Balanced activity of multiple FtsW and RodA proteins is crucial for maintaining cell shape and antibiotic resistance in Listeria monocytogenes

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 64
Bacterial cells are surrounded by a mesh of peptidoglycan (PG) that determines their shape 65 and also protects the cells from lysis due to their high internal turgor pressure (Weidel and66 Pelzer, 1964, Vollmer et al., 2008). Peptidoglycan is comprised of glycan strands that are 67 crosslinked by short peptides (Rogers et al., 1980). The glycan strands are composed of 68 alternating N-acetylglucosamine and N-acetylmuramic acid residues that are connected by a 69 ß-1,4 glycosidic bond (Ghuysen and Strominger, 1963). The synthesis of peptidoglycan 70 begins in the cytoplasm with the production of the PG precursor lipid II by the proteins 71  (Höltje, 1998, Sauvage et al., 2008, Goffin and Ghuysen, 1998. In addition, some species 80 such as Escherichia coli, Staphylococcus aureus and Streptococcus pneumoniae encode 81 monofunctional glycosyltransferases (MGTs) that can also incorporate lipid II into the 82 growing glycan strand (Park and Matsuhashi, 1984, Park et al., 1985, Karinou et al., 2018, 83 Wang et al., 2001, Hara and Suzuki, 1984. Interestingly, SEDS proteins and the class B PBPs are more conserved among different 92 bacterial species than class A PBPs (Meeske et al., 2016). 93 In rod-shaped bacteria, peptidoglycan is synthesized by two multiprotein complexes, the rod 94 complex that is essential for the cell elongation and the divisome that is crucial for the 95 formation of the division septum (Ricard andHirota, 1973, Nanninga, 1991, Carballido-96 Lopez and Formstone, 2007). RodA is part of the rod complex and is essential in many 97 bacteria including B. subtilis and S. pneumoniae (Liu et al., 2017, Henriques et al., 1998. 98 Depletion of RodA results in the production of enlarged, spherical cells in B. subtilis 99 (Henriques et al., 1998). In contrast, FtsW is essential for cell division and cells depleted for 100 FtsW grow as long filaments (Kobayashi et al., 2003, Boyle et al., 1997, Gamba et al., 2016. with high doses of ß-lactam antibiotics such as ampicillin, which inhibit the transpeptidase 113 activity of PBPs (Swaminathan and Gerner-Smidt, 2007). We demonstrate that the expression 114 of two SEDS proteins, FtsW2 and RodA3, is induced in the presence of ß-lactam antibiotics 115 likely to compensate for the inhibition of PBPs and that a rodA3 mutant is more sensitive to 116 the ß-lactam antibiotic cefuroxime. Antimicrobials inhibiting the activity of proteins of the 117 SEDS family could therefore potentially improve the treatment of Listeria infections in the 118 future. 119 120

Materials and Methods 121
Bacterial strains and growth conditions 122 All strains and plasmids used in this study are listed in Table S1. Strain and plasmid 123 constructions are described in the supplemental materials and method section and all primers 124 used in this study are listed in Table S2. E. coli strains were grown in Luria-Bertani (LB) 125 medium and L. monocytogenes strains in brain heart infusion (BHI) medium at 37°C unless 126 otherwise stated. If necessary, antibiotics and supplements were added to the medium at the 127 following concentrations: for E. coli cultures, ampicillin (Amp) at 100 µg/ml and kanamycin 128 (Kan) at 30 µg/ml, and for L. monocytogenes cultures, chloramphenicol (Cam) at 10 µg/ml, 129 kanamycin (Kan) at 30 µg/ml and Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 1 mM. 130 We used the L. monocytogenes strain 10403S and derivatives thereof. However, we refer to 131 (ANG5192), the strains were cultivated overnight in the presence of 1 mM IPTG. The next 141 day, cells were washed once with fresh medium, subsequently diluted 1:50 in 5 ml BHI 142 medium and grown for 8-10 h in the absence of the inducer. The cultures were diluted 1:100 143 into fresh BHI medium and grown until the next morning at 37°C. The depleted cells were 144 then diluted to an OD600 of 0.01 and grown in the presence or absence of 1 mM IPTG at 145 37°C. Averages and standard deviations from three independent experiments were plotted. 146 147

Determination of minimal inhibitory concentration (MIC) 148
