Function and contribution of two putative Enterococcus faecalis glycosaminoglycan degrading enzymes to bacteremia and catheter-associated urinary tract infection

ABSTRACT Enterococcus faecalis is a common cause of healthcare-acquired bloodstream infections and catheter-associated urinary tract infections (CAUTIs) in both adults and children. Treatment of E. faecalis infection is frequently complicated by multi-drug resistance. Based on protein homology, E. faecalis encodes two putative hyaluronidases, EF3023 (HylA) and EF0818 (HylB). In other Gram-positive pathogens, hyaluronidases have been shown to contribute to tissue damage and immune evasion, but the function in E. faecalis has yet to be explored. Here, we show that both hylA and hylB contribute to E. faecalis pathogenesis. In a CAUTI model, ΔhylA exhibited defects in bladder colonization and dissemination to the bloodstream, and ΔhylB exhibited a defect in kidney colonization. Furthermore, a ΔhylAΔhylB double mutant exhibited a severe colonization defect in a model of bacteremia while the single mutants colonized to a similar level as the wild-type strain, suggesting potential functional redundancy within the bloodstream. We next examined enzymatic activity, and demonstrate that HylB is capable of digesting both hyaluronic acid (HA) and chondroitin sulfate in vitro, while HylA exhibits only a very modest activity against heparin. Importantly, HA degradation by HylB provided a modest increase in cell density during the stationary phase and also contributed to dampening of lipopolysaccharide-mediated NF-κB activation. Overall, these data demonstrate that glycosaminoglycan degradation is important for E. faecalis pathogenesis in the urinary tract and during bloodstream infection.

infection, GAGs are present at many barrier sites, such as the endothelial glycocalyx and the urinary tract urothelial lining, where they are thought to play a protective role against pathogen invasion (11,12).For example, the GAG-rich glycocalyx of the bladder mucosa is believed to mask uroplakins and other surface proteins that are targeted by bacteria for adhesion and invasion (11)(12)(13).In support of this hypothesis, removal of GAGs from the bladder mucosa increases attachment of E. faecalis as well as other uropathogenic bacteria (14)(15)(16).
The most common GAGs in urothelium and endothelial glycocalyx are hyaluronic acid (HA), chondroitin sulfate (CS), and heparin (6,17).Several pathogenic bacteria produce enzymes that digest GAGs, which can destroy barrier functions, provide a carbohydrate source for microbial growth, and alter the innate immune response either by promoting inflammation or disrupting inflammatory signaling.For example, some Streptococcus species produce hyaluronidase enzymes that depolymerize HA and have been shown to promote tissue invasion, disrupt inflammatory signaling, and facilitate bacterial growth by liberating a new carbon source (18)(19)(20)(21).We therefore hypothesized that GAG degradation by E. faecalis could contribute to pathogenesis and invasive disease.
There are several conflicting reports pertaining to E. faecalis hyaluronidase activity.In 1964, Rosan and Williams reported hyaluronidase activity in oral Enterococcus isolates (22).However, this study was performed at a time when enterococcal taxonomy was in a state of flux, so it is unclear whether the hyaluronidase-positive strains were E. faecalis, E. faecium, or another species (23,24).A putative hyaluronidase was identified in Enterococcus faecium that is predominantly associated with vancomycin-resistant clinical isolates (25), and transfer of the plasmid encoding the putative hyaluronidase increased virulence of a fecal isolate in a mouse peritonitis model (26).However, no hyaluronidase activity was observed for any of the E. faecium strains (27), and deletion of the putative hyaluronidase itself did not reduce fitness in the mouse peritonitis model (28).
A recent assessment of in vitro GAG digestion by several E. faecalis clinical isolates found that none of the isolates degraded HA, CS, or heparin sodium salt (HS) (29).However, a homolog of the Streptococcus hyaluronidase gene is present in 90% of E. faecalis complete genome sequences available through the Bacterial and Viral Bioin formatics Resource Center (BV-BRC) (30) and 45% of isolates have a second putative hyaluronidase.E. faecalis strain V587 is one of the clinical isolates with both putative hyaluronidases.This strain is particularly notable as it is vancomycin-resistant and was isolated from the urine of a patient who later experienced sepsis with the same strain (31), making it relevant to two of the most prevalent types of E. faecalis infection.The purpose of this study was to characterize the enzymatic activity of both putative hyaluronidases and examine the contribution to CAUTI and bacteremia.

E. faecalis V587 encodes two putative hyaluronidases with differing predicted protein architectures
The genome of E. faecalis strain V587 in the BV-BRC (30) contains two putative hyaluroni dases located in different parts of the chromosome: ef3023 (hylA) is 4,119 bp at position at 2,884,596 bp (Fig. 1A), and ef0818 (hylB) is 3,015 bp at position 763,339 bp (Fig. 1B).The hylA gene is monocistronic, and no neighboring genes are predicted to be involved in oligosaccharide digestion or uptake.The hylB gene is similarly monocistronic but immediately downstream of an operon that encodes for a putative hyaluronateoligosaccharide phosphotransferase system with a divergently transcribed GntR-family transcriptional regulator (32).
Using the BV-BRC (33), we searched 646 complete genome sequences of E. faecalis for the presence of hylA and hylB.A total of 582 strains (90%) had at least one putative hyaluronidase with >95% amino acid identity and >80% query coverage: 470 strains (73%) had a homolog of hylA, 459 strains (71%) had a homolog of hylB, and 289 (45%) had both.Thus, hylA and hylB are well conserved across E. faecalis strains.Both are predicted to encode "polysaccharide lyase family 8" enzymes, and both are predicted to contain Sec pathway protein export signal peptides according to SignalP 5.0 (34).However, the amino acid sequences of HylA and HylB share only 36% identity with each other and 24%-30% identity with hyaluronidases from other bacterial species (Table S1).There are also substantial differences in the domain architecture of the two proteins: in addition to the GAGase domain, HylA contains a Discoidin (DS) and Bacterial immuno globulin-like fold (Big2) domains, a section of repeated amino acid residues annotated as a "Found In Various ARchitectures" (FIVAR) domain, and a predicted LPXTG domain for cell wall attachment (Fig. 1C) (35), while HylB lacks cell wall anchoring motifs and has only two small flanking domains with no predicted function by an NCBI Conserved Domain Search (36) (Fig. 1D).Structure predictions of HylA (Fig. 1E) and HylB (Fig. 1F) were generated using AlphaFold (37,38), with the GAGase domain shown in pink and other domains in different colors.Importantly, an alignment of the two proteins could not be achieved, which underscores the substantial differences in structure.

E. faecalis V587 does not exhibit hyaluronidase activity under in vitro conditions
Considering the prior literature regarding a lack of hyaluronidase activity in E. faecalis isolates (29), we first sought to examine the expression of hylA and hylB in strain V587.Notably, RNA sequencing data from the closely related V583 strain indicate that hylA and hylB are not expressed during growth in brain-heart infusion (BHI) (39).We therefore used RT-qPCR to examine the expression of hylA, hylB, and the housekeeping gene recA in V587 after 4 h of growth in BHI, 0.5× tryptic soy broth (TSB) (a condition previously found to support hyaluronidase activity in other species) (29), and human urine (Fig. 2).TSB and urine were also supplemented with either unfractionated high molecular weight HA or low molecular weight HA (~5 kDa) to determine if expression is induced by hyaluronic acid.The Cq values for recA were similar across all conditions, confirming that recA is an appropriate housekeeping gene for normalizing expression (Fig. 2A), and all samples were detected at least 10 cycles before the respective no-template controls (Fig. 2B).When growth in BHI was used as the calibrator, growth in 0.5× TSB resulted in a slight increase in expression of both hylA and hylB genes while growth in urine reduced expression (Fig. 2C).To examine the impact of HA on expression, we next used unsupplemented 0.5× TSB or urine as the calibrator.In TSB, supplementation with unfractionated HA slightly induced expression of hylA but had no effect on hylB (Fig. 2D).In contrast, unfractionated HA had no impact on expression in urine, but supplementation with 5 kDa HA induced the expression of both hylA and hylB (Fig. 2E).Thus, hylA and hylB are expressed by E. faecalis V587 under multiple growth conditions, and expression can be stimulated to some extent by HA.
We generated markerless deletion mutants of hylA and hylB, as well as a double deletion mutant (ΔhylAΔhylB).All of the mutants grew similarly to V587 in BHI, TSB, and human urine (Fig. 3A through C).To examine in vitro hyaluronidase activity, V587 and the ΔhylAΔhylB mutant were incubated on agar plates containing HA and examined for the development of a zone of clearance around the colony (Fig. 3D).Consistent with prior studies, no hyaluronidase activity was detected for either strain, suggesting that while hylA and hylB are expressed in vitro, active protein is not produced under these conditions.To determine if the lack of activity is specifically due to lack of expres sion or function under these conditions, hylB was cloned into pBAD and transformed into Lactococcus lactis, a Gram-positive species that lacks endogenous hyaluronidases.Notably, expression of the V587 hylB gene in L. lactis resulted in modest hyaluronidase activity, suggesting that the E. faecalis HylB can indeed function as a hyaluronidase and the lack of activity in V587 is likely due to regulatory control of expression and activity.

