-eslABC (ANG5661) as a donor strain, yielding strains 10403SΔeslA pPL3e-PeslA-eslABC (LJR34, short 10403SΔeslA compl.) and 10403SΔeslC pPL3e-PeslA-eslABC (LJR21, short 10403SΔeslC compl.).
For the construction of bacterial two hybrid plasmids, eslA, eslB and eslC were amplified by PCR using primer pairs JR44/45, JR46/47 and JR48/49, respectively. The resulting eslA and eslC fragments were cut with XbaI and KpnI and ligated into pKT25, pKNT25, pUT18 and pUT18C that had been cut with the same enzymes. The eslB fragment was cut with XbaI and BamHI and ligated into XbaI/BamHI cut pKT25, pKNT25, pUT18 and pUT18C. The resulting plasmids were recovered in E. coli XL1-Blue yielding strains XL1-Blue pKNT25-eslA (EJR4), XL1-Blue pKT25-eslA (EJR5), XL1-Blue pUT18-eslA (EJR6), XL1-Blue pUT18C-eslA (EJR7), XL1-Blue pKNT25-eslB (EJR8), XL1-Blue pKT25-eslB (EJR9), XL1-Blue pUT18-eslB (EJR10), XL1-Blue pUT18C-eslB (EJR11), XL1-Blue pKNT25-eslC (EJR12), XL1-Blue pKT25-eslC (EJR13) and XL1-Blue pUT18C-eslC (EJR15). Using this approach, we were unable to construct pUT18-eslC without acquiring mutations in eslC. In a second attempt to generate pUT18-eslC, plasmid pKT25-eslC (from strain EJR13) was cut with XbaI and KpnI, the eslC fragment extracted and ligated into XbaI/KpnI cut pUT18. The resulting plasmid was recovered in E. coli CLG190 yielding strain CLG190 pUT18-eslC (EJR14).
For the localization of an early cell division protein, the N-terminus of ZapA was fused to mNeonGreen. For this purpose, mNeonGreen and zapA genes were amplified using primer pairs JR73/JR39 and JR40/JR74, respectively. The resulting PCR products were fused in a second PCR using primers JR73/JR74, the product was cut with NcoI and SalI and ligated with pIMK2 that had been cut with the same enzymes. pIMK2-mNeonGreen-zapA was recovered in E. coli XL1-Blue and transformed into E. coli S17-1 yielding strains EJR39 and EJR60, respectively. S17-1 pIMK2-mNeonGreen-zapA was used as a donor strain to transfer the plasmid pIMK2-mNeonGreen-zapA by conjugation into L. monocytogenes strains 10403S (ANG1263) and 10403SΔeslB(2) (ANG5662) resulting in the construction of strains 10403S pIMK2-mNeonGreen-zapA (LJR28) and 10403SΔeslB(2) pIMK2-mNeonGreen-zapA (LJR29).
Bacterial two-hybrid assays
Interactions between EslA, EslB and EslC were analyzed using bacterial adenylate cyclase two-hybrid (BACTH) assays (30). For this purpose, 15 ng of the indicated pKT25/pKNT25 and pUT18/pUT18C derivatives were co-transformed into E. coli strain BTH101. Transformants were spotted on LB agar plates containing 25 μg/ml kanamycin, 100 μg/ml ampicillin, 0.5 mM IPTG and 80 μg/ml X-Gal and the plates incubated at 30°C. Images were taken after an incubation of 48 h.
Whole genome sequencing
Genomic DNA of L. monocytogenes was extracted using the FastDNA™ Kit (MP Biomedicals) and libraries for sequencing were prepared using the Illumina Nextera DNA kit. The samples were sequenced at the London Institute of Medical Sciences using an Illumina MiSeq instrument and a 150 paired end Illumina kit. The reads were trimmed, mapped to the L. monocytogenes 10403S reference genome (NC_017544) and single nucleotide polymorphisms (SNPs) with a frequency of at least 80% and small deletions (zero coverage) identified using the CLC workbench genomics (Qiagen).
Growth analysis
L. monocytogenes strains were grown overnight in 5 ml BHI medium at 37°C with shaking. The next day, these cultures were used to inoculate 15 ml fresh BHI medium or BHI medium containing 0.5 M sucrose, fructose, glucose, maltose, galactose or sodium chloride to an OD600 of 0.05. The cultures were incubated at 37°C with shaking at 180 rpm, OD600 readings were taken every hour for 8 h.