The minimal inhibitory concentration for bacitracin, penicillin and moenomycin and 149 lysozyme was determined using a microbroth dilution assay in 96-well plates. Approximately 150 10 4 L. monocytogenes cells were used to inoculate 200 µl BHI containing two-fold dilutions 151 of the different antimicrobials. The starting antibiotic concentrations were: 1 mg/ml for 152 bacitracin A, 1 µg/ml for penicillin G, 0.8 or 1.6 µg/ml for moenomycin, 8 µg/ml cefuroxime 153 and 10 mg/ml for lysozyme. The OD600 was determined after incubating the 96-well plates 154 for 24 h at 37°C with shaking at 500 rpm in a plate incubator (Thermostar, BMG Labtech). 155 The MIC value refers to the antibiotic concentration at which bacterial growth was inhibited 156 by >90%. 157 158 Determination of antibiotic susceptibility using a spot plating assay 159 Overnight cultures of the indicated L. monocytogenes strains were adjusted to an OD600 of 1 160 and 5 µl of serial dilutions were spotted on BHI agar plates or BHI agar plates containing 1 161 µg/ml cefuroxime. Plates were photographed after incubation at 37°C for 24 h. 162

Fluorescence and phase contrast microscopy 163
For bacterial cell length measurements, 100 µl of mid-log cultures were mixed with 5 µl of 164 100 µg/ml nile red solution to stain the cell membrane. After an incubation of 20 min at 165 37°C, the cells were washed once with 1x PBS and resuspended in 50 µl 1x PBS. 1.5 µl of 166 the different samples were spotted on microscope slides covered with a thin agarose film (1.5 167 % agarose in distilled water), air-dried and covered with a cover slip. Phase contrast and 168 fluorescence images were taken at 100x magnification using the Zeiss Axio Imager.A1 169 microscope coupled to the AxioCam MRm and processed using the Zen 2012 software (blue 170 edition). For the detection of nile red fluorescence signals, the Zeiss filter set 00 was used. 171 For the determination of the β-galactosidase activity, overnight cultures of strains 10403S 192 pPL3e-Plmo2689-lacZ, 10403S∆rodA1 pPL3e-Plmo2689-lacZ and 10403S∆rodA1∆rodA2 pPL3e-193 Plmo2689-lacZ were diluted 1:100 in fresh BHI medium and grown for 6 h at 37°C. Sample 194 collection and preparation were performed as described previously (Gründling et al., 2004). Plmo2689-lacZ was diluted 1:100 in fresh BHI medium and the culture incubated with shaking 202 at 37°C until an OD600 of 0.5-0.6. The culture was divided into several flasks and incubated 203 for two hours at 37°C in the presence or absence of the following substances: 0.5 µg/ml 204 ampicillin, 0.05 µg/ml penicillin, 0.5 µg/ml vancomycin, 4 µg/ml cefuroxime, 0.05 µg/ml 205 moenomycin, 0.5 mg/ml lysozyme, 1% ethanol, 300 µg/ml MgSO4 or 300 µg/ml EDTA. 206 After 2 h, bacterial cultures were harvested and frozen as described above. 207 Samples were thawed and 1:10 dilutions were prepared in ABT buffer. 50 µl of the 1:10 208 The L. monocytogenes protein Lmo1071 is the closest homolog to B. subtilis FtsW with a 234 sequence identity of 48% (Table 1). Furthermore, L. monocytogenes lmo1071 and B. subtilis 235 ftsW are found in the same chromosomal context. More specifically, lmo1071 is located 236 between genes lmo1070, which encodes a protein with homology to the B. subtilis YlaN 237 protein, and pycA coding for the pyruvate carboxylase. This analysis suggests that gene 238 lmo1071 encodes the cell division protein FtsW. However, L. monocytogenes encodes a 239 second protein, Lmo2688, that shares a higher degree of homology to the B. subtilis FtsW as 240 compared to the B. subtilis RodA protein (Table 1). Due to these similarities and additional 241 data presented in this study, we refer to Lmo1071 and Lmo2688 as FtsW1 and FtsW2, 242

respectively. 243