HylA and HylB contribute to E. faecalis bladder colonization and bacteremia
Prior to further probing the enzymatic activity of HylA and HylB, we first sought to determine contribution to E. faecalis pathogenesis in our established mouse model of CAUTI.Briefly, female CBA/J mice were inoculated transurethrally with 1 × 10 5 CFU of either V587, ΔhylA, ΔhylB, or ΔhylAΔhylB.During inoculation, a small piece of silicone tubing was left in the bladder to simulate a urinary catheter, as previously described (40).At 48 h post infection (hpi), the mice were sacrificed, and the bladders, kidneys, and spleens were collected for quantitation of CFUs, with spleen CFU used as an indicator of bacteremia (Fig. 4A).Inoculation with the ΔhylA mutant significantly reduced bladder and spleen colonization compared to infection with wild-type V587, and also reduced the overall incidence of bacteremia.In contrast, inoculation with the ΔhylB mutant resulted in similar bladder colonization as wild-type V587 but a significant defect in the kidneys.Therefore, HylA and HylB both contribute to E. faecalis CAUTI but may be differentially expressed in different host niches or have differing roles in pathogenesis.Interestingly, infection with the ΔhylAΔhylB double mutant phenocopied the mean bladder colonization of infection with ΔhylA and the mean kidney colonization of ΔhylB, but the only statistically significant defect compared to wild-type V587 was a reduction in spleen colonization and incidence of bacteremia.
As we observed a reduced incidence of secondary bacteremia in the CAUTI model and E. faecalis is a common cause of CLABSI, we next sought to determine whether HylA and HylB contribute to primary bacteremia.CBA/J mice were inoculated via tail vein injection with 1 × 10 8 CFU of V587, ΔhylA, ΔhylB, or ΔhylAΔhylB, mice were sacrificed 24 hpi, and CFUs were quantified in the liver, spleen, and kidneys (Fig. 4B).While the ΔhylA and ΔhylB mutants exhibited similar overall colonization to V587 in this model, the ΔhylAΔhylB double mutant had a significant colonization defect in the kidneys and the spleen.Thus, HylA and HylB may be functionally redundant during primary bacteremia.

Constitutive expression of hylB confers hyaluronidase and chondroitinase activity
With a contribution to pathogenesis confirmed, we next sought to define the enzymatic properties of HylA and HylB in E. faecalis V587.To bypass any issues due to endogenous gene expression, we opted to constitutively express hylA or hylB in the ΔhylAΔhylB double mutant.HylB is predicted to be fully secreted and lacks complex domain architecture, so the full-length sequence of hylB was cloned under the control of the ermB promoter in pAOJ84, and exported protein was detected as the expected molecular weight of 109 kDa (Fig. 5A).HylA is predicted to be anchored to the cell wall, which presents a potential challenge for assays requiring unattached secreted enzymes and could also impact cell wall integrity when overexpressed.We therefore designed vector pAOJ86 to contain the full-length hylA sequence lacking the LPXTG anchor, and exported protein was again detected at the expected molecular weight of 146 kDa (Fig. 5A).
Using an established semi-quantitative method for measuring GAG digestion with live bacteria (29), we incubated E. faecalis WT, ΔhylAΔhylB, and ΔhylAΔhylB containing the constitutive expression vectors for 24 h at 37°C under low oxygen conditions in 0.5× TSB containing 2.5 mg/mL of hyaluronic acid (Fig. 5B).In agreement with the literature and our prior results, V587 did not digest any of the GAGs and had an identical profile as the negative control strain ΔhylAΔhylB (Fig. 5B through D), confirming that E. faecalis does not exhibit GAGase activity under in vitro conditions (29).Unexpectedly, ΔhylAΔhylB expressing hylA (pAOJ86) did not exhibit any HA degradation, but ΔhylAΔhylB expressing hylB (pAOJ84) fully degraded HA, confirming that HylB is indeed a hyaluronidase.
Four additional variants of HylA were generated for constitutive expression to account for potential issues in protein folding, including (i) full-length HylA with the LPXTG anchor intact (pAOJ83), (ii) HylA lacking both the LPXTG and FIVAR/repeat domains (pAOJ87), (iii) HylA lacking the non-enzymatic conserved domains at the Nterminus (pAOJ88), and (iv) a HylA chimera in which the LPXPTG anchor was removed and the N terminal region upstream of the predicted GAGase domain was replaced with the N terminal region upstream of the GAGase domain of HylB (pAOJ90).All resulted in secreted protein at the correct molecular weight except for pAOJ87 (Fig. S1), which may indicate a requirement for the FIVAR/repeat domain for proper protein expression, stability, or secretion.However, none of the HylA expression vectors conferred HA degradation, indicating that either none of the HylA constructs produce catalytically active enzymes or that HylA acts on a different substrate.
To determine if either HylA or HylB acts on other GAGs, we repeated the assay using CS (Fig. 5C) and HS (Fig. 5D).The strain expressing HylB degraded the majority of the CS, which was not entirely unexpected as hyaluronidases from other bacteria have been shown to exhibit activity against CS (41).In contrast, the only phenotype observed for the strain expressing HylA was a slight though statistically significant decrease in HS levels.
An important consideration when studying GAG degradation is the final product size.Host hyaluronidases and other factors such as oxidative damage (42) can cleave HA into intermediate-sized fragments (<500 kDa) or oligosaccharides that act as damageassociated molecular patterns and stimulate inflammation via TLR-2 and/or TLR-4 (10,43,44), while hyaluronidases from some pathogenic bacteria can cleave terminal disacchar ides from HA which are anti-inflammatory (18,45).Since the semi-quantitative GAG degradation assay is based on precipitation of high molecular weight GAGs, it would not detect subtle decreases in GAG fragment size.We therefore incubated HA directly with The indicated E. faecalis strains were grown in BHI for 24 h at 37°C, then filter-sterile supernatant was concentrated on a 10 kDa mol wt cutoff filter, and the retentates were incubated with 1 mg/mL HA in reaction buffer for 24 h before subjecting the samples to electrophoresis.live E. faecalis V587, ΔhylAΔhylB, or ΔhylAΔhylB harboring each of the constitutive hyaluronidase constructs or a vector control (pAOJ55) for 24 h and used agarose gel electrophoresis to examine HA degradation product size (Fig. 5E).An uninoculated control was incubated under the same conditions.Since E. faecalis acidifies BHI to a pH of ~5.7 after 24 h, an additional BHI control was adjusted to pH 5.7 with lactic acid to account for any HA degradation that might occur due to acidification alone.Incubation of HA with commercially available hyaluronidases from Streptomyces hyalurolyticus (HylSh) and Bovine Testicular Hyaluronidase Type I-S (BovHyl), that both digest HA down to short oligosaccharides (46), were utilized as positive controls.
Incubation with HylSh and BovHyl fully degraded the HA to a small band at ~5 kDa, while no degradation was observed for either BHI control (Fig. 5E).Importantly, HylSh is highly specific for HA (46), confirming the identity of the bands stained by this method.The strain containing pAOJ84 (hylB) displayed obvious digestion of HA and left only a low molecular weight smear of degradation products visible on the gel, albeit larger than the ~5 kDa products of HylSh or BovHyl (Fig. 5E).No degradation was observed for any of the HylA constructs.We hypothesized that HylA may only act on lower molecular weight HA fragments produced through the action of HylB, and further digest them to even smaller oligo-or disaccharides.To test this hypothesis, we incubated HA with a combination of ΔhylAΔhylB carrying pAOJ83 (full-length hylA, in case the LPXTG cell wall anchor was required for full activity) and ΔhylAΔhylB carrying pAOJ84 (hylB).The combination still only resulted in a low molecular weight smear of HA similar to that of ΔhylAΔhylB carrying pAOJ84 alone (Fig. 5E), indicating that HylA does not provide any additional depolymerization of HA digested by HylB.
A puzzling observation was that all E. faecalis strains including ΔhylAΔhylB partially depolymerized HA, resulting in a large smear that ran lower on the agarose gel than the BHI negative controls at either neutral pH or pH 5.7 (Fig. 5E).This observation suggests that E. faecalis V587 has an additional mechanism to partially degrade HA that would not have been apparent in the semi-quantitative assay (Fig. 5B) as large fragments would still precipitate and appear as undigested HA.To determine if E. faecalis exported protein was responsible for the observed basal level of HA degradation, HA was incubated with cell-free supernatants of E. faecalis ΔhylAΔhylB carrying pAOJ84 (hylB), pAOJ86 (hylA), or pAOJ55 (vector control) that had been filter sterilized and concentrated using a 10 kDa molecular weight cutoff filter (Fig. 5F).Concentrated supernatants from the strain constitutively expressing hylB showed partial digestion of high molecular weight HA, possibly suggesting that continual production of HylB is required for reaction comple tion.However, no HA degradation was observed for any of the other strains, indicating that the partial HA degradation that occurs during incubation with live bacteria is not mediated by a soluble, secreted product ≥10 kDa.
Intrigued by this result, we searched the genome of V587 and the related V583 for other potential GAGases and noted the presence of a putative heparinase, EF2268, that could be responsible for the weak digestion.No signal peptide was predicted in the EF2268 amino acid sequence by SignalP 5.0, which would align with our observa tion that HylB was the only large, secreted product that depolymerized HA (Fig. 5E).We therefore deleted ef2268 from the ΔhylAΔhylB strain background to generate a triple GAGase mutant.Surprisingly, partial depolymerization was still observed during incubation with the triple mutant, demonstrating that EF2268 is not responsible for the basal HA degradation (Fig. S2).

