Rhomboid protease GlpG regulates type 1 pili quality control and virulence in pathogenic E. coli

Domain: chemistry
Published: 2025-12-30
Editorial outcome: major_revision

Abstract
With the rise in antimicrobial resistance, understanding the virulence factors utilized by pathogenic E. coli is essential for the development of alternative therapeutics. While previous work has shown that disruption of the E. coli rhomboid protease gene glpG leads to defects in bacterial colonization, here we provide mechanistic insight into the loss of fitness. We show GlpG is essential for the assembly of type 1 pili, a virulence factor required for the colonization of eukaryotic cells. Since pili are critical for biofilm formation and bacterial persistence, the absence of GlpG proteolytic activity reduces the production of biofilm. Working towards new potential antimicrobial targets for treating infections, we show that biofilm formation is hampered by GlpG inhibition. Our data demonstrates that GlpG plays a key role in protein quality control of type 1 pili and alters the paradigm for GlpG proteolysis, previously implicated in the cleavage of only membrane embedded substrates.

Introduction
Escherichia coli is a diverse bacterial species with the ability to colonize and persist in various niches within animal hosts, often forming a symbiotic relationship by providing nutrients, keys signals for homeostatic regulation, and protection against foreign pathogens1–3. Within the gastrointestinal tract, resident E. coli are typically commensal; however, these strains can take on a pathogenic course, causing disease outside the gut through combinations of acquired virulence factors such as adhesins, secreted toxins, or lipopolysaccharides4–6. Pathogenic strains are broadly categorized as either intestinal pathogenic E. coli, causing infection within the gastrointestinal tract, or extraintestinal pathogenic E. coli (ExPEC), as they have retained the ability to survive in the gut, but also wield virulence factors with the capacity to disseminate and colonize other sites, including the blood, brain, and urinary tract7–9. In addition, there are three unclassified pathogenic groups: adherent-invasive E. coli involved in Crohn’s disease, necrotoxic E. coli, which is associated with disease in humans and other mammals, and cell-detaching E. coli that involved in hemolysin production. Among ExPEC, the uropathogenic E. coli (UPEC) pathotype accounts for 80–90% of all urinary tract infections (UTI), one of the most common bacterial infections globally10–12. About 50% of women in the United States are expected to have at least one UTI in their lifetime with half experiencing a recurrent infection within 12 months of the initial episode13,14. The global death rate due to UTIs has increased by 140% from 1990 to 2019, largely attributed to the rise of antimicrobial resistance to classes of commonly prescribed antibiotics such as β-lactams, cephalosporins, and fluoroquinolones, ultimately making UPEC infections more challenging to treat15–20. Consequently, this places an urgent need for the development of alternate treatment strategies to combat these infections. However, in order to explore alternative therapeutics, a better understanding of bacterial virulence factors regulation is required.

Virulence factors play a crucial role in bacterial survival and persistence, largely through biofilm initiation and formation. Biofilms are surface-attached communities of bacteria embedded in a matrix of biopolymers such as proteins and polysaccharides, and are one of the critical properties responsible for most chronic infections by UPEC21. Pili and flagella, along with other adhesins and extracellular factors such as toxins, siderophores, enzymes, and polysaccharide coatings, are among the main virulence factors necessary for initial adhesion and colonization of host mucosal surfaces22–24. Once formed, biofilms are remarkably difficult to eradicate as these communities are more resistant to antimicrobial agents, surviving transient antibiotic treatment and regrowing when antibiotic is withdrawn, leading to infection persistence and recurrence25–27.

The expression, assembly, and turnover of virulence factors reflect a prokaryote’s ability to disseminate and infect a host, but how the proteins involved regulate these processes is not completely understood. The rhomboid protease superfamily are intramembrane proteases that are ubiquitously expressed across all kingdoms of life, with the eukaryotic members implicated in various functions such as mitophagy, growth factor signaling, amyloid precursor protein processing, and more28–32. Meanwhile, the roles of prokaryotic rhomboid proteases have been linked to functions such as quorum sensing with AarA in Providencia stuartii, biofilm formation with Rho1/2 in Haloferax volcanii, ion homeostasis with YqgP in Bacillus subtilis and protein quality control with GlpG in Shigella sonnei33–36. GlpG is a conserved and widely represented member of the rhomboid protease superfamily located in the plasma membrane of all known strains of E. coli as well as other prokaryotes37. Moreover, it has been shown that the glpG gene is essential for ExPEC persistence in the murine intestine and by knocking-out glpG, bacterial colonization is significantly reduced, however, by an unknown mechanism38. Additionally, while GlpG in S. sonnei targets and cleaves orphan proteins to prevent aggregation in the cell, a potential association to bacterial pathogenicity remains to be shown35.

Here, we provide a mechanistic insight into the link between bacterial colonization and proteostasis in pathogenic E. coli. Using proteomics, we sought to reveal physiological binding partners of GlpG and identified type 1 pili subunit, FimA, as well as periplasmic serine endoproteases DegQ and DegP39. We demonstrate that GlpG proteolytic activity is essential for type 1 pili formation in pathogenic pathotype UPEC, affecting the ability of E. coli to colonize and infect host cells. As adherence is essential for the initiation of biofilm formation, we show that the absence of GlpG catalysis, as well as GlpG inhibition, significantly reduces biofilm production. Our data demonstrates that GlpG regulates FimA protein quality control and turnover by cleaving FimA aggregates during type 1 pili formation. Moreover, proteomic analysis revealed GlpG association with housekeeping proteases DegP and DegQ. We demonstrated that these proteases are also involved in cleaving aggregated or misfolded FimA subunits, assisting in the clearance of aggregates accumulated in the periplasm. Thus, GlpG is part of an important quality control checkpoint of FimA subunits, ensuring assembly fidelity of type 1 pili. Overall, our study demonstrates that GlpG plays an essential role in UPEC virulence and may represent a viable drug target for the treatment of bacterial infections, such as UTIs, to combat the rise of antimicrobial resistant bacteria.

Results
Proteomic analyses of GlpG identified substrates associated with bacterial virulence
Rhomboid proteases were discovered in the early 2000’s, and E. coli rhomboid protease, GlpG, quickly became a key model for understanding the structure and mechanism of this family of intramembrane proteases40–42. Despite being the first family member to have the structure solved, the interacting partners, physiological substrate(s), and hence, the function of E. coli GlpG remained unknown. Our first focus was to identify binding partners/substrates of GlpG using a 3×FLAG tag genomically inserted downstream of the glpG gene to enable affinity pulldown and whole cell proteomics in the presence of non-denaturing detergent. This revealed that GlpG interacted with several proteins with roles in intracellular secretion and protein turnover when assigned to Clusters of Orthologous Groups (COGs) (Fig. 1a and Supplementary Data 1), in particular with SecY and G of the SecYEG translocon, its associated ATP-dependent motor protein SecA as well as its ancillary proteins YidC, SecD, and F. Together they drive Sec-mediated translocation of unfolded proteins across the membrane, a tightly regulated process that exports most secretory proteins in bacteria. GlpG may associate with the Sec translocon to readily cleave misfolded or aggregated proteins to prevent the toxic accumulation of aggregates, as part of a role in protein quality control, a function shared by other rhomboid proteases studied in the current literature. We also demonstrate GlpG association with DegQ and DegP (Fig. 1a), proteases with central roles in protein quality control that are closely involved in clearing unproductive or harmful protein intermediates, particularly under stress conditions. There is increasing evidence that both DegP and DegQ interact functionally with the SecYEG translocon43. DegP, in particular, has been shown to degrade preproteins that are stalled or improperly inserted into the membrane, suggesting a surveillance role at or near the translocon44.

The list of interacting partners from whole cell proteomics led us to take a narrower approach to analyze only the resident proteins in the periplasmic space. The active site of GlpG in E. coli faces the periplasm, allowing potential substrates to be identified when comparing isolated periplasmic proteins in the presence of active or inactive GlpG using LC-MS/MS (Fig. 1b). In addition, this minimizes cytosolic proteins that would otherwise be identified as non-physiological substrates45. Notably, the periplasmic fraction was isolated from a ΔglpG mutant of K12 substr. MG1655 E. coli to ensure any identifiable proteins were not prematurely cleaved by endogenous GlpG. We hypothesized that proteolysis by GlpG would lead to the generation of smaller protein fragments potentially leading to apparent decrease in protein abundance due to the fragment's degradation, precipitation in the lysate or running out of the gel. Therefore, proteins identified as significantly decreased in the presence of active GlpG, relative to inactive GlpG, would represent potential cleavage by GlpG (Fig. 1c). Several proteins were identified, including those involved in cell division, stress response, and chaperone mediated folding, such as FtsA, RcnB, and PpiD (Supplementary Fig. 1 and Supplementary Data 2). Importantly, virulence factor FimA, a subunit of type 1 pili, was identified among the proteins significantly decreased in the presence of active protease, drawing our attention in accordance with previous literature, describing colonization defects of pathogenic E. coli with a glpG knockout strain38.

To further investigate the role of GlpG in proteostasis, an in-gel digestion was performed whereby periplasmic proteins isolated from the K12 ΔglpG mutant strain were incubated with active or inactive GlpG, separated by SDS-PAGE and followed by gel excision at different molecular weight ranges to be analyzed using LC-MS/MS. In line with the function of other rhomboid proteases that cleave aggregated substrates to prevent their accumulation34,35, we sought for a more detailed examination of which proteins may be forming aggregates in the absence of GlpG and could be cleaved by the re-addition of GlpG (Supplementary Data 3). When periplasmic proteins were incubated with inactive GlpG, FimA was found to be at a higher molecular weight (>135 kDa) instead of its monomeric size (18 kDa), whereas in the presence of active GlpG, this oligomeric form of FimA was not present (Fig. 1d and Supplementary Fig. 2). In wild-type (WT) E. coli FimA monomers were rarely detected, likely due to their incorporation into pili or rapid turnover (Fig. 1d, e).

In WT E. coli, homeostasis of FimA protein in periplasmic space is maintained, as evidenced by the lack of FimA aggregation when performing immunoblot detection by western blot. By contrast, FimA aggregation was detected in the periplasm of K12 MG1655 ΔglpG, even under SDS-denaturing conditions (Fig. 1e), suggesting that in the absence of GlpG catalytic activity, proteostasis of FimA is dysregulated, causing its accumulation and aggregation within the periplasm during its translocation. When K12 MG1655 ΔglpG was transformed with the plasmid bearing active GlpG, we observed notably decreased amount of aggregates, confirming GlpG’s role in protein quality control as seen in many other rhomboid proteases34,35. To test the hypothesis that periplasmic proteases DegQ and DegP found associated with GlpG (Fig. 1a), may also be involved in the biogenesis of type 1 pili, we detected FimA in the samples of periplasmic proteins isolated from single gene knockout of degQ and degP in K12 MG1655. Indeed, immunoblot analysis demonstrated aggregated FimA in the periplasm in the absence of either ΔdegQ and ΔdegP (Fig. 1e), identifying a link between DegQ and DegP proteases and quality control of type 1 pili for the first time. The more profound effect of ΔdegQ on FimA aggregation could be explained by the fact that ΔdegP protease is not constitutively expressed under normal growth conditions46, as opposed to ΔdegQ, which has relatively constant level regardless of specific environmental conditions47. Moreover, the K12 MG1655 ΔdegP and ΔdegQ mutant transformed with plasmid expressing active GlpG exhibited reduced amount of FimA aggregates. This suggests that there may be interplay between GlpG and DegQ/P that reflects a specialized proteostasis module that monitors and responds to the periplasmic aggregation of pilin subunits.