The BLAST search with the B. subtilis RodA sequence as a query sequence yielded the L. 244 monocytogenes protein Lmo2428 as the closest homolog with a sequence identity of 40% 245 (Table 1). In addition to Lmo2428, two additional RodA homologs are present in L. 246 monocytogenes, namely Lmo2427 and Lmo2687. As presented below, Lmo2427, Lmo2428 247 and Lmo2687 are likely bona-fide RodA homologs and were therefore renamed RodA1, 248 RodA2 and RodA3, respectively. rodA1 is located adjacent to rodA2, but despite their To our knowledge, all bacteria analyzed to date possess only one FtsW protein that is 272 essential for cell survival. We identified a second potential FtsW protein, Lmo2688, in L. 273 monocytogenes. In contrast to ftsW1, a L. monocytogenes ftsW2 deletion strain could be 274 constructed and no significant growth or cell morphology phenotypes could be observed for 275 the ∆ftsW2 deletion strain (data not shown). In a previous study, it was reported that the 276 operon comprised of genes lmo2689-lmo2686 is only minimally expressed when L. 277 monocytogenes 10403S is grown in BHI broth (Lobel and Herskovits, 2016). We reasoned 278 that if ftsW2 does indeed code for a second FtsW protein, it should be possible to delete ftsW1 279 in a strain in which ftsW2 is artificially expressed from an IPTG-inducible promoter. Indeed, 280 strain 10403S∆ftsW1 iftsW2 could be generated in the presence of IPTG. In contrast, we were 281 unable to generate strain 10403S∆ftsW1 when any of the other FtsW/RodA homologs 282 Lmo2427 (RodA1), Lmo2428 (RodA2) or Lmo2687 (RodA3) were expressed from the same 283 IPTG-inducible promoter system. While prolonged depletion of FtsW2 in strain 284 10403S∆ftsW1 iftsW2 had again no impact on the growth, the cells were significantly 285 elongated in the absence of the inducer compared to wildtype or bacteria grown in the 286 presence of inducer (Fig. 3). These data strongly suggest that ftsW2 encodes a second FtsW 287 enzyme while the remaining three proteins Lmo2427, Lmo2428 and Lmo2687 likely function 288 as RodA proteins. 289 290

L. monocytogenes encodes three RodA homologs 291
We were able to assign roles for two of the FtsW/RodA homologs as FtsW-like proteins. 292 However, L. monocytogenes encodes three additional homologs, which show a higher 293 similarity to the B. subtilis RodA protein as compared to the B. subtilis FtsW protein (Tab. 1). 294 As described above, expression of none of the enzymes Lmo2427, Lmo2428 or Lmo2687 295 was able to rescue the growth of an ftsW1 deletion strain, indicating that these enzymes likely 296 function as RodA proteins in L. monocytogenes and hence they were renamed RodA1, 297 RodA2 and RodA3, respectively. All attempts to construct a rodA1-3 triple mutant failed 298 further corroborating that these proteins function as RodA proteins and at least one of them 299 needs to be present for cell viability. To determine whether the different RodA homologs 300 have distinct functions or whether they are merely duplications, single and double mutant 301 strains were generated. No significant differences with regards to growth and cell length 302 could be observed between the wildtype strain 10403S and single rodA1, rodA2 or rodA3 303 deletion strains (Fig. S2). Similar observations were made with the rodA double mutant 304 strains 10403S∆rodA1∆rodA2 and 10403S∆rodA2∆rodA3 (Fig. 4)  protein levels of RodA2 might be sufficient to maintain cell viability but too low to maintain 334 the rod shape. To determine which of these possibilities might be the case, a strain was 335 constructed which lacks rodA1 and rodA3, but carries pIMK3-rodA2 to allow for IPTG-336 inducible expression of rodA2 in addition to the expression of rodA2 from its native locus 337 (10403S∆rodA1∆rodA3 irodA2). In the absence of the inducer, the cells had a median cell 338 length of 1.2±0.03 µm (data not shown). However, the cell length of strain 339 10403S∆rodA1∆rodA3 irodA2 increased to 1.71±0.8 µm when the strain was grown in the 340 presence of IPTG (Fig. 4F). Therefore, additional expression of RodA2 can partially 341 complement the cell length phenotype of the rodA1/rodA3 deletion strain, suggesting that 342 RodA2 has a similar function as RodA1 and RodA3, but that it has either a lower activity or 343 is not expressed in sufficient amounts. constructed which lacks all three rodA genes from its genome, but harbors pIMK3-rodA1 to 353 enable IPTG-inducible expression of RodA1. Prolonged depletion of RodA1 in strain 354 10403S∆rodA1-3 irodA1 led to a growth defect that was not seen when the strain was grown 355 in the presence of the inducer (Fig. 5A). However, the depletion was not efficient enough to 356 see a complete growth