HylB purified from E. coli retains hyaluronidase activity
To characterize the activity of purified HylB enzyme, we cloned hylB into the E. coli expression plasmid pBAD/myc his A, transformed the plasmid into E. coli BL21(DE3) pLysS, and purified the recombinant HylB-Myc-6x His protein.The extracted protein was of high purity (Fig. S3).While E. coli BL21(DE3) pLysS is not suspected to encode any enzymes that could degrade HA, we used lysate from a culture of E. coli BL21(DE3) pLysS expressing unrelated proteins in a pBAD/myc his A background to serve as a negative control.Hyaluronidase activity was measured in a turbidimetric assay designed for use with purified protein rather than live bacteria.In this assay, HylB-Myc-6x His achieved complete HA digestion in less than 15 min, compared with ~40% HA remaining with bovine hyaluronidase at equimolar protein concentration (Fig. 6A).Digestion product size was also examined by agarose gel electrophoresis.Incubation of a 601 kDa HA fragment with Streptomyces hyaluronidase (HylSh) resulted in complete digestion of HA, such that no remaining product was visible on the gel (Fig. 6B).Incubation with recombinant HylB-Myc-6x His resulted in near complete HA digestion with only a low molecular weight smear around ~30 kDa remaining, mirroring the results obtained with live E. faecalis cells (Fig. 6B).Importantly, incubating HA with the E. coli protein lysate showed no digestion in either the gel or turbidometric assay (Fig. 6A and B), confirming that all activity was derived from HylB-Myc-6x His.Combined, these data confirm HylB as a hyaluronidase, and demonstrate that purified HylB enzyme is sufficient for complete digestion of HA.While a similar approach was tried for expression and purification of HylA, all attempts were unsuccessful.Thus, the function of HylA remains to be defined.

E. faecalis does not use HA as a nutrient source during growth in vitro
Some bacteria can use HA as a nutrient source after breaking down the repeating polymers into N-acetyl-glucosamine and glucuronic acid monomers.For example, the hyaluronate lyase activity of S. pneumoniae allows this species to use HA as a carbon source in minimal media (21).We therefore sought to determine whether HA degrada tion by HylB could support growth of E. faecalis in a simplified defined media (SDM) (47) in which E. faecalis is unable to grow without a supplied carbohydrate source  (47).E. faecalis V587 exhibited robust growth in SDM supplemented with glucose, but failed to grow in SDM with HA as the sole carbohydrate (Fig. 7A).However, HylB may not be active in WT E. faecalis under these conditions, even when HA is supplied as the sole carbohydrate source.Since HylB-mediated degradation of HA was clearly observed in the semi-quantitative GAG degradation assay in 0.5× TSB, we next asked whether HA supplementation enhanced growth when HylB was constitutively expressed.V587, ΔhylAΔhylB, and ΔhylAΔhylB with pAOJ84 (constitutively expressing hylB) all grew similarly in 0.5× TSB (Fig. 7B) and they all achieved stationary phase at a similar rate and density when supplemented with HA (Fig. 7C).The addition of unfractionated HA caused a slight reduction in the cell density at which the stationary phase was achieved for all strains.However, constitutive expression of hylB resulted in an increase in ΔhylAΔhylB culture density from 8 to 24 h that was not observed for the other strains (Fig. 7C), indicating that HylB activity either provides E. faecalis with an additional nutrient source or alleviates any inhibition imposed by the presence of high molecular weight HA.

HylA and HylB contribute to suppression of LPS-induced inflammation but do not dampen inflammation during CAUTI
Hyaluronidases from other Gram-positive bacteria have been shown to mediate inflammatory regulation (18).For example, S. agalactiae digests HA to disaccharides that facilitate pathogenesis by interfering with TLR-2/TLR-4 signaling ( 18) and subverting neutrophil and macrophage killing (19,48,49).Since E. faecalis is well-known for the ability to modulate the immune response, including suppression of lipopolysaccharide (LPS) stimulated inflammation (50,51), we sought to determine whether HylA and HylB contribute to the modulation of the innate inflammatory response using a well-estab lished NF-κB macrophage-like reporter cell line, RAW-Blue.This cell line expresses a secreted embryonic alkaline phosphatase (SEAP) under the control of an NF-κB inducible promoter, allowing for a readout of NF-κB induction via an alkaline phosphatase assay.
To determine if HA disaccharides dampen NF-κB activation in this cell line, RAW-Blue cells were incubated for six hours with increasing concentrations of HA disaccharides, LPS, and each combination thereof (Fig. 8A).Interestingly, the HA disaccharides appear to be pro-inflammatory at 0 and 0.1 ng/mL of LPS, but dampened NF-κB induction at higher concentrations of LPS.While these effects were all modest, the data confirm that HA disaccharides can dampen LPS-stimulated activation of NF-κB in RAW-Blue cells.
We next examined NF-κB activation in response to infection.In preliminary experi ments, we observed that multiplicities of infection (MOIs) greater than 10 resulted in substantial acidification of the culture media before assay completion.Since low pH has been shown to interfere with the NF-κB pathway (52)(53)(54), an MOI of 10 was chosen for all experiments.RAW-Blue cells were incubated for 6 h with wild-type E. faecalis V587, ΔhylA, ΔhylB, ΔhylAΔhylB, ΔhylAΔhylB with pAOJ83 (constitutively expressing the full-length HylA protein), or ΔhylAΔhylB with pAOJ84 (constitutively expressing the full-length HylB protein) with or without 10 ng/mL LPS and 100 µg/mL of high molecular weight HA (~700 kDa).In uninfected cells, LPS supplementation resulted in a ~5-fold increase in NF-κB activation, HA alone had no impact, and LPS+HA had lower NF-κB activation than LPS alone (Fig. 8B).
Infection with any of the E. faecalis strains resulted in a >6-fold increase in NF-κB activation over Dulbecco's modified Eagle's medium (DMEM) alone (Fig. 8C through H), clearly demonstrating that strain V587 is capable of inducing inflammation at an MOI of 10.The addition of HA during infection with V587 resulted in a slight though statistically significant decrease in NF-κB activation, which was also observed during infection with ΔhylB or ΔhylAΔhylB constitutively expressing the full-length HylA protein but not during infection with any strain lacking hylA.These data suggest that HylA may provide a small degree of immune suppression in the absence of other stimuli.
Supplementation with LPS during infection resulted in much greater NF-κB activity for all strains compared to infection in DMEM alone (~10-fold induction vs ~6-fold), demonstrating that E. faecalis V587 does not suppress LPS-stimulated NF-κB activation under these experimental conditions.This is in contrast to prior reports and likely due to differences in MOI and subsequent media acidification.The addition of HA slightly mitigated NF-κB activation during infection, as HA + LPS resulted in a modest though significant decrease in NF-κB activation for all strains except the ΔhylAΔhylB double mutant.Since loss of either gene alone or complementation with either gene alone allowed for a decrease in NF-κB activation, these data suggest that digestion of HA by either HylA or HylB is sufficient to modestly suppress LPS-stimulated inflammation.
We next sought to determine whether HylA or HylB contributes to immune modula tion during CAUTI.Since individual mutants exhibited colonization defects at 48 hpi in the CAUTI model, we conducted three additional infection studies with V587 and the ΔhylAΔhylB mutant to examine colonization at 6 hpi and two studies at 24 hpi (Fig. 9A  and B).No significant differences in colonization were observed at these earlier time points, allowing us to pinpoint the potential contribution of HylA and HylB on immune modulation.
For the initial assessment of the innate immune response, bladder homogenates were pooled by experiment date and infection group, resulting in three biological samples per inoculum at 6 hpi and two biological samples at 24 hpi.Homogenates from mock-infec ted [phosphate-buffered saline (PBS)] catheterized mice were also included for each time point.Samples were then analyzed by Luminex to quantify the levels of 32 cytokines, chemokines, and growth factors, of which 14 were above the limit of detection (Fig. 9C and D; Fig. S4).Unexpectedly, the global profiles of the catheterized mock-infected PBS mice were not significantly different from either infection group at either time point, suggesting that the majority of the inflammation was driven by the presence of the catheter.Thus, E. faecalis V587 does not appear to substantially dampen catheter-driven inflammation, and also does not further stimulate the immune response in this model.
To determine the potential contribution of HylA and HylB to inflammation, the profile of bladders from mice infected with V587 was compared to those infected with the ΔhylAΔhylB double mutant.Interestingly, the only analyte that was significantly different at any time point was a reduction in IL-6 at 6 hpi for mice infected with ΔhylAΔhylB (P = 0.0004).This observation was unexpected, as we hypothesized that HylA and HylB would most likely dampen the immune response, resulting in an increase in pro-inflammatory

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Infection and Immunity cytokines during infection with the mutant strain.To confirm whether HylA and HylB might contribute to IL-6 induction, an additional set of mice were inoculated as above along with an extra control group lacking the catheter segment, and IL-6 was quantified by ELISA (Fig. 9E).As expected, the presence of the sterile catheter segment alone resulted in ~8-fold induction of IL-6 in the absence of infection, but no further increase in IL-6 was observed during infection with V587.These data confirm the hypothesis that the catheter segment drives the majority of inflammation in this model at 6 hpi.Further, IL-6 levels were significantly lower in mice inoculated with the ΔhylAΔhylB double mutant compared to V587, confirming that HylA or HylB contribute to stimulation of IL-6.It is important to note that the effect is modest at 6 hpi and appears to be resolved by 24 hpi, and likely does not substantially contribute to the differences in dissemination that are observed in this model.In summary, HylA and HylB may contribute to immune modulation but do not appear to be the primary mediators of immune suppression during CAUTI.

