Glucose decoration on wall-teichoic acid is required for phage adsorption and InlB-mediated virulence in Listeria ivanovii

Listeria ivanovii (Liv) is an intracellular Gram-positive pathogen that primarily infects ruminants, but also occasionally causes enteric infections in humans. Albeit rare, this bacterium possesses the capacity to cross the intestinal epithelium of humans, similar to its more frequently pathogenic cousin, Listeria monocytogenes (Lmo). Recent studies in Lmo have shown that specific glycosyl modifications on the cell wall-associated glycopolymers (termed wall-teichoic acid, or WTA) of Lmo are responsible for bacteriophage adsorption and retention of the major virulence factor, Internalin B (InlB). However, the relationship between InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-deficient mutant became resistant to phage B025 infection due to an inability of the phage to adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation of liv1070 restored the characteristic phenotypes, including glucose decoration, phage adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-off between phage resistance and virulence attenuation may be a general feature in the Listeria genus. Importance Listeria ivanovii is a Gram-positive bacterial pathogen known to cause enteric infection in rodents and ruminants, and occasionally in immunocompromised humans. Recent investigations revealed that, in its better-known cousin Listeria monocytogenes, strains develop resistance to bacteriophage attack due to loss of glycosylated surface receptors, which subsequently resulting in disconnection of one of the bacterium’s major virulence factors, InlB. However, the situation in L. ivanovii remains unclear. Here, we show that L. ivanovii acquires phage resistance following deletion of a unique glycosyltransferase. This deletion also leads to dysfunction of InlB, making the resulting strain unable to invade host cells. Overall, this study suggests that the interplay between phage, bacteria and the host may be a feature common to the Listeria genus.

