A Cell-based Screen in Actinomyces oris to Identify Sortase Inhibitors

Sortase enzymes are attractive antivirulence drug targets that attach virulence factors to the surface of Staphylococcus aureus and other medically significant bacterial pathogens. Prior efforts to discover a useful sortase inhibitor have relied upon an in vitro activity assay in which the enzyme is removed from its native site on the bacterial surface and truncated to improve solubility. To discover inhibitors that are effective in inactivating sortases in vivo, we developed and implemented a novel cell-based screen using Actinomyces oris, a key colonizer in the development of oral biofilms. A. oris is unique because it exhibits sortase-dependent growth in cell culture, providing a robust phenotype for high throughput screening (HTS). Three molecules representing two unique scaffolds were discovered by HTS and disrupt surface protein display in intact cells and inhibit enzyme activity in vitro. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris may be a useful platform for discovery efforts targeting sortase.

but in some instances these enzymes perform lysine-isopeptide transpeptidation reactions that construct pili virulence factors 16 .
Given its potential as a drug target, because sortase mutants are attenuated in virulence, considerable effort has been put forth to discover small molecule Sa SrtA inhibitors 9,[17][18][19] . Previously reported studies have searched for inhibitors by monitoring the activity of the purified Sa SrtA enzyme in vitro using a Förster resonance energy transfer (FRET) assay. The FRET assay has been used to screen small-molecule compound libraries [20][21][22][23] and to assess the potency of rationally designed peptidomimetics [24][25][26][27] , natural products [28][29][30][31][32][33][34][35][36][37][38] , and small molecules identified using virtual screening approaches [39][40][41][42] . While several inhibitors have been discovered, to the best of our knowledge, none have advanced into clinical trials. It is possible that some of these compounds are unable to effectively inhibit the enzyme in its natural context, the extra-cellular bacterial membrane where it may associate with components of the protein secretion and cell wall synthesis machinery. A cell-based assay for high-throughput screening (HTS) for sortase inhibitors could overcome this limitation, but has yet to be implemented in S. aureus because inhibiting Sa SrtA activity does not significantly affect the growth or morphology of this microbe in cell culture 6 . Sa SrtA activity can be detected in cells, but these methods are cumbersome and require antibody detection of sortase-displayed proteins 41 , cell adhesion assays 43 or incubation of cells with fluorogenic peptidyl sortase substrates that can be slow to label cells 44 .
Recently, Wu et al. made the surprising discovery that the viability of the oral bacterium Actinomyces oris MG-1 in cell culture depends on the activity of its sortase ( A°S rtA) 45,46 . Ao SrtA anchors the glycosylated surface protein A (GspA) to the cell wall. In this process, GspA is first glycosylated by the LCP enzyme and then attached to the cell wall by Ao SrtA via lipid II (Fig. 1A, top). Interestingly, reducing Ao SrtA expression causes cell arrest, presumably due to glycol-stress caused by accumulation of glycosylated GspA in the membrane (Fig. 1A, bottom). To the best of our knowledge, A. oris is the only known bacterium that exhibits a sortase-dependent growth The effects of small molecules on wild-type A. oris MG-1 was determined for several compound libraries (left). Preliminary hits (1,904) that impaired growth were counter-screened by determining their ability to affect the growth of a ΔsrtA/ΔgspA strain, whose viability is dependent upon the activity of Ao SrtA (middle). Strain-specific growth inhibitors were then validated for sortase inhibitor activity using biochemical and cellular approaches (right). Adobe Illustrator Version: 15.0.0 (https:// www.adobe.com/products/illustrator.html).

Development and implementation of the cell-based screen.