DISCUSSION
E. faecalis infections are very common and impose a high burden on the healthcare system, ranking as the fifth most commonly encountered healthcare-acquired pathogen in the United States overall, both in pediatric patients and adults (1, 2).E. faecalis can cause a variety of infections, including CAUTI and CLABSI (2,3).In this study, we revealed HylA and HylB as two new virulence factors for CAUTI and bacteremia caused by vancomycin-resistant E. faecalis.
Even though we detected hylA and hylB mRNA transcripts in WT E. faecalis V587 during growth in vitro, we did not detect any hyaluronidase activity in the WT strain.These data confirm prior work indicating a lack of HA degradation by E. faecalis.However, by cloning hylB under a constitutive promoter in E. faecalis strain V587, we conclusively demonstrated that HylB (EF0818) is a bona fide hyaluronidase with substantial chondroi tinase activity.This enzyme appears to act specifically on HA and CS, as we did not detect any evidence of HS digestion.We further demonstrated that the expression and purification of HylB in E. coli also provide robust hyaluronidase activity, showing that HylB is both necessary and sufficient to digest HA.As the amino acid sequence of HylB in E. faecalis V587 shows low identity with previously characterized Gram-positive bacterial hyaluronidases (55)(56)(57)(58)(59), structural studies of this enzyme could provide critical information for GAGases in general.
In contrast to HylB, the only in vitro evidence of enzymatic activity for HylA (EF3023) was a very subtle degradation of HS and a potential role in dampening NF-κB activation in the presence of HA.However, hylA contributed to bladder colonization and dissem ination to the bloodstream in the mouse model of CAUTI and appeared to provide functional redundancy to hylB in the bacteremia model.There are many potential explanations for the discrepancy between the in vitro and in vivo phenotypes of HylA.One possibility is that none of the HylA expression constructs resulted in properly folded or active enzymes.Alternatively, HylA may require processing by additional factors not present under our in vitro conditions to become a catalytically active enzyme.For example, the HylB enzyme of S. agalactiae requires post-translational proteolytic cleavage to be active (60).Another possibility is that the substrate of HylA may be a specific glycosaminoglycan, glycoprotein, or sulfonation pattern that was not directly tested in this study but is present in the urinary tract and circulatory system.This would not be the first time that a predicted bacterial hyaluronidase turned out to have a different target substrate; Streptococcus pyogenes Spy1600 was initially labeled as a possible hyaluronidase but experimentally confirmed to act as a β-N-acetylgluco saminidase (61).Experimentation with additional host polysaccharides and expression constructs will be needed to either confirm or disprove HylA as a glycosaminoglycandegrading enzyme.
While some bacteria such as S. pneumoniae can use HA as a carbon source (21), this was not the case for E. faecalis in vitro.However, constitutive expression of hylB did allow the ΔhylAΔhylB double mutant to achieve higher cell density during stationary phase with HA supplementation than even the WT strain.Considering the proximity of hylB to a putative oligosaccharide PTS system, there is likely additional regulation that must occur for E. faecalis to potentially use HA degradation products as a nutrient.Our data also do not rule out a possible role for HA catabolism to fuel growth during infection.
The contribution of hyaluronidases to microbial pathogenesis has been studied in S. agalactiae in the context of pre-term birth (48,49) and systemic infection (19).During systemic infection in mice, disruption of hyaluronidase by mutation of hylB decreased tissue colonization and lethality (19).In a nonhuman primate model, hyaluronidase activity decreases neutrophil bactericidal activity by interfering with TLR2/ TLR4 signaling, and a hylB mutant deficient in hyaluronidase activity was deficient in establishing fetal invasion and bacteremia (48).In mice, the hylB mutant colonized the vaginal tract to a similar level as the parental isolate but exhibited defects in ascend ing infection and in the immune-subversion (19).Vaginal colonization has also been examined in E. faecalis strain OG1RF using a genome-wide transposon mutant screen for factors that allow E. faecalis to persist within the vaginal tract (62).Intriguingly, the homolog of hylB in this strain (OG1RF_10550) exhibited reduced colonization at all time points (62).However, OG1RF_10550 was just one of many genes identified as important for vaginal colonization in this study, and we are unaware of any follow-up studies on this gene.Considering that our studies demonstrate that E. faecalis HylA primarily contributes to bladder colonization and dissemination to the bloodstream while HylB contributes to kidney colonization and 45% of E. faecalis isolates possess homologs of both genes, expression and activity are likely highly regulated.This is supported by the recent observation that HylA expression is regulated by a BglG/SacY antiterminator homolog in E. faecalis strain V19, a plasmid-cured derivative of V583 (63).Further exploration into the conditions and signaling pathways required for each protein to be expressed and active is expected to provide further insight into how E. faecalis is able to adapt to many different infection niches.
Hyaluronic acid also modulates inflammatory signaling, with the outcome being largely dependent on HA fragment size.Hyaluronidases from S. agalactiae and other Gram-positive pathogens were shown to digest HA down to disaccharides, which block TLR-2 and TLR-4 signaling (18).In contrast, Streptomyces hyaluronidase produces HA oligosaccharides that stimulate inflammation, as do mammalian hyaluronidases (18).Our data indicate that infection of RAW-Blue cells with E. faecalis V587 activates NF-κB regardless of condition or infecting strain, but constitutive expression of HylB can modestly counteract LPS-stimulated activation of NF-κB.Thus, HA degradation by E. faecalis V587 could contribute to immune modulation during infection.
We also examined a broad array of cytokines, chemokines, and growth factors in the bladders and the kidneys of mice infected via our CAUTI model.Strikingly, we did not observe drastic differences between infected and mock-infected catheterized mice, which led us to confirm that the majority of inflammation during CAUTI with E. faecalis V587 is due to the insertion of the catheter itself.We also did not observe a dramatic difference in immune response between infection with wild-type V587 and the ΔhylAΔhylB strain, other than a slight reduction in IL-6 in the bladders of mice infected with ΔhylAΔhylB at 6 hpi.Infection with E. faecalis as well as catheter implantation are both known to stimulate IL-6 (64)(65)(66), so this observation may suggest a role for HylA or HylB in tissue invasion and damage.
In summary, this study highlights the importance of in vivo validation of in silico functional predictions.We have confirmed HylB as a hyaluronidase/chondroitinase enzyme in vancomycin-resistant E. faecalis, despite having low amino acid identity to other bacterial hyaluronidases.We have further demonstrated that active HylB can be produced in vitro, both by E. faecalis and by E. coli, and that hylA and hylB both contribute to E. faecalis pathogenesis.Further examination of the substrate and functions of HylA and the distribution of its target in mammalian hosts may reveal novel host-pathogen interactions or new information about GAG distribution in mammalian tissues.

Animal model
Mice were anesthetized with a weight-appropriate dose (0.1 mL for a mouse weighing 20 g) of ketamine/xylazine (80-120 mg/kg ketamine and 5-10 mg/kg xylazine) by intraperitoneal injection and euthanized by CO 2 with vital organ removal.

Bacterial strains and culture conditions
Vancomycin-resistant E. faecalis strain V587 (NR-31979) (31) was obtained through BEI Resources, NIAID, NIH.This strain was chosen for its clinical history that mirrors our mouse infection models, and for containing a large number of known E. faecalis virulence genes (67), including Enterococcal Surface Protein (esp) previously implicated in urinary tract colonization (68).E. faecalis V587 and isogenic mutants were routinely cultured from frozen glycerol stocks in 5 mL of BHI broth (Dot Scientific) or (TSB with 500 µg/mL gentamicin sulfate (ACROS Organics, catalog #455310250) in tightly capped 14 mL polypropylene tubes at 37°C with shaking at 300 RPM.E. faecalis harboring plasmids were cultured with 20 µg/mL chloramphenicol (Research Products Interna tional, catalog #C61000) instead of gentamicin.