Determination of minimal inhibitory concentration (MIC)
The minimal inhibitory concentration for the cell wall-acting antibiotics penicillin and moenomycin and the cell wall hydrolase lysozyme was determined in 96-well plates using a microbroth dilution assay. Approximately 104 L. monocytogenes cells were used to inoculate 200 μl BHI containing two-fold dilutions of the different antimicrobials. The starting antibiotic concentrations were: 0.025 μg/ml for penicillin G, 0.2 μg/ml for moenomycin and 10 mg/ml or 0.25 mg/ml for lysozyme. The 96-well plates were incubated at 37°C with shaking at 500 rpm in a plate incubator (Thermostar, BMG Labtech) and OD600 determined after 24 hours of incubation. The MIC value refers to the antibiotic concentration at which bacterial growth was inhibited by >90%.
Plate spotting assay
Overnight cultures of the indicated L. monocytogenes strains were adjusted to an OD600 of 1 and serially diluted to 10−6. 5 μl of each dilution were spotted on BHI agar plates or BHI agar plates containing 100 μg/ml lysozyme, both containing 1 mM IPTG. Images of the plates were taken after incubating them for 20-24 h at 37°C.
Peptidoglycan isolation and analysis
Overnight cultures of 10403SΔeslB(1) and 10403SΔeslB(1) compl. were diluted in 1 L BHI broth (supplemented with 1 mM IPTG for strain 10403SΔeslB(1) compl.) to an OD600 of 0.06 and incubated at 37°C. At an OD600 of 1, bacterial cultures were cooled on ice for 1h and the bacteria subsequently collected by centrifugation. The peptidoglycan was purified, digested with mutanolysin and the muropeptides analyzed by HPLC using an Agilent 1260 infinity system, as previously described (31, 32). Peptidoglycan of the wildtype L. monocytogenes strain 10403S was purified and analyzed in parallel. The chromatogram of the same wild-type control strain was recently published (33) and also used as part of this study, since all strains were analyzed at the same time. The major peaks 1-6 were assigned according to previously published HPLC spectra (18, 34), with peaks 2, 4, 5 and 6 corresponding to N-deacetylated GlcNAc residues. Peaks 1-2 correspond to monomeric and peaks 4-6 to dimeric (crosslinked) muropeptide fragments. The Agilent Technology ChemStation software was used to integrate the areas of the main muropeptide. For quantification, the sum of the peak areas was set to 100% and the area of individual peaks was determined. The sum of values for peaks 3-6 corresponds to the % crosslinking, whereas the deacetylation state was calculated by adding up the values for peaks 4, 5 and 6. Averages values and standard deviations were calculated from three independent extractions.
O-acetylation assay
Peptidoglycan of strains 10403S, 10403SΔeslB(1) and 10403SΔeslB(1) compl., which had not been treated with hydrofluoric acid and alkaline phosphatase to avoid removal of the O-acetyl groups, was used for the O-acetylation assays. O-acetylation was measured colorimetrically according to the Hestrin method described previously (35) with slight modifications. Briefly, 800 μg of PG (dissolved in 500 μl H2O) were incubated with an equal volume of 0.035 M hydroxylamine chloride in 0.75 M NaOH for 10 min at 25°C. Next, 500 μl of 0.6 M of perchloric acid and 500 μl of 70 mM ferric perchlorate in 0.5 M perchloric acid were added. The color change resulting from the presence of O-acetyl groups was quantified at 500 nm. An assay reaction with 500 μl H2O was used as a blank for the absorbance measurement.
Autolysis assays
L. monocytogenes strains were diluted in BHI or BHI medium supplemented with 0.5 M sucrose to an OD600 of 0.05 and grown for 4 h at 37°C. Cells were collected by centrifugation and resuspended in 50 mM Tris-HCl, pH 8 to an OD600 of 0.7-0.9 and incubated at 37°C. For penicillin& and lysozyme-induced lysis, 25 μg/ml penicillin G or 2.5 μg/ml lysozyme was added to the cultures. Autolysis was followed by determining OD600 readings every 15 min.