InlB and WTA in Liv remains unclear. Here, we report the identification of the unique gene, 23 liv1070 that encodes a putative glucosyltransferase in the polycistronic WTA gene cluster of 24 the Liv WSLC 3009 genome. We found that in-frame deletion of liv1070 led to loss of the 25 glucose substitution on WTA, as revealed by UPLC-MS analysis. Interestingly, the glucose-26 deficient mutant became resistant to phage B025 infection due to an inability of the phage to 27 adsorb to the bacterial surface, a binding process mediated by the receptor-binding protein 28 B025_Gp17. As expected, deletion of liv1070 led to loss of InlB retention to the bacterial cell 29 wall, which corresponded to a drastic decrease in cellular invasion. Genetic complementation 30 of liv1070 restored the characteristic phenotypes, including glucose decoration, phage 31 adsorption, and cellular invasion. Taken together, our data demonstrate that an interplay 32 between phage, bacteria, and host cells also exists in Listeria ivanovii, suggesting the trade-33 off between phage resistance and virulence attenuation may be a general feature in the 34 Listeria genus. 35 36 Introduction 48 The Listeria genus contains two species that are capable of causing disease in 49 mammals. L. monocytogenes (Lmo) is by far the more common of the two, which is capable 50 of invading and replicating within mammalian cells, and has a mortality rate of up to 30% (1). 51 While Lmo is capable of causing disease in both animals and humans, L. ivanovii (Liv) does 52 so almost exclusively in ruminants (2) and rodents (3). Liv has on occasion demonstrated its 53 ability to cause disease in humans (4)(5)(6)(7), showing that while it may be less adapted to cause 54 established infection in humans, it possesses all the necessary virulence factors and has the 55 capability given the right patient circumstances. There are two subspecies within Liv, 56 including spp. ivanovii and spp. londoniensis. Both have been described and categorized 57 based on the ability to metabolize N-acetyl-mannosamine and ribose. Liv spp. ivanovii is 58 generally sensitive to infection by many phages, while strains of spp. londoniensis appear to 59 be quite resistant to phage attack due to the presence of a functional type II-A CRISPR-Cas 60 system (8). To date, only Liv spp. ivanovii has been shown to cause listeriosis in human and 61 animals (3,9). Like Lmo, Liv infections are thought to be food-borne, as bacteria have been 62 isolated from both the feces and blood from infected human patients (4). From this, it can be 63 hypothesized that Liv also possesses the capability to cross membrane barriers during 64 infection, and indeed, has been shown in cell culture to be internalized in both human and 65 bovine cell lines, and possesses the major pathogenicity island similar to that in Lmo (6). Liv 66 is also thought to be rarer in the environment, suggesting that host tropism and lower 67 pathogenicity may not be the only reasons for its infrequent disease occurrence. 68 All species in the Listeria genus can be characterized by serotyping, a serological 69 process that relies upon the structural variation of the bacterial cell surface. Of the many 70 serovars (SVs) within the Lmo species, 1/2 and 4b cause the vast majority of disease in 71 humans, suggesting that there may be a clinical relevance for the surface structures that 72 confer serovar identity (10), which are primarily the wall-teichoic acids (WTAs) (11). WTAs 73 are complex carbohydrate molecules that make up a majority (up to ~60%) of the dry weight 74 of the bacterial cell wall and are covalently conjugated to the peptidoglycan and extend 75 outward (12). Beyond serving as the major antigenic determinants for serotyping, WTAs are 76 involved in several key physiological functions, including maintaining osmotic pressure, 77 antibiotic resistance, virulence, and interaction with host cells and bacteriophages (13)(14)(15)(16)(17)(18). 78 WTAs consist of a single glycosylated glycerol-based linkage unit, and a chain of 20-30 79 repeating units which can vary in structure between individual strains (19). In Listeria, the 80 WTA repeating units are generally made up of ribitol phosphate (type I WTA), but in some 81 serovars contain an N-acetylglucosamine (GlcNAc) residue integrated into the chain (type II 82 WTA) (20). Further structural variation stems from the carbon position at which GlcNAc is 83 linked to the ribitol residue, or further glycosylation or other modifications, termed 84 Liv1073 possesses a GT-2 (type 2 glycosyltransferase) domain, and it is conserved across 121 Listeria subspecies (Fig. S1) featuring type II WTA with an integrated GlcNAc in the primary 122 WTA chain (20). In our hands, previous attempts to delete the Liv1073's homolog in 4b Lmo 123 did not yield a viable mutant, which led us to speculate that this gene is involved in the 124 addition of GlcNAc onto the polymer chain, a process required for cell growth and 125 development. Other members of this gene cluster are annotated OatT (22) (47% AA 126 sequence identity), IspC (31) (limited sequence identity, but contains conserved glycine-127 tryptophan modules and the amidase motif), GttA (25) (34% AA identity), and GalU (89% AA 128 identity), which have recently been described in Lmo SV 4b (22). Domain homology 129 searches did not reveal any functional domains in Liv1067, yet Liv1070 was found to be 130 conserved in L. ivanovii and some other Listeria subspecies (Table. S1). The encoded 131 protein bears an N-terminal GT-2 domain (pfam00535), and a glycerol phosphotransferase 132 domain at its C-terminus (Fig. 1B). We therefore hypothesized that Liv1070 is responsible 133 for the glucose decoration on WTA (Fig. 1C). To test this hypothesis, we first generated the 134 knock-out mutant 3009Δliv1070 and found that this mutant strain did not show any growth 135 defects relative to the WT strain (Fig. 1D). 136

In-frame deletion of liv1070 results in loss of glucose decoration on WTA 137
To further verify that liv1070 confers WTA glucosylation in the parent strain 3009, 138 WTA was purified from the WT and mutant strains using previously-described analytical 139 technique (20). The structure of the WTA repeating unit was determined using ultra-140 performance liquid chromatography, coupled to mass spectrometry (UPLC-MS) (Fig. 2). The 141 structure of the WT strain showed two major peaks, one with m/z 354 (representing the 142 GlcNAc-Rbo fragment), and the other at 516 (with the Glc decoration) (see Fig. 2). As is 143 consistent with previous findings, this shows in the WT 3009 strain that only a portion of WTA 144 repeating unit structures are glycosylated, in this case with glucose. In the 3009Δliv1070 145 strain however, the peak with m/z 516 is completely missing, indicating that no Glc 146 decoration exists on the WTA of this strain. As expected, the chromatogram of the 147 3009Δliv1070::pPL2(liv1070) complemented strain (see Methods section) appears identical 148 to the WT strain, indicating that the phenotype is fully restored. 149

