Abstract
Rod-shaped bacteria have two modes of peptidoglycan synthesis: lateral synthesis and synthesis at the cell division site. These two processes are controlled by two macromolecular protein complexes, the Rod complex and divisome, respectively. Recently, it has been shown that the Bacillus subtilis RodA protein, which forms part of the Rod complex, has peptidoglycan glycosyltransferase activity. The cell division specific RodA homolog FtsW fulfils a similar role at the divisome. The human pathogen Listeria monocytogenes encodes up to six FtsW/RodA homologs, however their functions have not yet been investigated. Analysis of deletion and depletion strains led to the identification of the essential cell division-specific FtsW protein, FtsW1. Interestingly, L. monocytogenes encodes a second FtsW protein, FtsW2, which can compensate for the lack of FtsW1, when expressed from an inducible promoter. L. monocytogenes also possesses three RodA homologs, RodA1, RodA2 and RodA3 and their combined absence is lethal. Cells of a rodA1/rodA3 double mutant strain are shorter and have increased antibiotic and lysozyme sensitivity, probably due to a weakened cell wall. Results from promoter activity assays revealed that expression of rodA3 and ftsW2 is induced in the presence of antibiotics targeting penicillin binding proteins. Consistent with this, a rodA3 mutant was also more susceptible to the ß-lactam antibiotic cefuroxime. Taken together, our study highlights that L. monocytogenes encodes a multitude of functional FtsW and RodA enzymes to produce its rigid cell wall and that their expression needs to be tightly regulated to maintain growth, cell division and antibiotic resistance.
Importance The human pathogen Listeria monocytogenes is usually treated with high doses of ß-lactam antibiotics, often combined with gentamicin. However, these antibiotics only act bacteriostatically on L. monocytogenes and the immune system is needed to clear the infection. Therefore, individuals with a compromised immune system are at risk to develop a severe form of Listeria infection, which can be fatal in up to 30% of cases. The development of new strategies to treat Listeria infections is therefore necessary. Here we show that the expression of some of the FtsW and RodA enzymes of L. monocytogenes is induced by the presence of ß-lactam antibiotics and their combined absence makes bacteria more susceptible to this class of antibiotics. The development of antimicrobials that inhibit the activity or production of FtsW/RodA enzymes might therefore help to improve the treatment of Listeria infections and thereby lead to a reduction in mortality.
Introduction
Bacterial cells are surrounded by a mesh of peptidoglycan (PG) that determines their shape and also protects the cells from lysis due to their high internal turgor pressure (Weidel and Pelzer, 1964, Vollmer et al., 2008). Peptidoglycan is comprised of glycan strands that are crosslinked by short peptides (Rogers et al., 1980). The glycan strands are composed of alternating N-acetylglucosamine and N-acetylmuramic acid residues that are connected by a ß-1,4 glycosidic bond (Ghuysen and Strominger, 1963). The synthesis of peptidoglycan begins in the cytoplasm with the production of the PG precursor lipid II by the proteins MurABCDEF, MraY and MurG (Blumberg and Strominger, 1974, van Heijenoort, 2001, Scheffers and Pinho, 2005, Pinho et al., 2013). Lipid II is then transported across the cytoplasmic membrane by the flippase MurJ and Amj (Meeske et al., 2015, Sham et al., 2014, Ruiz, 2008) and subsequently incorporated in the growing glycan strand by glycosyltransferases. The polymerization and the crosslinking of the glycan strands are facilitated by the activity of glycosyltransferases and transpeptidases, respectively. Class A penicillin binding proteins (PBPs) are bifunctional enzymes that possess glycosyltransferase and transpeptidase activity, whereas class B PBPs only contain a transpeptidase domain (Höltje, 1998, Sauvage et al., 2008, Goffin and Ghuysen, 1998). In addition, some species such as Escherichia coli, Staphylococcus aureus and Streptococcus pneumoniae encode monofunctional glycosyltransferases (MGTs) that can also incorporate lipid II into the growing glycan strand (Park and Matsuhashi, 1984, Park et al., 1985, Karinou et al., 2018, Wang et al., 2001, Hara and Suzuki, 1984).
B. subtilis encodes four class A PBPs and no MGT, however, deletion of all class A PBPs only manifests in small PG changes (McPherson and Popham, 2003). Recently, it has been shown that members of the SEDS (shape, elongation, division, sporulation) family of proteins, namely RodA and FtsW, also act as glycosyltransferases (Meeske et al., 2016, Cho et al., 2016, Emami et al., 2017, Taguchi et al., 2019). Both, RodA and FtsW, form complexes with cognate class B PBPs to enable polymerization and crosslinking of glycan strands (Cho et al., 2016, Taguchi et al., 2019, Leclercq et al., 2017, Fraipont et al., 2011). Interestingly, SEDS proteins and the class B PBPs are more conserved among different bacterial species than class A PBPs (Meeske et al., 2016).
