Enterococcal quorum-controlled protease alters phage infection

Increased prevalence of multidrug resistant bacterial infections has sparked interest in alternative antimicrobials, including bacteriophages (phages). Limited understanding of the phage infection process hampers our ability to utilize phages to their full therapeutic potential. To understand phage infection dynamics we performed proteomics on Enterococcus faecalis infected with the phage VPE25. We discovered numerous uncharacterized phage proteins are produced during phage infection of Enterococcus faecalis. Additionally, we identified hundreds of changes in bacterial protein abundances during infection. One such protein, enterococcal gelatinase (GelE), an fsr quorum sensing regulated protease involved in biofilm formation and virulence, was reduced during VPE25 infection. Plaque assays showed that mutation of either the fsrA or gelE resulted in plaques with a “halo” morphology and significantly larger diameters, suggesting decreased protection from phage infection. GelE-associated protection during phage infection is dependent on the murein hydrolase regulator LrgA and antiholin-like protein LrgB, whose expression have been shown to be regulated by GelE. Our work may be leveraged in the development of phage therapies that can modulate the production of GelE thereby altering biofilm formation and decreasing E. faecalis virulence.


INTRODUCTION:
Enterococcus faecalis is a Gram-positive bacterium and a member of the gut microbiota of diverse animals, including humans (1-3).Following prolonged antibiotic therapy, E. faecalis can outgrow other members of the microbiota and disseminate to the bloodstream, leading to life threating disease such as sepsis and endocarditis (4)(5)(6)(7).Additionally, E. faecalis is a common cause of healthcare-associated infections (8)(9)(10).Treatment of enterococcal infections is complicated by the increasing prevalence of multi-drug resistant (MDR) E. faecalis strains, including those resistant to "last-resort" antibiotics (11)(12)(13)(14).With the ongoing antibiotic discovery gap and rising incidence of MDR infections, it is estimated that over 10 million individuals may die of antibiotic resistant infections per year by 2050, nearly ten times the current yearly mortality (15,16).Therefore, the development of innovative antimicrobial therapies is crucial to combating antibiotic resistant bacteria.
Bacteriophages (phages) are viruses that infect and kill bacteria.They have reemerged as potential therapeutics due to their diversity and abundance in nature, making them readily available for medical applications (17,18).Despite the discovery of phages over 100 years ago, we know little about the function of most phage-encoded genes (19,20).Additionally, we have only a rudimentary understanding of how phages interact with their target bacteria, particularly in non-model hosts (21).Understanding these fundamental aspects of phage biology is an important milestone toward the development of phages as antimicrobials.
During infection, phages co-opt host cellular processes to support their genome replication and translation of proteins responsible for virion assembly (22,23).One way that phages are known to influence the bacterial response to infection is by modulating the cell density-dependent transcriptional program known as quorum sensing (24)(25)(26)(27).Phage-mediated changes in quorum sensing gene regulation within a bacterial population can create an environment that is more permissible for infection (24,25).For example, changes in quorum sensing can result in the modification of wall teichoic acids, a major phage receptor, which .CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It is made The copyright holder for this preprint (which this version posted May 11, 2024.; https://doi.org/10.1101/2024.05.10.593607 doi: bioRxiv preprint 4 contributes to phage infectivity (28,29).In E. faecalis, high levels of the quorum-sensing peptide AI-2 are associated with release of prophages, possibly resulting in the transfer of virulence genes (26).Additionally, E. faecalis quorum sensing itself is interrupted during phage infection, with decreased transcription of quorum-sensing-induced genes and increased transcription of quorum-sensing-repressed genes (30).
Additionally, GelE and SprE have both been shown to be involved in the post-processing of the autolysin AtlA, although the role of SprE in this context has been contested (41,46).
In this paper, we used a proteomic approach to explore the interaction between the enterococcal phage VPE25 and its E. faecalis host.We found time-dependent trends in phage protein production and identified correlations between gene expression and protein abundance levels during infection.Investigation of the changes in bacterial protein abundance during phage predation revealed large scale trends in the abundance of a wide variety of proteins.We show that decreased levels of the quorum-sensing-regulated protein GelE, and subsequent downstream changes in the production of the murein hydrolase modulator LrgA and antiholinlike protein LrgB, influence the outcome of phage infection.