Our previous studies have shown that reducing srtA gene expression in A. oris leads to cell death and that this phenotype is conditionally dependent on gspA expression 45,46 . To exploit this unique dependence, we developed a cell-based assay to screen for sortase inhibitors that compares the growth-inhibitory effects of small molecules cultured with A. oris MG-1 (wild-type) versus a ΔsrtA/ΔgspA strain. In the assay, it is presumed that a small molecule sortase inhibitor will selectively impair growth of A. oris MG-1 by causing GspA to accumulate in the membrane, whereas growth of the ΔsrtA/ ΔgspA strain should be unaffected. Prior to implementing the assay in HTS, we optimized conditions for A. oris growth in 384-well plate format. Both MG-1 and ΔsrtA/ΔgspA strains had similar growth rates and final cell densities in brain-heart infusion broth. Control growth experiments using media containing 1% dimethyl sulfoxide (DMSO) confirmed that both strains are tolerant to this solvent at concentrations used for screening. The growth phenotype of both A. oris strains was sufficient for use in a HTS, as a Z' score of 0.81 (MG-1) and 0.71 (ΔsrtA/ ΔgspA) was determined when positive (1 µg mL −1 penicillin) and negative (media only) control experiments are performed 47 .
The overall workflow for the HTS is shown in Fig. 1B. Initially, small molecules were screened for their ability to kill the wild-type MG-1 strain in 384-well format. Molecules that reduced growth by more than 2.75 standard deviations from the average growth were considered preliminary hits. These molecules were then tested in high-throughput for their ability to impair ΔsrtA/ΔgspA growth, which serves as a counter-screen. Small molecules that exhibited differential growth effects in MG-1 but not ΔsrtA/ΔgspA strains were considered hits and carried forward for further analysis. These hits were validated by determining their minimum inhibitory concentrations (MICs) in cell culture, measuring the in vitro half maximal inhibitory concentration of each compound (IC 50 ) against the isolated Ao SrtA and Sa SrtA enzymes, and their ability to affect protein display in A. oris cells.
In the primary screen, a total of 200,834 small molecules were tested for their ability to impair A. oris MG-1 growth. Cells were added to an optical density (OD 600 ) of 0.01 in media containing 10 µM of each molecule, followed by a 15 hour incubation at 37 °C and end-point OD 600 measurement. The growth effect of each molecule was normalized and expressed as a percent growth relative to the DMSO-only control (see Methods). A total of 1,904 molecules were classified as preliminary hits (0.95% of the molecules tested), as they reduced MG-1 growth after 15 hours by at least 34%, or 2.75 standard deviations below the average. The preliminary hits were then subjected to a counter-screen in which their growth effects on ΔsrtA/ΔgspA were determined. In order to eliminate potentially erroneously identified preliminary hits, the counter-screen and preliminary screen was performed in duplicate for the 1,904 preliminary hit molecules. A molecule was deemed as a hit if it caused greater than 15% differential growth effect in duplicate when the MG-1 and ΔsrtA/ΔgspA strains were compared. Figure 2 shows a plot of each preliminary hit molecule's growth effect against the MG-1 and ΔsrtA/ΔgspA strains. Data is plotted as the percentage of growth inhibition for each molecule (see Methods).
A total of six compounds preferentially affected MG-1 growth in duplicate, constituting 0.3% of the preliminary hits identified from the primary screen and 0.003% of the total number of compounds tested. The six compounds were termed 1-6 and carried forward for further analysis (Fig. 3). R Version: 3.3.3 (https://www.r-project. org/) Evaluation of the sortase inhibitor candidates. Two validation assays were performed for each hit molecule that measured: (i) the minimum inhibitory concentration (MIC) required to prevent growth of the MG-1 and ΔsrtA/ΔgspA strains, and (ii) their in vitro inhibitory activity against the enzymatic activity of the A. oris and S. aureus sortase enzymes. Initially, the selective growth effect originally observed in the HTS that used 384-well plates was more rigorously defined by determining each molecule's MIC value for the MG-1 and ΔsrtA/ΔgspA strains. Cells were cultured in 100 µL media containing 2-fold dilutions of each small molecule between 320 µM and 0.039 µM (approximately 134 µg mL −1 to 0.16 µg mL −1 ). Each of the six preliminary hit molecules (1-6) has a lower MIC for the MG-1 strain (between 16.4 µg mL −1 (40 µM) and 1.0 µg mL −1 (2.6 µM) than for ΔsrtA/ΔgspA (Table 1). Two additional molecules exhibited activity in only one replicate of the high-throughput counter-screen assay; however, we chose to interrogate their activity in proceeding assays to better appreciate the reliability of the HTS. Neither exhibited marked differences in their MIC values when tested in MG-1 and ΔsrtA/ΔgspA strains, which affirms the strength of the duplicate experiment cutoff. Thus, the MIC data confirm the results of the HTS by demonstrating that only the duplicate hit molecules preferentially impair the growth of A. oris MG-1.
