Exploiting phage receptor binding proteins to enable endolysins to kill Gram-negative bacteria

Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since the outer membrane prevents access to the peptidoglycan. Here, we present Innolysins, an innovative concept for engineering endolysins to exert antibacterial activity against Gram-negative bacteria. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As proof-of-concept, we constructed 12 Innolysins by fusing phage T5 endolysin and RBP Pb5 in different configurations. One of these, Innolysin Ec6 displayed antibacterial activity against Escherichia coli only in the presence of Pb5 receptor FhuA, leading to 1.22 ± 0.12 log reduction in cell counts. Accordingly, other bacterial species carrying FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa were sensitive to Innolysin Ec6. To enhance the antibacterial activity, we further constructed 228 novel Innolysins by fusing 23 endolysins with Pb5. High-throughput screening allowed to select Innolysin Ec21 as the best antibacterial candidate, leading to 2.20 ± 0.09 log reduction in E. coli counts. Interestingly, Innolysin Ec21 also displayed bactericidal activity against E. coli resistant to third-generation cephalosporins, reaching a 3.31 ± 0.53 log reduction in cell counts. Overall, the Innolysin approach expands previous endolysin-engineering strategies, allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria.


Strategy for construction of innolysins.
To construct E. coli-specific Innolysins (Innolysins Ec), we combined the RBP of phage T5 (Pb5) with the phage T5 endolysin (T5 Lys) for targeted delivery of the endolysin. The binding domain of Pb5 has previously been shown to be located in the N-terminus (488aa) of the protein 26 .
To determine whether the binding domain of the Pb5 was sufficient to enable the endolysin to exert antibacterial activity, we fused T5 endolysin with both the entire Pb5 and with the Pb5 binding domain (Pb5 1-488 ). We anticipated that antibacterial activity of an Innolysin Ec requires both that Pb5 is able to bind to the outer membrane protein FhuA, and that the fused phage T5 endolysin remains active to degrade the peptidoglycan. Thus, to ensure that the joined domains remain functional after fusion, we either fused them directly or added linkers in between. We used flexible linkers composed of small non-polar amino acids, glycine and alanine, providing a certain degree of flexibility of the fused domains 27 . To optimize the two-domain cooperation, we used linkers of two different sizes, L1 composed of six amino acids and L2 that consisted of 14 amino acids. In addition, to investigate the optimal orientation of the endolysin and RBP domains, we constructed the Innolysins in two directions with and without linkers. As such, a total of 12 Innolysins Ec were initially constructed (Fig. 1).
Scientific RepoRtS | (2020) 10:12087 | https://doi.org/10.1038/s41598-020-68983-3 www.nature.com/scientificreports/ innolysins show muralytic activity. To demonstrate conservation of the muralytic activity of the endolysin after fusion with the RBP and a linker, the fused proteins were expressed in E. coli BL21 and cleared cell lysates were tested for muralytic activity. A standardized assay for analysis of the muralytic activity of endolysins against Gram-negative bacterial peptidoglycan was used. This assay is based on outer membrane permeabilized P. aeruginosa cells, which share a common peptidoglycan chemotype (Α1γ) with E. coli 28 . The majority of Innolysins (nine out of 12) were active with enzymatic activity ranging between 126-771 U/ml. Innolysin Ec9 (Pb5-L1-T5Lys) showed the highest activity per ml cleared lysate, which was similar to the muralytic activity of phage T5 endolysin alone (795 U/ml) (Fig. 2). Although expression was confirmed for all constructs, three of the Innolysins (Ec1, Ec3 and Ec12) and Pb5 alone did not show any significant activity compared to the negative control (muralytic activity of cleared cell lysates carrying the empty vector, pVTD). Yet, as expression yield was not taken into account, some of these constructs might have muralytic activity. Here, we showed that phage T5 endolysin fused with Pb5 in different configurations could maintain its muralytic activity.