RNA-seq analysis
A previously published RNA-seq dataset, generated in a parallel experiment, was reanalyzed for phage transcript abundances (EMBL-EBI ArrayExpress database, accession number E-MTAB-8546) (30,59).RNA sequencing reads were analyzed for quality using FastQC v0.12.0 (60).Reads were then mapped to the VPE25 open reading frames (ORFs) by Salmon v1.10.2 (61).The output read quantities, as reported in the nascent quant.sffile, were subject to relative abundance calculations by dividing the transcripts per million (TPM) for a given ORF by the total number of TPMs for a sample.ORFs were ranked by their abundance for each timepoint, and this ranking was used to inform heatmap rings in Figure 1A, generated using the R package Circlize (62).

Preparation of samples for proteomics
Subcultures of E. faecalis overnight cultures were infected with VPE25 as described previously (30).Four samples of 4 mL each were taken at 0, 10, 20, and 40 minutes after 6 VPE25 treatment and pelleted.Pelleted samples were resuspended in 300 µL of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl, 0.1 M DTT).Cells were lysed by bead-beating using a Bead Ruptor Elite (OMNI) with Matrix Z beads (MP Biomedicals) for two cycles of 45 s at 6 m/s.
Samples were incubated at 95°C for 10 min.Tryptic digests of protein extracts were prepared following the filter-aided sample preparation (FASP) protocol described previously, with minor modifications as described in Kleiner et al. (63,64).Lysate was not cleared by centrifugation after boiling the sample in lysis buffer.The whole lysate was loaded onto the filter units used for the FASP procedure.Centrifugation times were reduced to 20 minutes as compared to Kleiner et al. (64).Peptide concentrations were determined with the Pierce Micro BCA assay (Thermo Scientific) using an Epoch2 microplate reader (Biotek) following the manufacturer's instructions.

LC-MS/MS
All samples were analyzed by 1D-LC-MS/MS as described previously, with the modification that a 75 cm analytical column and a 140-minute long gradient were used (65).For each sample run, 400 ng peptide were loaded with an UltiMateTM 3000 RSLCnano Liquid Chromatograph (Thermo Fisher Scientific) in loading solvent A (2% acetonitrile, 0.05% trifluoroacetic acid) onto a 5 mm, 300 µm ID C18 Acclaim® PepMap100 pre-column (Thermo Fisher Scientific).Separation of peptides on the analytical column (75 cm x 75 µm analytical EASY-Spray column packed with PepMap RSLC C18, 2 µm material, Thermo Fisher Scientific) was achieved at a flow rate of 300 nl min 1 using a 140 min gradient going from 95% buffer A (0.1% formic acid) to 31 % buffer B (0.1% formic acid, 80% acetonitrile) in 102 min, then to 50% B in 18 min, to 99% B in 1 min and ending with 99% B. The analytical column was heated to 60°C and was connected to a Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) via an Easy-Spray source.Eluting peptides were ionized via electrospray ionization (ESI).Carryover was reduced by one wash run (injection of 20 µl acetonitrile, 99% eluent B) between samples.Full scans were acquired in the Orbitrap at 60,000 resolution.The 15 most abundant precursor ions were selected for fragmentation and MS/MS scans were acquired at 15,000 resolution.The mass (m/z) 445.12003 was used as lock mass.
Ions with charge state +1 were excluded from MS/MS analysis.Dynamic exclusion was set to 18 s.Roughly 120,000 MS/MS spectra were acquired per sample.

Protein identification
A database containing protein sequences from E. faecalis (RefSeq accession: NC_017316.1)and the phage VPE25 (GenBank accession: LT615366.1),both downloaded from NCBI, were used (66,67).Sequences of common laboratory contaminants were included by appending the cRAP protein sequence database (http://www.thegpm.org/crap/).The final database contained 2,893 protein sequences.Searches of the MS/MS spectra against this database were performed with the Sequest HT node in Proteome Discoverer version 2.2.0.388 (Thermo Fisher Scientific) as previously described (68).Only proteins identified with medium or high confidence were retained resulting in an overall false discovery rate of <5%.