Loss of glucosylated WTA renders Liv 3009 cells insensitive to phage adsorption 150
Bacteriophage B025 is thought to utilize the Glc decoration on the WTA for binding 151 and recognition, as it features a specificity for SV 5 Liv strains. To determine whether 152 deletion of liv1070 confers phage resistance, a pulldown assay was performed. As expected, 153 far fewer B025 phage particles adsorbed to the surface of the 3009Δliv1070 strain (Fig. 3A). 154 To further verify that the Glc moiety was missing from the WTA in strain 3009Δliv1070, the 155 fluorescently labeled receptor binding protein B025_Gp18-GFP was utilized for glycotyping 156 (32,33). We previously demonstrated that this protein binds and recognizes Listeria strains 157 possessing WTA monomers with the GlcNAc moiety linked to the C2 position of ribitol, and 158 decorated with Glc or Gal (33). Thus, loss of Glc decoration from the WTA of strain 3009 159 would lead to an inability of the protein to recognize the bacterial cell surface. Indeed, 160 incubation of 3009Δliv1070 with the B025_Gp18-GFP fusion protein showed no fluorescence 161 signal relative to that of the 3009 WT strain (Fig. 3B). The complemented strain showed a 162 restored B025_gp18-GFP protein binding and phage adsorption, demonstrating that lmo1070 163 is sufficient to confer this phenotype. Together, these data strongly suggest that the gene 164 lmo1070 is responsible for WTA glucosylation in the Liv strain 3009, a structure which 165 mediates bacteriophage adsorption and susceptibility. 166