In rod-shaped bacteria, peptidoglycan is synthesized by two multiprotein complexes, the rod complex that is essential for the cell elongation and the divisome that is crucial for the formation of the division septum (Ricard and Hirota, 1973, Nanninga, 1991, Carballido-Lopez and Formstone, 2007). RodA is part of the rod complex and is essential in many bacteria including B. subtilis and S. pneumoniae (Liu et al., 2017, Henriques et al., 1998). Depletion of RodA results in the production of enlarged, spherical cells in B. subtilis (Henriques et al., 1998). In contrast, FtsW is essential for cell division and cells depleted for FtsW grow as long filaments (Kobayashi et al., 2003, Boyle et al., 1997, Gamba et al., 2016). B. subtilis harbors a sporulation specific member of the SEDS family, SpoVE in addition to RodA and FtsW. SpoVE is dispensable for growth, however, it is essential for the synthesis of the spore cortex peptidoglycan (Henriques et al., 1992, Ikeda et al., 1989). Other Bacillus species such as B. cereus and B. anthracis possess 4 to 5 FtsW/RodA proteins and strains of different serotypes of the human pathogen Listeria monocytogenes encode even up to 6 FtsW/RodA homologs in their genome. However, their functions have not yet been investigated.
Here, we determine the role of the different FtsW and RodA homologs for the growth and cell morphology of L. monocytogenes. Our results show that L. monocytogenes encodes two FtsW and three RodA enzymes. Absence of either FtsW1 or of all three RodA proteins is lethal under standard laboratory conditions. L. monocytogenes infections are usually treated with high doses of ß-lactam antibiotics such as ampicillin, which inhibit the transpeptidase activity of PBPs (Swaminathan and Gerner-Smidt, 2007). We demonstrate that the expression of two SEDS proteins, FtsW2 and RodA3, is induced in the presence of ß-lactam antibiotics likely to compensate for the inhibition of PBPs and that a rodA3 mutant is more sensitive to the ß-lactam antibiotic cefuroxime. Antimicrobials inhibiting the activity of proteins of the SEDS family could therefore potentially improve the treatment of Listeria infections in the future.
Materials and Methods
Bacterial strains and growth conditions
All strains and plasmids used in this study are listed in Table S1. Strain and plasmid constructions are described in the supplemental materials and method section and all primers used in this study are listed in Table S2. E. coli strains were grown in Luria-Bertani (LB) medium and L. 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 and kanamycin (Kan) at 30 μg/ml, and for L. monocytogenes cultures, chloramphenicol (Cam) at 10 μg/ml, kanamycin (Kan) at 30 μg/ml and Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 1 mM. We used the L. monocytogenes strain 10403S and derivatives thereof. However, we refer to L. monocytogenes EGD-e gene and locus tag numbers as this was the first fully sequenced L. monocytogenes strain.
Growth curves
Overnight cultures of wildtype L. monocytogenes 10403S and the indicated deletion strains were diluted to an OD600 of 0.01 or 0.05 in 15 ml BHI medium and the cultures were incubated at 37°C with shaking. Growth was monitored by determining OD600 readings at hourly intervals. For growth curves with the IPTG-inducible depletion strains 10403SΔftsW1 iftsW (ANG4314), 10403SΔftsW1 iftsW2 (ANG5119) and 10403SΔrodA1-3 irodA1 (ANG5192), the strains were cultivated overnight in the presence of 1 mM IPTG. The next day, cells were washed once with fresh medium, subsequently diluted 1:50 in 5 ml BHI medium and grown for 8-10 h in the absence of the inducer. The cultures were diluted 1:100 into fresh BHI medium and grown until the next morning at 37°C. The depleted cells were then diluted to an OD600 of 0.01 and grown in the presence or absence of 1 mM IPTG at 37°C. Averages and standard deviations from three independent experiments were plotted.
Determination of minimal inhibitory concentration (MIC)
The minimal inhibitory concentration for bacitracin, penicillin and moenomycin and lysozyme was determined using a microbroth dilution assay in 96-well plates. 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: 1 mg/ml for bacitracin A, 1 μg/ml for penicillin G, 0.8 or 1.6 μg/ml for moenomycin, 8 μg/ml cefuroxime and 10 mg/ml for lysozyme. The OD600 was determined after incubating the 96-well plates for 24 h at 37°C with shaking at 500 rpm in a plate incubator (Thermostar, BMG Labtech). The MIC value refers to the antibiotic concentration at which bacterial growth was inhibited by >90%.
Determination of antibiotic susceptibility using a spot plating assay
Overnight cultures of the indicated L. monocytogenes strains were adjusted to an OD600 of 1 and 5 μl of serial dilutions were spotted on BHI agar plates or BHI agar plates containing 1 μg/ml cefuroxime. Plates were photographed after incubation at 37°C for 24 h.
Fluorescence and phase contrast microscopy
For bacterial cell length measurements, 100 μl of mid-log cultures were mixed with 5 μl of 100 μg/ml nile red solution to stain the cell membrane. After an incubation of 20 min at 37°C, the cells were washed once with 1x PBS and resuspended in 50 μl 1x PBS. 1.5 μl of the different samples were 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 100x magnification using the Zeiss Axio Imager.A1 microscope coupled to the AxioCam MRm and processed using the Zen 2012 software (blue edition). For the detection of nile red fluorescence signals, the Zeiss filter set 00 was used. For the determination of the cell lengths, 300 cells were measured for each experiment and the median cell length was calculated. Averages and standard deviations of the median cell length of three independent experiments were plotted.