Protein quantification and statistical analyses
For quantification of bacterial proteins, normalized spectral abundance factors (NSAFs) were calculated and multiplied by 100% to obtain relative protein abundance (69).The NSAF values were loaded into Perseus version 1.6.2.3 and log 2 transformed (70).Only proteins with valid values in four replicates of at least one treatment were considered for further analysis.
Missing values were replaced by a constant number that was lower than any value across the experiment.Differentially abundant proteins between conditions were calculated using student's t-tests corrected for multiple hypothesis testing using a permutation-based false discovery rate of 5%.
As the number of phage proteins is small and their relative abundances are heavily influenced by the overall abundance of the bacterial proteins in the cultures, we used a centered-log ratio (CLR) transformation for more robust analysis of compositional data (71).The CLR was calculated for all proteins, both phage and bacterial.CLR values for phage proteins were loaded into Perseus 1.6.2.3 where t-tests were performed as described above (70).Since the spectral counts at t0 (0 minutes post-infection) were much lower than the other time points, only proteins with 70% of total values across conditions with more than 20 PSMs were considered for comparisons with t0.For comparisons between the 10, 20, and 40 minutes postinfection time points, no PSM cut-off was employed.Replacement of missing values and t-tests were performed as described above.

Plaque assays
One mL of overnight bacterial culture was pelleted at 21,000 × g for 1 minute and resuspended in 2 mL SM-plus phage buffer (100 mM NaCl, 50 mM Tris-HCl, 8 mM MgSO 4 , 5 mM CaCl 2 [pH 7.4]) (55).Approximately fifteen plaque forming units (PFU) of phage were added to 120 μL of resuspended bacterial culture and incubated statically at room temperature for 5 minutes.Following incubation, 5 mL of molten THB top agar (0.35% agar, unless otherwise noted) supplemented with 10mM MgSO 4 was added to the suspension and poured over a 1.5% THB agar plate supplemented with 10 mM MgSO 4 .Plates were incubated upright at 37°C for 24 hours unless otherwise noted.Photos of plates were taken on an iPhone 12, and plaque diameters were measured using ImageJ v1.53t (72).A standard ruler was included in each image to determine scale.

Assessment of VPE25 transcript and protein abundances during E. faecalis infection
To understand global protein regulation and abundances during phage infection of E. faecalis, we infected E. faecalis OG1RF with the siphophage VPE25 (55).Mid-log cultures of E. faecalis were infected with VPE25 at a multiplicity of infection (MOI) of 10.Samples were taken at 0, 10, 20, and 40 minutes post-infection and subjected to LC-MS/MS-based proteomics analyses.We compared the phage-encoded protein abundances to a previously generated RNA-Seq dataset during VPE25 infection of E. faecalis OG1RF, where the experimental parameters were identical (30).From the RNAseq, we identified 131 phage transcripts throughout infection (Supplemental Table 2).Proteomic analysis identified 93 of those transcripts as proteins (Supplemental Table 3).
Figure 1A shows the VPE25 transcript and protein abundances throughout infection.
Few proteins were detected at the initiation of infection, and it is likely that those detected were either present in the virion or rapidly expressed following DNA entry.Many replication proteins, such as DNA polymerase and DNA helicases, were significantly differentially abundant (DA) after only 10 minutes post-infection (Supplemental Table 3).By 20 minutes post-infection, we saw increases in abundance of additional replication machinery, such as DNA gyrase subunits A and B. Virion components, primarily proteins involved in assembly of the tail, were also DA at this time, as was the phage lysin.Final virion components, including the portal protein and the head maturation protease, were DA by 40 minutes post-infection.Notably, the most abundant phage protein at each timepoint was the major capsid protein, whose relative abundance continued to increase throughout the course of infection (Figure 1B, Supplemental Table 3).The major capsid transcript levels reached the top ten most abundant phage transcripts at the 10minute timepoint and remained among the most abundant transcripts throughout infection (Supplemental Table 2).
A high number of detected phage transcripts and proteins were annotated as genes of unknown function.Of the 133 putative genes in the VPE25 genome, more than 60% are hypothetical.Many of these uncharacterized proteins were expressed at a level equal to or above proteins with characterized functions.For example, SCO93409.1 is an uncharacterized phage protein whose abundance at 40 minutes was third only to the major capsid and major tail proteins (Figure 1B).In silico analysis of these hypothetical proteins failed to provide insight on their potential function, with more than half having no conserved domains identified via InterProScan (73).Together these data show that the majority of VPE25 proteins are expressed during infection, many of which are proteins of unknown function.