GlpG activity is essential for pili formation
FimA protein is the structural subunit of type 1 pili, comprising of up to 3000 subunits in a single pilus. It is an abundant and essential component for bacterial adherence to host cells, therefore maintaining its protein homeostasis is crucial in pathogenic E. coli. The main function of adhesion virulence factors, such as type 1 pili, is to facilitate attachment of the bacterium to the host cell, often as an initial step in invasion or uptake. To determine if the lack of GlpG catalysis affects type 1 pili formation, transmission electron microscopy (TEM) was used to visualize surface pili. UPEC strain NU149 was chosen to represent a clinically relevant isolate from a patient presenting cystitis48. We show that significantly more surface pili are present on NU149 wild-type relative to a genomically inactive glpG strain generated using CRISPR/Cas9, which harbors a Ser201Ala point mutation at the catalytic serine (Fig. 2a, b and Supplementary Fig. 3). A genomically inactive glpG mutant, hereby denoted as glpG-S201A, was used to gain a more physiological insight into the role of the protease compared to a gene knock-out. The growth curve and glpG mRNA expression of the resulting strain endogenously expressing catalytically inactive GlpG was unaffected compared to its wild-type counterpart (Supplementary Fig. 4). Importantly, it is known that mutation of the catalytic serine does not disrupt the folding of GlpG protease49. The significant impairment of pili formation observed in glpG-S201A mutant was rescued when the active GlpG protease was reintroduced to NU149 glpG-S201A using an inducible plasmid, with an apparent increase in not only pili but flagella, which was unexpected. The same trends were observed when fim protein chaperone, FimC, was overexpressed, assisting the folding, binding, and translocation of FimA (Fig. 2b and Supplementary Fig. 5) (discussed in detail below). To test if GlpG expression may be elevated in pathogenic E. coli relative to commensal E. coli, we quantified glpG mRNA transcript levels using RT-qPCR, and indeed detected higher expression in NU149 and J96 UPEC strains compared to K12 MG1655. This result was not affected by different nutrient environments such as LB, high nutrient terrific broth (TB), or M9 minimal media that could influence bacterial growth rate, biomass yield, gene expression, and protein production (Fig. 2c).

To confirm that the surface organelles detected using TEM were indeed type 1 pili, we performed a classical functional test for the presence of type 1 pili: a mannose-sensitive hemagglutination (MSHA) assay on guinea pig erythrocytes. Type 1 pili mediate the hemagglutination (HA) that occurs when piliated bacteria are mixed with erythrocytes of certain species. Since type 1 pili on UPEC bind to mannosylated proteins on host cells, particularly mannose found on uroplakins and integrins by FimH adhesion, the HA reaction can be inhibited by addition of 2% mannose50. The NU149 strains described above were grown under the same conditions as for TEM imaging and assessed for HA (Fig. 2d). NU149 wild-type and glpG-S201A strain overexpressing active GlpG protease were MSHA positive (type 1 piliated), whereas inactive glpG mutant as well as the cells transformed with an empty pBad vector did not exhibit HA, suggesting the absence of type 1 pili, in agreement with the TEM results. Overall, this data shows that the catalytic activity of GlpG influences type 1 pili, a critical virulence factor in E. coli, through the quality control of FimA.

GlpG activity influences pathogenic E. coli invasion
UPEC can infect both bladder and kidneys cells and account for the vast majority of all UTI infections12. To determine whether loss of pili observed in glpG-S201A strains results in reduced host colonization, both an in vitro adherence and invasion (gentamicin protection) assays were used to quantify bacterial adhesion and entry into epithelial cells (Fig. 3a). The glpG-S201A mutants of UPEC NU149 and commensal K12 substr. MG1655 showed significantly reduced invasion relative to their wild-type counterparts in both T24 human bladder (Fig. 3b) and mIMCD-3 mouse kidney cell lines (Fig. 3c). Notably, this reduction in invasion was rescued to wild-type levels when active GlpG was reintroduced using an inducible plasmid, confirming the loss of GlpG catalysis results in reduced host colonization in uropathogenic and commensal strains of E. coli. We also assessed invasion indices of UPEC strains against kidney and bladder host cells. The invasion index represents the ratio of invaded bacteria to adherent bacteria, quantifying invasion efficiency independent of adhesion. The invasion indices were not significantly different between NU149 WT and glpG-S201A mutant, therefore GlpG does not cause defect in initial attachment, but specifically impairs the ability to invade host cells (Supplementary Fig. 6). As observed by other groups, other adhesins besides pili, such as the Afa/Dr family of nonfimbrial adhesions, or curli can also facilitate attachment playing roles in different host environments during different stages of infection51,52.

Proximal and distal tubular epithelial cells of the kidney have the capability to phagocytose E. coli present in the tubular lumen, causing peritubular inflammation53. To determine if the GlpG protease impacts the E. coli infectivity for tubular epithelial cells, human kidney organoids differentiated to express segments of the proximal tubule, were infected with NU149 wild-type or NU149 glpG-S201A E. coli. Kidney organoids infected with wild-type NU149 had E. coli-FITC stained structures present within LTL-Cy5 positive regions, which label proximal tubules (Fig. 4a, b and Supplementary Fig. 7). Meanwhile, a 3.5-fold reduction of detectable bacteria at 469 NB100-FITC per mm2 of LTL-Cy5 for NU149 glpG-S201A was observed compared to wild-type infected kidney organoids which had 1650 anti-E. coli-FITC per mm2 of LTL-Cy5 (Fig. 4c). Consistent with our results of the gentamicin protection assay, proximal tubular cell invasion for NU149 glpG-S201A was significantly lower compared to wild-type, further highlighting the importance of GlpG for bacterial pathogenicity.

GlpG influences biofilm formation of uropathogenic E. coli
Biofilms are a major cause in recurrent bacterial infections as it protects the pathogen from the host immune response as well as administered antibiotics, leading to persistence, chronic infection, and poorer patient outcomes54,55. Previous work showed that pathogenic E. coli can form biofilms on the surface of epithelial cells, which promote their persistence in vivo56. Importantly, surface pili were shown to play a key role in driving the formation of E. coli biofilms56–58. Given its role in the homeostasis of type 1 pili, we reasoned that GlpG is required for the assembly of biofilms by UPEC on biotic surfaces. To test this, we infected mIMCD-3 kidney cells with NU149 wild-type or glpG-S201A isogenic strains for 6h to allow biofilm formation. To quantify these structures, we tallied the number of bacterial aggregates larger than 100 µm2, which was previously shown to correspond to the sizes of biofilms made by pathogenic E. coli56. Interestingly, we observed a significant reduction in biofilm formation (~25-fold) by the mutant relative to the wild-type (Fig. 5a, b). This was rescued by overexpressing GlpG in the mutant strain, confirming the role of this protease in facilitating biofilm assembly, likely through its role in FimA proteostasis.

Another method to validate the effect GlpG has on the formation of bacterial biofilm is safranin staining, used to quantify biofilm mass produced in UPEC on an abiotic surface. Indeed, the NU149 glpG-S201A mutant, which displays impaired pili formation, showed significant reduction in biofilm production compared to the wild-type (Fig. 6a). Interestingly, the glpG-S201A strain overexpressing active GlpG protease showed a significant increase in biofilm biomass. These results demonstrate a correlative relationship between the catalytic activity of GlpG, pili formation, and biofilm forming abilities of E. coli. Indeed, negatively stained electron micrographs of NU149 demonstrated increased amount of GlpG protease in the cell significantly affected the amount of pili on the surface.

GlpG inhibition affects the expression of surface virulence factors
To investigate whether the expression of surface virulence factors can be blocked by inhibiting E. coli GlpG rhomboid protease, we used commercially available serine protease inhibitor, tosyl phenylalanyl chloromethylketone (TPCK). We determined the inhibitory concentration of TPCK for GlpG in vitro using a peptidic FRET substrate (Mca-FATA*AFGSP-Dpn) designed by our team based on the cleavage sequence of the transmembrane domain of TatA protein from P. stuartii—a known bacterial substrate of rhomboid protease AarA that E. coli GlpG is also able to cleave59. We measured the IC50 value of TPCK for GlpG to be 296 µM (Supplementary Fig. 8), consistent with previous observations60.

To explore the effect of TPCK on virulence factor expression, bacterial adherence, and formation of biofilm, biofilm inhibition and biofilm eradication assays were performed. In parallel, electron micrographs of E. coli cells treated with TPCK were taken to observe changes in surface organelles presentation. Biofilm inhibition was assessed by adding the inhibitor at the same time as the re-addition of active GlpG to the glpG-S201A strain (NU149 glpG-S201A + GlpG) and assessing the biofilm production. Overall, we observed a decrease in biofilm formation in the presence of TPCK for NU149 wild-type, but a more profound inhibitory effect was observed in NU149 glpG-S201A + GlpG, which we show produces more biofilm than the wild-type (Fig. 6a and d). We then explored whether inhibition of GlpG can aid in biofilm eradication, or the removal of pili that have already formed. To test this, biofilms were first pre-established with bacterial growth before the addition of TPCK inhibitor. When quantified, we did not observe a significant difference in adhered biomass for wild-type NU149 treated with TPCK; however, the NU149 glpG-S201A + GlpG strain, again, demonstrated a significant decrease in biofilm mass in the presence of TPCK (Fig. 6b and e). To ensure the reduction in biofilm was not due to toxic effects of TPCK, a metabolic activity assay was performed in parallel. Using tetrazolium salt dye, 2,3,5-triphenyl tetrazolium chloride (TTC), detecting cell redox activity and viability, we did not observe the decrease in metabolism for any of the strains used, both with and without inhibitor treatment (Fig. S9), indicating that biofilm eradication was not in fact due to cell death.

These observations were confirmed with negatively stained electron micrographs of NU149 glpG-S201A + GlpG. When TPCK was added along with the inducing agent for GlpG expression, the cells had a drastic decrease in pili (Fig. 6d), whereas when TPCK was added after surface elements were already formed, we observed a fragmented pili network (Fig. 6e). Pili are known to be fragile and constantly turned over and if assembly is faulty for reasons such as GlpG inhibition, the resulting pilus rods may have structural defects and reduced stability, making them more prone to dissociation. This indicates that GlpG is important for maintaining the integrity of the pili in pathogenic bacteria, a crucial feature for retaining the biofilm.

GlpG cleaves aggregated FimA to prevent accumulation in the periplasm
To confirm the mechanism of GlpG and its contribution to the quality control of FimA, we performed an in vitro endpoint activity assay capturing GlpG-mediated cleavage of FimA aggregates. Recombinantly expressed active GlpG, either dodecyl maltoside (DDM) solubilized or reconstituted into E. coli lipid proteoliposomes (PLS) to mimic the physiological membrane, was added to the purified periplasmic fraction of the K12 MG1655 ΔglpG mutant containing aggregated FimA. The time course cleavage was visualized by immunoblotting with FimA antibody (Fig. 7a, b). Indeed, upon incubation with active GlpG protease in DDM and in PLS, an observed decrease in FimA aggregates was evident over time confirmed by densitometry analysis of the corresponding bands (Fig. 7c, d and Supplementary Fig. 10), demonstrating GlpG can cleave FimA aggregates in the lipid bilayer.

During FimA translocation through the Sec pathway, FimA has an exposed, hydrophobic C-terminal tail that is prone to aggregation in an aqueous periplasm. This C-terminal tail of FimA must immediately bind to FimC chaperone in the periplasm for its eventual transport to the outer membrane. Thus, if after SecYEG-export to periplasm, FimA is improperly folded, or when insufficient FimC chaperone molecules are available to bind and transport FimA through the periplasm, pilins will misfold and aggregate and are targeted by DegP and DegQ, the housekeeping periplasmic proteases. GlpG most likely influences this pathway by processing or regulating misfolded pilins, cleaving the C-terminus of FimA subunits and “untangling” big oligomers prior to its degradation upstream of DegP/Q-mediated degradation. To verify the cleavage site in the FimA molecule, we designed a FRET-FimA peptidic substrate (DABCYL-QARYFATGAATPGAANADATFKVQYQ-Glu(EDANS) based on the sequence of the FimA peptide identified in LC-MS/MS analysis of periplasmic proteins. The identified sequence is located on the C-terminus of FimA, residues 157 to 182, which forms the hydrophobic groove and binding site for FimC chaperone during translocation (Fig. 7e). Due to the hydrophobic nature of the region, the FRET-FimA peptide was prone to aggregation and had limited solubility, as expected. Cleavage of the peptide was demonstrated using purified GlpG in DDM and PLS with no cleavage by catalytically inactive GlpG as a control (Fig. 7f).