Peptidoglycan isolation and analysis
Overnight cultures of L. monocytogenes 10403S, 10403SΔrodA1ΔrodA3 and 10403SΔrodA1ΔrodA3 pIMK3-rodA1 were used to inoculate 1 L BHI broth (with 1 mM IPTG for the complementation strain 10403SΔrodA1ΔrodA3 pIMK3-rodA1) to a starting OD600 of 0.06. The cultures were grown at 37°C until they reached an OD600 of 1, at which point the cultures were cooled on ice for 1 h. The bacteria were subsequently collected by centrifugation and peptidoglycan was purified and digested with mutanolysin as described previously (de Jonge et al., 1992, Corrigan et al., 2011). Digested muropeptides were analyzed by high-performance liquid chromatography (HPLC) and recorded at an absorption of 205 nm as described previously (de Jonge et al., 1992). For quantification, the areas of the main muropeptide peaks were integrated using the Agilent Technology ChemStation software. The sum of the peak areas was set to 100% and individual peak areas were determined. Averages and standard deviations from three independent extractions were calculated.
β-galactosidase assay
For the determination of the β-galactosidase activity, overnight cultures of strains 10403S pPL3e-Plmo2689-lacZ, 10403SΔrodA1 pPL3e-Plmo2689-lacZ and 10403SΔrodA1ΔrodA2 pPL3e-Plmo2689-lacZ were diluted 1:100 in fresh BHI medium and grown for 6 h at 37°C. Sample collection and preparation were performed as described previously (Gründling et al., 2004). Briefly, OD600 readings were determined (for the final β-galactosidase unit calculations) for the different cultures after 6 h of growth and cells from 1 ml culture were pelleted by centrifugation for 10 min at 13,200 x g, resuspended in 100 μl ABT buffer (60 mM K2HPO4, 40 mM KH2PO4, 100 mM NaCl, 0.1% Triton X-100, pH 7.0, filter sterilized), snap frozen in liquid nitrogen and stored at −80°C until use. For the identification of substances inducing the expression of the lmo2689-lmo2686 operon, an overnight culture of strain 10403S pPL3e-Plmo2689-lacZ was diluted 1:100 in fresh BHI medium and the culture incubated with shaking at 37°C until an OD600 of 0.5-0.6. The culture was divided into several flasks and incubated for two hours at 37°C in the presence or absence of the following substances: 0.5 μg/ml ampicillin, 0.05 μg/ml penicillin, 0.5 μg/ml vancomycin, 4 μg/ml cefuroxime, 0.05 μg/ml moenomycin, 0.5 mg/ml lysozyme, 1% ethanol, 300 μg/ml MgSO4 or 300 μg/ml EDTA. After 2 h, bacterial cultures were harvested and frozen as described above.
Samples were thawed and 1:10 dilutions were prepared in ABT buffer. 50 μl of the 1:10 diluted samples were mixed with 10 μl of 0.4 mg/ml 4-methyl-umbelliferyl-ß-D-galactopyranoside (MUG) substrate prepared in DMSO and incubated for 60 min at room temperature (RT). A reaction with ABT buffer alone was used as negative control. Following this incubation step, 20 μl of each reaction was diluted into 180 μl of ABT buffer in a black 96-well plate and fluorescence values were measured using an HIDEX Sense Microplate Reader at 355 nm excitation and 460 nm emission wavelengths. 0.125-20 μM of the fluorescent 4-methylumbelliferone (MU) standard were used to obtain a standard curve. β-galactosidase units were calculated as (pmol of substrate hydrolyzed x dilution factor)/(ml culture volume x OD600 x minute). The amount of hydrolyzed substrate was determined from the standard curve as (emission reading – y intercept)/slope.
Results
L. monocytogenes 10403S encodes six FtsW/RodA homologs
So far, FtsW and RodA proteins of the human pathogen L. monocytogenes have not been studied. FtsW and RodA are members of the SEDS (shape, elongation, division, sporulation) family of proteins and are multispanning membrane proteins with 8-10 transmembrane helices and a large extracellular loop (Fig. 1A). Using BLAST, six proteins with homology to the B. subtilis FtsW and RodA proteins could be identified in the genome of L. monocytogenes 10403S (Tab. 1). The protein encoded by lmo0421 has the weakest homology to B. subtilis FtsW and RodA (Fig. S1, Tab. 1). lmo0421 is part of the sigC operon, which is comprised of lmo0422 encoding the PadR-like repressor LstR and lmo0423 coding for the ECF-type sigma factor SigC (Fig. 1B). The sigC operon acts as a lineage II specific heat shock system (Zhang et al., 2005) and is therefore not encoded in all L. monocytogenes genomes. Due to the weak homology to FtsW and RodA and its absence in L. monocytogenes strains of lineage I and III, Lmo0421 was excluded from further analysis.