Fluorescence and phase contrast microscopy
Overnight cultures of the indicated L. monocytogenes strains were diluted 1:100 in BHI medium and grown for 3 h at 37°C. For staining of the bacterial membrane, 100 μl of these cultures were mixed with 5 μl of 100 μg/ml nile red solution and incubated for 20 min at 37°C. The cells were washed twice with PBS and subsequently suspended in 50 μl of PBS. 1-1.5 μl of the different samples were subsequently spotted on microscope slides covered with a thin agarose film (1.5 % agarose in distilled water), air-dried and covered with a cover slip. Phase contrast and fluorescence images were taken at 1000x magnification using the Zeiss Axio Imager.A1 microscope coupled to an AxioCam MRm and processed using the Zen 2012 software (blue edition). The nile red fluorescence signal was detected using the Zeiss filter set 00. The length of 300 cells was measured for each experiment and the median cell length was calculated.
For ZapA-localization studies, overnight cultures of the indicated L. monocytogenes strains were grown in BHI medium at 37°C to an OD600 of 0.3-0.5. The staining of the bacterial membrane with nile red was performed as described above. After nile red staining, cells were fixed in 1.2% paraformaldehyde for 20 min at RT. 1-1.5 μl of the different samples were spotted on microscope slides as described above. Phase contrast and fluorescence images were taken at 1000x magnification using the Zeiss Axioskop 40 coupled to an AxioCam MRm and processed using the Axio Vision software (release 4.7). The nile red and mNeonGreen fluorescence signals were detected using the Zeiss filter set 43 and 37, respectively.
Transmission electron microscopy
Overnight cultures of L. monocytogenes strains 10403S, 10403SΔeslB(2) and 10403SΔeslB(2) compl. were used to inoculate 25 ml BHI broth or BHI broth supplemented with 0.5 M sucrose to an OD600 of 0.05. Bacteria were grown at 37°C and 200 rpm for 3.5 h (BHI broth) or 6 h (BHI broth containing 0.5 M sucrose). 15 ml of the cultures were centrifuged for 10 min at 4000 rpm, the cell pellet washed twice in phosphate-buffered saline (127 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and fixed overnight in 2.5 % (w/v) glutaraldehyde at 4°C. Cells were then mixed with 1.5 % (w/v, final concentration in PBS) molten Bacto-Agar, kept liquid at 55°C. After solidification, the agar block was cut into pieces with a volume of 1 mm3. A dehydration series was performed (15 % aqueous ethanol solution for 15 min, 30 %, 50 %, 70 % and 95 % for 30 min and 100 % for 2x 30 min) at 0°C, followed by an incubation step in 66 % (v/v, in ethanol) LR-white resin mixture (Plano) for 2 h at RT and embedded in 100 % LR-white solution overnight at 4°C. One agar piece was transferred to a gelatin capsule filled with fresh LR-white resin, which was subsequently polymerized at 55°C for 24 h. A milling tool (TM 60, Reichert & Jung, Vienna, Austria) was used to shape the gelatin capsule into a truncated pyramid. An ultramicrotome (Reichert Ultralcut E, Leica Microsystems, Wetzlar, Germany) and a diamond knife (Delaware Diamond Knives, Wilmington, DE, USA) were used to obtain ultrathin sections (80 nm) of the samples. The resulting sections were mounted on mesh specimen grids (Plano) and stained with 4 % (w/v) uranyl acetate solution (pH 7.0) for 10 min. Microscopy was performed using a Jeol JEM 1011 transmission electron microscope (Jeol Germany GmbH, Munich) at 80 kV. Images were taken at a magnification of 30,000 and recorded with an Orius SC1000 CCD camera (Pleasanton, CA, USA). For each replicate, 20 cells were photographed and cell wall thickness was measured at three different locations using the ImageJ software (36). The average of the three measurements was calculated and the average and standard deviation of 20 cells plotted. The experiment was performed twice.
Cell culture
Bone marrow-derived macrophages (BMMs) were extracted from female C57BL/6 mice as described previously (37). BMMs were a gift from Charlotte S. C. Michaux and Sophie Helaine. 5×105 BMMs were seeded per well of a 24-well plate and grown overnight in 500 μl high glucose Dulbecco’s Modified Eagle Medium (DMEM) at 37°C and 5% CO2. L. monocytogenes strains were grown overnight without shaking in 2 ml BHI medium at 30°C. The next morning, bacteria were opsonized with 8% mouse serum (Sigma-Aldrich) at room temperature for 20 min and BMMs were infected for one hour at a multiplicity of infection (MOI) of 2. BMMs were washed with PBS and 1 ml DMEM containing 40 μg/ml gentamycin was added to kill extracellular bacteria. After 1 h, cells were washed with PBS and covered with 1 ml DMEM containing 10 μg/ml gentamycin. The number of recovered bacteria was determined 2, 4, 6 and 8 h post infection. To this end, BMMs were lysed using 1 ml PBS containing 0.1% (v/v) triton X-100 and serial dilutions were plated on BHI agar plates. The number of colony forming units (CFUs) was determined after incubating the plates overnight at 37°C. Three technical repeats were performed for each experiment and average values calculated. Average values and standard deviations from three independent experiments were plotted.