To confirm the role of GlpG in maintaining the fine balance between free FimA that is prone to aggregation, and FimA bound to FimC for pilin assembly, we investigated the effect of increasing the ratio of FimC relative to FimA in the NU149 glpG-S201A strain on cell invasion and biofilm formation. NU149 glpG-S201A, which we show has reduced pili and invasion of host cells, had pili (Fig. 2), as well as host invasion (Fig. 7g, h), return to wild-type levels when overexpressing FimC and. This is further supported by the partial rescue of biofilm formation upon overexpressing FimC in the glpG-S201A mutant where we observed ~75% restoration of biofilm biogenesis, suggesting that overexpression of this chaperone can prevent the aggregation of pilin subunits when GlpG is absent (Fig. 7i, j). This change in equilibrium, increasing available FimC to bind free FimA, rescues the pili-expressing, invasive phenotype. Since FimA requires FimC to reach the outer membrane, having excess chaperone available to bind and transport FimA during translocation limit the presence of unbound FimA and potential aggregation in the absence of GlpG proteostasis (Fig. 8). By contrast, when the cell has less FimC relative to FimA during type 1 pili formation, GlpG performs its role in protein homeostasis, cleaving FimA at the exposed, hydrophobic C-terminal tail. By identifying FimA as a physiological substrate of E. coli GlpG, our data supports the role of GlpG in maintaining type 1 pili quality control by cleaving FimA aggregates for subsequent degradation by other proteases such as DegP and DegQ that were identified as interactors of GlpG in whole cell proteomics. This prevents the accumulation of FimA in the periplasm and obstruction of the translocation pathway during type 1 pili assembly.

Methods
LC-MS/MS proteomics on endogenously FLAG-tagged GlpG purified from whole cells
E. coli strain DY330 harboring a triple FLAG-tag (SPA-tag) inserted at the N-terminus of the genomic glpG gene was generated by targeted homologous recombination into the E. coli genome. For purification of endogenously expressed SPA-tagged GlpG, E. coli was grown in LB media with kanamycin and lysed in buffer containing 50 mM Tris, pH 8.0, 200 mM NaCl, 20% glycerol, and detergent, either 1% Triton X-100, DDM or C12E8. No significant differences in pulldown data were observed therefore the three detergents samples were treated as replicates. Interactions in the presence of Triton X-100 are presented for clarity. Cleared fractions containing solubilized GlpG bait and associated proteins were purified on anti-FLAG and calmodulin columns, following the SPA method67, except that after the first three washing steps, calmodulin wash buffer (30 mM Tris-HCl pH 7.9, 150 mM NaCl, 2 mM CaCl2, 0.1–0.5 mM Tris (2-carboxyethyl) phosphine (TCEP)) with 0.1% Triton was used, with the final two washes performed without detergent. Negative controls were performed in wild-type DY330 E. coli with anti-FLAG beads68. Purified protein preparation was resuspended in 25 μL of 8 M urea (1.5 M final), 20 mM HEPES (pH 8.0), reduced with 5 mM TCEP for 45 min at room temperature and alkylated with 15 mM iodoacetamide for 60 min in the dark. Modified trypsin (0.3 mg, Promega) was added, and digestion performed overnight at room temperature with gentle shaking. Peptide mixtures were acidified with formic acid and desalted using C18 Top Tips (Glygen). Resulting peptides were loaded via autosampler onto a 75-μm inner diameter fused silica column packed with ~10 cm C18 resin (3 μm, 100 Å, Phenomenex) coupled to a Proxeon EASY nLC 1000 (Proxeon) and Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptides were eluted by electrospray ionization with a 100 min water/acetonitrile gradient at ~0.30 µl min-1. Precursor ions [350–2000 m/z] underwent data-dependent collision-induced dissociation, with one full mass scan followed by 15 tandem mass scans of intense precursor ions, with dynamic exclusion.

The Orbitrap Elite mass spectrometer was operated with Thermo XCalibur software. All RAW files were converted to mzXML using ReAdW-4.3.1 and submitted for database searching using SEQUEST-PVM v.27 (rev. 9)69 under standard workflow conditions with a non-redundant E. coli proteome sequence FASTA file from Ecocyc. Search parameters were set to allow for dynamic modification of methionine oxidation and fixed modification of cysteine carbamidomethylation using precursor ion tolerance of 20 ppm. A stringent false discovery rate of 1% (P < 0.01) was used to filter candidate peptide and protein identifications. Precursor mass tolerance was set to 3 Da (daughter mass ion tolerance set to the default of 0), while enabling partial tryptic enzyme and single site missed cleavages. The STATQUEST filtering algorithm70 was then applied to all putative SEQUEST search results to assign statistical confidence. High confidence (>90% likelihood) spectral counts from SEQUEST/STATQUEST were used to calculate Normalized Spectral Abundance Factor (NSAF) score71. To be more conservative, the proteins were then filtered on median NSAF score or STATQUEST probability of 99%. Proteins localized in the periplasm and inner membrane, belonging to the COG categories O and U72 were visualized as Cytoscape network73. All raw data is deposited and publicly available in the MassIVE repository (MSV000097249) and Supplementary Data 1.

Periplasmic protein isolation
Periplasmic proteins were isolated using cold osmotic chock and a Tris-Sucrose-EDTA buffer45. K12 substr. MG1655 ΔglpG mutant was grown to OD 1.0 and centrifuged at 5000 × g for 10 min at 4°C. Cells were resuspended in a Tris-Sucrose-EDTA buffer (200 mM Tris pH 8.0, 500 mM sucrose, 1 mM EDTA, 1 mM PMSF) with a cOmplete EDTA-free protease inhibitor cocktail tablet (Roche, Cat. # 11836170001) and incubated on ice for 30 min. The sample was centrifuged 20,000 × g for 30 min at 4°C where the final supernatant contains the periplasmic proteins.

GlpG purification
Recombinantly expressed E. coli GlpG and inactive mutant GlpG Ser201Ala were purified following standard membrane protein purification protocols74. Briefly, E. coli GlpG-TEV-6×His or GlpG-S201A-TEV-6×His in a pBAD vector was expressed in TOP10 cells with 0.002% arabinose at OD 0.8 and collected by centrifugation at 5000 × g for 15 min at 4°C. The pellet was resuspended in lysis buffer containing 50 mM Tris, pH 8.0, 200 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail tablet (Roche Cat. #), and lysed using high-pressure with an Emulsiflex. The proteins were separated from cellular debris at 13,000 × g for 30 min at 4°C and the resulting supernatant was centrifuged at 40,000 × g for 2 h at 4°C to isolate the membrane fraction. Membranes were resolubilized in a lysis buffer containing 1% Dodecyl-β-D-maltoside, incubated with Ni-NTA resin and eluted with increasing concentrations of imidazole in the lysis buffer. Fractions containing GlpG-TEV-6×His were further incubated with TEV protease and incubated in Ni-NTA resin again. The flow-through contains full-length GlpG while TEV-6×His remains bound to the resin. The resulting protein fraction was concentrated with a 30 MWCO Amicon spin concentrator (Millipore, Cat. #UFC9030) and injected into a HiLoad 16/600 Superdex 200pg column (Cytiva, Cat. #28-9893-33).

Reconstitution of GlpG into proteoliposomes
400 µg E. coli polar lipids (Avanti) suspended in chloroform were dried under nitrogen stream in a glass tube, followed by overnight incubation in a desiccator to yield a thin film of lipid. 50 µl of water and DDM detergent was added to the lipid film for resuspension at room temperature for 10 min, followed by the addition of purified GlpG protease (400 µg) in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 20% glycerol, 0.1% DDM, to yield the final weight ratio of 1:1:2, GlpG:lipid:detergent. The detergent was slowly removed by the addition of SM2 Biobeads (Bio-Rad) while stirring on ice for 6 h to allow for the generation of proteoliposomes (PL); the process was controlled by the regimen of Bio-bead addition. 50%:20% sucrose density gradient ultracentrifugation was used to purify PL. The concentration of reconstituted GlpG was assessed by BCA assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific) and the purity was confirmed with Coomassie staining after SDS-PAGE.

LC-MS/MS proteomics on periplasmic proteins
Periplasmic proteins isolated from K12 substr. MG1655 ΔglpG mutant were incubated with 5 µM of active or inactive GlpG and separated based on molecular weight using 4–20% Mini-PROTEAN TGX Precast Gels (Bio-Rad, Cat. # 4561094) at either (1) 100 V for 5 min, or a migration of 1.5 cm or (2) 150 V for 30 min where proteins were analyzed separately based on molecular weight range. Gels were fixed for 20 min (50% ethanol, 2% phosphoric acid), washed for 20 min in MilliQ H2O and stained overnight with a blue silver Coomasie stain (10% phosphoric acid, 20% ethanol, 750 mM ammonium sulfate, 13 mM Coomassie blue G-250) and washed for 20 min in MilliQ H2O. The band or bands were excised from the gel and further cut into 1 mm pieces. The gel pieces were transferred to a round bottom 96-well plate for destaining (50 mM ammonium bicarbonate, 50% acetonitrile). The plate was incubated for 10 min at 37°C. The solution was removed from the wells and the destaining procedure was repeated 4 times. The solution was removed and replaced by acetonitrile for dehydration. The samples were incubated again at 37°C for 10 min. Dehydration was repeated until the gel pieces became white (3 times). The samples were dried at 37°C for 10 min, rehydrated in a reducing solution (100 mM ammonium bicarbonate and 10 mM β-mercaptoethanol), and incubated for 30 min at 37°C. The reducing solution was removed. The samples were alkylated with iodoacetamide (10 mg/mL of iodoacetamide and 100 mM ammonium bicarbonate) and incubated for 30 min at 37°C. The gel bands were subsequently washed with 100 mM ammonium bicarbonate and incubated for 10 min at 37°C (repeated twice). The samples were then dehydrated in acetonitrile for 10 min at 37°C (repeated twice). Acetonitrile was removed and the samples were dried at 37°C for 10 min. The gel bands were then trypsinized (0.3 μg of mass-spec grade trypsin per well, Promega) overnight at room temperature. The solutions containing tryptic peptides were transferred to a V-bottom 96-well plate. Tryptic peptides were further extracted from the gel with 2% acetonitrile and 1% formic acid followed by incubation at 37°C for 1 h. The extraction was repeated using 50% acetonitrile and 0.5% formic acid followed by incubation at 37°C for 1 h. The samples were dried with vacuum and resuspended in 20 mL of 0.1% formic acid for analysis on liquid chromatography tandem mass spectrometry (LC-MS/MS). Peptides were separated using a nanoflow-HPLC (Thermo Fisher Scientific EASY-nLC 1200 or 1000 System) coupled to a Orbitrap Fusion Lumos or Q Exactive mass spectrometer (Thermo Fisher Scientific).