The L. monocytogenes protein Lmo1071 is the closest homolog to B. subtilis FtsW with a sequence identity of 48% (Table 1). Furthermore, L. monocytogenes lmo1071 and B. subtilis ftsW are found in the same chromosomal context. More specifically, lmo1071 is located between genes lmo1070, which encodes a protein with homology to the B. subtilis YlaN protein, and pycA coding for the pyruvate carboxylase. This analysis suggests that gene lmo1071 encodes the cell division protein FtsW. However, L. monocytogenes encodes a second protein, Lmo2688, that shares a higher degree of homology to the B. subtilis FtsW as compared to the B. subtilis RodA protein (Table 1). Due to these similarities and additional data presented in this study, we refer to Lmo1071 and Lmo2688 as FtsW1 and FtsW2, respectively.
The BLAST search with the B. subtilis RodA sequence as a query sequence yielded the L. monocytogenes protein Lmo2428 as the closest homolog with a sequence identity of 40% (Table 1). In addition to Lmo2428, two additional RodA homologs are present in L. monocytogenes, namely Lmo2427 and Lmo2687. As presented below, Lmo2427, Lmo2428 and Lmo2687 are likely bona-fide RodA homologs and were therefore renamed RodA1, RodA2 and RodA3, respectively. rodA1 is located adjacent to rodA2, but despite their proximity, rodA1 and rodA2 are likely not transcribed as part of the same operon (Toledo-Arana et al., 2009). In contrast, rodA3 and ftsW2 are part of the four-gene operon lmo2689-lmo2686. Lmo2689 is similar to a Mg2+-type ATPase, whereas lmo2686 encodes a protein of unknown function.
lmo1071 encodes FtsW1 and is essential for the survival of L. monocytogenes
The cell division protein FtsW is essential for growth in the Gram-negative and Gram-positive model organisms E. coli and B. subtilis (Boyle et al., 1997, Ikeda et al., 1989, Khattar et al., 1994, Kobayashi et al., 2003). Depletion of FtsW in these organisms leads to a block in cell division and formation of elongated cells (Boyle et al., 1997, Gamba et al., 2016). All our attempts to delete the ftsW1 gene in L. monocytogenes 10403S remained unsuccessful, suggesting that FtsW1 is also essential for growth in Listeria. Next, strain 10403SΔftsW1 iftsW1 was constructed, in which the expression of ftsW1 is controlled by an IPTG-inducible promoter. While no difference in the growth was observed between the wildtype and FtsW1-depletion strain (likely due to leakiness of the inducible promoter) (Fig. 2A), cells depleted for FtsW1 were significantly elongated (Fig. 2B-C). Bacteria depleted for FtsW1 had a median cell length of 3.41±0.16 μm, while wildtype and 10403SΔftsW1 iftsW1 bacteria grown in the presence of 1 mM IPTG had a median cell length of 1.85±0.16 μm and 1.93±0.07 μm, respectively (Fig. 2C). These data indicate that lmo1071 encodes the cell division specific SEDS protein FtsW.
L. monocytogenes encodes a second FtsW protein
To our knowledge, all bacteria analyzed to date possess only one FtsW protein that is essential for cell survival. We identified a second potential FtsW protein, Lmo2688, in L. monocytogenes. In contrast to ftsW1, a L. monocytogenes ftsW2 deletion strain could be constructed and no significant growth or cell morphology phenotypes could be observed for the ΔftsW2 deletion strain (data not shown). In a previous study, it was reported that the operon comprised of genes lmo2689-lmo2686 is only minimally expressed when L. monocytogenes 10403S is grown in BHI broth (Lobel and Herskovits, 2016). We reasoned that if ftsW2 does indeed code for a second FtsW protein, it should be possible to delete ftsW1 in a strain in which ftsW2 is artificially expressed from an IPTG-inducible promoter. Indeed, strain 10403SΔftsW1 iftsW2 could be generated in the presence of IPTG. In contrast, we were unable to generate strain 10403SΔftsW1 when any of the other FtsW/RodA homologs Lmo2427 (RodA1), Lmo2428 (RodA2) or Lmo2687 (RodA3) were expressed from the same IPTG-inducible promoter system. While prolonged depletion of FtsW2 in strain 10403SΔftsW1 iftsW2 had again no impact on the growth, the cells were significantly elongated in the absence of the inducer compared to wildtype or bacteria grown in the presence of inducer (Fig. 3). These data strongly suggest that ftsW2 encodes a second FtsW enzyme while the remaining three proteins Lmo2427, Lmo2428 and Lmo2687 likely function as RodA proteins.