RNA extraction and qRT-PCR
E. faecalis V587 was grown overnight in BHI plus gentamicin 500 µg/mL, 0.6 mL aliquots of the overnight culture were centrifuged and washed once with PBS (pH 7.4), the cell pellet was resuspended in 0.6 mL of PBS, then diluted 1:100 in one of the following media: BHI, 0.5× TSB (TSB diluted to 50% concentration with sterile water), 0.5× TSB with 2.5 mg/mL ~5 kDa size fractionated HA (Lifecore Biomedical, catalog #HA5K), 0.5× TSB with 2.5 mg/mL unfractionated HA sodium salt from Streptococcus equi (Sigma-Aldrich), filter sterilized human urine (Lee BioSolutions catalog #991-03-P-FTD, lot #01J5567), human urine with 2.5 mg/mL ~5 kDa size fractionated HA, or human urine with 2.5 mg/mL unfractionated HA.About 3 mL cultures was used for samples with rich media as the base, and 6 mL cultures was used for the samples grown in urine to account for lower growth density.Cultures were incubated with shaking for 4 h at 37°C, pelleted by centrifugation, washed once with 1 mL of TE buffer (pH 8), then suspended in 50 µL RLT Buffer with BME (Qiagen RNeasy Mini Kit).About 1.5 mL safe-lock microfuge tubes (Eppendorf ) was filled halfway with 0.5 mm glass disruption beads (RPI Research Products International), the suspended pellet was added to the bead tube, and samples were homogenized using a Bullet Blender Gold (Next Advance) at max speed for 5 min.After bead beating, 250 µL of RLT Buffer with BME was added and samples were vortexed to mix.A small hole was punched in the bottom of the tube with an 18G needle (BD Precision Glide), and the tube was immediately placed inside a 1.5 mL microfuge tube to collect the homogenate while leaving the beads behind.The collected homogenate was centrifuged at 21,000 rcf for 10 min at 4°C, supernatant was removed, and 1 vol of 70% ethanol was added and vortexed to mix.The sample was then transferred to an RNeasy Mini spin column for on-column DNase digestion kit (Qiagen), and RNA was eluted with 40 µL of RNase-free water.A second DNase digestion was then performed off-column by adding 0.1 vol of 10× DNase buffer and 1 µL of DNase (Invitrogen) and incubating at 37°C for 30 min.The digestion was terminated by adding 0.1 volumes of DNase inactivation reagent, incubating at room temperature for 2 min, and centrifuging for 90 s at 8,000 rcf.cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad) and qRT-PCR was performed using PCRBio SyGreen Blue Mix Lo-Rox (PCR Biosystems) and a Bio-Rad CFX-Connect Real-Time System.Data were normalized to recA as the reference gene and analyzed via the ∆∆CT method.

Generation of E. faecalis deletion mutants
Markerless deletion mutants of E. faecalis V587 were constructed by allelic exchange using the plasmids designated in the Supplemental primer table.Roughly 1,000 base pairs upstream and downstream of the desired deletion location were amplified via PCR and assembled into allelic exchange vector pIMAY (Addgene plasmid #68939, gifted by Tim Foster) (69) via either restriction digest and ligation or NEBuilder HiFi DNA Assembly Mastermix (NEB), as indicated in the Supplemental vector table.Assembled vectors were propagated in E. coli Top10 and verified a combination of restriction digest and sequencing (Plasmidsaurus).
Plasmids were electroporated into E. faecalis, recovered and plated at 30°C, and isolated colonies were struck onto pre-warmed BHI-chloramphenicol plates and incubated overnight at 37°C to induce plasmid integration (70).Chloramphenicol-resist ant colonies were then struck on BHI agar containing 1 µg/mL anhydrotetracycline hydrochloride (Cayman Chemical catalog #10009542) to induce expression of the counter selection marker, and incubated overnight at 30°C.Large, isolated colonies from these plates were then re-struck on anhydrotetracycline BHI plate and incubated at 37°C overnight to ensure loss of the plasmid backbone.Mutants were PCR verified by checking for the presence of the desired mutation, the absence of the original gene, the absence of the pIMAY backbone, and retention of natural V587 plasmids using the primers described in the Supplemental primer table.The esp gene was also amplified using esp11 and esp12 from a previous publication (71).Verified colonies were cultured in BHI-gentamicin broth overnight to generate glycerol stocks and PCR-verified a second time.The ΔhylAΔhylB double mutant was generated by sequential deletion of hylB from the ΔhylA strain using the method described above, and was verified by PCR as described above and by sequencing of ~1,200 base pairs up and downstream of each gene (Eton Biosciences).

Hyaluronidase and chondroitinase plate assay
HA plates were made using a published recipe (18).A hyaluronic acid solution was first generated by suspending 300 mg unfractionated HA sodium salt and 8 g of bovine serum albumin (BSA) fraction V at pH 5.2 (Sigma-Aldrich) in 200 mL of water and slowly adjusting to pH 7.5 using 0.1 M sodium hydroxide.The HA solution was then filter sterilized and warmed to 37°C.The agar base (10 g Noble agar, 10 g yeast extract, 30 g Todd-Hewitt broth powder, and 800 mL of RO water) was autoclaved, cooled to ~56°C, supplemented with the warmed hyaluronic acid solution, and 16 mL was distributed into petri dishes.Once cooled, plates were inoculated with 10 µL of overnight cultures of E. faecalis or L. lactis and incubated overnight at 37°C.After overnight incubation, plates were cooled to ambient temperature and flooded with 2 M acetic acid to precipitate high molecular weight GAGs, and incubated for at least 10 min.The acetic acid was then aspirated and the plates were imaged on an Axygen Gel Documentation System (Corning) with blue light illumination.

Mouse infection models
For establishment of CAUTI, 6-8-week-old female CBA/J mice (The Jackson Laboratory, strain #000656) were inoculated transurethrally with 50 µL of a suspension of 2 × 10 6 colony forming units (CFU)/mL in PBS of either wild-type V587, ΔhylAΔhylB, ΔhylA, ΔhylB, or mock-infected using PBS.A 4 mm segment of silicone tubing was placed in the bladder during inoculation to simulate a urinary catheter, as previously described (40,72).At 6, 24, or 48 h post-infection, mice were sacrificed, and the bladders, kidneys, and spleens were homogenized in 5 mL tubes (Eppendorf ) containing 500 µL of 3.2 mm stainless steel beads (Next Advance) and 1 mL of sterile PBS in a Next Advance 5 E Gold Bullet Blender.Organ homogenates were plated for CFU counts and an aliquot of each homogenate was flash-frozen and stored at −80°C for cytokine and chemokine measurements as described below.
For establishment of bacteremia, 7-week-old female CBA/J mice were inoculated via tail vein injection of 100 µL of a suspension of 5 × 10 8 CFU/mL of either wild-type V587, ΔhylA, ΔhylB, or ΔhylAΔhylB.Twenty-four hours post-infection, the mice were sacrificed, and the livers, spleens, and kidneys were harvested, homogenized and plated for CFU counts.
Due to technical issues encountered while propagating the constitutive expression constructs in E. coli, all ligation products were transformed into Lactococcus lactis NZ9000 (MoBiTec catalog #VS-ELS09000-01) for plasmid amplification.L. lactis competent cells were made and transformed using the previously described Lithium acetate-Dithiothrei tol (DTT) method (77).All constructs were sequence-verified via Plasmidsaurus before transformation into E. faecalis V587 ΔhylAΔhylB.

Secreted protein profiles of E. faecalis V587, ΔhylAΔhylB, and constitutive expression strains
E. faecalis overnight cultures were diluted 1:200 into 50 mL conical tubes containing 20 mL BHI.Cultures were further supplemented with 20 µg/mL chloramphenicol for ΔhylAΔhylB with pAOJ83, pAOJ84, pAOJ86, pAOJ87, pAOJ88, and pAOJ90, or 500 µg/mL gentamicin sulfate for V587 WT and V587 ΔhylAΔhylB.The tubes were tightly capped and incubated with shaking for 4 h, at which point the cells were pelleted and the supernatant filter sterilized using a 0.22-µm PES filter.The 4-h time point was chosen to balance bacterial cell density while limiting contact time with the broad-spectrum E. faecalis proteases GelE and SprE, which exhibit peak expression in the late exponential phase (78).About 20 mL of filter-sterilized supernatant was placed on ice to cool, mixed with 5 mL of cold 6.1 N trichloroacetic acid (TCA), and incubated at 4°C overnight for protein precipitation.Tubes were then centrifuged at 3,000 rcf for 30 min, supernatants discarded, and protein pellets washed two times with 90% acetone saturated with Tris base and once with 100% acetone.Pellets were air-dried, resuspended in 0.5 mL of 2× Laemmli Sample Buffer (Bio-Rad), and incubated at 95°C for 10 min.Samples were then cooled to room temperature and centrifuged at 21,130 rcf for 10 min to remove the remaining insoluble material for better gel resolution.About 10 µL of each sample was loaded into the lanes of a Bio-Rad Any kD SDS-PAGE gel.Gels were washed and stained with Coomassie Brilliant Blue R-250 and destained with water/methanol/acetic acid 50:40:10 until protein bands were clearly distinguishable from background.Gels was photographed with a Nikon Z30 on white background.Photographs were adjusted using Adobe Photoshop 2022 for exposure, levels, hue, saturation and/or contrast until fainter bands, easily visible by eye, were also distinguishable from background in the captured image.These adjustments were applied to the entire photograph equally.