Raw data were analyzed using Proteome Discoverer (v2.4.1.15, Lumos) or Protein Prospector (v5.22.1, QExactive) software against the E. coli proteome (https://www.uniprot.org). Search parameters include a precursor and a fragment mass tolerance of 15 ppm and 0.8 Da (Lumos) or 10 ppm and 0.01 Da (QExactive), respectively. Searches include up to two missed trypsin cleavages, and charge states of 2, 3, and 4. Static modification was set to carbamidomethylation of cysteine and dynamic modifications include oxidation of methonines, deamidated glutamine and asparagine, and acetylated protein N-termini. 895 proteins were quantified with high reproducibility (~95%) between biological replicates n=2. Protein abundance changes between periplasmic proteins incubated with active and inactive GlpG with a significant p-value below 0.05 (−log10 p-value > 1.30) and a fold change (FC) greater than two (log2 FC > 1), are presented. Identified peptides were searched against a random decoy protein database to evaluate the false-positive rate (set to 1%). All raw data is deposited and publicly available in the MassIVE repository (MSV000097236) and Supplementary Data 2 and 3. Gene ontology was performed using https://metascape.org against the E. coli proteome using: GO biological processes, KEGG pathway, Reactome Gene Sets, CORUM, TRRUST, PaGenBase, and WikiPathways.

Hemagglutination assay
E. coli strains were grown statically at 37°C until OD 600 = 1.0; for strains harboring an inducible plasmid, cells were grown till OD 600 = 0.6, the gene was induced and grown for another 2 h. The OD 600 then was adjusted to 1.0 for comparison between different strains. Guinea pig erythrocytes (Innovative Research, Inc., USA) were washed with PBS and diluted to the final concentration of 3% (v/v). In a 96-well round bottom plate, 50 µL of bacterial suspension was mixed with 50 µL of 3% red blood cell (RBC) suspension. The plate was incubated at room temperature for 60 min without agitation. Absence of type 1 pili is indicated by a “button” formed by the settled RBCs at the round bottom well, whereas presence of type 1 pili binds the RBCs forming a diffuse mixture. For MSHA, 2% mannose was added to bacterial suspension to selected wells before adding RBC and incubated for 15 min to inhibit type 1 pili-mediated hemagglutination.

FimA cleavage time point assay
Periplasmic proteins were dialyzed in GlpG activity buffer containing 50 mM MES, pH 6.0, 150 mM NaCl, 20% glycerol, 0.1% DDM, as determined by our group75. The periplasmic proteins were incubated with 5 µM of E. coli GlpG or the catalytically inactive mutant (Ser201Ala) at 37°C for 6 h, taking fractions at various time points for western blot analysis.

Western blot
To measure the level of FimA over time, fractions of FimA incubated with GlpG were analyzed by standard western blot methods and probed with rabbit anti-FimA antibody (dilution=1:2000; Biomatik, CAC11912) and HRP conjugated goat anti-rabbit antibody (dilution=1:5000; Thermo Fisher Scientific, Cat. #31460). Amersham Western Blot Detection Reagent (Cytiva, Cat. #45-002-401) was used and the blot was imaged on the Li-COR using the ImageJ program.

In vitro cleavage assay
FimA FRET peptide, DABCYL-QARYFATGAATPGAANADATFKVQYQ-Glu(EDANS) (BioMatik) was incubated either GlpG in DDM or in PLS. For GlpG in DDM, activity buffer consisting of 50 mM MES, pH 6.0, 150 mM NaCl, 20% glycerol, 0.1% DDM. The reaction was started with 0.5 µM of purified GlpG solubilized in DDM and was monitored for 3 h at 37°C. The activity assay with GlpG reconstituted into PLS was performed the same way as for the protease in DDM with the only difference being the activity buffer, where DDM was omitted (50 mM MES pH 6.0, 150 mM NaCl, 20% glycerol). Prism 9.0, GraphPad Software, was used to determine cleavage of the peptide.

Construction of mutant strains
Ser201Ala point mutations in the glpG gene were generated in E. coli K12 substr. MG1655 and NU149, denoted as glpG-S201A, by method of CRISPR/Cas9 gene-editing76. Briefly, electrocompetent cells harboring pCas (Addgene: #62225) were co-transformed with pTarget GlpG and 500 ng of donor template DNA containing the glpG gene with a S201A mutation. Cells were recovered at 30°C for 2 h and spread on LB agar containing kanamycin (50 µg/mL) and spectinomycin (50 µg/mL) and incubated overnight at 30°C. Transformants were identified by colony PCR and Sanger sequencing.

Negatively stained transmission electron microscopy of E. coli
Different strains of E. coli were grown statically to preserve pili integrity in LB for 48 h at 37°C. Strains harboring a GlpG plasmid were induced after the first 8 h of growth. 7 µL of cell culture were gently added to glow-discharged carbon-coated copper grids (Electron Microscopy Sciences, Cat. # CF200-Cu-50) and left at room temperature for 5 min. Excess sample was wicked away using Wattman paper, washed with nuclease-free water and stained with 2% (w/v) uranyl acetate. Grids were imaged on a Hitachi H-7650 Transmission Electron Microscope.

RT-qPCR of glpG
E. coli strains were grown in LB media, TB media, or M9 minimal media to an OD 600 of 1.0 and centrifuged at 5000 × g. RNA was extracted using TRIzol™ Max Bacterial RNA Isolation kit (Invitrogen, Cat. #16096020). cDNA was amplified using iScript Reverse Transcription Supermix (Bio-RAD, Cat. #1708840) and RT-qPCR was performed with PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Cat. #A25741) and analyzed for mRNA gene expression using the QuantStudio 5 system. Both glpG and housekeeping gene rrsB were measured with glpG mRNA expression was measured as ΔΔCt.

Mammalian cell culture conditions
Mouse inner medullary collecting duct (mIMCD-3; ATCC, Cat. #CRL-2123) cells were grown in DMEM:F12 medium (Gibco, # Cat. #11320033) containing 10% fetal bovine serum (Sigma-Aldrich, Cat. #12103C) at 37°C, 5% CO2. Human bladder (T24; ATCC, Cat. #HTB-4) cells were grown in McCoy’s medium (Sigma-Aldrich, Cat. #30-2007) containing 10% fetal bovine serum at 37°C, 5% CO2.

Gentamicin protection assay
Various strains of E. coli were grown to an OD 600 of 1.0 and added to a confluent layer of mouse inner medullary collecting duct (mIMCD-3; ATCC, Cat. #CRL-2123) or human bladder (T24; ATCC, Cat. #HTB-4) cell line to facilitate infection for 4 h at 37°C. Cells were washed with sterile PBS and grown in medium containing 100 μg/mL of gentamicin for 2 h to kill all remaining extracellular bacteria, leaving invasive bacteria unaffected. Invasive bacteria were collected by lysing mammalian cells with 2% (v:v) Triton X-100. Serial dilutions of lysate were plated on LB agar plates and grown for 18 h at 37°C to quantify bacterial invasion by manual counting of colony-forming units.

Generation of kidney organoids
Kidney organoids were differentiated using the Takasato protocol with minor modifications as previously described by the Chun group77,78. Briefly, induced pluripotent stem cells were grown in 6 well plates coated with Matrigel matrix (Corning, Cat. #354277). Cells were treated with 8 μM CHIR99021 (R&D Systems, Cat. #4423) for 4 days followed by recombinant human FGF-9 (200 ng/mL; R&D Systems, Cat. #273-F9-025) and heparin (1 μg/mL, Millipore Sigma, Cat. #H4784) for an additional 3 days in APEL2 medium (Stem Cell Technologies, Cat. #05275) supplemented with 5% PFHM-II (Thermo Fisher, Cat. #12040077). On day 7, cells were dissociated using trypsin-EDTA (0.05%) for 2 min and 100,000 cells were transferred onto a six-well transwell plate (Corning, Cat. #07-200-170) with nine pellets per well without an aggregation step. Cell clusters were incubated with a pulse of 5 μM CHIR99021 in APEL2 + PFHM-II medium for 1 h at 37°C. After 1 h, the medium was changed to APEL2 + PFHM supplemented with 200 ng/mL FGF-9 and 1 μg/mL heparin for an additional 5 days and then were maintained in APEL2 + PFHM-II medium for 14 days with medium change every other day.

Infecting kidney organoids with E. coli
UPEC E. coli NU149 wild-type and NU149 glpG-S201A were grown overnight in 5 ml LB at 37°C, 225 RPM. 300 μl of overnight culture for each strain were transferred to 2.7 ml LB and grown for 4 h at 37°C, 225 RPM. Cultures were centrifuged at 2000 × g for 2 min with the supernatant aspirated and discarded. Each pellet of bacteria was resuspended in 1 mL APEL2. On day 26 of differentiation, kidney organoids were infected in triplicates with 1:10 and 1:100 concentrations of E. coli strain or vehicle control and infected at 37°C, 5% CO2 for 6 h with agitation every 1 h. After 6 h, supernatant was discarded, and kidney organoids were washed 2 times with PBS for 5 min with rocking for each wash. To inactivate E. coli, kidney organoids were treated with an APEL2 solution containing 2× Pen/Strep (200 units/mL penicillin and 200 μg/mL streptomycin) (Gibco; Cat. #15140122) at 37°C, 5% CO2 for 2 h. The organoids were then washed 2 times with sterile PBS for 5 min and fixed with 2% paraformaldehyde for 20 min at 4°C. After two washes with PBS, kidney organoids were stored at 4°C until processed for immunofluorescence and imaging.

Immunofluorescence and confocal microscopy of kidney organoids
Kidney organoids were processed prior to immunofluorescence microscopy78. In brief, kidney organoids were blocked in a blocking buffer (10% donkey serum and 0.05% Triton-X in PBS) for 2 h at room temperature. Primary antibodies to E. coli (anti-E. coli-FITC; Novus Cat. #NB10064448, 1:25), anti-NPHS1 (R&D Systems, Cat. #AF4269, 1:200), Lotus tetragonolobus lectin-Cy5 (GlycoMatrix Cat. #21761117-1; 1:200) were incubated at 4°C overnight. Following the overnight incubation, organoids were washed 3 times with wash buffer (0.3% Triton X-100 in PBS) for 10 min each wash followed by incubation in wash buffer with fluorescent conjugated secondary antibodies that include Alexa Fluor donkey anti-sheep 594 (Thermo Fisher Scientific, 1:250) for 2 h at room temperature. After 3 washes with PBS and 10 min labeling with 5 μg/ml Hoescht 33342 (Thermo Fisher Scientific, Cat. #H3570), confocal images were acquired with a Zeiss LSM880 confocal microscope.

Quantification of bacteria infected proximal tubules
LTL-Cy5 labeled proximal tubules were identified and isolated for quantification of E. coli infected proximal tubules of the kidney organoids. Confocal images of E. coli-FITC positive structures within the LTL-Cy5 positive regions were analyzed using ImageJ as were described previously for SARS-CoV-2 infected kidney organoids79.

GlpG inhibition assay
Inhibition was calculated by using 0.2 µM of purified GlpG that was incubated with tosyl phenylalanyl chloromethylketone (TPCK) in a concentration range of 0.1 µM to 5 mM at 37°C for 60 min in activity buffer consisting of 50 mM MES, pH 6.0, 150 mM NaCl, 20% glycerol, 0.1% DDM. The reaction was started with 15 µM of modified TatA-FRET substrate (K M for modified TatA-FRET is 10 µM; Mca-FATA*AFGSP-Dpn, Biomatik, 98% purity) and was monitored for 3 h at 37°C. Prism 9.0, GraphPad Software, was used to determine the IC50 value.