inhibition, which would be expected for a strain lacking all three 357 RodA homologs. Cells of the L. monocytogenes strain 10403S∆rodA1-3 irodA1 that were 358 grown without IPTG were also significantly shorter with a cell length of 1.18±0.08 µm as 359 compared to cells of the double mutant 10403S∆rodA1∆rodA3 or the wildtype strain 10403S 360 (Fig. 5B, D). Interestingly, different cell morphologies could be observed for strain 361 10403S∆rodA1-3 irodA1 after prolonged RodA1 depletion (Fig. 5C). The placement of the 362 division septum was affected in some cells and daughter cells of different size or cells with 363 two septa were observed (Fig. 5C). These morphological defects could be complemented and 364 the cell length increased to 1.95±0.04 µm upon addition of IPTG and expression of RodA1 365 (Fig. 5D). These data highlight that RodA1 alone is sufficient to maintain the cell shape of L.  No significant differences could be observed in terms of resistance against penicillin, 379 bacitracin or moenomycin for the FtsW1 depletion strain 10403S∆ftsW1 iftsW1. This is 380 presumably due to basal level expression of ftsW1 even in the absence of the inducer (data 381 not shown). Simultaneous deletion of rodA1 and rodA3 resulted in a slight decrease in the 382 MIC for penicillin, however, this difference was not significant (Fig. 6A). However, strain 383 10403S∆rodA1∆rodA3 was 2-4-fold more sensitive to the antibiotic bacitracin (Fig. 6B). This 384 phenotype could be complemented by expressing either RodA1, RodA2 or RodA3 from an 385 IPTG-inducible promoter (Fig. 6B). 386 As described above, moenomycin inhibits the transglycosylase activity of PBPs leading to a 387 decreased activity of these enzymes. In the absence of RodA1 and RodA3, cells are more 388 susceptible to a reduced activity of PBPs manifesting in a 4-fold reduced resistance to 389 moenomycin ( Fig 6C). Induction of RodA1 expression in the 10403S∆rodA1∆rodA3 deletion 390 strain resulted in a significantly higher resistance to moenomycin as compared to the 391 wildtype strain 10403S and expression of RodA2 or RodA3, led to partial or complete 392 complementation of the moenomycin sensitivity (Fig. 6C). 393 Moreover, resistance to lysozyme, an enzyme that cleaves the linkage between N-acetyl 394 muramic acid and N-acetylglucosamine residues of the peptidoglycan, was drastically 395 decreased in strain 10403S∆rodA1∆rodA3 and could be fully restored by expression of 396 RodA1, RodA2 or RodA3 (Fig. 6D). deacetylated monomeric muropeptides, respectively, whereas peak 3 and peaks 4-6 are 408 acetylated and deacetylated muropeptide dimers, respectively. Deletion of rodA1 and rodA3 409 led to a reduction of both monomeric muropeptides and therefore to an increase in 410 crosslinked peptidoglycan fragments by approximately 2% as compared to the wildtype strain 411 10403S, in which 65% of the peptidoglycan was cross-linked (Fig. 6F). However, no 412 significant difference with regards to the deacetylated muropeptides could be observed 413 between the wildtype 10403S, the 10403S∆rodA1∆rodA3 and the 10403S∆rodA1∆rodA3 414 irodA1 complementation strain (Fig. 6F). These results suggest that the lysozyme sensitivity 415 phenotype of strain 10403S∆rodA1∆rodA3 is not caused by changes in the peptidoglycan 416 deacetylation, but rather due to general defects in the peptidoglycan structure. 417 418 Cell wall-acting antibiotics induce the promoter of lmo2689 419 The operon lmo2689-lmo2686, that contains the genes encoding FtsW2 and RodA3, is only 420 minimally expressed under standard laboratory conditions (Lobel and Herskovits, 2016). β-galactosidase activity could be measured for cells that had been grown in the presence of 431 sub-inhibitory concentrations of the β-lactam antibiotics ampicillin, penicillin and cefuroxime 432 and the phosphoglycolipid moenomycin (Fig. 7A). In contrast, no increase in β-galactosidase 433 activity could be detected upon addition of vancomycin, lysozyme or ethanol as compared to 434 untreated control cells (Fig. 7A). We also tested whether the presence of MgSO4 or EDTA 435 has an impact on the lmo2689 promoter activity since lmo2689 encodes a putative Mg 2+ -type 436 ATPase. However, the β-galactosidase activity of cells grown in the presence of MgSO4 or 437 EDTA was comparable to the β-galactosidase activity seen for untreated cells (Fig. 7A). 