Innolysin Ec6 inhibits E. coli growth. To assess whether binding of Pb5 allowed the fused endolysin to exert antibacterial activity, we screened the muralytically active Innolysins for their ability to inhibit growth of the phage T5 bacterial host E. coli ATCC11303. This strain was mixed with cleared lysates of cells expressing Innolysins, and bacterial growth was measured spectrophotometrically after 18 h at 37 °C (Fig. 3). Growth inhibition was determined as the lack of growth of a start inoculum treated with an Innolysin compared to growth of ATCC11303 cells treated with cleared lysates of cells carrying the empty vector, pVTD (negative control). When E. coli ATCC11303 was treated with Innolysin Ec6 a significant inhibitory activity was noticed, which was similar to that of Art-175, an engineered endolysin that was previously shown to have both an inhibitory and bactericidal effect against various E. coli isolates 29,30 . The remaining eight Innolysins, Pb5 or endolysin alone did not significantly affect the E. coli growth compared to the negative control (Fig. 3). Out of the 12 initially constructed Innolysins, this screening resulted in one promising antibacterial candidate that could inhibit E. coli growth, indicating that domains may have to be fused in a specific order and with a specific linker to acquire antibacterial activity after fusion. However, we could not exclude that a potential antibacterial candidate might not have been identified during our screen due to low protein expression yield. www.nature.com/scientificreports/ harbor the Pb5 receptor, FhuA. FhuA uptake of ferrichrome requires energy from the cytoplasmic proton motive force transduced to the outer membrane via the TonB protein, but phage T5 interacts with FhuA independent of TonB 14,31 . To determine whether activity of Innolysin Ec6 required such energy, antibacterial activity was tested against an E. coli tonB deletion mutant (ECOR4ΔtonB) and the wild type ECOR4 as a positive control (Fig. 4). Approximately 1 log reduction was demonstrated in both ECOR4ΔtonB and the wild type ECOR4 in cell numbers after 30 min of treatment with Innolysin Ec6 (Fig. 4), supporting that TonB was not required for Innolysin to work as an antibacterial. Our combined data demonstrate that Innolysin Ec6 requires the presence of FhuA but not TonB-provided energy to exert antibacterial activity.  www.nature.com/scientificreports/

Morphological changes of cells treated with Innolysin Ec6. Transmission electron microscopy
(TEM) analysis was performed to determine the effects of Innolysin Ec6 on cell morphology and viability. The morphology of both E. coli BL21 and E. coli BL21ΔfhuA was visualized after incubation with 0.2 mg/ml Innolysin Ec6 for 15 min and compared to cells treated with 20 mM HEPES-NaOH (pH 7.4) as a negative control (Fig. 5). Almost all cells treated with HEPES-NaOH were intact with normal cell envelope morphology. In contrast, treatment of E. coli BL21 with Innolysin Ec6 led to cell integrity damage in the majority of cells with cytosol leakage occurring mainly at the poles. Furthermore, cell debris could be detected possibly due to cell lysis. This dramatic effect on cell morphology was not observed on E. coli BL21ΔfhuA treated with Innolysin Ec6, where only a few damaged cells were noticed compared to the cells treated with HEPES-NaOH. These observations demonstrate that Innolysin Ec6 acts rapidly by interfering with membrane integrity and reconfirms that the presence of FhuA is essential for Innolysin Ec6 to exert high antibacterial activity.