The ability of the hit molecules to inhibit sortase activity was measured in vitro using an established FRET-based assay 13 that measures the ability of each enzyme to cleave a fluorogenic peptide substrate; Abz-LAQTG-Dap(Dnp)-NH 2 and Abz-LPETG-Dap(Dnp)-NH 2 , substrates for Ao SrtA and Sa SrtA, respectively. Each enzyme cleaves between the threonine and glycine bond in these peptides, leading to a measurable increase in fluorescence (see Methods). The IC 50 value of each compound was determined for both the Ao SrtA and Sa SrtA enzymes. Compounds 3, 4 and 6 are bona fide enzyme inhibitors, as each inhibits both the Ao SrtA and Sa SrtA enzymes with IC 50 values ranging from 30-70 µg mL −1 (73-170 µM) ( Table 1). In contrast, molecules 1, 2 and 5 failed to inhibit Ao SrtA in vitro, suggesting that they selectively affect A. oris MG-1 growth through a mechanism not involving sortase inhibition.
Sortase inhibitors affect pilus assembly in A. oris. The MIC and IC 50 data suggest that at least three of the hit molecules are capable of inhibiting Ao SrtA either within the context of the cell or as an isolated (2020) 10:8520 | https://doi.org/10.1038/s41598-020-65256-x www.nature.com/scientificreports www.nature.com/scientificreports/ transpeptidase. To understand the physiological effects that these molecules have on the display of pili and surface proteins on the surface of A. oris, we grew cells with 10 µM of each hit molecule and assessed sortase activity by western blot and electron microscopy as previously reported 45 . To avoid a confounding problem that inhibiting Ao SrtA causes cell arrest, we performed the experiments in a mutant devoid of gspA; the aforementioned genetic suppressor of sortase lethality. Consistent with both MIC and IC 50 data, when samples were immunoblotted with antibodies against the type 2 pilus shaft protein FimA, both molecules 3 and 4 decreased FimA polymers as compared to the control ( Fig. 4; compare the first two lanes with lanes containing molecules #3 and #4).
To examine if these molecules inhibit cell wall anchoring of surface proteins (e.g. GspA), we similarly treated cells devoid of lcpA, i.e. ΔlcpA, another suppressor of srtA 46 . When blotted with antibodies against GspA, no GspA polymers were observed in samples treated with molecules 3, 4 and 6 ( Fig. 4B). To corroborate these observations, we subjected A. oris cells of ΔgspA and Δlcp strains to 10 μM of each molecule (3, 4 and 6) and studied pilus display with transmission electron microscopy (TEM), whereby A. oris cells were immobilized on nickel grids and stained with 1% uranyl acetate prior to viewing by an electron microscope. Intriguingly, compared to the untreated control, treatment of molecules 3, 4 and 6 caused significant reduction of pilus assembly at the cell www.nature.com/scientificreports www.nature.com/scientificreports/ pole in either strain background, i.e. ΔgspA or ΔlcpA (Fig. 5). The results suggest that these molecules target the nascent pilus assembly machine.