Biofilm formation assay on biotic surface
mIMCD-3 cells were grown in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12 1:1, Gibco). Cells were seeded on coverslips pre-coated with poly-L-lysine and grown to 90% confluency. UPEC NU149 strains were grown overnight at 37°C in lysogeny broth (LB). The following day, cells were subcultured in LB at 37°C until the OD 600 reached 0.2. Protein expression was induced by adding 0.002% arabinose or 1 mM IPTG for 2 h. mIMCD-3 cells were infected with NU149 strains at a multiplicity of infection of 1:25 for 1.5 h. Non-adherent bacteria were washed off with 1× PBS twice, then cells were incubated with fresh media for an additional 4 h. At the end of the infection period, cells were washed twice with 1× PBS, then fixed with 3% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature. Coverslips were blocked overnight with 3% goat serum (Invitrogen) and 1% bovine serum albumin in PBS (BioShop) at 4°C. To stain the cells and bacteria, samples were incubated with rabbit anti-O-Ag conjugated to FITC (1:50 dilution, Novus Biologicals) and phalloidin Alexa Fluor 568 (1:40 dilution, Invitrogen, Cat. #A12380) for 1 h at room temperature. Coverslips were washed three times in 1× PBST, then stained with 300 nM DAPI (Invitrogen, Cat. #D1306) for 5 min at room temperature and washed three times with 1× PBST prior to mounting on glass slides. All images were captured with BioTek Cytation 5 Imager using a 20× objective. Samples were imaged blindly and processed with BioTek Gen5 software for biofilm quantification. Acquired images were preprocessed then analyzed to quantify aggregates with surface area of 100 µm2 or more, shown previously to represent biofilms formed by pathogenic E. coli56. Images were obtained from three independent experiments.

Biofilm formation assay on abiotic surface
E. coli NU149 wild-type, E. coli NU149 glpG-S201A strain and E. coli NU149 glpG-S201A + GlpG, the inactive glpG strain transformed with pBad plasmid bearing active GlpG protease, were streaked on LB agar with the appropriate antibiotic. Bacteria cultures were grown in LB medium from colonies and grown for 16–18 h at 37°C with shaking. Overnight cultures were diluted to OD 600 of 0.01 where 200 µl of bacterial sample was added per well on a 96-well plate. LB media alone was always included as a negative control of contamination. Bacterial cultures were grown at 37°C under static conditions. After 3 h, arabinose was added to the wells with NU149 glpG-S201A + GlpG strain to the final concentration of 0.02%. After 24 h, the optical density at 600 nm was recorded on a BioTeK Cytation 5 microplate reader to quantify the overall bacterial growth in each well. Media with planktonic cells was discarded and wells were rinsed three times with PBS. Once excess PBS was removed, adhered biomass was stained by adding 230 µl of 0.1% Safranin solution to each well and placing the plate on an orbital shaker for 30 min at room temperature (Ommen et al., 2017). The stain was discarded, the wells were rinsed with PBS three times again and the excess of the buffer was removed. Safranin dye bound to the adhered biofilm was solubilized in 230 µl of 30% acetic acid per well. The absorbance at 595 nm was recorded to evaluate the amount of biomass adhered.

Biofilm inhibition assay
TPCK inhibitor was added at the same time as a freshly prepared bacterial suspension with OD 600 of 0.01 at the final concentration of 400 µM and then incubating for a certain time followed by quantification of biofilm biomass by Safranin the same as described above.

Biofilm eradication assay
A requirement is pre-establishing consistent biofilms in the well prior to inhibition; thus biofilm was allowed to be formed as described above. After incubation for 24 h the adhered biomass was rinsed with fresh LB media, treated with TPCK (400 µM final concentration), incubated for another 24 h and quantified with Safranin dye.

Metabolic activity assay
The 96-well plate for metabolic activity assay was set up the same way as the biofilm eradication assay plate above. After pre-establishing biofilms, washed with fresh LB media and treated with TPCK inhibitor, TTC dye (TTC, 0.05% final concentration) was added to the wells and the plate was incubated for 24 h under static conditions at 37°C. The wells were rinsed three times with PBS, and 230 µl of methanol was added to each well followed by quantification by reading the absorbance at 500 nm.