Innolysin Ec6 targets FhuA homologs of species other than E. coli. To investigate whether the
constructed Innolysin Ec6 could target FhuA homologs in other species, we tested the antibacterial activity of the purified Innolysin against Shigella sonnei and Pseudomonas aeruginosa (Fig. 6). These bacteria carry FhuA homologs with identity to the FhuA of E. coli BL21 ranging between 22.6 and 99.6% (Supplementary Table S1). The Innolysin displayed antibacterial activity against S. sonnei (99.6% FhuA identity) and P. aeruginosa PAO1 (less than 39% of overall FhuA identity), leading to average log reductions in cell number of 1.52 ± 0.14 and 1.03 ± 0.25, respectively. To give support that the antibacterial activity of Innolysin Ec6 against the tested strains is specific for the Innolysin, we further tested the antibacterial activity of either T5 endolysin or the Pb5 1-488 . Treatment with T5 endolysin did not significantly reduce the cell counts of either P. aeruginosa (0.08 ± 0.02 log   Table S4). To assess whether LysEc8 alone could exert antibacterial activity, the killing efficiency of LysEc8 was tested against E. coli BL21. No significant effect on the cell numbers was detected with log reduction reaching to 0.08 ± 0.07. In contrast, application of purified Innolysin Ec21 (0.2 mg/ml) on E. coli BL21 led to 2.20 ± 0.09 log reduction in cell numbers, thus increasing the antibacterial activity compared to Innolysin Ec6 that led to 1.22 ± 0.12 log reduction (Fig. 4). To determine whether Innolysin Ec21 was also effective against antibiotic-resistant E. coli strains, the antibacterial activity was tested against six E. coli resistant to third-generation cephalosporins isolated from production animals and meat (Supplementary Table S5). Interestingly, three out of the six of such E. coli strains were sensitive to Innolysin Ec21 with the maximum bactericidal activity reaching to 3.31 ± 0.53 log reduction in cell number (Fig. 4). In contrast, no reduction of cell counts was observed when the remaining three E. coli strains were treated with Innolysin Ec21, displaying resistance. Therefore, our results indicate a variable sensitivity of antibiotic-resistant E. coli resistant to third-generation cephalosporins to Innolysin Ec21.
To understand the variability in antibacterial spectrum of the E. coli strains tested, we investigated whether the sensitivity pattern correlates with differences in the FhuA protein sequence, potentially affecting the interaction and activity of Innolysin Ec21. Alignment of the FhuA protein sequences showed that Innolysin sensitivity was not correlated with the presence of specific loops of the FhuA barrel, including the L4 loop previously shown to be one of the binding targets of phage T5 Pb5 15,17 . Some sensitive strains even lacked the amino acids responsible for the formation of L4 loop in FhuA (Fig. 7), suggesting that the L4 loop is not required for the antibacterial activity of Pb5-based Innolysins. In addition, sequence variation was observed in other FhuA regions between strains, but with no correlation to the sensitivity profile. Thus, variations in FhuA cannot explain the Innolysin Ec21 killing spectrum of antibiotic-resistant E. coli. Overall, by shuffling endolysin components we enhanced www.nature.com/scientificreports/ the antibacterial activity of Innolysins and showed that Innolysins can be used as antibacterials to kill at least a subset of antibiotic-resistant E. coli.

Discussion
Phages have developed unique and complex mechanisms to infect and lyse bacteria. In the first stage of infection, phages utilize RBPs to target specific bacterial host receptors on the cell surface, whereas at later stages phage endolysins degrade peptidoglycan, inducing lysis and progeny release. Here, we exploited the binding capacity of a phage RBPs to enable an endolysin to exert antibacterial activity. As proof of concept, we used the phage T5 endolysin and its RBP Pb5 and constructed 12 Innolysins by fusing the endolysin to the whole Pb5 or the binding domain of Pb5 in different orientations, with or without linkers. The majority of the novel Innolysins maintained their muralytic activity and Innolysin Ec6 also reduced the E. coli cell viability by approximately 1 log.
To improve the antibacterial activity of Innolysins, we redundantly screened a library of 228 novel Innolysins each consisting of one out of 23 different endolysins fused with Pb5 in four distinct configurations. Growth inhibition was obtained for Innolysins from all four classes, indicating that Pb5 could be fused with different endolysins in several configurations and still could enable the fused endolysins to exert antibacterial activity. Interestingly, high-throughput screening for the best antibacterial candidate allowed the identification of Innolysin Ec21, that increased the reduction of E. coli cells compared to Innolysin Ec6, reaching to approximately 2 log. The two Innolysins share the same configuration (endolysin-Linker 2-Pb5 1-488 ), but differ in the endolysin component, as Innolysin Ec6 and Ec21 contain phage T5 endolysin and LysEC8, respectively.