Semi-quantitative in vitro measurement of E. faecalis GAGase
A recently published semi-quantitative turbidimetric assay of GAGase activity was used to examine the profiles of wild-type E. faecalis V587, ΔhylAΔhylB, and ΔhylAΔhylB with each constitutive expression construct (29).E. faecalis strains for GAG degradation assays were cultured overnight at 37°C in 3 mL of BHI with 20 µg/mL of chloramphenicol for plasmid containing strains.Cultures were then diluted 1:4 in BHI, adjusted to an OD 600 nm of 0.04, pelleted at 6,010 rcf for 8 min, and the supernatant was removed.Cell pellets were resuspended in 1 mL of 0.5× TSB containing 2.5 mg/mL of either hyaluronic acid (Sigma-Aldrich, catalog #53747), chondroitin sulfate A (Sigma-Aldrich, catalog #C9819), or heparin sodium salt (Fisher BioReagents, catalog #BP2425), and 300 µL aliquoted in triplicate into 96-well plates.Plates were incubated in a hypoxia chamber for 24 h at 35°C with 10% CO 2 and 5% O 2 , followed by a semi-quantitative GAG degradation measurement as previously described (29).Briefly, the OD 600 nm of all replicates was measured, bacteria were pelleted by centrifugation at 3,214 rcf for 10 min, 180 µL of supernatant was transferred from each well to a new 96-well plate, and supernatants were then serially diluted in 1× PBS. 10 µL of 10% BSA was then added to all wells and OD 600 was measured.Finally, 40 µL of 2M acetic acid was added to all wells, mixed by gentle stirring with the pipette tip, and OD 600 was measured again.The percentage of GAG remaining was calculated by comparing the post-acetic acid OD 600 values of all experimental conditions to bacteria-free controls (29).Percentage of GAG remaining was calculated using the following formula using the OD 600 reading of the post-acetic acid serial dilution falling in the center of the linear range of the assay for each condition.

=
Bacteria Post Acetic Acid OD 600 − Average Pre Acetic Acid OD 600 Control Post Acetic Acid OD 600 − Average of Pre Acetic Acid OD 600 × 100

Agarose gel electrophoresis for determination of GAG degradation product size
About 100 µL of indicated E. faecalis overnights was diluted into 20 mL of BHI contain ing appropriate antibiotics and 1 mg/mL HA (Sigma-Aldrich), incubated for 24 h at 37°C with shaking, pelleted, and supernatants were filter sterilized.Two uninoculated HA BHI tubes with 20 µg/mL chloramphenicol were incubated the same way was included as a negative control, one of which had no pH adjustment, and one was acidified to pH 5.7 using 85%-90% lactic acid (BeanTown Chemical) to simulate E. faecalis fermentation.Hyaluronidase-positive controls were made by incubating HA BHI with either 12.5 µg/mL Bovine Hyaluronidase (Sigma-Aldrich) or Streptomyces hyalurolyticus hyaluronidase (Sigma-Aldrich) at 3 units/mL.Agarose gel electrophoresis was followed using a modified protocol from Echelon Biosciences (79) to determine HA size ranges.About 750 µL of each supernatant was added to 250 µL of loading buffer [0.02% Bromophenol blue, 2 M sucrose in 1× Tris Borate EDTA (TBE) buffer], 10 µL of which was added to each well of a 2% (wt/vol) agarose gel and electrophoresis was performed at 100 V in TBE buffer.Gels were washed with 30% (vol/vol) ethanol for ~15 min to fix the HA fragments within the gel prior to staining, and then washed in 50% (vol/vol) ethanol for ~1 h to remove the aqueous buffer as Stains-All dye has low water solubility.Gels were transferred to 0.01% Stains-All in 50% ethanol (Alfa Aesar) overnight, then destained with 50% ethanol until bands were clearly distinguishable from background staining.Gels were imaged on a ChemiDoc MP (Bio-Rad) using the DyLight 680 setting.
The gel assay was also utilized to determine what HA size ranges were produced by recombinant HylB.For these experiments, 1 mg/mL of 601 kDa HA (Echelon Biosciences, catalog #HYA-601KEF-1) was incubated with either 27.5 µg/mL of recombinant HylB, 3 units/mL of Streptomyces hyaluronidase, or 50 µg/mL E. coli protein overnight at 37°C, and gels were prepared as above.

Production of E. faecalis concentrated cell-free supernatants for determina tion of GAG degradation by secreted products
To collect E. faecalis protein supernatants, 50 mL conical tubes containing 20 mL of BHI were inoculated with 100 µL of ΔhylAΔhylB with pAOJ86, pAOJ84, or pAOJ55 and incubated for 4 h with shaking at 37°C.After incubation, cultures were centrifuged and supernatants were filter sterilized and concentrated using a 10 kDa molecular weight cutoff filter.Retentates were then mixed with an equal proportion of 2 mg/mL HA in 20 mM sodium phosphate (pH 7) with 77 mM sodium chloride.A blank containing only the HA in phosphate buffer mixed 1:1 with PBS was also used.All samples were incubated for 24 h statically at 37°C then subjected to agarose gel electrophoresis as described above.

Cloning, expression, and purification of HylB Myc-6x His
The HylB expression vector pAOJ66 was generated by amplifying the ef0818 gene with primers AOJ_651 and AOJ_652 and cloning into NcoI-HF/ApaI digested pBAD/myc his A (Invitrogen).The vector was propagated in E. coli TOP10, then transformed into E. coli BL21(DE3) pLysS (80) and cultured overnight in 50 mL LB with 100 µg/ml carbenicillin (Research Products International catalog #C46000) and 20 µg/mL chloramphenicol.The following day, 10 mL aliquots were subcultured into four 2 L flasks containing 900 mL pre-warmed Terrific Broth (81) supplemented with 10 mM of MgCl 2 and 100 µg/mL carbenicillin.Flasks were incubated with shaking at 37°C until the OD 600 reached 0.4-0.5 (~3 h post-inoculation), after which 100 mL of 200 mM arabinose was added to each flask (20 mM final concentration) to induce protein expression (82).Cultures were incubated for 4 h at 37°C with shaking, centrifuged at 8,000 rcf for 10 min, and pellets were stored at −80°C overnight.
Cell pellets were thawed on ice, suspended in 200 mL total volume of lysis buf fer [PBS pH 7.4 with 35 µM polymyxin B, 2% Triton X-100, 2 SIGMAFAST EDTA-free protease inhibitor tablets (Sigma-Aldrich, catalog #S8830-20TAB), 20 µL Benzonase nuclease (Sigma-Aldrich), and 50 mg of egg white lysozyme (Gold Biotechnology)], then incubated at 37°C for 30 with gentle agitation to lyse the cells.Lysates were centrifuged at 8,000 rcf at 4°C for 30 min to remove insoluble material.The supernatant was filtered through a 0.22-µm PES membrane, and mixed with cold ammonium sulfate solution in PBS (pH 6) to a final concentration of 20% (vol/vol) ammonium sulfate.Samples were then centrifuged at 14,500 rcf for 45 min at 4°C to remove higher molecular weight contaminants, supernatants were mixed with ammonium sulfate solution to a final concentration of 75% (vol/vol), and protein was precipitated overnight at 4°C.The next day, supernatants were centrifuged for 30 min at 14,500 rcf at 4°C, the resulting protein pellet was dissolved in 100 mL of cold PBS pH 7.4 with 10 mM imidazole, and passed through a 3 mL His-Pur Ni-NTA column (Thermo Scientific, catalog #88226) according to the manufacturer's instruction.The column-bound protein was eluted with 10 mL of 250 mM of imidazole in PBS pH 7.4, washed with PBS on a 10 kDa molecular weight cutoff column to remove imidazole, further washed with PBS + 5% glycerol, and the retentate was snap frozen and stored at −80°C.
Concentration and purity of the recombinant protein were estimated using the Blue Dry Western technique (83) via a Trans-Blot Turbo RTA Transfer Kit, PVDF (Bio-Rad, catalog #1704272), and Trans-Blot Turbo transfer apparatus (Bio-Rad) on the "large protein" pre-programmed setting.The blot was stained with the primary mouse α-c-Myc Monoclonal 9E10 antibody (Invitrogen, catalog #13-2500) and Goat anti-mouse IgG1 Cross-Adsorbed Horseradish Peroxidase secondary antibody (Invitrogen, catalog #A10551), imaged colorimetrically (for Coomassie Brilliant Blue R-250), treated with Clarity Western ECL Substrate (Bio-Rad, catalog #170-5060), and imaged for chemilumi nescence on a Bio-Rad Chemi Doc MP imager.The chemiluminescence and colorimetric images were overlaid with the multi-image setting on the Chemi Doc MP to find bands containing the c-Myc tag of the recombinant protein.Densitometry was performed using Adobe Photoshop on raw colorimetric images to determine protein concentration by comparison to a BSA dilution series of known concentrations loaded on the same gel.