Discussion
Sortase enzymes are promising drug targets as they mediate the display of important virulence factors in several medically significant bacteria. The S. aureus Sa SrtA enzyme is particularly interesting because it plays an important role in MRSA infections that are a major cause of human mortality. Despite over fifteen years of effort by a number of research groups, antivirulence agents that work by inhibiting Sa SrtA have yet to be discovered. This may be because nearly all screens for Sa SrtA inhibitors have used the isolated enzyme that is truncated at its N-terminus to increase solubility. Searching for inhibitors using a cell-based assay may yield better results as it could discover small molecules that are capable of traversing the cell wall and targeting the intact enzyme in the microbial membrane. Moreover, it could overcome screening limitations that are caused by the slow enzyme kinetics of the isolated protein. To the best of our knowledge, a robust cell-based sortase assay has yet to be developed, presumably because current methods used to monitor Sa SrtA activity in intact cells are laborious, requiring multi-step immunolabeling and fluorescence-detection experiments. To overcome this limitation, we developed and implemented in high throughput a cell-based assay to identify sortase inhibitors. Our approach exploits the unique growth dependence of A. oris on the activity of its SrtA enzyme, which is conditionally dependent on glycosylation of the GspA surface protein by an LCP enzyme (Fig. 1A). First, a primary screen was performed in which>200,000 small molecules were tested for their ability to impair the growth of wild-type A. oris MG-1 (Fig. 1B). Preliminary hit molecules were then counter-screened against the A. oris ΔsrtA/ΔgspA mutant strain to eliminate small molecules that impaired A. oris growth via processes unrelated to sortase. In the primary screen,  (1-6) from the A. oris screen. These molecules exhibit statistically significant strain-specific effects on growth, impairing the wild-type more than the ΔsrtA/ΔgspA strain. Compounds 3, 4 and 6 were validated sortase inhibitors, whereas 1, 2 and 5 selectively impair growth through an unknown, non-sortase dependent mechanism. ChemDraw Professional Version:19.0.1.28 (https://www.perkinelmer.com/product/chemdrawprofessional-chemdrawpro). www.nature.com/scientificreports www.nature.com/scientificreports/ most molecules did not affect A. oris MG-1 growth when present in the cell culture at 10 μM, as the mean and median percent growth of cultures containing small molecules relative cells grown in standard growth culture containing DMSO was 99.3% and 100.9%, respectively. The interquartile range (IQR) is 12.1%, meaning that 75% of molecules cells grow to 94.2% the average DMSO-only density or better. A total of 1,904 molecules in the screen reduced MG-1 growth to 66% growth (2.75 standard deviations below the average percent growth) and were subsequently counter-screened using the ΔsrtA/ΔgspA mutant strain. For these molecules, growth effects on each strain were tested in duplicate on separate days to rigorously identify molecules that selectively impaired MG-1 growth. Six preliminary hit molecules were identified, 1-6 (0.32% of the 1,904 MG-1 growth effecting molecules). Each of the six molecules inhibit the growth of the MG-1 strain more than the ΔgspA/ΔsrtA strain when tested in duplicate. The finding that few molecules cause selective growth effects is not surprising, as most molecules can be expected to be generally cytotoxic by inhibiting other essential non-sortase pathways within cell, e.g. division and respiration.
Repeating the counter-screen in duplicate significantly eliminated false positive results. This is evident by our finding that the six preliminary hits also exhibit strain-specific growth effects when they were re-purchased and tested in larger culture volumes ( Table 1); each of the preliminary hits (1-6) exhibited lower MIC values against www.nature.com/scientificreports www.nature.com/scientificreports/ MG-1 than against ΔgspA/ΔsrtA. The MIC values against MG-1 fall within a similar range, around 10 μM for all hits, which is expected, as the primary screen and counter-screen monitored growth effects at a single concentration. It is worth noting that the growth screen may have failed to detect potential sortase inhibitors with MIC values significantly greater than 10 μM, meaning higher concentrations of the small molecule are necessary for enzyme inhibition. In principle, this limitation could be overcome by repeating the HTS using higher concentrations of the small molecules in the screen, but this was not performed here because it was cost-prohibitive.