438 These results indicate that the expression of ftsW2 and rodA3, that are part of the lmo2689-439 lmo2686 operon, are induced in the presence of various cell wall-acting antibiotics, 440 suggesting that FtsW2 and RodA3 might be important for the intrinsic resistance of L. 441 monocytogenes against these antibiotics. However, no significant differences in MICs for 442 penicillin and moenomycin could be observed between wildtype 10403S, the ftsW2 or rodA3 443 single mutant strains or the ftsW2/rodA3 double mutant (Fig. S3). However, there was a slight 444 reduction in the resistance of the rodA3 single mutant against cefuroxime as compared to the 445 wildtype (Fig. S3). To further assess whether there is a difference in the cefuroxime 446 resistance between the L. monocytogenes wildtype strain 10403S and the rodA1, rodA2 and 447 rodA3 single mutant strains, dilutions of overnight cultures were spotted on BHI agar plates 448 with or without 1 µg/ml cefuroxime. Deletion of rodA1 or rodA2 results in a slightly reduced 449 ability of these strains to grow on BHI plates supplemented with 1 µg/ml cefuroxime as 450 compared to the wildtype 10403S strain (Fig. 7B). However, deletion of rodA3 leads to a 451 stronger reduction of growth on BHI plates containing 1 µg/ml cefuroxime as compared to 452 the rodA1 and rodA2 single mutants (Fig. 7B). Our results therefore suggest that L. The rod-shape determining protein RodA is part of the elongation machinery. The data 482 presented in this study suggest that L. monocytogenes encodes not one but three RodA 483 proteins and depletion of the three RodA enzymes leads to a decreased cell length (Fig. 5). 484 Simultaneous deletion of rodA1 and rodA3 already results in the formation of shorter cells, 485 whereas cells of strains deleted for rodA1/rodA2 or rodA2/rodA3 have a cell length that is 486 comparable to the wildtype strain 10403S. Taking into consideration that rodA3 is only 487 minimally expressed under standard laboratory growth conditions in L. monocytogenes 488 10403S (Lobel and Herskovits, 2016), the results presented in this study suggest that rodA3 489 expression gets induced upon inactivation of RodA1, since we observed morphological 490 differences between the rodA1 single and the rodA1/3 double mutant strains. Indeed, β-491 galactosidase assays confirmed that deletion of rodA1 or rodA1/2 increases the activity of the 492 promoter from which rodA3 is expressed. The data presented in this study also indicate that 493 RodA1 is the "main" RodA enzyme in L. monocytogenes as no significant phenotypic 494 changes with regards to growth and cell division could be observed as long as RodA1 was 495 present. On the other hand, RodA2 was only able to compensate for the loss of RodA1 and 496 RodA3, when overproduced from an inducible promoter. This suggests that either RodA2 has 497 a reduced activity compared to RodA1 or RodA3, which can be overcome by overproducing 498 the enzyme, or the expression levels of RodA2 are too low to maintain the cell shape. RodA3, leads to a drastic reduction in cell length (Fig. 5). A similar phenotype was observed 520 for a L. monocytogenes strain depleted for the essential class B PBP, PBP B1 (Rismondo et 521 al., 2015). In contrast, the absence of either FtsW1 (Fig.2)        iftsW2. For depletion of FtsW2, strain 10403S∆ftsW1 iftsW2 was grown as described in   Bacteria from mid-logarithmic cultures of strain 10403S pPL3e-Plmo2689-lacZ were exposed for 2 h at 37°C to different stressors. The activity of the lmo2689 promoter was subsequently determined by performing β-galactosidase activity assays as described in the method section.
Bacteria that had been grown in the absence of a stressor were included as negative (-) control. The averages of the β-galactosidase activity units and standard deviations from three independent experiments were plotted. For statistical analysis, a one-way ANOVA coupled with a Dunnett's multiple comparisons test was used (*** p≤0.001, **** p≤0.0001). (B) Absence of RodA3 results in decreased cefuroxime resistance. Dilutions of overnight cultures