Statistics and reproducibility
All statistical analyses for in vitro experiments were performed using the one-way ANOVA test except for the analysis biotic biofilm formation, which was performed with the Kruskal–Wallis test, using GraphPad v10.6.1 for Windows. Figures 1b, 3a, and 8 were created using BioRender.com. No statistical method was used to predetermine sample size.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The work by Lu et al utilizes a novel technique to identify substrates for the E. coli rhomboid protease GlpG, by adding purified GlpG to periplasmic extracts isolated from a glpG deficient mutant followed by LC/MS/MS. One of the putative substrates identified is FimA, a protein required for type I pili formation. The authors go on to show that in the absence of glpG FimA aggregates accumulate in the periplasm, a phenomenon detrimental to pili formation. Subsequent downstream consequences include decrease in cell invasion and reduced biofilm formation.\n\nMajor comments\nWhile the authors have established a connection between GlpG and FimA, which agrees with previous findings that demonstrated a colonization defect for a glpG mutant strain, it is still unclear if FimA is directly cleaved by GlpG in vivo and whether it is part of a cleavage cascade with other proteases to clear misfolded proteins from the periplasm. Indeed, the authors performed a pull-down assay with Flag-tagged GlpG and identified proteases, peptidases, chaperones, and transportation machinery that closely associated with the rhomboid protease. One of the top associated factors with GlpG was DegQ, a periplasmic protein broadly understood to harbor chaperone and peptidase activity towards aggregated proteins. Another associated factor, DegP, has been shown to cleave pili (PapA, structurally related to FimA) in E. coli, which could also be a factor in FimA cleavage. As a potential component of the proteostasis degradome machinery, a better understanding of GlpG is required. For example, if GlpG serves as a site-1 protease, a FimA cleavage product should accumulate in the absence of DegP and/or DegQ. Alternatively, if GlpG acts on cleavage products generated by DegP and/or DegQ, then GlpG would not cleave FimA in a degP/degQ mutant.\n\nAnother concern relates to the lack of proper controls for complementation. While mutant strains lacking glpG are complemented with glpG expressed from the pBAD plasmid in the presence of arabinose, the WT and glpG mutant strains do not contain a plasmid. They should contain empty pBAD vector and should similarly be grown in the presence of antibiotic and arabinose (Figures 2-7).\n\nIn addition, statistical analysis is limited, and figure legends need additional information.\n\nSpecific comments\nFigure 1B; a better description of the experimental setup and data is required. How can the amount of some proteins be “increased” when the starting material is a set amount of protein content?\nFigure 1D needs an antibody control (a fimA knockout mutant). Whole cell extracts of WT and glpG mutant are also needed to verify total FimA levels.\nFigure 2, quantification of pili given here, but not in other places in the text.\nFigure 2B, statistical test? How many cells were included in this analysis?\nFigure 2D, unclear why different growth conditions were used to quantify fimA and glpG expression levels.\nFigure 3, results from the gentamycin protection assay should be displayed as percent cells detected following gentamycin treatment compared to total cells detected in the absence of gentamycin treatment.\nFigure 4, additional description would help interpretation. Unclear in the figure description what NC100-FITC is detecting.\nFigure 6, A and B unclear what statistical test is shown.\nFigure 6, is the effect observed FimA dependent? I.e. what would happen if GlpG is overexpressed in a fimA mutant?\nFigure 7F, an empty pBAD vector/arabinose control is missing.\nFigure 7, panel H is labeled as B in the text and no statistical test is shown even though indicated in text. Panel F, indicating residues in figure would help interpretation as would N- and C- terminal labeling for FimA.\nSup. Figure 3, statistics\nSup. Figure 4, empty pBAD vector/arabinose control\nSup. Figure 5, very hard to interpret without additional labels in the figure\nSup. Figure 7, statistics? How are data normalized if there are reduced cells but equivalent metabolic activity?", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "In this paper, Lu at al provide compelling evidence that the well-conserved GlpG inner membrane protease can help prevent aggregation of the major type 1 pilus subunit FimA during pilus assembly. The authors also present some data suggesting that GlpG can affect flagella biosynthesis. Biochemical data indicating that purified GlpG can promote the dissolution of FimA aggregates in vitro are backed up by functional assays suggesting that the proteolytic activity of GlpG is important for bacterial invasion of kidney and bladder cells, as well as biofilm formation. One of the most interesting results in support of the authors’ model is that overexpression of the FimA periplasmic chaperone FimC can compensate for loss of GlpG activity, at least in some assays. This is an important study, as it provides the first description of a physiologically relevant substrate for GlpG in E. coli and helps explain the decreased host colonization phenotype observed with a glpG mutant in uropathogenic E. coli (UPEC). Though this study has many strengths, there are a few problems that should be addressed, including some missing controls and some potentially misleading text, as noted below:\n\nMajor issues:\n1. The authors use TEM a lot to examine levels of type 1 pili and flagella in the various mutant and complemented strains throughout the manuscript. However, these organelles are only quantified in Fig 2B for one K12 and one UPEC isolate. The authors should ideally quantify numbers of type 1 pili and flagella by either counting these structures (ideally in a blinded fashion) in random EM fields or by alternate assays such as mannose-sensitive hemagglutination assays (type 1 pili) or motility plates (flagella). It seems that they quantified some of the images in Fig 2 (based on lines 214-215), but it is not clear how this was done and it does not seem like quantification was consistently performed for all of the EM-based assays. The methods do not seem to include a description of how bacteria were imaged by EM. This should include growth conditions for the bacteria prior to processing for imaging. The pili observed by TEM are not easy to see. Use of immunogold labeling, or use of a fim KO mutant would help confirm the identity of the observed pili.\n2. As referenced by the authors, UPEC lacking glpG have a defect in colonization of the mouse gut. The authors focus most of their functional experiments (host cell invasion and biofilm formation) on assays that are more relevant to UTI. Does GlpG affect the fitness of UPEC within the urinary tract in vivo? It seems likely based on the in vitro studies presented, but the authors should either test this directly or note this caveat in the text.\n3. The authors show that the proteolytic activity of GlpG promotes invasion of mouse kidney and human bladder cells (Fig 3). Is bacterial adherence affected by GlpG? Might the decrease in invasion reflect a decrease in adherence?\n4. For the TPCK experiments (Fig. 6), time of TPCK incubations should be made clearer. The text and caption for Fig 2 are a bit confusing. Maybe a diagram would help. Does TPCK affect the growth rates or size (e.g. filamentation) of the bacteria in the biofilm assays?\n5. One of the authors’ main conclusions, based mostly on biofilm assays and EM, is that “GlpG is important for maintaining the integrity of the pili and flagella in pathogenic bacteria, a crucial feature for retaining the biofilm.” This sentence, along with Fig 6E and associated text indicating that TPCK caused formation of a “fragmented pili network” is intriguing, as they suggest that TPCK is affecting the stability of pili after they have already been assembled. Though interesting, it is not clear how this observation fits with the authors’ model (which is well substantiated by other data presented) that GlpG helps prevent formation of FimA aggregates during pilus assembly. Might TPCK, which likely has numerous targets in the bacteria, have other GlpG-independent effects on the assembled pili or envelope? Would be interesting, though admittedly not trivial, to see if induced ablation of GlpG expression after type 1 pili assemble leads to fragmentation, similar to TPCK effects (Fig 6E). Without this or related data, the authors may want to re-phrase the text or at least provide some possible explanation(s) in the Discussion.\n\nMinor issues:\n1. For Fig 2D and the methods, TB should be defined.\n2. Considering the effects of GlpG mutants on flagella expression (based on EM), it would be worth noting if the authors’ proteomics data picked up flagellin or other motility-related proteins as potential GlpG substrates.\n3. From the Methods, it appears that the bacteria were often grown shaking prior to use in invasion assays and other experiments. Shaking does not promote type 1 pili expression as much as growth in static culture, which is more commonly used for invasion assays.\n4. The abstract states that “Our data demonstrates that GlpG plays a key role in protein quality control of virulence factors…” The paper really only nails down GlpG effects on type 1 pili, and starts to address effects on flagella. Both of these factors are better defined as colonization or fitness factors, rather than ‘virulence factors’. Minor point.\n5. Lines 39-43, text gives the impression that all E. coli strains residing in the gut are either commensals or ExPEC, but overlooks diarrheagenic strains that may also persist asymptomatically within the gut. Might want to rephrase text for accuracy.\n6. In the Introduction and elsewhere in the text (e.g. lines 191-192), it is stated that biofilm formation is the most critical factor for development of chronic and rUTI. This may be true, or might be true for only a subset of individuals, but whether or not it is true for all, or even most, individuals with chronic/rUTI problems is not at all clear. Biofilms have been reported in the kidneys, but the contributions of these communities to rUTI or chronic UTI is not clear. Text should be adjusted/qualified to reflect these ambiguities.\n7. Line 106: Might be good to briefly mention some of the “challenges of membrane protein proteomics” for non-experts. Solubility issues?\n8. Based on the author’s findings with FimA, can they comment (maybe in the /Discussion) on whether or not GlpG is likely to have similar quality control functions with other pili (e.g. P pili or others)?", "reviewer_4": "This study by Jimmy Lu et al. investigates the role of the rhomboid protease GlpG in regulating virulence factors, particularly type 1 pili, in pathogenic Escherichia coli. The research provides valuable insights into the mechanisms underlying bacterial pathogenicity and potential targets for therapeutic intervention.\n\nStrengths:\n1. Comprehensive approach: The study employs a wide range of techniques, including proteomics, genetic manipulation, microscopy, and in vitro assays, to elucidate GlpG's role in virulence factor regulation.\n2. Novel findings: The identification of FimA as a target protein of GlpG using proteomics advances our understanding of bacterial virulence mechanisms.\n3. Method: The use of a genomically inactive glpG strain (glpG-S201A) generated by CRISPR/Cas9 for transmission electron microscopy studies demonstrates a thoughtful experimental design.\n4. Functional relevance: The study effectively links GlpG activity to pili formation, pathogenic E. coli invasion, and biofilm formation, highlighting its significance in bacterial pathogenicity.\n5. Translational potential: The demonstration that GlpG inhibition affects the expression of surface virulence factors suggests potential therapeutic applications.\n\nWeaknesses and Suggestions for Improvement:\n1. Context: While the study builds on previous knowledge of GlpG's role in bacterial colonization, more context on how this work advances the field beyond existing literature would be beneficial.\n2. In vitro assay conditions: The in vitro assay demonstrating GlpG's ability to cleave aggregated FimA uses detergent-solubilized GlpG. To better mimic physiological conditions, it would be preferable to incorporate GlpG into a membrane system, such as large unilamellar vesicles (LUVs) containing an E. coli lipid mixture.\n3. FRET-FimA peptide assay: Similar to the in vitro assay, the FRET-based assay could be improved by using a membrane-incorporated GlpG to more accurately represent the periplasmic environment.\n4. Cleavage site specificity: The inability to determine the exact cleavage position of GlpG on FimA (only narrowed down to residues 157-182 at the C-terminus) leaves some mechanistic details unresolved.\n5. Physiological relevance: While the study demonstrates GlpG's ability to cleave aggregated FimA in vitro, further evidence is needed to confirm that this activity occurs in the periplasmic space under physiological conditions.\n\nConclusion:\nThis study provides valuable insights into the role of GlpG in regulating virulence factors in pathogenic E. coli. The comprehensive approach and novel findings contribute significantly to our understanding of bacterial pathogenicity mechanisms. However, some aspects of the in vitro assays could be improved to better reflect physiological conditions. Despite these limitations, the research opens new avenues for exploring potential therapeutic targets in bacterial infections."}, "author_response": "We thank all reviewers for the time taken to carefully review our manuscript. All edits have improved the quality and readership of this paper. Please find detailed responses below:\n>Reviewer #1 (Remarks to the Author)\nThe work by Lu et al utilizes a novel technique to identify substrates for the E. coli rhomboid protease GlpG, by adding purified GlpG to periplasmic extracts isolated from a glpG deficient mutant followed by LC/MS/MS. One of the putative substrates identified is FimA, a protein required for type I pili formation. The authors go on to show that in the absence of glpG FimA aggregates accumulate in the periplasm, a phenomenon detrimental to pili formation. Subsequent downstream consequences include decrease in cell invasion and reduced biofilm formation.\n\nMajor comments\nWhile the authors have established a connection between GlpG and FimA, which agrees with previous findings that demonstrated a colonization defect for a glpG mutant strain, it is still unclear if FimA is directly cleaved by GlpG in vivo and whether it is part of a cleavage cascade with other proteases to clear misfolded proteins from the periplasm. Indeed, the authors performed a pull-down assay with Flag-tagged GlpG and identified proteases, peptidases, chaperones, and transportation machinery that closely associated with the rhomboid protease. One of the top associated factors with GlpG was DegQ, a periplasmic protein broadly understood to harbor chaperone and peptidase activity towards aggregated proteins. Another associated factor, DegP, has been shown to cleave pili (PapA, structurally related to FimA) in E. coli, which could also be a factor in FimA cleavage. As a potential component of the proteostasis degradome machinery, a better understanding of GlpG is required. For example, if GlpG serves as a site-1 protease, a FimA cleavage product should accumulate in the absence of DegP and/or DegQ. Alternatively, if GlpG acts on cleavage products generated by DegP and/or DegQ, then GlpG would not cleave FimA in a degP/degQ mutant.\n\nResponse: Thank you for the suggestion. We tested FimA aggregation pattern in degQ and degP knockout K12 E. coli strains. We detected the FimA accumulation in the periplasmic space in the absence of both, DegQ and DegP proteases, although at lower levels compared ot the glpG KO. This is the first evidence in the literature that DegP/Q are involved into FimA (type 1 pili) quality control. Moreover, after we transformed these mutants with recombinant active GlpG, we were able to demonstrate significant reduction of aggregates suggesting that GlpG is the first step of the cleavage cascade and its proteolytic reaction is likely the rate-limiting step in proteins degradation. We added the detailed description of our data in Results and in Discussion sections.\n\nAnother concern relates to the lack of proper controls for complementation. While mutant strains lacking glpG are complemented with glpG expressed from the pBAD plasmid in the presence of arabinose, the WT and glpG mutant strains do not contain a plasmid. They should contain empty pBAD vector and should similarly be grown in the presence of antibiotic and arabinose (Figures 2-7).