Drosophila melanogaster infections
Fly injections were carried out with microinjection needles produced from borosilicate glass capillaries (World Precision Instruments TW100-4) and a needle puller (Model PC-10, Narishige). Injections were performed using a Picospritzer III system (Parker Hannifin), and the injection volume was calibrated by expelling a drop of liquid from the needle into a pot of mineral oil and halocarbon oil (both Sigma). The expelled drop was measured using the microscope graticule to obtain a final injection volume of 50 nanolitres (nl). Flies were then anesthetized with CO2 and injected with either 50 nl of bacterial suspension in PBS or sterile PBS. 5-7-day old age matched male flies were used for all experiments. Flies were grouped into uninjected control, wounding control (injection with sterile PBS), and flies infected with L. monocytogenes. Each group consisted of 58-60 flies. All survival experiments were conducted at 29°C. Dead flies were counted daily. Food vials were placed horizontally to reduce the possibility of fly death from flies getting stuck to the food, and flies were transferred to fresh food every 3-4 days. For the quantification of the bacterial load, 16 flies per condition and per bacterial strain were collected at the indicated time points. The flies were homogenised in 100 μl of TE-buffer pH 8 containing 1% Triton X-100 and 1% Proteinase K (NEB, P8107S). Homogenates were incubated for 3 h at 55°C followed by a 10 min incubation step at 95°C. Following incubation, qPCR was carried out using the actA gene specific primers EGD-E_ActA_L1 and EGD-E_ActA_R1 to determine the number of bacterial colony forming units. PCR was performed with Sensimix SYBR Green no-ROX (Bioline) on a Corbett Rotor-Gene 6000. The cycling conditions were as follows: Hold 95°C for 10 min, then 45 cycles of 95°C for 15 s, 57°C for 30 s, 72°C for 30 s, followed by a melting curve. Gene abundances were calculated as previously described (38).
RESULTS
EslC interacts with the transmembrane protein EslB
Previously it has been shown that L. monocytogenes strains with mutations in the eslABCR operon (Fig. 1A) display decreased resistance towards the cell wall hydrolase lysozyme (18, 19). The esl operon encodes the ATP binding protein EslA and the transmembrane proteins EslB and EslC, which are proposed to form an ABC transporter. However, it is currently unknown if EslC forms part of the ABC transporter as depicted in Figure 1B and if it is required for the function of the transporter. To gain insights into the composition of the ABC transporter, we assessed the interaction between EslA, EslB and EslC using the bacterial adenylate cyclase-based two-hybrid system. In addition to self-interactions of EslA, EslB and EslC, we observed an interaction between EslB and EslC (Fig. 1C), indicating that EslC might be part of the ABC transporter.
Deletion of eslB in L. monocytogenes leads to lysozyme sensitivity and an altered peptidoglycan structure
An eslA in-frame deletion mutant and an eslB transposon insertion mutant were shown to be more sensitive to lysozyme compared to the wildtype strain (18, 19). However, it is still unknown how the function of an ABC transporter is linked to this phenotype. To investigate this further, strains with markerless in-frame deletions in eslA, eslB and eslC were constructed in the L. monocytogenes strain background 10403S. First, the lysozyme resistance of these mutants was assessed using a plate spotting assay. The eslA and eslB mutants showed reduced growth on BHI plates containing 100 μg/ml lysozyme compared to the wildtype and eslA and eslB complementation strains (Fig. 2A). On the other hand, no phenotype was observed for the eslC mutant (Fig. 2A). Since deletion of eslA and eslB resulted in a decreased lysozyme resistance, and an eslA mutant has already been characterized in previous work (19), we focused here on the characterization of the eslB deletion strain.