Assaying hyaluronidase activity of HylB Myc-6x His
A turbidimetric assay similar to the protocol developed by Sigma-Aldrich (84) was used to measure HA digestion by recombinant HylB.The assay consists of four reagents: Reagent A, the phosphate buffer solution, consisting of 20 mM sodium phosphate (pH 7) with 77 mM sodium chloride; Reagent B, the substrate solution, was HA sodium salt (Sigma-Aldrich) dissolved to a concentration of 0.3 mg/mL in reagent A; Reagent C, the enzyme diluent, consisted of Reagent A with 0.01% (wt/vol) BSA; and Reagent D, an acidic stop solution, was made by dissolving 0.1% (wt/vol) BSA into a buffer consisting of 24 mM sodium acetate and 79 mM glacial acetic acid adjusted to a pH of 3.75 with 5 M hydrochloric acid.Bovine Hyaluronidase Type I-S (Sigma-Aldrich, catalog #H3506-500MG) was utilized for comparison as a positive control, and was diluted to a concentration of 12.5 µg/mL in Reagent C. HA incubated with BSA at 15 µg/mL was used as the negative control for calculation of HA degradation, and soluble E. coli protein lysate from BL21(DE3) pLysS (pBAD-PL-Lux) at 50 µg/mL in Reagent C was included as an additional control.Protein samples were mixed with an equal volume of Reagent B and incubated at 37°C for 15 min, then the reaction was stopped by adding an equal volume of Reagent D. Remaining high molecular weight HA was precipitated during a 10-min incubation at ambient temperature, the OD 600 nm was read on a Synergy H1 plate reader (BioTek), and the percentage of remaining HA was estimated by dividing the OD 600 nm of the BSA sample and multiplying by 100.
To determine if E. faecalis V587 could use HA as a sole source of carbon, the SDM recipe was used (47).Overnight cultures of E. faecalis V587 were centrifuged, washed once with an equal volume of PBS to remove traces of BHI, and diluted 1:100 in SDM with one of the following carbohydrate sources: 12 mM glucose (21), a twofold dilution series of HA (Sigma-Aldrich) starting at 2.5 mg/mL, or water (no carbon source).Growth was assessed as above.
Growth in human urine was assessed as previously described (40).Briefly, overnight cultures of E. faecalis were washed with PBS and diluted to an approximate cell density of 2 × 10 7 CFU/mL in sterile, pooled urine from healthy female donors (Lee BioSolutions catalog #991-03-P-FTD, lot #01J5567) and incubated at 37°C with shaking.CFU/mL was determined by dilution and plating every hour for 7 h.

RAW-Blue NF-κB reporter assay
NF-κB activation by E. faecalis was assessed using RAW-Blue cells as previously descri bed (50).RAW-Blue cells were obtained from InvivoGen (catalog #raw-sp) and routinely cultured at 37°C in a 5% CO 2 in pre-warmed DMEM (Corning catalog #10-017-CM) with 4.5 g/L glucose, 0.584 g/L L-glutamine, 10% fetal bovine serum (Sigma-Aldrich), and no pyruvate.For each experiment, low-passage vials of cells were thawed and incubated in Corning Falcon 75 cm 2 vented culture flasks at a density of 2 × 10 6 /15 mL of DMEM with 100 µg/mL Normocin (InvivoGen catalog #ant-nr) for approximately 72 h.Culture media were then removed, cells were dislodged with a cell scraper and suspended in 10 mL of DMEM with 200 µg/mL Zeocin (InvivoGen catalog #ant-zn), counted on a Beckman Coulter Z2 Particle Counter and Size Analyzer set to a 10-20 µm size range, diluted to 5 × 10 5 cells/mL in DMEM with 200 µg/mL Zeocin and 200 µL (1 × 10 5 cells), and 100 µL were seeded into a Corning Falcon 96-well flat bottom polystyrene tissue culture treated plate and incubated overnight.The following day, the media were removed, the RAW-Blue cells were washed once with Dulbecco's Phosphate-Buffered Saline (DPBS) and then incubated with either LPS and/or HA disaccharide sodium salt (Biosynth catalog #OH11537) or infected with E. faecalis at an MOI of 10 (1 × 10 6 CFU/well) in either DMEM or DMEM plus additives.MOI 10 was chosen as values higher than 10 were observed to acidify the media in a dose-dependent manner, low extracellular pH has been shown by to interfere with the NF-κB pathway (52)(53)(54).For infection experiments, media conditions used were: DMEM, DMEM with 100 µg/mL HA sodium salt at a molecular weight range of 500-749 kDa (Lifecore Biomedical, catalog #HA700K), DMEM with 10 ng/mL ultrapure LPS from E. coli O111:B4 (InvivoGen, catalog #tlrl-3pelps), or DMEM with both HA and LPS.
For all experiments, 200 µL of the treatment media or inoculum was added to the washed RAW-Blue cells and incubated for 6 h.The plate was then centrifuged at 3,000 rcf for 10 min, and 20 µL of supernatant was added to 180 µL of QUANTI-Blue Alkaline Phosphatase Detection Medium (InvivoGen catalog #rep-qbs), incubated at 37°C with shaking, and OD 640 was read every 15 min for up to 16 h to identify the optimal linear range.Under our experimental conditions, a 2-h incubation was found to produce reads near the end of the linear range of the reporter assay, so this time point was used for reporting NF-κB activation.

Analysis of cytokines, chemokines, and growth factors from mouse bladders and kidneys
Flash-frozen tissue homogenates were thawed on ice and pooled by infection date and inoculation group (WT, ΔhylAΔhylB, or mock-infected with PBS).Pooled samples were extracted using the Thermo Scientific Tissue Protein Extraction (T-PER) reagent bundle (Fisher Scientific catalog #B90002).About 600 µL of total pooled sample volume was lysed with 700 µL T-PER lysis buffer with 1 Pierce Protease Inhibitor Mini Tablet per 7 mL of T-PER lysis buffer.Vortexed samples were centrifuged for 5 min at 10,000 rcf at 4°C and the supernatant was removed.The protein concentration of 20 µL of supernatant was determined with Pierce Rapid Gold BCA Kit.Individual pooled samples were diluted to obtain 10 µg total protein in 50% T-PER buffer, 50% PBS.
Quantification of 32 different analytes in the adjusted bladder and kidney pools was provided by the Flow and Immune Analysis Shared Resource at the Roswell Park Comprehensive Cancer Center.The following analytes were measured: Eotaxin, G-CSF, GM-CSF, IFNγ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG, MIP-1α, MIP-1β, MIP-2, RANTES, TNFα, and VEGF, using a commercially available kit (MCYTMAG-70K-PX32, Millipore Sigma, Burlington, MA, USA).Data were acquired on a Luminex 200 instrument (Luminex Corporation, Austin, TX, USA).The experiment and instrument set-up were performed based on the manufacturer's kit instructions.Briefly, serially diluted standards were analyzed in duplicate wells, while the experimental samples were tested in single wells.The plate was incubated overnight with the multiplex beads on a plate shaker, in the dark, at 4°C and processed with the reporter reagents the next day, as per the manufacturer's instructions.Analyte concentrations were determined by extrapolating individual experimental fluorescence intensity values against each analyte's standard curve using the BeadView multiplex data analysis software, version 1.0 (Upstate Cell Signaling Solutions, Lake Placid, NY, USA).The analytical performance was checked using high-concentration and low-concentration quality controls provided with the kit, of which the determined concentration for each analyte needs to be within the manufac turer-determined concentration range.
Radar charts were developed for select Luminex analytes to visualize the multivariate and multi-scale data simultaneously on a single plot.Separate plots were made for 6 and 24 h using the same grid.The upper bound of the range (outer ring) for each variable was set as the log of the maximum average pg/mL values across replicates for V587, ΔhylA ΔhylB, and PBS.The inner rings represent 25%, 50%, and 75% of the maximum, respectively.Radar charts were developed in the R programming language using the "fmsb" packagev (85,86).

IL-6 ELISA
A colorimetric Mouse IL6 ELISA kit (Novus Biologicals NBP1-92668) was used to measure IL-6 from mouse tissue homogenates following the manufacturer's instructions.Briefly, the ELISA plate was washed, bladder homogenates were thawed on ice and gently vortexed, and 50 µL of homogenate was mixed with 50 µL of diluent.50 µL of Biotin conjugate was added to all wells (samples, blanks, and standards), the plate was incubated at room temperature for two hours with shaking at 400 rpm, then washed five times with a multichannel pipette.Strep-HRP was added and incubated for 1 h at room temperature with shaking at 400 rpm.The plate was then washed, developer was added, and OD 620 was monitored every minute on a BioTeck Synergy H1 until Standard 1 reached OD 0.9.Stop solution was then added and absorbance was read at 450 nm.