In vitro enzyme activity testing reveals that compounds 3, 4 and 6 inhibit both the Ao SrtA and Sa SrtA enzymes with IC 50 values ranging from 30 to 70 μg mL −1 (73-170 µM). Compounds 3 and 4 are bioisosteres and share related N-(1,3-benzothiazol-2-yl)−5-nitrofuran-2-carboxamide chemical scaffold. In compound 3 the scaffold is elaborated with N-3-pyridylmethyl and 6-ethyl-2-benzothiazole moieties, whereas 4 features benzyl and 5-methoxybenzothiazole. Effectively these compounds are bioisosteres, as the benzene is an isostere with pyridine, as are the ethyl and methoxy groups. Interestingly, molecules containing 5-nitrofuran functional groups are particularly potent at eliciting selective growth effects as they are enriched in our primary hits; 1% of the 1,904 hits from the cell based screen contained this functional group even though it is present in only 0.09% of 200,834 molecules that we screened. Compound 6 is unique, exhibiting a N-methyl-(4,5,6,7-tetrahydro-1,3-benzothiazol-2-yl)-1,3-thiazole-5-carboxamide scaffold. Importantly, compound 6 would not likely have been discovered using conventional FRET-based HTS as it is intrinsically fluorescent and presumably would have been disregarded as a hit. The IC 50 values for the inhibitors against Ao SrtA are higher than their measured MIC values. This suggests that they may also kill A. oris cells through non-sortase related mechanisms and/or that they more efficiently inhibit the A. oris enzyme when it is embedded in the microbial membrane. Preliminary hits 1, 2 and 5 also effect growth www.nature.com/scientificreports www.nature.com/scientificreports/ in a strain-specific manner, but do not inhibit Ao SrtA in vitro ( Table 1). The origins of their selective effects on growth remain to be determined. To the best of our knowledge, none of the hit molecules have been described as antimicrobials, however benzothiazole-based DNA gyrase B inhibitors have been shown to have modest growth inhibitor properties against Gram-positive Entereoccocus faecalis when dosed at high concentrations (50 μM) 48 .
Cellular studies indicate that sortase inhibitors 3, 4 and 6 alter the ability of A. oris to display GpsA and to assemble pili on its surface. A. oris uses three distinct sortases to elaborate its cell envelope. Ao SrtA is a class E housekeeping sortase that attaches GspA and other proteins containing the LAQTG sorting signal to the cell wall 49 . Ao SrtA catalyzes a transpeptidation reaction that attaches GspA to lipid II, a cell wall precursor. A. oris also assembles surface pili (fimbriae) using two class C sortases, SrtC1 and SrtC2. These sortases catalyze transpeptidation by linking protein subunits of the pilus together via lysine-isopepide bonds. Subsequent cell wall anchoring of pilus polymers to peptidoglycan requires Ao SrtA 49 . SrtC1 produces type 1 fimbriae that are comprised of the fimbrial shaft FimP protein and the tip fimbrillin FimQ protein. This structure mediates bacterial adherence to the tooth surface via FimQ interactions with salivary proline-rich protein deposits. The SrtC2 sortase produces type 2 fimbriae, made of the fimbrial shaft FimA protein and tip fimbrillin FimB; these fimbriae are required for bacterial adherence to host cells, biofilm formation, and bacterial coaggregation 50,51 . We performed cell fraction and immunoblot studies to gain insight into how inhibitors 3, 4 and 6 affect SrtC1 and Ao SrtA sortase activity in intact cells (Fig. 4). Molecule 6 inhibits Ao SrtA in cells as its presence significantly diminishes GspA display (Fig. 4B). In contrast, it has little effect on type 2 pilus production by the SrtC2 sortase as judged by immunoblots of the FimA shaft protein in fractionated cells. The diminished potency of molecule 6 against pili display indicates that it lacks inhibitory activity against class C sortases. This is substantiated by TEM images, which show that molecule 6 only modestly effects pili display in the ΔgspA strain (Fig. 5A-D). Since Ao SrtA has been implicated in anchoring of the pilus to the peptidoglycan, one might still expect to see diminished pili display in the absence of the housekeeping sortase; however, it is not uncommon for class C enzymes to compensate for loss of the housekeeping sortase and independently display pili as SrtC2 is capable of catalyzing pilus polymerization and cell wall anchoring of pilus polymers 49,52 . Thus, the data supports molecule 6 as being more selective for Ao SrtA in intact cells.
Cellular studies suggest that compounds 3 and 4 inhibit both class E Ao SrtA and class C SrtC2 sortases. Our interrogation of type 2 pilus assembly reveals that 3 and 4 not only decrease the abundance of pili, but also reduce the amount of higher molecular weight FimA-containing polymers that are being formed (Fig. 4A, compare molecular weight bands ≥150 kDa). Decreased pilin-polymerase activity suggests that SrtC2 is being inhibited. This is substantiated by TEM images of cells treated with these compounds 3 and 4, which show either completely absent or diminished pili at their poles, respectively (Fig. 5B,C). Inspection reveals that the pili in these cells become gradually shorter toward the poles, suggesting that the SrtC2 enzyme is being inhibited during the 3 hour period of growth. Moreover, cells treated with compounds 3 and 4 have decreased amounts of cell wall associated GspA, compatible with the fact that these molecules also inhibiting the housekeeping Ao SrtA sortase (Fig. 4B). Consistent with the lower IC 50 against Ao SrtA and Sa SrtA and the lower MIC against MG-1 relative to 4, we note that 3 appears to more severely limit GspA display than 4 at 10 μM (Figs. 4B and 5B,C). This suggests that 3, an analog of 4, is the more potent molecule. It is important to note that these inhibitors were added to actively growing cell cultures that already produced pili. Given the presence of pili at the septal area and significantly reduced pili at the pole (Fig. 5), we surmise that these inhibitors target the new sortase machine at the pole, where the nascent peptidoglycan is synthesized and modified.