E. faecalis responds to VPE25 infection by altering its gene expression and protein abundances
Our understanding of bacterial gene expression levels in response to phage infection and how this corresponds to protein abundances is limited.While transcript data is often used as a stand-in for shifts in proteomic abundances, comparative analyses show that these are often not fully concordant (74,75).In fact, many of the most differentially abundant bacterial proteins in our analysis are relatively unchanged at the transcript level when compared to our previously published RNA-Seq dataset (Table 1, Supplemental Table 4) (30).
Of the 2,647 genes in the E. faecalis OG1RF genome, 2,550 were detected as transcripts (30).Of those transcripts, 1,225 proteins were detected using proteomics.A total of 680 proteins were differentially abundant (DA) between infected and uninfected E. faecalis by 40 minutes (Figure 1C, Supplemental Table 4).There was no statistical difference in bacterial protein relative abundances at the 10-minute timepoint, and at 20 minutes post-infection only 6 proteins were differentially abundant (Supplemental Figure 1A).The limited differences in protein abundances at the earlier timepoints reflect a lag time between the transcriptional response, which begins by the 10-minute timepoint, and the time it takes to translate those transcripts into proteins (30).All of the DA proteins at 20 minutes remained DA at the 40-minute timepoint with the exception of OG1RF_12042, a CYTH domain-containing protein.
The differential abundance ratio, or DAR, was calculated as the relative change in abundance of a protein between the infected and uninfected samples at each timepoint.The DAR at 40 minutes (DAR 40 ) was used to identify potential key effectors of bacteria-phage interactions during infection (Table 1).Proteins with high DAR values, such as SecY, are at an increased level during phage infection, while proteins with low DAR values, like AtpF, are found in decreased abundance during phage infection.This up-and down-regulation of protein abundances can be attributed to the bacterium responding to phage infection or when phageencoded proteins hijack bacterial host processes leading to changes in protein abundances.To determine which, if any, of these proteins may be integral to phage virulence during infection, we used mutants from an arrayed E. faecalis OG1RF transposon library and screened them for changes in phage infectivity (48).Mutants selected represent available transposon insertions in genes whose proteins were most over-or underrepresented at 40 minutes as indicated by DAR (Table 1).While no differences were seen in the number of viable PFU on each strain tested (Supplemental Figure 1B), a change in plaque morphology was noted on the gelE-Tn mutant, a protein whose expression is undetected at 40 minutes (Figure 1D).

E. faecalis gelatinase alters phage infection
VPE25 forms clear plaques with a well-defined border approximately 1 mm in diameter on E. faecalis OG1RF.However, after 24 hours of plaque formation on an in-frame gelE deletion strain (OG1RF∆gelE), VPE25 forms a central zone of clearing surrounded by a large turbid ring which more than doubles the overall diameter (Figure 2A).This phenotype suggests that when GelE is absent, bacterial cells are more susceptible to phage infection associated factors.This "halo" morphology is rescued through the addition of gelE on a constitutive expression vector (pLZ12A::gelE) (Figures 2B, 2C).The gelE gene encodes the enterococcal gelatinase, a secreted protease that targets a variety of substrates and is regulated by the Fsr quorumsensing system (31,35,44,45,(76)(77)(78)(79)(80).GelE is one of five proteins, which became undetectable in the phage-infected sample by 40 minutes post-infection and thus has a DAR of 0, supporting our previous finding that the transcription of quorum-sensing regulated genes, including gelE, are significantly lower during phage infection (Table 1, Supplemental Table 4) (30).
To further investigate this phenotype, we assessed phage plaque morphology on E. faecalis OG1RF∆fsrA, a strain lacking the quorum-sensing master regulator fsrA.Again, plaques showed a halo morphology, and plaque diameter was significantly larger than wildtype (Figure 2D).GelE cleaves the target proteins EntV and SprE, which are both under the control of FsrA, as well as AtlA, an autolysin that is involved in biofilm formation (31,36,37,41,(44)(45)(46).
We tested deletion mutants of entV, sprE, and atlA for plaque morphology during VPE25 infection.Deletion of entV or atlA showed no change in plaque morphology or size, but a sprE transposon mutant strain formed haloed plaques similar to OG1RF∆gelE and OG1RF∆fsrA (Figure 2E).GelE-dependent changes in the viability of extracellular virions and ability of VPE25 to adsorb were also tested; however, we observed no significant differences regardless of the presence or absence of GelE (Supplemental Figures 2B, 2C).
The top agar overlay plaque assay supports the diffusion of both phage and secreted factors, such as GelE, through the top agar.We varied the agar density to look for changes in plaque size to determine if this phenotype is associated with diffusion.Plaque assays were performed using either double (0.7% agar) or half (0.175% agar) the normal concentration of soft agar in the overlay, while all other parameters remained constant.As the agar concentration within the overlay increased, the size of VPE25 plaques and their halos on OG1RF∆gelE decreased, suggesting that a diffusible factor is indeed responsible for this phenotype (Figure 2F).When wild type OG1RF is infected with VPE25, the average plaque diameter does not change regardless of the agar concentration.