In the course of the study, we determined the genome sequence of the originally constructed eslB mutant (10403SΔeslB(1)) by whole genome sequencing (WGS) and identified an additional small deletion in gene lmo2396 coding for an internalin protein with a leucine-rich repeat (LRR) and a mucin-binding domain (Table S3). While to the best of our knowledge, the contribution of Lmo2396 to the growth and pathogenicity of L. monocytogenes has not yet been investigated, other internalins are important and well-established virulence factors (39, 40). Our WGS analysis also revealed a single point mutation in gene lmo2342, coding for a pseudouridylate synthase in the complementation strain 10403SΔeslB(1) compl. (Table S3). Since we identified an additional mutation in a gene coding for a potential virulence factor in the eslB mutant, we constructed a second independent eslB mutant, 10403SΔeslB(2). We also constructed a second complementation strain, strain 10403SΔeslB(2) PeslA-eslABC (or short 10403SΔeslB(2) compl.), in which the eslABC genes are expressed from the native eslA promoter from a chromosomally integrated plasmid. Our WGS analysis revealed that strain 10403SΔeslB(2) did not contain any secondary mutations (Table S3). A 1-bp deletion in gene lmo2022 encoding a predicted NifS-like protein required for NAD biosynthesis, was identified in strain 10403SΔeslB(2) compl. (Table S3), which if non-complementable phenotypes are observed needs to be kept in mind. We confirmed that our second eslB mutant strain 10403SΔeslB(2) showed the same lysozyme sensitivity phenotype and that this phenotype could be complemented in strain 10403SΔeslB(2) compl., in which eslB is expressed along with eslA and eslC from its native promoter (Fig. 2A). Since we only identified the genomic alterations in the course of the study, some experiments were performed as stated in the text with the original eslB mutant and complementation strains 10403SΔeslB(1) and 10403SΔeslB(1) compl., while other experiments were conducted with strains 10403SΔeslB(2) and 10403SΔeslB(2) compl.
Using microbroth dilution assays, we observed a 40-fold lower MIC for lysozyme for L. monocytogenes strain 10403SΔeslB(1) as compared to the wildtype strain (Fig. 2B and S1A) (18, 19). This phenotype could be complemented and strain 10403SΔeslB(1) compl., in which eslB is expressed from an IPTG-inducible promoter, is even slightly more resistant to lysozyme as compared to the wildtype strain (Fig. 2B). Next, we tested whether the resistance towards two cell wall-targeting antibiotics, namely penicillin and moenomycin, is changed upon deletion of eslB. The MIC values obtained for the wildtype, eslB deletion and eslB complementation strains were comparable (Fig. 2C-D), suggesting that the deletion of eslB does not lead to a general sensitivity to all cell wall-acting antimicrobials but is specific to lysozyme. In L. monocytogenes, lysozyme resistance is achieved by the modification of the peptidoglycan (PG) by N-deacetylation via PgdA and O-acetylation via OatA (9, 10). To assess whether deletion of eslB affects the N-deacetylation and crosslinking of PG, PG was isolated from wildtype 10403S, the eslB deletion and complementation strains, digested with mutanolysin and the muropeptides analyzed by high performance liquid chromatography (HPLC). This analysis revealed a slight increase in PG crosslinking in the eslB mutant strain (68±0.53%) compared to the wildtype (65.47±0.31%) and the complementation strain grown in the presence of IPTG (64.57±2.3%) (Fig. 3A-B). The GlcNAc residues of the PG isolated from the eslB deletion strain were also slightly more deacetylated (71.54±0.21%) as compared to the wildtype (67.17±0.31%) and the complementation strain (67±2.27%) (Fig. 3A-B), which should theoretically result in an increase and not decrease in lysozyme resistance. However, when we assessed the degree of O-acetylation using a colorimetric assay, the PG isolated from the eslB mutant was less O-acetylated compared to the wildtype and the complementation strain (Fig. 3C). Taken together, our results suggest that slight changes in the PG structure and in particular the observed reduction in O-acetylation likely contribute to the lysozyme sensitivity of the eslB deletion strain.