L. monocytogenes encodes three RodA homologs
We were able to assign roles for two of the FtsW/RodA homologs as FtsW-like proteins. However, L. monocytogenes encodes three additional homologs, which show a higher similarity to the B. subtilis RodA protein as compared to the B. subtilis FtsW protein (Tab. 1). As described above, expression of none of the enzymes Lmo2427, Lmo2428 or Lmo2687 was able to rescue the growth of an ftsW1 deletion strain, indicating that these enzymes likely function as RodA proteins in L. monocytogenes and hence they were renamed RodA1, RodA2 and RodA3, respectively. All attempts to construct a rodA1-3 triple mutant failed further corroborating that these proteins function as RodA proteins and at least one of them needs to be present for cell viability. To determine whether the different RodA homologs have distinct functions or whether they are merely duplications, single and double mutant strains were generated. No significant differences with regards to growth and cell length could be observed between the wildtype strain 10403S and single rodA1, rodA2 or rodA3 deletion strains (Fig. S2). Similar observations were made with the rodA double mutant strains 10403SΔrodA1ΔrodA2 and 10403SΔrodA2ΔrodA3 (Fig. 4). However, cells lacking RodA1 and RodA3 were shorter (1.3±0.03 μm) compared to wildtype cells (1.9±0.06 μm, Fig. 4B-C), indicating that either RodA1 or RodA3 needs to be present for L. monocytogenes to maintain its rod shape. RodA3 is part of the lmo2689-lmo2686 operon that is only minimally expressed when L. monocytogenes is grown in BHI broth (Lobel and Herskovits, 2016). The fact that we observe differences in cell morphology between a rodA1 single and the rodA1/rodA3 double mutant, suggests that rodA3 expression might increase upon deletion of rodA1. To test this hypothesis, we fused the promoter upstream of lmo2689 and driving rodA3 expression to lacZ and inserted this fusion into the chromosome of wildtype 10403S, the rodA1 and the rodA1/rodA2 deletion strains. The promoter activity was indeed 1.5- to 2- fold increased in the rodA1 and rodA1/rodA2 mutant strains as compared to the wildtype, as assessed by the increase in the ß-galactosidase activity (Fig. 4G). This result indicates that expression of the lmo2689-lmo2686 operon, which encodes FtsW2 and RodA3, is induced in the absence of RodA1, suggesting a coordination of the expression of the different RodA homologs.
To confirm that the decrease in cell length of the double mutant strain 10403SΔrodA1ΔrodA3 depends on the absence of RodA1 and RodA3, complementation strains with IPTG-inducible expression of rodA1 or rodA3 were constructed. Expression of RodA1 restored the cell length to 1.84±0.1 μm, which is comparable to the cell length of wildtype cells (1.76±0.15 μm, Fig. 4E). On the other hand, expression of RodA3 from the ectopic locus in strain 10403SΔrodA1ΔrodA3 led to the formation of longer cells with an average cell length of 2.47±0.01 μm (Fig. 4D-E). These results indicate that induction of RodA3 from the IPTG-inducible promoter likely results in an overproduction of the protein as compared to the expression from the native promoter, highlighting that in particular fine-tuning of RodA3 production is essential for cell-length determination in L. monocytogenes.
The observation that the rodA1/rodA3 double mutant forms shorter cells suggests that RodA2 is not sufficient to maintain the cell length of L. monocytogenes. There are several possible explanations for this: 1. RodA2 might have a reduced activity as compared to RodA1 or RodA3. 2. RodA2 might have a function that is different from RodA1 and RodA3. 3. The protein levels of RodA2 might be sufficient to maintain cell viability but too low to maintain the rod shape. To determine which of these possibilities might be the case, a strain was constructed which lacks rodA1 and rodA3, but carries pIMK3-rodA2 to allow for IPTG-inducible expression of rodA2 in addition to the expression of rodA2 from its native locus (10403SΔrodA1ΔrodA3 irodA2). In the absence of the inducer, the cells had a median cell length of 1.2±0.03 μm (data not shown). However, the cell length of strain 10403SΔrodA1ΔrodA3 irodA2 increased to 1.71±0.8 μm when the strain was grown in the presence of IPTG (Fig. 4F). Therefore, additional expression of RodA2 can partially complement the cell length phenotype of the rodA1/rodA3 deletion strain, suggesting that RodA2 has a similar function as RodA1 and RodA3, but that it has either a lower activity or is not expressed in sufficient amounts.
As stated above, several attempts to construct a strain inactivated for all three RodA homologs remained unsuccessful, suggesting that at least one of the proteins RodA1, RodA2 or RodA3 needs to be present for the viability of L. monocytogenes. The results presented so far indicate that RodA1 is the most important RodA homolog considering that RodA2 alone is not sufficient to maintain the rod shape and that RodA3 is only minimally expressed under standard laboratory conditions (Lobel and Herskovits, 2016). To understand the impact of RodA enzymes on cell growth and cell division in L. monocytogenes, a strain was constructed which lacks all three rodA genes from its genome, but harbors pIMK3-rodA1 to enable IPTG-inducible expression of RodA1. Prolonged depletion of RodA1 in strain 10403SΔrodA1-3 irodA1 led to a growth defect that was not seen when the strain was grown in the presence of the inducer (Fig. 5A). However, the depletion was not efficient enough to see a complete growth inhibition, which would be expected for a strain lacking all three RodA homologs. Cells of the L. monocytogenes strain 10403SΔrodA1-3 irodA1 that were grown without IPTG were also significantly shorter with a cell length of 1.18±0.08 μm as compared to cells of the double mutant 10403SΔrodA1ΔrodA3 or the wildtype strain 10403S (Fig. 5B, D). Interestingly, different cell morphologies could be observed for strain 10403SΔrodA1-3 irodA1 after prolonged RodA1 depletion (Fig. 5C). The placement of the division septum was affected in some cells and daughter cells of different size or cells with two septa were observed (Fig. 5C). These morphological defects could be complemented and the cell length increased to 1.95±0.04 μm upon addition of IPTG and expression of RodA1 (Fig. 5D). These data highlight that RodA1 alone is sufficient to maintain the cell shape of L. monocytogenes.