Statistical analysis
Statistical analysis was performed using GraphPad Prism Software version 9. Significance was determined using a P < 0.05.All P values are two-tailed at a 95% CI.For colonization data in both the CAUTI and bacteremia model, we performed two types of statistical analysis to assess fitness of wild type versus mutant strains.Data were first analyzed by Mann-Whitney U test (P MW < 0.05).Bacterial infection data do not necessarily follow distributions that are simple to perform standard statistical analysis on (87).To mitigate this issue, in addition to these tests, contingency tables of values above and below certain thresholds were constructed and analyzed for statistical significance by Fisher's exact test (P Fe <0.05).In the CAUTI model, a threshold of 1 × 10 5 CFU/g of tissue was chosen for the bladders/kidneys because it represented a middle point between the limit of detection (10 2 CFU) and the upper range of recovered CFU (10 8 ).The exception to this was at 6 hpi, in which the threshold was set to 1 × 10 4 CFU due to lower overall colonization at this time point.For the incidence of bacterial dissemination to the blood from CAUTI, the threshold was set at the limit of detection in the spleen (200 CFUs).For the bacteremia model in which mice were inoculated via tail-vein injection, statistical analysis was performed as in the CAUTI samples, with the exception that a cutoff of 1 × 10 5 CFU/g of tissue was used for all organs.
Statistical analysis of relative expression was performed with Wilcoxon signed-rank test against a hypothetical value of 1. Analysis of growth curves, the NF-κB reporter assay with diHA, and the Luminex assay was performed using a two-way ANOVA for overall trends, with Dunnett's multiple comparisons used to compare specific strains/conditions.The semi-quantitative GAG assays were analyzed with one-way ANOVA with Dunnett's multiple comparisons.

FIG 1
FIG 1 Genetic context and predicted protein architecture of HylA and HylB.Panels A and B show the genetic context of EF3023 (hylA) and EF0818 (hylB), respectively.Yellow box with dashed outline indicates a putative oligosaccharide phosphotransferase system upstream of hylB.Panels C and D show the domain architecture of HylA and HylB, respectively.SP indicates a signal peptide as predicted by SignalP 5.0.A dashed line indicates a predicted proteolytic cleavage site with either signal peptidase (SP) or sortase (LPXTG).The FIVAR/Repeat domain indicates a complete 69 amino acid segment with a predicted "FIVAR" domain, followed by three 71 amino acid repeats with partial FIVAR domain prediction."?" indicates a stretch of >40 amino acids which have no predicted conserved domains or other obvious features.Panels E and F show AlphaFold structure predictions of HylA and HylB, respectively.

FIG 2
FIG 2 Expression profiles of hylA and hylB in E. faecalis V587.E. faecalis strain V587 was cultured in BHI, 0.5× TSB, or human urine with or without supplementation with unfractionated high molecular weight HA or 5 kDa HA, and mRNA levels of recA, hylA, and hylB were determined by quantitative RT-PCR.(A) Cycle thresholds achieved for each gene compared to a no-template control (NTC).(B) Data display the number of cycles below the NTC at which each transcript was detected.(C) Expression of hylA and hylB were normalized to the recA housekeeping gene and expression across different growth media was compared to expression in BHI by the ∆∆CT method.(D and E) Induction of expression by the presence of HA was determined by comparison to expression in unsupplemented TSB (D) or urine (E).Error bars represent mean ± standard deviation (SD) for at least two independent experiments with two technical replicates each.*P < 0.05, **P < 0.01 by Wilcoxon signed-rank test against a hypothetical value of 1.0.

FIG 3
FIG 3 In vitro growth rates and hyaluronidase activity of E. faecalis strains.(A and B) E. faecalis V587 and isogenic mutants were grown in BHI broth (A) or TSB (B) at 37°C with shaking and OD 600 was measured every 15 min for 18 h.Graphs are representative of two independent experiments.Error bars indicate mean ± SD for three technical replicates.(C) E. faecalis V587 and isogenic mutants were grown in human urine at 37°C with shaking and CFUs were determined every hour for 7 h.Error bars indicate mean ± SD for three independent experiments.(D) Representative images of E. faecalis V875, ΔhylAΔhylB, Lactococcus lactis, and L. lactis expressing hylB after incubation on agar plates containing HA.A zone of clearance is only observed if the bacterium is producing active hyaluronidase.

FIG 5
FIG 5 HylA and HylB expression and GAG degradation.(A) Coomassie Brilliant Blue stained SDS-PAGE gel of soluble secreted protein from cell-free supernatants of ΔhylAΔhylB constitutively expressing hylB (pAOJ84), hylA (pAOJ86), or no plasmid (hylAhylB), with V587 included as a control.Gel is representative of at least three independent experiments.(B-D) E. faecalis strains were incubated with 2.5 mg/mL of HA (B), CS (C), or heparin sodium salt (HS, D) in 0.5× TSB for 24 h and supernatants were measured for remaining GAG by precipitation with BSA/acetic acid.Percentage of GAG remaining was calculated by comparison to a curve of undigested GAG standard concentrations in bacteria-free samples, and data were normalized to ΔhylAΔhylB for each individual experiment.Error bars indicate mean ± standard deviation (SD) for at least three independent experiments with two replicates each.*P < 0.05,****P < 0.0001 by one-way ANOVA with Dunnett's multiple comparison correction.(E) E. faecalis strains were incubated in BHI with 1 mg/mL HA for 24 h and HA degradation was examined by gel electrophoresis.The ladder was generated by combining 700 kDa and 5 kDa HA.Labels above each lane indicate E. faecalis strain, with the ΔhylAΔhylB strains containing expression constructs abbreviated by plasmid number, in which 55 refers to the vector control."Blank" indicates uninoculated BHI with HA and "5.7" indicates BHI with HA adjusted to pH 5.7."83 + 84" indicates inoculation with an equal amount of ΔhylAΔhylB (pAOJ83) and ΔhylAΔhylB (pAOJ84).HylSh and HylBov are Streptomyces and bovine hyaluronidase, respectively.(F) The indicated E. faecalis strains were grown in BHI for 24 h at 37°C, then filter-sterile

FIG 6
FIG 6 Characterization of purified HylB.(A) Turbidimetric assay of the remaining amount of HA post treatment with E. coli protein lysate, Bovine Hyaluronidase Type I-S, or HylB-Myc-6x His overexpressed and purified from E. coli after a 15-min incubation at 37°C.Protein concentrations were adjusted based on molecular weight to obtain approximately equimolar amounts to 12.5 µg/mL Bovine Hyaluronidase, with the exception of the mixed soluble E. coli proteins which were used at a concentration of 50 µg/mL.The percentage of HA remaining is based on the BSA negative control.Error bars indicate mean ± SD for three experimental replicates.(B) Agarose gel electrophoresis of a 601 kDa HA fragment digested with recombinant HylB.The 601 kDa HA fragment was incubated overnight at 37°C with either: assay buffer and no enzyme (HA); recombinant HylB-Myc-6x His (+HylB); soluble E. coli protein (+Ec); or commercially purchased Streptomyces hyaluronidase (+HylSh).Image was captured on a ChemiDoc MP, irrelevant lanes were cropped out, and levels were adjusted for better visibility with Adobe Photoshop 2022.

FIG 7 FIG 8
FIG 7 Contribution of HA degradation to E. faecalis growth.(A) E. faecalis V587 was incubated in simplified defined medium (SDM) supplemented with either 12 mM glucose or a series of HA concentrations and growth was measured by OD 600 every 15 min for 24 h.Graph is representative of two independent experiments.Error bars indicate mean ± SD for 10 technical replicates.(B and C) E. faecalis V587, ΔhylAΔhylB, and ΔhylAΔhylB, expressing hylB (pAOJ84) were incubated in 0.5× TSB (B) or 0.5× TSB supplemented with 2.5 mg/mL of unfractionated high molecular weight HA (C).Growth was measured by OD 600 every 15 min for 24 h.Graph is representative of two independent experiments.Error bars indicate mean ± SD for 10 technical replicates.***P < 0.001 by two-way ANOVA with Dunnett's multiple comparison correction.

FIG 9
FIG 9 Contribution of HylA and HylB to bladder cytokine and chemokine profiles.(A and B) Seven-week-old female CBA/J mice were inoculated via transurethral catheterization with 2 × 10 5 CFU of V587 or ΔhylAΔhylB, or inoculated with a PBS control and then sacrificed at 6 and 24 hpi.Each data point represents the CFU/g obtained from a single mouse, and bars indicate the geometric mean.(C and D) Radar graphs for select cytokines and chemokines in bladders from mice inoculated with V587, ΔhylAΔhylB, or PBS at (C) 6 h and (D) 24 h post-inoculation.Each ring in the graph represents quartiles of the given analyte, with the furthest ring representing the upper bound of the range of the log of mean abundance (pg/mL) for each metabolite.The scales between (A) and (B) are the same for ease of interpretation.(E) IL-6 levels were quantified by ELISA in bladder homogenates from mice that were either mock-inoculated with PBS, catheterized and mock-inoculated with PBS, or catheterized and infected with either V587 or ΔhylAΔhylB.Error bars represent mean ± SD. *P < 0.05, ***P < 0.001 by one-way ANOVA with Dunnett's multiple comparison correction.