Here we showed that neither the cell viability nor the morphology of BL21ΔfhuA cells was changed after application of Innolysin Ec6, indicating that antibacterial activity of the Innolysin is dependent on the presence of the phage T5 receptor, FhuA 11,12 . Since FhuA functions as a transport channel for uptake of ferrichrome, it is tempting to speculate that an Innolysin might be transported through the channel. Yet, it has previously been shown that Pb5 binding to FhuA did not open the channel 11,15 . Furthermore, our results demonstrate that Innolysin Ec6 activity is independent of energy provided by TonB, which is needed for the opening of the channel. www.nature.com/scientificreports/ Therefore, binding of an Innolysin might not provide the conformational changes, leading to the opening of FhuA channel. In addition, the size of the unplugged channel (2.5 nm) 13,14 appeared to be too narrow for Innolysin Ec6 (67.62 kDa) to pass 32 , similarly to what was previously suggested for a hybrid lysin consisting of the phage T4 lysozyme and the binding domain of pesticin targeting FyuA 19 . Thus, we hypothesize that Innolysins might overcome the outer membrane barrier by other means than passing through the FhuA channel. One possible way that Innolysins could access the peptidoglycan might be by interfering with the membrane integrity. FhuA has been proposed to function as an anchor for phage T5 by an irreversible binding of Pb5 to the FhuA receptor 15,17 . We combined Pb5 with phage T5 endolysin, which is a globular endolysin carrying a single enzymatically active domain (EAD) with limited intrinsic antibacterial activity 33 . By irreversible binding to FhuA 11 , Pb5 might bring the Innolysin in close proximity to the outer membrane. Therefore, the Innolysin could interfere with phosphates in the outer membrane lipopolysaccharides and displace the stabilizing Mg 2+ / Ca 2+ ions due to the basic isoelectric point of phage T5 endolysin (7.91), thus destabilizing the ionic forces in the outer membrane 34 . Outer membrane interference has also been described for Artilysins, endolysins engineered with polycationic or amphipathic peptides that destabilize the outer membrane and target the endolysins to peptidoglycan 21 . Similar to polycationic peptides, the positively charged N-terminal extension of Thermus phage 2,631 endolysin has been shown to be crucial for the enzyme to pass through the outer membrane and exert a strong antibacterial activity against Gram-negative bacteria 35,36 . This mode of action could also explain how substituting the T5 endolysin by other endolysin components with higher pI resulted in an increase of the antibacterial activity of Innolysin Ec21. LysEC8 endolysin, the endolysin component in Innolysin Ec21, displays a pI of 9.16 exceeding the T5 endolysin pI (7.91). This difference might influence the distribution of ions in the surrounding interfacial region, thereby destabilizing outer membrane ionic interactions more efficiently. Interestingly, we noticed that Innolysin Ec6 caused cytosol leakage mainly from the bacterial poles of the wild type E. coli carrying the FhuA. It has been shown that several phages preferentially adsorb at the bacterial poles, including E. coli phage φ80 that uses FhuA as receptor 37 . Therefore, Innolysins may bind to the bacterial surface in a similar manner as phages and interfere locally with the outer membrane integrity to allow the fused endolysins to target peptidoglycan. However, further insights in the exact mechanism by which the RBPs enable the fused endolysins to exert antibacterial activity are needed to clarify the mode of Innolysins action.
Interestingly, Innolysin Ec21 demonstrated bactericidal activity against three out of six tested E coli strains resistant to third-generation cephalosporins with killing efficiency reaching to 3.31 ± 0.53 log reduction in only 30 min. This diverse killing spectrum of Innolysin Ec21 could not be explained by differences in the binding ability of the Innolysin to FhuA since killing abilities were not correlated with differences in the FhuA protein sequence, including the L4 loop that is one of the binding targets of phage T5 31,38 . Therefore, other mechanisms such as receptor masking by lipopolysaccharides (LPS) or downregulation of receptor expression 39,40 could play a role in Innolysin resistance. Furthermore, if Innolysins indeed interfere with the ionic interactions, differences in LPS composition could affect membrane integrity and could explain the variation in sensitivity to Innolysins similarly. Therefore, the killing efficiency of an Innolysin might be affected not only by the endolysin component but also by the outer membrane composition of the specific strain.