Decreased moenomycin and lysozyme resistance in the absence of RodA homologs
Next, we wondered whether the absence of FtsW or RodA proteins affects the resistance of L. monocytogenes towards the antibiotics penicillin, bacitracin and moenomycin, which target different steps in the peptidoglycan biosynthesis process. Penicillin binds to the transpeptidase domain of PBPs and inhibits their function, leading to a reduced crosslinking of the peptidoglycan (Nakagawa et al., 1979, Korsak et al., 2010). Bacitracin inhibits the dephosphorylation of the bactoprenol carrier leading to a block in lipid II synthesis (Stone and Strominger, 1971). The phosphoglycolipid antibiotic moenomycin inhibits the glycosyltransferase activity of bifunctional PBPs and thereby prevents the polymerization of the glycan chain (van Heijenoort et al., 1987).
No significant differences could be observed in terms of resistance against penicillin, bacitracin or moenomycin for the FtsW1 depletion strain 10403SΔftsW1 iftsW1. This is presumably due to basal level expression of ftsW1 even in the absence of the inducer (data not shown). Simultaneous deletion of rodA1 and rodA3 resulted in a slight decrease in the MIC for penicillin, however, this difference was not significant (Fig. 6A). However, strain 10403SΔrodA1ΔrodA3 was 2-4-fold more sensitive to the antibiotic bacitracin (Fig. 6B). This phenotype could be complemented by expressing either RodA1, RodA2 or RodA3 from an IPTG-inducible promoter (Fig. 6B).
As described above, moenomycin inhibits the transglycosylase activity of PBPs leading to a decreased activity of these enzymes. In the absence of RodA1 and RodA3, cells are more susceptible to a reduced activity of PBPs manifesting in a 4-fold reduced resistance to moenomycin (Fig 6C). Induction of RodA1 expression in the 10403SΔrodA1ΔrodA3 deletion strain resulted in a significantly higher resistance to moenomycin as compared to the wildtype strain 10403S and expression of RodA2 or RodA3, led to partial or complete complementation of the moenomycin sensitivity (Fig. 6C).
Moreover, resistance to lysozyme, an enzyme that cleaves the linkage between N-acetyl muramic acid and N-acetylglucosamine residues of the peptidoglycan, was drastically decreased in strain 10403SΔrodA1ΔrodA3 and could be fully restored by expression of RodA1, RodA2 or RodA3 (Fig. 6D).
Lysozyme resistance in L. monocytogenes is mainly accomplished by two modifications of the peptidoglycan; deacetylation of N-acetylglucosamine residues by PgdA or O-acetylation of N-acetylmuramic acid residues by OatA (Boneca et al., 2007, Aubry et al., 2011). To determine whether the activity of PgdA is changed in the absence of RodA1 and RodA3, peptidoglycan was purified from the 10403SΔrodA1ΔrodA3 mutant strain, digested with mutanolysin and the resulting muropeptides analyzed by HPLC. Peptidoglycan samples isolated from the wildtype strain 10403S and the complementation strain 10403SΔrodA1ΔrodA3 irodA1, that had been grown in the presence of IPTG, were analyzed as controls (Fig. 6E). The main muropeptide peaks were assigned as described previously (Rismondo et al., 2015, Burke et al., 2014). Peaks 1 and 2 correspond to the acetylated and deacetylated monomeric muropeptides, respectively, whereas peak 3 and peaks 4-6 are acetylated and deacetylated muropeptide dimers, respectively. Deletion of rodA1 and rodA3 led to a reduction of both monomeric muropeptides and therefore to an increase in crosslinked peptidoglycan fragments by approximately 2% as compared to the wildtype strain 10403S, in which 65% of the peptidoglycan was cross-linked (Fig. 6F). However, no significant difference with regards to the deacetylated muropeptides could be observed between the wildtype 10403S, the 10403SΔrodA1ΔrodA3 and the 10403SΔrodA1ΔrodA3 irodA1 complementation strain (Fig. 6F). These results suggest that the lysozyme sensitivity phenotype of strain 10403SΔrodA1ΔrodA3 is not caused by changes in the peptidoglycan deacetylation, but rather due to general defects in the peptidoglycan structure.