Deletion of eslB results in a growth defect in high sugar media
The bacterial cell wall is an important structure to maintain the cell integrity and to prevent lysis due to high internal turgor pressure or when bacteria experience changes in the external osmolality. Alterations of the PG structure or other cell wall defects leading to an impaired cell wall integrity could affect the growth of bacteria in environments with high osmolalities, e.g. in the presence of high salt or sugar concentrations. Next, we compared the growth of the wildtype, the eslB mutant and complementation strains at 37°C in different media. No growth difference could be observed between the strains tested, when grown in BHI medium (Fig. 4A and S1B). However, the eslB deletion strain grew slower in BHI medium containing 0.5 M sucrose as compared to the wildtype and the eslB complementation strain (Fig. 4B and S1C). A similar growth phenotype could be observed when the strains were grown in BHI medium containing either 0.5 M fructose, glucose, maltose or galactose (Fig. S2). In contrast, the presence of 0.5 M NaCl did not affect the growth of the eslB deletion strain (Fig. 4C). These results suggest that the observed growth defect seen for the eslB mutant is not solely caused by the increase in external osmolality, but rather seems to be specific to the presence of high concentrations of sugars.
Deletion of eslB results in increased endogenous and lysozyme-induced lysis
The observed lysozyme sensitivity and the growth defect of the eslB deletion strain in media containing high concentrations of sucrose raised the question, whether the absence of EslB might also cause an impaired cell wall integrity and an increased autolysis due to this impairment. To test this, autolysis assays were performed. To this end, the L. monocytogenes wildtype strain 10403S, the eslB deletion and complementation strains were grown in BHI medium and subsequently transferred in a Tris-HCl buffer (pH 8). After 2 h incubation at 37°C, the OD600 of the suspensions of the wildtype and eslB complementation strain had dropped to 89.9±1.6% and 86.5±2.9% of the initial OD600, respectively (Fig. 5A). Enhanced endogenous cell lysis was observed for the eslB mutant strain and the OD600 of the suspensions dropped to 68.8±1.7% within 2 h (Fig. 5A). The addition of penicillin had no impact on the cell lysis of any of the strains tested (Fig. 5B). On the other hand, the addition of 2.5 μg/ml lysozyme increased the rate of cell lysis of all strains, but had a particularly drastic effect on the eslB mutant. After 30 min, the OD600 reading of a suspension of the eslB deletion strain had dropped to 50.3±10.2%. For the wildtype and eslB complementation strains, it took 90 min to see a 50% reduction in the OD600 readings (Fig. 5C).
Next, we wanted to determine what impact the growth in the presence of high levels of sucrose has on endogenous bacterial autolysis rates. To this end, the wildtype 10403S, eslB mutant and complementation strains were grown in BHI medium supplemented with 0.5 M sucrose, cell suspensions prepared in Tris-buffer and used in autolysis assays. While the wildtype and complementation strain showed similar autolysis rates following growth in BHI sucrose medium (Fig. 5D) as after growth in BHI medium (Fig 5A), the eslB mutant lysed rapidly following growth in BHI 0.5 M sucrose medium (Fig. 5E). The lysis of the eslB mutant strain could be further enhanced by the addition of 25 μg/ml penicillin, a concentration which only acts bacteriostatic on the wildtype L. monocytogenes strain 10403S (Fig. 5E). These findings indicate that the eslB mutant is sensitive to osmotic downshifts and we thus wondered whether in addition to the changes in the PG modifications and crosslinking, more general differences in the ultrastructure of the cell wall might be observed. To test this, cells of L. monocytogenes strains 10403S, 10403SΔeslB(2) and 10403SΔeslB(2) compl. were subjected to transmission electron microscopy. The eslB deletion strain produces a thinner PG layer of 15.8±1.9 nm, when grown in BHI broth as compared to the wildtype (20±3.4 nm) and the complementation strain (20±4.3 nm, Fig. 6A-B). This phenotype was even more pronounced when the strains were grown in BHI broth containing 0.5 M sucrose. The PG layer of the eslB mutant had a thickness of 15±2 nm, while wildtype and the complementation strain produced a PG layer of 21.4±3.1 and 23.3±2.8 nm, respectively (Fig. 6A-B). We hypothesize that the enhanced endogenous lysis of the eslB mutant is likely caused by a thinner PG layer combined with the observed alterations in PG structure such as reduced O-acetylation.