\n\nResponse: Thank you for the suggestion, as this was a clear omission on our part. We transformed the mutant bearing glpG-S201A gene with empty pBad vector, induced it with arabinose and incubated under the same conditions as the mutant complemented with pBad-glpG active plasmid. Using that strain, we performed invasion assay, hemagglutination assay and imaged the cells using TEM. We did not observe the effect of an empty vector on the phenotype of mutant strain. The controls are now added to the corresponding figures (Figures 2 and 3).\n\nIn addition, statistical analysis is limited, and figure legends need additional information.\n\nResponse:\nResponse: We have modified the figures and figure legends adding more rigorous statistical analysis and clarifying the figure legends.\n\nSpecific comments\nFigure 1B; a better description of the experimental setup and data is required. How can the amount of some proteins be “increased” when the starting material is a set amount of protein content?\n\nResponse: The term increased and decreased as a result of apparent increased or decreased cleavage events. We added more detailed explanation to the manuscript. The figure remains unchanged.\n\nFigure 1D needs an antibody control (a fimA knockout mutant). Whole cell extracts of WT and glpG mutant are also needed to verify total FimA levels.\n\nResponse: Thank you for the comment. We added fimA knockout and glpG inactive strains to our controls.\n\nFigure 2, quantification of pili given here, but not in other places in the text.\n\nResponse: More rigorous quantification of type 1 pili on WT, mutant, and rescue strains has been added to Figure 2 and also in the manuscript. The TME micrographs were blinded and set box size was used to map areas for manual pili counting.\n\nFigure 2B, statistical test? How many cells were included in this analysis?\n\nResponse: Statistical significance calculated using One-Way ANOVA. Type 1 pili was counted from n=30 E. coli per strain\n\nFigure 2D, unclear why different growth conditions were used to quantify fimA and glpG expression levels.\n\nResponse: Only glpG expression levels were quantified in this experiment. E. coli were grown under different conditions to determine if excess nutrients or a lack of nutrients (stress conditions) affected glpG mRNA expression.\n\nFigure 3, results from the gentamycin protection assay should be displayed as percent cells detected following gentamycin treatment compared to total cells detected in the absence of gentamycin treatment.\n\nResponse: It appears the reviewer is asking for an adhesion assay in addition to our invasion assay. We performed preliminary adhesion assays with WT and GlpG inactive mutant of both NU149 and K12 strains. Moreover, we tested how addition of 2% mannose influences adhesion to confirm the presence and involvement of type 1 pili in the process of adhesion/invasion. However, even though we observed the drop in the amount of adherent cells in the presence of 2% mannose, it didn’t block the adherence 100%, implying the involvement of other surface components and most likely in case of our assay - non-specific binding. Moreover, the difference between WT and inactive mutants of both strains was not as significant as for invasion assay. Therefore, we decided to present invasion assay in the paper, especially since UPEC form intracellular bacterial communities in bladder epithelial cells as a key strategy for persistence and immune evasion, especially in acute and recurrent urinary tract infections, ability to invade is a key characteristic of a successful pathogenic UPEC. This is an area our lab would like to study in more details in a future manuscript.\n\nFigure 4, additional description would help interpretation. Unclear in the figure description what NC100-FITC is detecting.\n\nResponse: NC100-FITC-conjugated antibody recognize all \"O\" and \"K\" antigenic serotypes of E. coli. This antibody was used to detect E. coli cells on the tissue. This has been added to the Figure legend (Figure 4) for clarification.\n\nFigure 6, A and B unclear what statistical test is shown.\n\nResponse: One-way ANOVA was used for calculating statistical significance. This has been added to the figure legend.\n\nFigure 6, is the effect observed FimA dependent? I.e. what would happen if GlpG is overexpressed in a fimA mutant?\n\nResponse: Yes, it was definitively FimA effect since we observe significant decrease in biofilm formation in GlpG inactive mutant, which, as it was shown by TEM and hemagglutination assay, lacks type 1 pili.\n\nFigure 7F, an empty pBAD vector/arabinose control is missing.\n\nResponse: Thank you for the suggestion. We added the pBad empty vector control to invasion assay.\n\nFigure 7, panel H is labeled as B in the text and no statistical test is shown even though indicated in text. Panel F, indicating residues in figure would help interpretation as would N- and C- terminal labeling for FimA.\n\nResponse: Thank you for the comment. We corrected the figure number and added the statistical analysis. Unfortunately, it wouldn’t be very helpful adding N- or C-terminals to the figure because they both are located close to each other and to FimC.\n\nSup. Figure 3, statistics\n\nResponse: One-way ANOVA was used for calculating statistical significance. This has been added to the figure legend.\n\nSup. Figure 4, empty pBAD vector/arabinose control\n\nResponse: The control has been added to the figure\n\nSup. Figure 5, very hard to interpret without additional labels in the figure\n\nResponse: Additional labels have been added to the figure.\n\nSup. Figure 7, statistics? How are data normalized if there are reduced cells but equivalent metabolic activity?\n\nResponse: One-way ANOVA was used for calculating statistical significance. This has been added to the figure legend. Biofilm assay measures the amount of attached cells on the plate. Metabolic assays (TTC staining) measure the amount of live cells in both planktonic and attached populations. It aids to distinguish between the biofilm compounds which kill the cells and those that just get cells detach. Therefore, if a compound inhibits FimA assembly the cells will loose ability to attach to the surface but stay alive.\n\n>Reviewer #2 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\n\nResponse: We thank you for helping to review this manuscript.\n\n>Reviewer #3 (Remarks to the Author)\nIn this paper, Lu at al provide compelling evidence that the well-conserved GlpG inner membrane protease can help prevent aggregation of the major type 1 pilus subunit FimA during pilus assembly. The authors also present some data suggesting that GlpG can affect flagella biosynthesis. Biochemical data indicating that purified GlpG can promote the dissolution of FimA aggregates in vitro are backed up by functional assays suggesting that the proteolytic activity of GlpG is important for bacterial invasion of kidney and bladder cells, as well as biofilm formation. One of the most interesting results in support of the authors’ model is that overexpression of the FimA periplasmic chaperone FimC can compensate for loss of GlpG activity, at least in some assays. This is an important study, as it provides the first description of a physiologically relevant substrate for GlpG in E. coli and helps explain the decreased host colonization phenotype observed with a glpG mutant in uropathogenic E. coli (UPEC). Though this study has many strengths, there are a few problems that should be addressed, including some missing controls and some potentially misleading text, as noted below:\n\nMajor issues:\n1. The authors use TEM a lot to examine levels of type 1 pili and flagella in the various mutant and complemented strains throughout the manuscript. However, these organelles are only quantified in Fig 2B for one K12 and one UPEC isolate. The authors should ideally quantify numbers of type 1 pili and flagella by either counting these structures (ideally in a blinded fashion) in random EM fields or by alternate assays such as mannose-sensitive hemagglutination assays (type 1 pili) or motility plates (flagella). It seems that they quantified some of the images in Fig 2 (based on lines 214-215), but it is not clear how this was done and it does not seem like quantification was consistently performed for all of the EM-based assays. The methods do not seem to include a description of how bacteria were imaged by EM. This should include growth conditions for the bacteria prior to processing for imaging. The pili observed by TEM are not easy to see. Use of immunogold labeling, or use of a fim KO mutant would help confirm the identity of the observed pili.\n\nResponse: The methods section has been updated to clarify how the bacteria were imaged by EM, as well as the growth conditions. Type 1 pili have been quantified at random EM fields in 30 or more of E. coli cells to show statistical significance in Figure 2. Additionally, a hemagglutination assay was performed (Figure 2) clearly showing loss of type 1 pili in the glpG mutant strain.\n\n2. As referenced by the authors, UPEC lacking glpG have a defect in colonization of the mouse gut. The authors focus most of their functional experiments (host cell invasion and biofilm formation) on assays that are more relevant to UTI. Does GlpG affect the fitness of UPEC within the urinary tract in vivo? It seems likely based on the in vitro studies presented, but the authors should either test this directly or note this caveat in the text.\n\nResponse: This is a great question and one we did not explore. We do however observed changes in the invasion of human kidney organoids. Further studies will be conducted in mice to see if the GlpG mutation affects UTI infections, which will be the focus of a follow up manuscript.\n\n3. The authors show that the proteolytic activity of GlpG promotes invasion of mouse kidney and human bladder cells (Fig 3). Is bacterial adherence affected by GlpG? Might the decrease in invasion reflect a decrease in adherence?\n\nResponse: We performed adhesion assays with WT and GlpG inactive mutant of both NU149 and K12 strains. Moreover, we tested how addition of 2% mannose influences adhesion to confirm the presence and involvement of type 1 pili in the process of adhesion/invasion. However, even though we observed the drop in the amount of adherent cells in the presence of mannose, it didn’t block the adherence 100%, implying the involvement of other surface components and most likely in case of our assay - non-specific binding. Moreover, the difference between WT and inactive mutants of both strains was not as significant as for invasion assay. Therefore, we decided to present invasion assay in the paper, especially since UPEC form intracellular bacterial communities in bladder epithelial cells as a key strategy for persistence and immune evasion, especially in acute and recurrent urinary tract infections, ability to invade is a key characteristic of a successful pathogenic UPEC.\n\n4. For the TPCK experiments (Fig. 6), time of TPCK incubations should be made clearer. The text and caption for Fig 2 are a bit confusing. Maybe a diagram would help. Does TPCK affect the growth rates or size (e.g. filamentation) of the bacteria in the biofilm assays?\n\nResponse: The times of TPCK incubations are given in the Materials and Methods. To make it more clear we also added it to the text. For biofilm inhibition assay the inhibitor was added at the same time as the bacterial culture was added to the 96 well plates and then biofilm formation was observed for the next 24 hours (Fig.2A). For biofilm eradication assay (Fig.2B) biofilm was allowed to be formed for 24 hours and then TPCK was added and biofilm was assessed after another 24 hours of incubation. In order to confirm that the inhibitor didn’t affect the growth/viability metabolic assays (TTC staining) were performed showing no difference in cell metabolism (Fig. S7).\n\n5. One of the authors’ main conclusions, based mostly on biofilm assays and EM, is that “GlpG is important for maintaining the integrity of the pili and flagella in pathogenic bacteria, a crucial feature for retaining the biofilm.” This sentence, along with Fig 6E and associated text indicating that TPCK caused formation of a “fragmented pili network” is intriguing, as they suggest that TPCK is affecting the stability of pili after they have already been assembled. Though interesting, it is not clear how this observation fits with the authors’ model (which is well substantiated by other data presented) that GlpG helps prevent formation of FimA aggregates during pilus assembly. Might TPCK, which likely has numerous targets in the bacteria, have other GlpG-independent effects on the assembled pili or envelope? Would be interesting, though admittedly not trivial, to see if induced ablation of GlpG expression after type 1 pili assemble leads to fragmentation, similar to TPCK effects (Fig 6E). Without this or related data, the authors may want to re-phrase the text or at least provide some possible explanation(s) in the Discussion.\n\nResponse: Thank you for your input and we agree, this effect is very intriguing. Pili are known to be fragile and constantly replaced and if assembly is faulty (which might be after GlpG full or, more likely, partial inhibition), the resulting pilus rods can have structural defects and reduced stability, making them more prone to dissociation. Our lab is planning on studying this effect in more detail in a future manuscript.\n\nMinor issues:\n1. For Fig 2D and the methods, TB should be defined.\n\nResponse: Terrific broth (TB) has been defined in the main text.\n\n2. Considering the effects of GlpG mutants on flagella expression (based on EM), it would be worth noting if the authors’ proteomics data picked up flagellin or other motility-related proteins as potential GlpG substrates.\n\nResponse: The N-terminomics method will not only pick up substrates but other protein in close association. The method used did not pick up flagellin, however it did find other chaperone such as DegP and DegQ. We have added studies to show these proteases work together in a cascade to facilitate protein quality control.\n\n3. From the Methods, it appears that the bacteria were often grown shaking prior to use in invasion assays and other experiments. Shaking does not promote type 1 pili expression as much as growth in static culture, which is more commonly used for invasion assays.\n\nResponse: Cultures were always grown statically. This is indicated in the methods.\n\n4. The abstract states that “Our data demonstrates that GlpG plays a key role in protein quality control of virulence factors…” The paper really only nails down GlpG effects on type 1 pili, and starts to address effects on flagella. Both of these factors are better defined as colonization or fitness factors, rather than ‘virulence factors’. Minor point.\n\nResponse: Thank you for suggestion. The title has also been adjusted to reflect the focus on pili. We based our statement on several papers, which considered surface structures, like pili and flagella, virulence factors, because they contribute to a pathogen's ability to cause disease by aiding in attachment to host cells, motility, and biofilm formation. And the ability to adhere to host epithelial cells in the urinary tract represents the most important determinant of pathogenicity (Terlizzi ME, Gribaudo G, Maffei ME. UroPathogenic Escherichia coli (UPEC) Infections: Virulence Factors, Bladder Responses, Antibiotic, and Non-antibiotic Antimicrobial Strategies. Front Microbiol. 2017 Aug 15;8:1566. doi: 10.3389/fmicb.2017.01566).\n\n5. Lines 39-43, text gives the impression that all E. coli strains residing in the gut are either commensals or ExPEC, but overlooks diarrheagenic strains that may also persist asymptomatically within the gut. Might want to rephrase text for accuracy.\n\nResponse: Thank you for the valuable suggestion. We added a sentence to the manuscript, explaining the classification of pathogenic E. coli.\n\n6. In the Introduction and elsewhere in the text (e.g. lines 191-192), it is stated that biofilm formation is the most critical factor for development of chronic and rUTI. This may be true, or might be true for only a subset of individuals, but whether or not it is true for all, or even most, individuals with chronic/rUTI problems is not at all clear. Biofilms have been reported in the kidneys, but the contributions of these communities to rUTI or chronic UTI is not clear. Text should be adjusted/qualified to reflect these ambiguities.\n\nResponse: The phrase, “and are one of the most critical properties responsible for chronic infections by UPEC”, has been changed to “and are one of the critical properties responsible for some chronic infections by UPEC\n\n7. Line 106: Might be good to briefly mention some of the “challenges of membrane protein proteomics” for non-experts. Solubility issues?\n\nResponse: Membrane protein proteomics is notoriously challenging, largely due to the unique biochemical and biophysical properties of membrane proteins. These proteins are hydrophobic and they require detergent for solubilisation, which interferes with downstream processes like digestion or MS ionization with low-abundance constituting only 20–30% of the proteome with low copy numbers. A sentence describing this was added to the main text.\n\n8. Based on the author’s findings with FimA, can they comment (maybe in the /Discussion) on whether or not GlpG is likely to have similar quality control functions with other pili (e.g. P pili or others)?