In conclusion, we have exploited the unique sortase-dependent growth phenotype of A. oris to screen small molecule compound libraries for sortase inhibitors. Three molecules, representing two unique scaffolds, inhibit sortase enzymes from both A. oris and S. aureus were discovered. This represents the first HTS for sortase inhibitors that relies on the simple metric of cellular growth and suggests that A. oris is a promising platform for sortase-targeted drug discovery. Future work will need to establish structure-activity relationships for the hit molecules to further increase their potency for potential use as novel anti-infectives, which are urgently needed to treat infections caused by MRSA and other drug-resistant bacterial pathogens.

Materials and Methods
Antibiotics, media and consumables. Brain Heart Infusion Broth, modified (BHI) was purchased from Fisher Scientific and prepared as directed within one week of its use. Penicillin G and kanamycin were purchased from Fisher Scientific and stored as directed. 1000x antibiotic stocks were prepared in water, filtered with 0.2 µm syringe filters, and stored at -20 °C until thawed immediately before use. Greiner 384-well plates (EK-30162) and universal lids (EK-2079) were purchased from E&K scientific and used once before disposal as medical waste.
High-throughput screen. A total of 200,834 small molecule compounds (Molecular Screening Shared Resource, UCLA) were dissolved in 100% Omnisolv methylsulfoxide (MilliporeSigma MX1456P-6) and stored in 384-well polypropylene plates. Actinomyces oris MG-1 and ΔgspAΔsrtA were streaked out on BHI agar plates containing 50 µg/mL kanamycin and grown at 37 °C for 48 hours to allow single colonies to grow. Cultures were started from 2-3 colonies in BHI with 50 µg/mL kanamycin and allowed to grow until an OD 600 between 0.1-0.4. Meanwhile, 384-well plates were filled with 25 µL BHI broth with 100 µg mL −1 kanamycin (columns  or 200 µg mL −1 Penicillin G (positive control, columns [23][24]. Five hundred nanoliters of small molecules were transferred into the media (columns 3-22) using the Biomex FX P automated work station with a 384-well pin tool. Precultures were diluted to an OD 600 of 0.02 in 1 L BHI broth without antibiotics. Twenty five microliters of cell culture was immediately aliquoted into plates containing 25 µL media, antibiotics, and small-molecules using a Multidrop (Thermo LabSystems), resulting in 50 µg mL −1 kanamycin and 10 µM small molecules with 1% DMSO or 100 µg mL −1 Penicillin G. Plates were immediately lidded and placed into a humidified Cytomat 6001 incubator at 37 °C and allowed to grow for 15.5 hours. Following incubation, plates were removed from the Scientific RepoRtS | (2020) 10:8520 | https://doi.org/10.1038/s41598-020-65256-x www.nature.com/scientificreports www.nature.com/scientificreports/ Cytomat using a Thermo Spinnaker robotic arm on a rail, de-lidded, and placed into an EnVision high-speed plate reader for optical density measurement at 620 nm.