Glucosylated WTA is required for InlB cell wall association and Caco-2 and Hela cell 167 invasion 168
Because Liv is an invasive species causing disease in certain animals, their cells 169 likely harbor functional InlB on the surface. InlB has been shown to rely upon WTA rhamnose 170 decorations for its surface retention in SV. 1/2 strain in Lmo (26). In SV 4b strains, it is also 171 known that InlB relies upon the WTA galactose decoration for its surface retention, but not 172 the glucose decoration (25). Liv possesses a similar WTA structure to that of the Lmo SV 4b, 173 but differs in its GlcNAc connectivity, and features only Glc decoration instead of both Glc 174 and Gal in SV 4b. We thus hypothesized that Glc alone may be responsible for InlB retention 175 via Liv type II WTA. 176 To evaluate whether the loss of the Glc decoration on the WTA of strain 3009 affects 177 InlB surface retention, Western blot assays were performed using whole-cell protein extracts 178 and precipitated supernatant, and tested with anti-InlB antibody. As can be seen, the 179 3009Δliv1070 strain seems to lose the surface-associated InlB protein (Fig. 4A), similar to 180 what has been described for the gttA mutant in SV 4b Lmo (22). The phenotype was restored 181 in the complemented 3009Δliv1070::pPL2(liv1070) strain. Overall, it appears that 3009 182 expresses lower levels of InlB protein than Lmo strain 1042 (Fig. 4A), which may be 183 consistent with Liv being a somewhat less virulent species. To determine whether the loss of 184 InlB in this Glc-deficient mutant has an effect on the strain's ability to invade host cells, a 185 gentamicin protection assay was performed in both HeLa cells and the Caco-2 epithelial cell 186 line (Fig. 4B). Because the 3009Δliv1070 strain is severely deficient in its invasive abilities, it 187 can be assumed that InlB function is lost. This is supported by the observation that invasion 188 into HeLa cells was almost abolished. As HeLa cells do not express E-cadherin, invasion in 189 this cell line is known to be entirely InlB-dependent (34). The low invasion levels of 190 3009Δliv1070 in Caco-2 cells, which do express E-cadherin, is presumably mediated via the 191 InlA pathway. Invasion of the complemented strain 3009Δliv1070::pPL2(liv1070) is 192 insignificantly different from the WT strain, demonstrating that the Glc decoration on WTA is 193 sufficient to maintain proper invasion levels via its ability to retain InlB on the cell surface. 194 Together, these data clearly show that glucosylation of the WTA in SV 5 L. ivanovii mediates 195 phage resistance and maintains the function of one of the major Listeria virulence factors. 196 197 Discussion 198 In this investigation, we sought to determine whether the InlB virulence factor also 199 relies upon WTA decoration, and more specifically glucosylation in L. ivanovii. Previous 200 investigations in L. monocytogenes showed that InlB requires presence of galactose on the 201 WTA polymer for its surface retention, and this decoration is also essential for phage 202 recognition and binding (22,25,26). However, L. ivanovii possesses a somewhat different 203 WTA structure, with a different type of glycosylation, a structure which confers the unique SV 204 5 designation (20). Here, we show that the gene liv1070 is necessary for glucosylation of the 205 WTA monomer. Its deletion led to the loss of glucosylation, which was determined both 206 structurally and by glycotyping with specific WTA-binding phage proteins (33). As expected, 207 the glucose moiety is utilized by phage B025, which specifically infects strains possessing 208 glycosylated WTA with an integrated GlcNAc linked to the ribitol backbone at the C2 position. 209 Loss of glucosylation confers phage resistance and leads to a loss of surface InlB, together 210 showing that like Lmo, Liv must also face an evolutionary tradeoff: to maintain an important 211 virulence factor that mediates invasion of certain cell types, or be resistant to predation by 212 bacteriophages. 213 The WTA of Lmo SV 4b cells possesses both a glucose and galactose decoration, 214 but it was shown that only the galactose and not glucose decoration was responsible for InlB 215 surface retention (25). The data shown here strongly suggest that the conserved InlB SH3b 216 domain, which is the domain responsible for WTA binding, evolved to specifically recognize 217 different glycosylated WTA types (22,26). It may be the position and orientation of the sugar 218 moiety on the WTA that governs binding to InlB, as it has been shown that glucosylated 219 GlcNAc at the C4 position of ribitol or galactosylated GlcNAc linked at the C2 position failed 220 to retain InlB on the bacterial surface (22). How InlB interacts with different types of WTA is 221 still not fully understood, but it will be interesting to experimentally test in future specificity 222 studies. Data have suggested that most of the protein is buried within the bacterial cell wall, 223 and only a small portion is available to the outside. This can be demonstrated when 224 preparing Lmo for immunofluorescence, as the cell wall has to be partially digested with 225 lysozyme in order for the anti-InlB antibody to access its epitope (22). Evidence suggests 226 that the SH3b domain of InlB, which sits at the C-terminus at the protein (the N-terminus 227 contains the membrane-spanning portion of the protein), and may explain why it interacts 228 with the WTA, but not the LTA, which is likely more buried within the cell wall (25,35). 229 However, activation of the host cMet receptor requires both the SH3b domain as well as the 230 LRRs, which are more N-terminal, and thus further buried within the cell wall (36). How it is 231 that InlB, a membrane-associated protein which may only be partially exposed above the 232 surface of the cell wall activates the host cell receptor, is still not fully understood and 233 requires further study. 234 InlB functions by recognizing the host cell receptor cMet, and inducing the endocytic 235 pathway. Data presented here have shown that the phage-resistant strain lacking a WTA 236 glycosyltransferase does not express InlB on its cell surface and is consequently deficient in 237 its ability to invade host cells. InlB is involved in the invasion of the liver, spleen and placenta 238 (23, 37-39), meaning that its loss leads to a virulence attenuation. Lmo strains deficient in 239 WTA glycosylation lack the function of other proteins, namely ActA (40), and the autolysins 240 (26). Whether this is also the case in Liv remains to be seen, but it can be theorized that the 241 glucose-deficient 3009Δliv1070 discussed here would show a significant virulence 242 attenuation in an animal model of Liv infection, as it has been shown that Liv can heavily 243 colonize in the liver (41). 244 We have identified a putative enzyme (Liv1070) required for the glucosylation of WTA 245 in Liv. Based on in-silico predictions and the previous findings obtained in Lmo (25), we 246 propose a model (Fig. 5) for the WTA glucosylation process in Liv. In previous work, GttA 247 was shown to be involved in catalyzing the addition of Gal onto the undecaprenyl lipid carrier 248 (C 55 -P). Due to the close homology to GttA, Liv1068 is thus predicted to be a cytoplasmic 249 glycosyltransferase that transfers UDP-Glc to the lipid carrier. In addition, we presume that 250 Liv2596 functions as a putative flippase (84% identity to GtcA) that transports the C 55 -P-Glc 251 lipid intermediate across the membrane (42). We show that 3009Δliv1070 lacks Glc 252 decorations on WTA, suggesting that liv1070 might encode a GT-B fold glucosyltransferase 253 responsible for the transfer of the Glc residues onto the WTA chain. Since Liv1070 is 254 predicted to feature 2 transmembrane helices (43) (AA 121 to 141 and AA 585 to 713), we 255 speculate that this glucosylation process may also occur on the outside of the cell as 256 revealed in Lmo (44). Nevertheless, further biochemical evidence is still required to elucidate 257 this process. 258 Liv is thought to seldomly occur in the environment. In the dairy industry, a study from 259 Ireland showed that Liv isolates exist at a low prevalence of 1.4%, but these strains were 260 similarly capable (or even better) at invading Caco-2 cells compared to Lmo EGDe (45). 261 While the prevalence is lower than what is typically found for Lmo (46), it seems significant 262 nonetheless. This also suggests that Liv may be more prevalent in the environment and in 263 food processing plants than previously assumed, promoting a need for further research into 264 approaches for its containment. Bacteriophages have evolved as a viable option for 265 biocontrol of Listeria, although as with classical antimicrobials, the occurrence of resistant 266 strains may present a significant hurdle. However, evidence from this study and other recent 267 studies has shown that resistance to bacteriophages may often be accompanied by 268 physiological and virulence defects, which could present benefits to the host and decrease 269 pathogenicity (47,48