In this study, we used the binding specificity of the phage T5 RBP to its cognate FhuA receptor to enable an endolysin to exert antibacterial activity. FhuA is conserved in bacterial species, including Escherichia and Shigella, and is structurally homologous to other TonB-dependent outer membrane receptors involved in bacterial iron uptake 41 . Our results demonstrate that Innolysin Ec6, similar to bacteriocin-derived fusions with endolysins, can target isolates of bacterial species other than Escherichia coli, including Shigella sonnei and Pseudomonas aeruginosa. As opposed to some bacteriocins and traditional antibiotics targeting outer membrane proteins 42 , Innolysins exert antibacterial activity against Gram-negative bacteria independent of the energy provided by TonB. These results give support to the potential of using Innolysins against non-actively growing bacteria. Similar to antibiotics, it is expected that bacterial resistance to Innolysins may emerge, putatively by mutations on the binding targets of Innolysins. Yet, some mutations of receptors targeted by phages are known to affect the bacterial virulence. In the case of Innolysins Ec, mutations in FhuA may not be critical for the bacterial virulence because E. coli use at least seven iron acquisition systems 43 . However, having provided the proof of concept will now allow us to use the vast diversity of phage RBPs to design Innolysins able to target outer membrane structures that constitute virulence determinants such as OmpX in Escherichia coli and Ail in Yersinia pestis 44,45 . In summary, we have demonstrated a novel antibacterial concept and have shown the potential for optimization of Innolysins activity by shuffling. In future, this concept may be further expanded to target other Gram-negative bacteria exploiting phage RBPs binding specificity and the vast diversity of bacterial receptors.

Materials and methods
Bacterial strains. E. coli BL21(DE3) CodonPlus-RIL (Agilent Technologies) was used for expression of recombinant proteins. Inhibition assays were conducted with E. coli ATCC11303 (Leibniz Institute) and E. coli BL21(DE3) (Agilent Technologies). E. coli ECOR4 (STEC Center at Michigan State University), Shigella sonnei 46 and Pseudomonas aeruginosa PAO1 47 were also used for antibacterial activity of Innolysin Ec6. Deletions of the fhuA and tonB genes on the chromosomes of E. coli BL21(DE3) and E. coli ECOR4, respectively, were previously generated by the Lambda red recombination system as described before 48 . Antibacterial activity of Innolysin Ec21 was tested against six E. coli strains resistant to third-generation cephalosporins collected during surveillance of antibiotic resistance in indicator E. coli from production animals and food in Denmark. These E. coli strains exhibit an AmpC phenotype due to promoter mutations leading to upregulation of the chromosomal ampC and belong to different sequence types (ST-types) by Multi Locus Sequence Typing (Supplementary  Table S5). www.nature.com/scientificreports/ cloning of constructs. Linker L1 (AGA GAG ) or linker L2 (GAG AGA GAG AGA GA) were used for joining the coding sequences of Pb5 and the endolysins, whereas a His-tag was fused to the C-terminus of all constructs. Cloning of DNA fragments encoding endolysins, linkers and His-tag and their assembly to Innolysin encoding sequences was conducted as described elsewhere 49,50 . In brief, genomic material was purified from phage T5 (Leibniz Institute) and used as a template for amplification (Pfu polymerase; Thermo Fisher Scientific, Waltham, MA) of the genes encoding T5 endolysin (YP_006868.1) or Pb5 (YP_006985.1) with specific primers (Supplementary Table S6). The amplified DNA fragments were separately cloned into the pVTE vector by conducting 30 cycles of restriction with 10 U SapI type IIs restriction enzyme (37 °C, 2 min) and ligation with 3U T4 DNA ligase (16 °C, 3 min), followed by transformation of E. coli TOP10. Transformed cells were selected on LB (Lysogeny Broth) agar plates in the presence of ampicillin (100 μg/ml) and supplemented with 5% sucrose as a positive selection for recombinants. Subsequently, these pVTE plasmids were extracted and used for assembly of the different DNA fragments (50 ng/µl of each pVTE vector) into the concatenated sequence encoding an Innolysin. This assembly and the insertion into destination vector pVTD was done using 10U BsaI and 3U T4 DNA ligase and the same cyclic reaction as above. The assembly reaction mixture was used for transformation of E. coli BL21(DE3)-CodonPlus RIL cells by heat shock. Transformant cells were plated on LB agar plates supplemented with kanamycin (100 μg/ml) and chloramphenicol (50 μg/ml). For large-scale expression of the Innolysin Ec6 and Ec21, a 1 L expression culture (LB medium) was induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (Thermo Fisher Scientific) in the mid-logarithmic phase (OD 600 = 0.6). Incubation of the culture was performed at 16 °C for 18 h at 120 rpm. Cells were harvested by centrifugation (8,000 × g, 10 min, 4 °C) and the cell pellet was resuspended in 10 ml of lysis buffer (20 mM NaH 2 PO 4 -NaOH, 0.5 M NaCl, 50 mM imidazole, pH 7.4), followed by sonication (Bandelin Sonopul HD 2070 homogeniser) with 10 bursts of 30 s (amplitude of 50%) with 30 s intervals. Protein lysate was double-filtered using filters with pore size of 0.22 μm. His GraviTrap™ gravity flow columns (GE Healthcare) were used for His-tagged protein purification according to the manufacturer's instructions. Buffer exchange was performed against 20 mM HEPES-NaOH (pH 7.4) by using Amicon R Ultra-4 centrifugal filters with 50 kDa cutoff (Merck Millipore) and protein concentration was measured by Qubit™ Protein Assay Kit (Q33211) with a Qubit 2.0 Fluorometer (Invitrogen, Q32866).