Cell wall-acting antibiotics induce the promoter of lmo2689
The operon lmo2689-lmo2686, that contains the genes encoding FtsW2 and RodA3, is only minimally expressed under standard laboratory conditions (Lobel and Herskovits, 2016). However, a genome-wide transcriptional analysis performed in L. monocytogenes strain LO28 has shown that lmo2687, lmo2688 and lmo2689 are part of the CesR regulon (Nielsen et al., 2012). The cephalosporin sensitivity response regulator CesR is part of the CesRK two-component system that regulates the transcription of several cell envelope-related genes in response to changes in cell wall integrity, such as caused by the presence of cell wall-acting antibiotics or alcohols such as ethanol (Gottschalk et al., 2008, Kallipolitis et al., 2003, Nielsen et al., 2012). Therefore, we next used the lmo2689 promoter-lacZ fusion described above, to assess if expression of the lmo2689-lmo2686 operon is induced in the presence of antibiotics that target different processes of the PG biosynthesis or ethanol. Indeed, increased β-galactosidase activity could be measured for cells that had been grown in the presence of sub-inhibitory concentrations of the β-lactam antibiotics ampicillin, penicillin and cefuroxime and the phosphoglycolipid moenomycin (Fig. 7A). In contrast, no increase in β-galactosidase activity could be detected upon addition of vancomycin, lysozyme or ethanol as compared to untreated control cells (Fig. 7A). We also tested whether the presence of MgSO4 or EDTA has an impact on the lmo2689 promoter activity since lmo2689 encodes a putative Mg2+-type ATPase. However, the β-galactosidase activity of cells grown in the presence of MgSO4 or EDTA was comparable to the β-galactosidase activity seen for untreated cells (Fig. 7A). These results indicate that the expression of ftsW2 and rodA3, that are part of the lmo2689-lmo2686 operon, are induced in the presence of various cell wall-acting antibiotics, suggesting that FtsW2 and RodA3 might be important for the intrinsic resistance of L. monocytogenes against these antibiotics. However, no significant differences in MICs for penicillin and moenomycin could be observed between wildtype 10403S, the ftsW2 or rodA3 single mutant strains or the ftsW2/rodA3 double mutant (Fig. S3). However, there was a slight reduction in the resistance of the rodA3 single mutant against cefuroxime as compared to the wildtype (Fig. S3). To further assess whether there is a difference in the cefuroxime resistance between the L. monocytogenes wildtype strain 10403S and the rodA1, rodA2 and rodA3 single mutant strains, dilutions of overnight cultures were spotted on BHI agar plates with or without 1 μg/ml cefuroxime. Deletion of rodA1 or rodA2 results in a slightly reduced ability of these strains to grow on BHI plates supplemented with 1 μg/ml cefuroxime as compared to the wildtype 10403S strain (Fig. 7B). However, deletion of rodA3 leads to a stronger reduction of growth on BHI plates containing 1 μg/ml cefuroxime as compared to the rodA1 and rodA2 single mutants (Fig. 7B). Our results therefore suggest that L. monocytogenes induces the expression of rodA3 and ftsW2 in the presence of β-lactam antibiotics and moenomycin to compensate for the inhibition of the glycosyltransferase and transpeptidase activity of PBPs and that in particular the RodA homolog RodA3 has an important function for the intrinsic antibiotic resistance of L. monocytogenes.
Discussion
Bacterial cell elongation and cell division need to be tightly regulated to maintain the cell shape. This is accomplished by two multiprotein complexes, the rod complex and the divisome, which are coordinated by the actin homolog MreB and the tubulin homolog FtsZ, respectively (Ricard and Hirota, 1973, Nanninga, 1991, Carballido-Lopez and Formstone, 2007, Typas et al., 2012, Jones et al., 2001, Bi and Lutkenhaus, 1991). The SEDS protein FtsW is part of the divisome and essential for growth as shown for many bacteria including E. coli, B. subtilis and S. aureus (Boyle et al., 1997, Ikeda et al., 1989, Khattar et al., 1994, Kobayashi et al., 2003, Reichmann et al., 2019). Our experiments suggested that FtsW1 is also essential in L. monocytogenes, however, a second FtsW protein, FtsW2, can compensate for the loss of FtsW1 if it is expressed from an inducible promoter. FtsW2 is encoded in the lmo2689-lmo2686 operon that appears to be only minimally expressed when L. monocytogenes 10403S is grown under standard laboratory conditions (Lobel and Herskovits, 2016). The expression of the lmo2689-lmo2686 operon is regulated by the two-component system CesRK that is activated by cell envelope stress (Kallipolitis et al., 2003, Gottschalk et al., 2008, Nielsen et al., 2012). Using an L. monocytogenes strain carrying a Plmo2689-lacZpromoter fusion, we could detect increased β-galactosidase activity after incubation with sub-inhibitory concentrations of different β-lactam antibiotics including penicillin, cefuroxime, and moenomycin. However, the expression of the lmo2689-lmo2686 operon was not induced by other cell wall-targeting antibiotics such as vancomycin or the hydrolase lysozyme. This suggests that inhibition of the glycosyltransferase or transpeptidase activity of PBPs leads to activation of the lmo2689-lmo2686 operon, and hence, to the expression of ftsW2 as well as rodA3.