Bacterial strains, plasmids, phages and growth conditions 274
All bacterial strains, plasmids and phages used in this study are listed in Table S2. E. 275 coli XL1-Blue (Stargene) used for cloning and plasmid construction was routinely cultured in 276 Luria-Bertani (LB) broth at 37°C. The E. coli BL21-Gold strain was used for protein 277 expression. L. monocytogenes strains were grown in 1/2 brain heart infusion (BHI), at 30°C 278 with shaking when working with phages, or at 37°C when the cells were used for infection 279 studies. For a list of strains, plasmids and primers utilized in the study, see Table S2. 280 Propagation and purification of bacteriophage B025 were performed using L. ivanovii strain 281 3009 as previously described (25). For growth curve determination, overnight cultures were 282 diluted in full BHI to an OD of 0.05 in triplicate in a 96-well plate, and the OD 600nm was 283 measured for 16 h in a plate reader set to 37°C. 284

Production of mutant and complemented strains 285
Deletion knockouts were produced via allelic exchange. 500bp flanking regions of the 286 gene liv1070 from strain 3009 were produced by PCR, along with the pHoss1 plasmid 287 backbone. The pHoss1 plasmid contains a temperature-sensitive origin of replication, which 288 cannot replicate at higher temperatures. The primers used for this are listed above in Table  289 S2, and contained homologous overlap regions to allow for assembly. The three fragments 290 were assembled together using Gibson assembly, followed by transformation into E. coli 291 XL1-Blue. The pHoss1 plasmid was extracted from a single colony of XL-1 blue containing 292 the two 500bp flanking regions of gene liv1070, and transformed into Liv strain 3009. This 293 transformant was grown at permissive temperatures, allowing for gene deletion to proceed 294 The complete deletion of via allelic exchange.
liv1070 was confirmed by PCR and Sanger 295 sequencing. Complementation of 3009Δliv1070 with a functional copy of liv1070 was 296 performed by using the chromosome-integrating vector pPL2 (49). The liv1070 gene was 297 inserted by Gibson assembly into plasmid pPL2 under the control of the native promoter. 298

Phage pulldown assay 299
Pull down assay using bacteriophage B025 was performed using MOI 0.01 as 300 previously described(22), using WSLC 3009 as the propagation strain. Following serial 301 dilution of the phage, the number of phages adsorbed to the bacterial surface was evaluated 302 by phage overlays and expressed as the total plaque-forming units of phage adsorbed to 303 WT, mutant and complemented strain. 304 Listeria glycotyping assay 305 The abilities of GFP-tagged CBD500 and B025_Gp18 to bind to Listeria cell surface 306 were tested using a fluorescence binding assay as previously reported (33). Briefly, Listeria cells from log phase cultures were harvested by centrifugation, and resuspended in 1/5 308 volume of PBS (pH 7.4). 100 μl of cells were incubated with 5 μl of 1 mg/ml of GFP-CBDs 309 and incubated for 5 min at room temperature. The cells were spun down, washed three 310 times, and finally resuspended in PBS buffer. The samples were then subjected to confocal 311 laser scanning microscopy. 312

WTA extractions and structural analysis 313
WTA was purified from the indicated L. ivanovii strains as previously described (20). 314 Purified WTA polymers were depolymerized into monomeric repeating units by hydrolysis of 315 the phosphodiester bonds using 48% hydrofluoric acid for 20 h at 0°C. The purified WTA 316 monomers were lyophilized and subjected to UPLC-MS/MS for compositional and structural 317 analysis. 318