Diverse E. faecalis bacterial strains and phages show altered infectivity that is dependent on GelE
To determine if the haloed plaque morphology was unique to E. faecalis OG1RF and VPE25, we tested VPE25 plaque morphology on E. faecalis V583, a genetically distinct vancomycin-resistant isolate (47).In a gelE mutant of V583 (V583∆gelE), VPE25 plaques develop a similar halo morphology, and the plaque diameter is significantly larger when compared to wild type cells (Figure 3A).However, unlike OG1RF∆gelE, the halo morphology is not well-observed at 24 hours and is only prominent in the V583 background after 48 hours (Supplemental Figure 3A).The halos observed in V583∆gelE are also present in a double deletion of both gelE and sprE (V583∆gelE∆sprE), but not present in the sprE deletion alone (V583∆sprE) (Figure 3A).
To determine if this halo plaque morphology was unique to the phage VPE25, we used a panel of genetically distinct phages and measured plaque diameters on OG1RF and OG1RF∆gelE.In addition to VPE25, the phage G01 produced a halo phenotype and significantly larger plaques after 48 hours of infection (Figure 3B).G01 is a 41 kbp phage isolated from the Ganges River in India (56).While both VPE25 and G01 are siphophages with icosahedral heads, the two phages share greater than 30% nucleotide identity in only four genes as identified via clinker (Supplemental Figure 4A) (81).Specifically, these homologous genes encode a lysin, glutaredoxin, a DUF1140 domain-containing protein, and a hypothetical protein.Interestingly, while the VPE25 and G01 lysins share 65% identity across 66% coverage, most of the homology is focused in the amidase domain (Supplemental Figure 4B) (82).These data indicate that GelE-mediated plaque morphology is neither E. faecalis strain-or phagedependent.

GelE regulation of the murein hydrolase regulator lrgA and antiholin-like protein lrgB provides protection from phage-mediated inhibition
Previous work in E. faecalis V583 has demonstrated an upregulation of the lrgAB operon in the presence of gelE (35).While the function of the lrgAB locus is uncharacterized in E. faecalis, LrgA and LrgB are involved in repression of murein hydrolase activity and decreased autolysis in Staphylococcus aureus and their expression is dependent on the Agr quorumsensing system (83,84).We hypothesized that lrgA and/or lrgB play a role in limiting phage-mediated haloed plaque formation.The lrgAB locus is regulated by the LytSR two-component system, located directly upstream of its operon.LytSR is hypothesized to activate in the presence of extracellular GelE, after which it increases transcription of lrgAB (35).While the functions of lrgA and lrgB have not been studied extensively in enterococci, they have been shown to be upregulated during growth in blood (85).
Considering the role of LrgA and LrgB in limiting murein hydrolase activity, we hypothesized that they may be involved in protection against phage-mediated extracellular lysis.
VPE25 plaque diameters on lytS-Tn and lytR-Tn mutants were significantly larger than wild type (Figure 4A).Next, the plaque assay was repeated with lrgA-Tn and lrgB-Tn mutants.Data again show that plaque diameters are significantly larger than wild type OG1RF (Figure 4B).Thus, our data suggests that the murein hydrolase regulator LrgA and the anti-holin LrgB are ultimately responsible for the altered plaque morphology in the absence of GelE and supports the notion that this activity is mediated first by GelE and then by the LytSR two-component system.The underabundance of proteins like GelE during phage infection may be due in part to phagemediated changes in enterococcal quorum-sensing to facilitate an environment more permissible to phage infection.This is further supported by the increased phage-dependent inhibition seen on plates in the absence of these genes.