The rod-shape determining protein RodA is part of the elongation machinery. The data presented in this study suggest that L. monocytogenes encodes not one but three RodA proteins and depletion of the three RodA enzymes leads to a decreased cell length (Fig. 5). Simultaneous deletion of rodA1 and rodA3 already results in the formation of shorter cells, whereas cells of strains deleted for rodA1/rodA2 or rodA2/rodA3 have a cell length that is comparable to the wildtype strain 10403S. Taking into consideration that rodA3 is only minimally expressed under standard laboratory growth conditions in L. monocytogenes 10403S (Lobel and Herskovits, 2016), the results presented in this study suggest that rodA3 expression gets induced upon inactivation of RodA1, since we observed morphological differences between the rodA1 single and the rodA1/3 double mutant strains. Indeed, β-galactosidase assays confirmed that deletion of rodA1 or rodA1/2 increases the activity of the promoter from which rodA3 is expressed. The data presented in this study also indicate that RodA1 is the “main” RodA enzyme in L. monocytogenes as no significant phenotypic changes with regards to growth and cell division could be observed as long as RodA1 was present. On the other hand, RodA2 was only able to compensate for the loss of RodA1 and RodA3, when overproduced from an inducible promoter. This suggests that either RodA2 has a reduced activity compared to RodA1 or RodA3, which can be overcome by overproducing the enzyme, or the expression levels of RodA2 are too low to maintain the cell shape. Interestingly, cells of strain 10403SΔrodA1ΔrodA3 in which rodA3 is overexpressed from an ectopic locus have an increased cell length as compared to the wildtype. An explanation for this could be that elevated levels of RodA3 lead to the depletion of proteins needed at the cell division site, resulting in an extended synthesis of PG on the lateral wall. Another possibility could be that RodA3 directly inhibits FtsW1 or displaces FtsW1 at the cell division site, leading to a block in cell division and therefore resulting in the formation of elongated cells. Further experiments are needed to distinguish between these possibilities.
Recent studies have shown that SEDS proteins acts as glycosyltransferases (Meeske et al., 2016, Emami et al., 2017). The glycosyltransferase activity of PBPs and MGT can be inhibited by moenomycin, whereas RodA/FtsW enzymes are not affected by moenomycin and are therefore important for moenomycin resistance (Tamura et al., 1980, Emami et al., 2017). In good agreement with this, deletion of the genes encoding two of the three RodA enzymes, RodA1 and RodA3, resulted in an increased moenomycin sensitivity of L. monocytogenes (Fig. 7B).
In B. subtilis, RodA is in a complex with the class B PBP, PBP 2A (also named PBPH), and these two proteins act together to polymerize and crosslink the glycan strands (Wei et al., 2003, Henriques et al., 1998). Similarly, FtsW and PBP 2B form a subcomplex as part of the divisome (Daniel et al., 1996, Gamba et al., 2009). Recently it was shown that RodA-PBP3 and FtsW-PBP1 act as cognate pairs in the coccoid bacterium S. aureus (Reichmann et al., 2019). Depletion of all three RodA enzymes in L. monocytogenes, RodA1, RodA2 and RodA3, leads to a drastic reduction in cell length (Fig. 5). A similar phenotype was observed for a L. monocytogenes strain depleted for the essential class B PBP, PBP B1 (Rismondo et al., 2015). In contrast, the absence of either FtsW1 (Fig.2) or the class B PBP, PBP B2, in L. monocytogenes results in the formation of elongated cells (Rismondo et al., 2015). These observations suggest that RodA and FtsW might work in a complex with the cognate PBPs PBP B1 and PBP B2 during cell elongation and cell division, respectively. Further studies are necessary to confirm this hypothesis.
It is also interesting to note that L. monocytogenes does not only encode multiple FtsW/RodA enzymes, but also encodes two copies of the lipid II flippase MurJ, MurJ1 and MurJ2. RNAseq data suggest that murJ1 and murJ2 are both expressed in L. monocytogenes 10403S when grown in BHI broth (Lobel and Herskovits, 2016). murJ1 and murJ2 might therefore solely be gene duplications, however, MurJ1 and MurJ2 might have specific roles in different growth conditions, which needs to be assessed in future studies.
Taken together, L. monocytogenes has a repertoire of two lipid II flippases, several PBPs and multiple members of the SEDS family of proteins to produce its rigid cell wall. The expression and the activity of these enzymes need to be tightly regulated in L. monocytogenes to maintain its cell shape. Our results suggest that L. monocytogenes adapts the expression of two FtsW/RodA enzymes, FtsW2 and RodA3, to environmental stresses such as the presence of β-lactam antibiotics, thereby preventing defects in the peptidoglycan synthesis and subsequent cell lysis.
Acknowledgements
This work was funded by the Wellcome Trust grants 100289/Z/12/Z and 210671/Z/18/Z to AG and the German research foundation (DFG) grant RI 2920/1-1 to JR.