Gentamicin protection assay 319
The Caco-2 and HeLa cell lines used for in vitro assays were cultured at 37°C with 320 5% CO 2 in DMEM GlutaMAX (Gibco), supplemented with sodium pyruvate, 1% non-essential 321 amino acids and 10% FBS. Before infection, human cells were diluted to a concentration of 322 2-4x10 5 cells/mL and seeded onto 96-well plates in triplicate the day before performing the 323 experiment. On the day of infection, bacteria were grown at 37°C to an OD 600 of 0.8-1.0, 324 before being washed twice in PBS and diluted in DMEM lacking FBS to OD 600 0.01, which 325 corresponds to a multiplicity of infection (MOI) of ~100. Human cells were washed twice with 326 DPBS and bacterial suspensions were added on top. The co-culture was incubated for 2 h at 327 37°C. Cells were washed twice with DPBS and incubated a further 1 h in normal growth 328 medium (with FBS) supplemented with 40 μg/mL gentamicin. Cells were lysed with 0.5% 329 Triton-X-100, serially diluted, and 10 μL were spot-plated onto BHI agar plates. CFU counts 330 were determined the following day. The number of bacteria that had adhered or invaded was 331 expressed as a fraction relative to the invasion rate of strain WSLC 1042, which was 332 normalized to 1 for each replicate. 333

Western Blotting 334
To detect total InlB, total cell extracts were used instead of surface protein extracts in 335 order to obtain a better visualization for the loading control. One mL of overnight culture 336 grown in 1/2 BHI medium was mixed with 0.5 mL of 0.5 mm glass beads in a 2 mL tube and 337 shaken on a vortex at maximum speed for 30 minutes. The tubes were spun down for 1 min 338 at 200 x g and 500 μL of supernatant was transferred to a 1.5 mL Eppendorf, and spun again 339 for 15 min at maximum speed. The supernatant was discarded and the pellet resuspended in 340 50 μL SDS sample buffer for an initial OD of 2 (volume was adjusted accordingly if initial OD 341 varied) containing 5% β-mercaptoethanol, and boiled for 5 min. Samples were loaded onto 342 an SDS-PAGE gel and western blot was performed as previously described (22), using a 343 custom anti-InlB rabbit polyclonal antibody (1:5000), using a LLO antibody as the loading 344 control (1:5000; Abcam. Inc). Supernatant extracts were produced via a TCA precipitation 345 method using the supernatant from a 5mL culture, as previously described. 346 Western blot of Liv total and surface proteins of indicated strains, detected using an anti-InlB 540 antibody with anti-LLO (Listeriolysin) as a loading control. L. monocytogenes serovar 4b 541 strain WSLC 1042 and its ΔgttA KO derivative were compared to demonstrate the relative or 542 decreased amount of surface-associated InlB. (B) Relative invasiveness of the indicated 543 strains compared to that of Liv 3009 WT, in both Caco-2 and HeLa cell lines for 3 hours 544 (mean normalized to 3009 WT ±SEM, as determined by a gentamicin protection assay. For 545 both assays, n = 4. ***P < 0.001; ****P < 0.0001; ns, not significant relative to 3009). bioinformatic and genetic data presented in this study, we propose that WTA in L. ivanovii is 556 glucosylated with the aid of cytoplasmic GT Liv1069 (GalU, glucose-1-phosphate 557 uridyltransferase), which we predict produces UDP-Glc from UTP and glucose-1-phosphate 558 (step 1). Next, we hypothesize that Liv1068 catalyzes the addition of UDP-Glc residues onto 559 the undecaprenyl lipid carrier (C 55 -P) on the inner leaflet of the cell membrane due to its high 560 homology to GttA (step 2) as previously described in serovar 4b L. monocytogenes. The C-561 55-P-glucose intermediate is then transported across the membrane by Liv1069 (step 3), 562 which is 84% identical to the L. monocytogenes GtcA protein reported in a previous study. 563 The glucose residues are subsequently transferred onto the growing WTA chain outside the 564 cell (step 4), and we suggest that this step is catalyzed by glucosyl transferase Liv1070. The 565 WTA chain is conjugated by TarTUV homologue to the MurNAc of peptidolycan following 566 export by an ABC transporter TarGH. The glucosylated WTA confers the retention of the InlB 567 on the Listeria surface, which interacts with the host receptor cMet to activate receptor-568 mediated endocytosis, facilitating entry into host cells. 569 570 Figure S1. Comparison of genes encoding type II WTA biosynthesis pathways in the 571 indicated Listeria serovars and strains. 572 573