The eslB deletion strain is impaired in cell division, but not in virulence
The increased endogenous autolysis together with the observed changes in the PG structure of the eslB deletion strain could result in an increased sensitivity to autolysins. The major autolysins of L. monocytogenes are p60 and NamA, which hydrolyze PG and are required for daughter cell separation during cell division (41, 42). Absence of either p60 or NamA results in the formation of chains (41, 42). We thus wondered whether deletion of eslB causes changes in the cell morphology of L. monocytogenes. Microscopic analysis revealed that cells lacking EslB are significantly longer with a median cell length of 3.26±0.25 μm as compared to the L. monocytogenes wildtype strain, which produced cells with a length of 1.85±0.08 μm (Fig. 6C-D), highlighting that the absence of EslB results in a cell division defect. To test whether the assembly of the early divisome is affected by the absence of EslB, we compared the localization of the early cell division protein ZapA in the wildtype and the eslB mutant background. In L. monocytogenes wildtype cells, a signal was observed at midcell for cells, which have initiated the division process (Fig. 6E). While short cells of the eslB mutant also only possess a single fluorescent signal, several ZapA fluorescence foci could be observed in elongated cells (Fig. 6E), suggesting that early cell division proteins can still localize in the eslB mutant and that a process downstream seems to be perturbed in the absence of EslB.
Next, we wanted to assess whether the impaired cell integrity and the observed cell division defect would also affect the virulence of the L. monocytogenes eslB mutant. Of note, in a previous study, it was shown that deletion of eslA, coding for the ATP-binding protein component of the ABC transporter, has no effect on the cell-to-cell spread of L. monocytogenes (19). To determine whether EslB is involved in the virulence of L. monocytogenes, primary mouse macrophages were infected with wildtype 10403S, the eslB mutant 10403SΔeslB(2) and complementation strain 10403SΔeslB(2) compl.. All three strains showed a comparable intracellular growth pattern (Fig. 7A), suggesting that EslB does not impact the ability of L. monocytogenes to grow in primary mouse macrophages. Next, we assessed the ability of the eslB deletion strains to kill Drosophila melanogaster as lysozyme is one important component of its innate immune response (43). All uninfected flies (U/C) and 96.6% of the flies that were injected with PBS survived the duration of the experiment (Fig. 7B). No statistically significant difference could be observed for the survival and bacterial load of flies infected with the different L. monocytogenes strains (Fig. 7B-C). These results indicate that, while EslB does not impact the ability of L. monocytogenes to infect and kill mammalian macrophages or Drosophila melanogaster, it nonetheless impacts the cell division and cell wall integrity of L. monocytogenes and consistent with this we have identified changes in the composition and thickness of the peptidoglycan layer.
DISCUSSION
Over the past years, several determinants contributing to the intrinsic lysozyme resistance of L. monocytogenes have been described (9, 10, 18, 19). One of these is a predicted ABC transporter encoded as part of the eslABCR operon (18, 19). In this study, we aimed to further investigate the role of the ABC transporter EslABC in lysozyme resistance of L. monocytogenes. Using bacterial two hybrid assays, we could show that EslB and EslC interact with each other and hence it is tempting to speculate that the transmembrane component of the ABC transporter consists of a heterodimer of EslB and EslC. However, analysis of different deletion mutants revealed that only EslA and EslB are required for lysozyme resistance of L. monocytogenes, suggesting that EslC is not required for the function of the ABC transporter under our assay conditions. Surprisingly, we did not observe an interaction between EslA and EslB using bacterial two hybrid assays, thus, further experiments are required to determine the composition of the ABC transporter and its interaction partners.
Next, we analyzed the PG structure of the eslB deletion strain and found that the PG isolated from the eslB mutant was slightly more crosslinked and also the fraction of deacetylated GlcNAc residues was slightly increased as compared to the PG isolated from the wildtype strain 10403S. Deacetylation of GlcNAc residues in PG is achieved by the N-deacetylase PgdA and has been shown to lead to increased lysozyme resistance (9). Since we saw a slight increase in the deacetylation of GlcNAc residues in the eslB mutant strain, our results indicate that the lysozyme sensitivity phenotype of the eslB deletion strain is independent of PgdA and that this enzyme functions properly in the mutant strain. A second enzyme required for lysozyme resistance in L. monocytogenes is OatA, which transfers O-acetyl groups to MurNAc (10, 44, 45). Using a colorimetric O-acetylation assay, we were able to show that PG isolated from the eslB mutant is less O-acetylated and we assume that this reduction in O-acetylation contributes to the lysozyme sensitivity of strain 10403SΔeslB.
Growth comparisons in different media revealed that the absence of EslB results in a reduced growth in BHI broth containing high concentrations of mono- or disaccharides. One could speculate that the EslABC transporter might be a sugar transporter with a broad sugar spectrum. However, we could not identify a potential substrate binding protein encoded in the esl operon, which is important for substrate recognition and delivery to ABC importers. EslABC could also be involved in the export of PG components and thus affecting cell wall biosynthesis in L. monocytogenes. Indeed, we could show that the eslB mutant produces a thinner PG layer as compared to the wildtype strain, suggesting that EslABC affects PG biosynthesis. Future studies will aim to determine how the ABC transporter EslABC influences the biosynthesis and subsequent modification of PG in L. monocytogenes.