DISCUSSION:
The continued rise in antibiotic resistant bacterial infections has led to renewed interest in alternative antimicrobials, including phages.However, gaps in our understanding of phage biology and infection dynamics limit the full potential of emerging phage therapeutics.In this study, we used proteomics to probe the complexities of phage infection and understand the interplay between phages and E. faecalis.The resulting dataset provided insights in the timing of phage protein expression during infection, as well as identifying the altered abundance of hundreds of bacterial proteins during infection, primarily those involved in cell wall remodeling and bacterial metabolism.Additionally, this work revealed a role for the FsrA-regulated protease GelE, and to a lesser extent the protease SprE, in driving phenotypic changes in phage plaque morphology.This phenotypic change is due in part to ablated activity of the murein hydrolase regulator LrgA and the anti-holin LrgB, which we hypothesize aid in protection from increased phage-mediated attack (Figure 5).Future development of phages for clinical application may exploit this phenotype to allow for phage-mediated modulation of the virulence factor GelE to reduce E. faecalis virulence.
GelE was first investigated due to its significant under-abundance during phage infection (Figures 1C, 1D, Table 1, Supplemental Table 4).This observation is consistent with the repression of quorum-sensing-regulated transcripts during infection, including gelE and sprE (30).Gelatinase has been previously characterized as a virulence factor and has a role in the formation of biofilms, which are well-penetrated by phage (41,42,(86)(87)(88).These data suggest that, in addition to aiding in the dispersal of biofilms, phage can limit biofilm initiation through repression of gelE.Further research may elucidate the phage factor responsible for mediating the repression of enterococcal quorum-sensing during infection.Inclusion of an identified gene or genes in a therapeutic phage may aid treatment by repressing the expression of fsr-regulated virulence factors (32,45,79).
While the absence of gelE is associated with the development of a haloed plaque in both E. faecalis OG1RF and V583, deletion of sprE results in changes to plaque morphology only in OG1RF (Figures 2C, 2E, 3A).These strains share 98.82% and 99.30% amino acid identity across the full sequences of GelE and SprE, respectively (82).With such high similarity and a well-characterized role for GelE in post-processing of SprE, it remains unclear why interruption of sprE does not result in haloed plaques in V583 (45).It is possible that SprE, in addition to GelE, is required for processing a downstream factor which is processed by GelE alone in V583, or in conjunction with an alternative protease.Post-processing by GelE is known to impact the localization of the autolysin AtlA on the cell surface (46).Bacteria often acquire phage resistance through mutations that result in changes to the structure of cell wallassociated molecules (54,55).While the haloed plaques reported here are not dependent on AtlA, characterization of additional changes to the cell surface in the absence of gelE, sprE, or both can provide insight into the mechanism of the phenotypic change (Figure 2E).
Interruption of lrgA and lrgB, which are positively regulated in the presence of gelE, also resulted in the haloed plaque morphology (Figure 4B) (35).LrgA and LrgB are known to repress murein hydrolase activity and autolysis in S. aureus, suggesting a potential role for these proteins in limiting phage-mediated extracellular lysis in this model (83).However, the exact role of these proteins has not been studied in Enterococcus.Elucidation of the mechanism of LrgABconferred protection can inform our understanding of phage infection and enterococcal function.
The similarity between the amidase domains of the G01 and VPE25 lysins, as well as the role of LrgA and LrgB in repression of murein hydrolase activity, suggests a possible protection against extracellular lysis conferred by the lrgAB operon (Figure 4B, Supplemental Figure 4B).Similar haloed plaque morphologies have been associated with the presence of phage depolymerases (89,90).These phage-encoded proteins can degrade a variety of bacterial polysaccharides, including capsular, extracellular, and lipopolysaccharides (91)(92)(93).While phage depolymerases have been previously associated with similar plaque phenotypes, analysis using InterProScan does not reveal any Pfam matches for such depolymerases within the genomes of phages VPE25 or G01 (73).While the mechanism of GelE-conferred protection from phagemediated inhibition is still unknown, phages which are able to modulate the production of virulence factors such as GelE should be considered for their potential role in future therapeutic applications.Taken together, our work here provides an understanding of phage infection at the protein level and demonstrates a protective role for GelE, LrgA, and LrgB during phage infection (Figure 5).
shown on the x-axis.Negative log10-transformed q-value is shown on the y-axis with a line at a significance cut-off of q < 0.05.Points colored in blue are significantly underrepresented in the infected sample when compared to the uninfected sample.Points in red are significantly overrepresented.Proteins with a q-value of zero were excluded from the chart.
(D) %NSAF of GelE over time in both the infected and uninfected samples.Points represent an average of four biological replicates.Significance was determined using student's t-tests corrected for multiple hypothesis testing using a permutation-based false discovery rate of 5% (see Materials and Methods: Protein quantifications and statistical analyses).Error bars represent standard deviation.* FDR ≤ 5%.

Figure 2 :
Figure 2: Phage VPE25 plaque morphology is altered in the absence of gelE.(A) VPE25

Figure 3 :
Figure 3: Infection by genetically distinct phages and bacterial species is impacted by

Figure 4 :
Figure 4: Mutation of lrgA and lrgB genes results in altered plaque morphology.(A)