\n\nResponse: We could not answer this question with the bacterial model we used in the manuscript, since NU149 expresses type 1 pili. But since it was shown that DegP is involved in P pilus biogenesis by cleaving pilin, PapA subunits, we feel the mechanism quality control of pili could be similar in many bacteria. We are planning on studying the possible universal role of GlpG in pili proteostasis and using bacteria expressing other pili types.\n\nReviewer #4\nWeaknesses and Suggestions for Improvement:\n1. Context: While the study builds on previous knowledge of GlpG's role in bacterial colonization, more context on how this work advances the field beyond existing literature would be beneficial.\n\nResponse: More context has been added with regards to other examples of rhomboid proteases and their involvement in bacterial colonization. This is seen in a recent paper describing rhomboid proteases having a role in adherence, motility and biofilm formation published in Frontiers of Microbiology. Furthermore we have added a line at the end of the discussion linking GlpG and its potential for the development of therapeutics to treat E. coli infections.\n\n2. In vitro assay conditions: The in vitro assay demonstrating GlpG's ability to cleave aggregated FimA uses detergent-solubilized GlpG. To better mimic physiological conditions, it would be preferable to incorporate GlpG into a membrane system, such as large unilamellar vesicles (LUVs) containing an E. coli lipid mixture.\n\nResponse: We thank the reviewer for the comment. We reconstituted GlpG into proteoliposomes consisting of E. coli lipids and repeated cleavage assays with FimA aggregates and fluorescent FimA peptide. We demonstrated that PLS-GlpG was able to cleave FimA even with better rate, which is in accordance with our previously published papers (Arutyunova E, Jiang Z, Yang J, Kulepa AN, Young HS, Verhelst S, O'Donoghue AJ, Lemieux MJ. An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases. Biol Chem. 2018 Nov 27;399(12):1389-1397. doi: 10.1515/hsz-2018-0255; Lysyk L, Brassard R, Arutyunova E, Siebert V, Jiang Z, Takyi E, Morrison M, Young HS, Lemberg MK, O'Donoghue AJ, Lemieux MJ. Insights into the catalytic properties of the mitochondrial rhomboid protease PARL. J Biol Chem. 2021 Jan-Jun;296:100383).\n\n3. FRET-FimA peptide assay: Similar to the in vitro assay, the FRET-based assay could be improved by using a membrane-incorporated GlpG to more accurately represent the periplasmic environment.\n\nResponse: We responded to this comment above. We do observe cleavage of the peptide when GlpG is embedded in proteoliposomes.\n\n4. Cleavage site specificity: The inability to determine the exact cleavage position of GlpG on FimA (only narrowed down to residues 157-182 at the C-terminus) leaves some mechanistic details unresolved.\n\nResponse: The cleavage site is found in the peptide fragments analysis from proteomics. We did not include the precise site as this was not the focus of the manuscript, but to be comprehensive we added this to the results section.\n\n5. Physiological relevance: While the study demonstrates GlpG's ability to cleave aggregated FimA in vitro, further evidence is needed to confirm that this activity occurs in the periplasmic space under physiological conditions.\n\nResponse: We agree with the reviewer and therefore we performed the experiment where we transformed NU149-glpGS201A strain (the strain with genomically inactivated rhomboid protease which leads to accumulation of FimA aggregates in periplasmic space) with plasmid bearing active GlpG. The WB (included in Fig.2) confirmed that addition of active GlpG significantly reduces FimA aggregates, confirming the role of the protease in clearing the aggregates.\n\nConclusion:\nThis study provides valuable insights into the role of GlpG in regulating virulence factors in pathogenic E. coli. The comprehensive approach and novel findings contribute significantly to our understanding of bacterial pathogenicity mechanisms. However, some aspects of the in vitro assays could be improved to better reflect physiological conditions. Despite these limitations, the research opens new avenues for exploring potential therapeutic targets in bacterial infections."}, {"round": 2, "reviewer_comments": {"reviewer_1": "While the revised manuscript is improved, some of the previously raised concerns have not been fully addressed. In addition, figure 1 has been updated to include GspN pulldown data (list of proteins) that were previously shown in the Supplemental material but barely discussed. With its presence up front and the strong claims made regarding GlpN’s interaction with other proteins in the revised manuscript (Fig. 1B), new concerns are raised that need addressing. The pulldown protocol is not described, and no information is included on how the authors arrived at the list of proteins pulled down with the FLAG-tagged GspN. No control, such as pulldown in the absence of GspN is included, and the number of replicas used is lacking.\n\nAdditional comments\nFigure 1A does not add to the understanding of the experiments or data.\nFigure 1F: the ability of overexpressed GlpG to complement the DegQ and DegP mutants suggests a proteolytic mechanism for DegQ and DegP that is independent of GlpN cleavage and not necessarily a regulated proteolysis cascade where GlpG initially cleaves followed by DegQ or DegP cleavage.\nFigure 3: The authors assert that there is a minimal effect on adhesion for the glpG mutant. We suggest including these findings to support the authors’ claim that the defect in invasion is specifically due to reduced cell entry and not to lack of cell adhesion.\nIn the supplement, there is a general need for additional information in the legends for figures S5-S8, which will aid in interpretation of the data.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors do a good job of addressing my previous concerns/questions. I have two minor issues that I think should be remedied.\n\n1. Based on the rebuttal and text, the authors have clearly performed adherence assays with glpGmutant. It is not clear why the authors do not want to show the adherence data. It is always better, and should be standard, to present adherence data with invasion data, as the two are related (adherence precedes invasion). Part of the problem may be that the authors state (lines 190-191) that the type 1 pilus is “an abundant and essential component for bacterial adherence to host cells…”. T1P promote adherence, but they are not always ‘essential’ and the inhibition of T1P expression rarely completely abrogates bacterial adherence. As reported in numerous studies, it is not uncommon for mutants or inhibitors that affect T1P expression or functionality to have more pronounced effects on invasion than on adherence (possibly due to involvement of alternate/multiple adhesins, interactions between FimH and multiple host receptors, or other issues). This does not lessen the relevance of the invasion results. Showing results from the adherence assays, or at least showing the invasion results as a percentage of adherent cells (invasion index), is good practice and provides a more complete and relevant picture of how glpG affects UPEC-host interactions.\n\n2. Line 232 “To determine whether loss of pili observed in glpG-S201A strains results in reduced pathogenicity…”. The host cell invasion assays described in this section do not directly measure effects on pathogenicity (ability to cause disease), though invasion can impact pathogenicity. “host colonization” is probably a better way to describe what is being tested in these assays."}, "author_response": "We thank all reviewers for the time taken to carefully review our manuscript. All edits have improved the quality and readership of this paper. Please find detailed responses below:\n>Reviewer #1 (Remarks to the Author)\nMajor comments\nWhile the revised manuscript is improved, some of the previously raised concerns have not been fully addressed. In addition, figure 1 has been updated to include GspN pulldown data (list of proteins) that were previously shown in the Supplemental material but barely discussed. With its presence up front and the strong claims made regarding GlpN’s interaction with other proteins in the revised manuscript (Fig. 1B), new concerns are raised that need addressing. The pulldown protocol is not described, and no information is included on how the authors arrived at the list of proteins pulled down with the FLAG-tagged GlpN. No control, such as pulldown in the absence of GlpG is included, and the number of replicas used is lacking.\n\nResponse: We thank the reviewer for bringing this to our attention. We included the pulldown protocol in the Methods section and added the final proteomics data table in the Supplemental Information. All raw data is deposited and publicly available in the MassIVE repository (MSV000097249) (mentioned in the Methods section).\n\nWe tested several detergents for membranes solubilisation (Triton, DDM and C12E8) to account for solubilisation efficiency, however, we did not see the significant differences in the results. Therefore, we treated these samples as replicates and present the data where we used Triton for solubilisation. We also included the line about negative control in the Methods section.\n\nAdditional comments\nFigure 1A does not add to the understanding of the experiments or data.\n\nResponse: Thank you for the suggestion. We removed Figure 1A and enlarged Figure 1B in its place.\n\nFigure 1F: the ability of overexpressed GlpG to complement the DegQ and DegP mutants suggests a proteolytic mechanism for DegQ and DegP that is independent of GlpN cleavage and not necessarily a regulated proteolysis cascade where GlpG initially cleaves followed by DegQ or DegP cleavage.\n\nResponse: Thank you for your comment. We agree that all three proteases work on FimA cleavage, however, the fact that the amount of endogenous GlpG in the cell is much lower in comparison to the amount of DegQ, which is abundant periplasmic protease, but upon deletion of GlpG we still see aggregated FimA in the amount comparable to what we observe in case of DegQ led us to hypothesis that GlpG cleavage might be a rate-limiting step in this process. If the clearance of aggregates was independent of GlpG cleavage, the endogenous DegP and DegQ should clear the aggregates in the absence of GlpG. However, we do agree that we don’t have enough evidence to prove this hypothesis and while it will be the focus of our next paper, we removed several strong statements from this paper.\n\nFigure 3: The authors assert that there is a minimal effect on adhesion for the glpG mutant. We suggest including these findings to support the authors’ claim that the defect in invasion is specifically due to reduced cell entry and not to lack of cell adhesion.\n\nResponse: We thank the reviewer for their insight. The data showing percentage of invaded cells versus adherent cells (invasion index) was added to the Supplementary Information Figure S6 with a brief explanation in the manuscript outlining our observations.\n\nIn the supplement, there is a general need for additional information in the legends for figures S5-S8, which will aid in interpretation of the data.\n\nResponse: Additional context has been added to supplemental figures S5-S8\n\n>Reviewer #2 (Remarks to the Author)\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\n\nResponse: We thank you for helping to review this manuscript.\n\n>Reviewer #3 (Remarks to the Author)\n1. Based on the rebuttal and text, the authors have clearly performed adherence assays with glpG mutant. It is not clear why the authors do not want to show the adherence data. It is always better, and should be standard, to present adherence data with invasion data, as the two are related (adherence precedes invasion). Part of the problem may be that the authors state (lines 190-191) that the type 1 pilus is “an abundant and essential component for bacterial adherence to host cells…”. T1P promote adherence, but they are not always ‘essential’ and the inhibition of T1P expression rarely completely abrogates bacterial adherence. As reported in numerous studies, it is not uncommon for mutants or inhibitors that affect T1P expression or functionality to have more pronounced effects on invasion than on adherence (possibly due to involvement of alternate/multiple adhesins, interactions between FimH and multiple host receptors, or other issues). This does not lessen the relevance of the invasion results. Showing results from the adherence assays, or at least showing the invasion results as a percentage of adherent cells (invasion index), is good practice and provides a more complete and relevant picture of how glpG affects UPEC-host interactions.\n\nResponse: We thank the reviewer for their insight. We agree that it is a good addition to the paper. The data showing percentage of invaded cells versus adherent cells (invasion index) was added to the Supplementary Information Figure S6 with a brief explanation in the manuscript outlining our observations.\n\n2. Line 232 “To determine whether loss of pili observed in glpG-S201A strains results in reduced pathogenicity…”. The host cell invasion assays described in this section do not directly measure effects on pathogenicity (ability to cause disease), though invasion can impact pathogenicity. “host colonization” is probably a better way to describe what is being tested in these assays.\n\nResponse: Line 232 has been corrected to better reflect the results of our assay."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have satisfactorily addressed all my comments and submitted a revised manuscript that has greatly improved since the original submission.\n\nI have no further comments besides an issue with the proteomics data shown in supplemental table 1 and summarized in figure 1A. It may have been an oversight by the authors, but it appears that a few of the proteins highlighted to interact with GlpG (Tpx, AnsB, HlpA and SurA) in figure 1A are found at higher level in the control sample than in the GlpG-3XFLAG pulldown sample according to supplemental table 1. I recommend removing these proteins from figure 1A.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors addressed my prior concerns, for the most part, but there are a few minor and easily addressed points that should be addressed.\n\n1.Inclusion of data for the invasion assays is appreciated, but these data (Supp fig 6) do not include any error bars, or indicators of variance in the data. These should be included in the graphs\n2. Type-o, second to last line of legend for Fig 2. “lack” not needed.\n3. Line 264: “Biofilms are the main cause of recurrent bacterial infections, largely because their environment protects the pathogen from administered antibiotics (Flemming et al., 2016).” Sentence should be qualified, as this is not demonstrated for all recurrent infections. More correct to note that “Biofilms are a major cause…”."}, "author_response": "Reviewer #1 (Remarks to the Author):\nThe authors have satisfactorily addressed all my comments and submitted a revised manuscript that has greatly improved since the original submission.\n\nI have no further comments besides an issue with the proteomics data shown in supplemental table 1 and summarized in figure 1A. It may have been an oversight by the authors, but it appears that a few of the proteins highlighted to interact with GlpG (Tpx, AnsB, HlpA and SurA) in figure 1A are found at higher level in the control sample than in the GlpG-3XFLAG pulldown sample according to supplemental table 1. I recommend removing these proteins from figure 1A.\n\nResponse: We thank the reviewer for bringing this to our attention. The proteins mentioned have been removed from Figure 1A.\n\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\n\nResponse: We thank you for helping to review this manuscript.\n\nReviewer #3 (Remarks to the Author):\nThe authors addressed my prior concerns, for the most part, but there are a few minor and easily addressed points that should be addressed.\n\n1.Inclusion of data for the invasion assays is appreciated, but these data (Supp fig 6) do not include any error bars, or indicators of variance in the data. These should be included in the graphs\n\nResponse: We thank the reviewer for bringing this to our attention. Error bars have been added to Supplementary Figure 6.\n\n2. Type-o, second to last line of legend for Fig 2. “lack” not needed.\n\nResponse: The type-o has been fixed.\n\n3. Line 264: “Biofilms are the main cause of recurrent bacterial infections, largely because their environment protects the pathogen from administered antibiotics (Flemming et al., 2016).” Sentence should be qualified, as this is not demonstrated for all recurrent infections. More correct to note that “Biofilms are a major cause…”.\n\nResponse: The sentence has been changed to reflect this. “Biofilms are a major cause in recurrent bacterial infections as it protects the pathogen from the host immune response as well as administered antibiotics, leading to persistence, chronic infection and poorer patience outcome.”"}]

Decision letter