Absence of EslB leads to the formation of elongated cells, however, it is currently not clear how the function of EslABC is linked to cell division of L. monocytogenes. It seems unlikely that the activity or levels of the autolysins p60 and NamA are affected by the absence of EslB. While iap and namA mutants also form chains of cells, the cell length of individual cells is still similar to wild-type cells, however the bacteria are just unable to separate (41, 42, 46). This is in contrast to the eslB mutant, in which the cell length of individual cells is increased suggesting that cell division is blocked at an earlier step. In elongated cells of the eslB mutant, we could observe several ZapA foci, suggesting that really early cell division proteins can still be recruited in this strain. Thus, a process downstream of ZapA localization but before the construction of the actual cell septum is perturbed in the absence of EslB. EslABC could potentially affect the activity of cell division proteins or the localization of late cell division-specific proteins. Hence, deletion of eslB could lead to a delayed assembly of an active divisome, which could lead to an altered PG biosynthesis at the division site and an impaired cell integrity. Indeed, cells of the eslB mutant lysed more rapidly as compared to the L. monocytogenes wildtype strain 10403S when shifted from BHI broth to Tris-buffer. The autolysis of cells lacking EslB was strongly induced following growth in BHI supplemented with 0.5 M sucrose prior to the incubation in Tris-buffer. These results indicate that the eslB mutant is sensitive to an osmotic downshift and we hypothesize that this is due to the production of a thinner PG layer and a resulting impaired cell integrity.
Reduced lysozyme resistance is often associated with reduced virulence. An E. faecalis strain with a deletion in the gene coding for the peptidoglycan deacetylase PgdA, showed a reduced ability to kill Galleria mellonella (11). Similarly, a S. pneumoniae pgdA mutant showed a decreased virulence in a mouse model of infection (13). In our study, we found that inactivation of EslB does not affect the intracellular growth of L. monocytogenes in primary mouse macrophages or the ability to kill Drosophila melanogaster. These observations are consistent with a previous report that another component of the EslABC transporter, EslA, is dispensable for the ability of L. monocytogenes to spread from cell to cell (19). Previously, it was also shown that combined inactivation of PgdA and OatA reduced the ability of L. monocytogenes to grow in bone-marrow derived macrophages, whereas inactivation of PgdA alone had no impact on the virulence of L. monocytogenes (44). We therefore reason that the changes in PG structure and associated reduction in lysozyme resistance caused by deletion of eslB are not sufficient to affect the ability of L. monocytogenes to grow and survive in primary macrophages and flies.
Taken together, we could show that EslB is not only important for the resistance towards lysozyme, its absence also affects the autolysis, cell division and the ability of L. monocytogenes to grow in media containing high concentrations of sugars. Our results indicate that the ABC transporter EslABC has a direct or indirect impact on peptidoglycan biosynthesis and maintenance of cell integrity in L. monocytogenes.
DATA AVAILABILITY
The Illumina reads for the L. monocytogenes strains 10403SΔeslB(1), 10403SΔeslB(2), 10403SΔeslB(1) compl. and 10403SΔeslB(2) compl. were deposited in the European Nucleotide Archive under the accession number PRJEB40123.
ACKNOWLEDGEMENTS
We thank Ivan Andrew and Jaspreet Haywood from the CSC Genomics Laboratory, Hammersmith Hospital, for their help with the whole genome sequencing and Annika Gillis for help with the genome sequence analysis. We would also like to thank Charlotte S. C. Michaux and Sophie Helaine for the bone marrow-derived macrophages and Neil Singh for the support during the transmission electron microscopy experiments. We are grateful to Prof. Jörg Stülke for providing JR and LMS with laboratory space, equipment and consumables. This work was funded by the Wellcome Trust grant 210671/Z/18/Z and MRC grant MR/P011071/1 to AG, the German research foundation (DFG) grants RI 2920/1-1 and RI 2920/2-1 to JR, and the Wellcome Trust grant 207467/Z/17/Z and MRC grant MR/R00997X/1 to MSD. LMS was supported by the Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences (GGNB, DFG grant GSC226/4).