Raw data obtained from the screen was formatted in-house to upload to the Collaborative Drug Discovery Vault (www.collaborativedrug.com). Individual optical density readings were converted to a percent growth value ([OD sample -OD Positive control ]/[OD Negative Control Average -OD Positive control ]) and percent growth inhibition (1-percent growth). The Z-factors for the individual molecules were determined. Molecules with a Z-factor less than or equal to -2.75 were considered preliminary hits to advance forward a generous collection of preliminary hit molecules. Five microliters of each of the 1904 preliminary hit molecules were re-arrayed into new 384-well polypropylene plates. Each of the 1904 preliminary hits were assayed against both the MG-1 and ΔgspAΔsrtA strains in duplicate, on two separate days. Percent difference of growth was calculated according to the equation 100-100[(OD MG-1, Sample -OD MG-1, Positive Control )/OD ΔgspAΔsrtA, Sample -OD ΔgspAΔsrtA, Positive Control )]/[(OD MG-1, Average -OD MG-1, Positive Control )/OD ΔgspAΔsrtA, Average -OD ΔgspAΔsrtA, Positive Control )]. A score of 100 indicates that a molecule causes extreme difference in cell survivability between the two strains whereas a score of 0 indicates no differential effect on cellular growth. Molecules were ranked according to their relative growth.
Hit validation assays. Minimum inhibitory concentration (MIC) values were determined for molecules 1-6 according to the Clinical Laboratory Standard Institutes' (CLSI) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Approved standards-ninth edition; M07-A9 Vol. 32 No. 2 with the following alterations. Overnight cultures of MG-1 and ΔgspAΔsrtA were diluted to OD 600 values of 0.01 before being added to BHI containing the appropriate molecule in 96-well plates. Mueller-Hinton broth was not usable because A. oris is a fastidious bacterium. Plates were sealed with Breatheasy seals and incubated for 18 hours, as A. oris has a doubling time of 2 hours. Plate seals were removed and OD 600 values measured. Each plate was run with a positive control (cells, media, and Penicillin G), negative control (media and cells only), and contamination control (media only).
IC 50 values for the six hit molecules against the Ao SrtA and Sa SrtA enzymes were determined as previously described, with some modifications. Briefly, molecules 1-6 were serial diluted 2-fold (from 1.25 mM to 2.44 µM, 500 µM to 0.98 µM final assay concentration) into 25 µM Ao SrtA or Sa SrtA (final assay concentration 10 µM) in buffer A (20 mM HEPES pH 7, 5 mM CaCl 2 , 0.05% TWEEN, 30% DMSO). The final DMSO concentration was 18% in the assay. Samples were incubated 1 hour at room temperature and aliquoted into a 384-well plate (EK-30892). Thirty microliters of FRET peptide (Peptide 2.0) in buffer B (20 mM HEPES pH 7, 5 mM CaCl 2 , 0.05% TWEEN), Abz-LPATG-Dap(Dnp)-NH 2 for Sa SrtA or Abz-LAQTG-Dap(Dnp)-NH 2 for Ao SrtA was added and the plate was immediately placed into a Flexstation II plate reader (Molecular Devices) and fluorescence was recorded at 335/420 nm excitation/emission wavelengths after 5 seconds of plate shaking to mix the reaction components. Plate fluorescence was measured over the course of 30 minutes.
Cell fractionation and western blotting. Cell fractionation and Western blotting were followed according to a published protocol 53 . Briefly, the cultures of A. oris strains grown in BHI at 37 °C until OD 600 of 0.25 were aliquoted, and bacterial aliquots were treated with 10 µM of individual sortase inhibitors for 3 hours. Bacterial cultures were then normalized to equal OD 600 , and cells were fractionated into culture medium (S), cell wall (W), and protoplast fractions. Isolated fractions were subjected to protein precipitation by 7.5% trichloroacetic acid, followed by washing with cold acetone, except for the protoplast fractions. Protein samples were dissolved in hot sodium dodecyl sulfate (SDS)-containing sample buffer, separated by 3-12% Tris-glycine gradient gels, and subjected to immunoblotting with specific antisera (α-FimA, 1:10,000 dilution; α-GspA, α-MdbA, α-SrtC2 and α-SrtA, 1:4000 dilution), followed by chemo-luminescence detection.
Transmission electron microscopy. To observe cell morphology by negative staining, A. oris cells harvested after inhibitor treatment were washed once with PBS and suspended in 0.1 M NaCl. A drop of 7 µL of bacterial suspension was placed onto carbon-coated nickel grids and stained with 1% uranyl acetate. Samples were examined using a JEOL JEM1200.

Data availability
All data from this study are available from the corresponding author.