Muralytic assay. Analysis of the muralytic activity was conducted as described before 21,51 using outer membrane permeabilized P. aeruginosa PAO1 cells as substrate. Briefly, exponentially growing cells (OD 600 = 0.6) were harvested by centrifugation (3,200 × g, 30 min, 4 °C) and permeabilized by resuspension in chloroform-saturated 0.05 M Tris-HCl (pH 7.7) and gentle shaking for 45 min. To remove chloroform traces, cells were washed twice with phosphate-buffered saline (PBS, pH 7.4) and further concentrated to OD 600 = 1.5. 30 μl of the cleared lysates was added on top of 270 μl of the substrate. Cleared lysates of cells expressing the phage T5 endolysin or carrying an empty vector (pVTD) were used as positive and negative controls, respectively. Turbidities were measured spectrophotometrically at 655 nm every 30 s for one hour by a Microplate Reader 680 system (Bio-Rad). Muralytic activities were calculated by a previously described standardized method 52 .
Growth inhibition assay. E. coli cells were used for the growth inhibition assay. Overnight cell cultures prepared in Mueller Hinton (MH) broth were adjusted to OD 600 = 0.1 in 2×MH and further 100-fold diluted in 2×MH. 50 µl of the cell suspension was mixed with 50 μl of the soluble lysate fraction. Art-175 (0.1 mg/ml) and the soluble lysate fraction of cells carrying an empty vector pVTD were mixed with cells as positive and negative controls, respectively. Endpoint measurement was performed spectrophotometrically at 655 nm after exactly 18 h incubation at 37 °C. All experiments were done in biological triplicate.
Antibacterial assay. Overnight  www.nature.com/scientificreports/ transmission electron microscopy. Exponentially growing cells (1 ml) were harvested by centrifugation (10,000 rpm, 5 min) and resuspended in 200 μl of 20 mM HEPES-NaOH (pH 7.4). This washing procedure was repeated 3 times, after which the cell pellets were resuspended in either 50 μl of HEPES buffer or the Innolysin Ec6 and incubated for 15 min at 20 °C. Samples were negatively stained with 2% uranyl-acetate on glow-discharged continuous carbon-coated 300-mesh copper grids (EM Resolutions Ltd). Transmission electron microscopy was performed on a Philips CM100 (Tungsten emitter) electron microscope operating at 100 kV. Images were recorded on a side-mounted Olympus Veleta (2048 × 2048 pixels) charge-coupled device camera via the iTEM software (Olympus SIS, Muenster, Germany).

Bioinformatic analysis.
To identify FhuA homologs, we used the FhuA protein sequence of E. coli BL21 (WP_000124402.1) to search homologous proteins in the National Center for Biotechnology Information (NCBI) genome database through BLASTP 53 . Homologous proteins with different levels of identity in species other than E. coli were selected and aligned by the multiple sequence alignment tool Clustal Omega 54 , through which the percent identity was obtained (Supplementary Table S1). Clustal Omega was also used to align FhuA or Ferrichrome Iron Receptor (FIR) protein sequences obtained by in silico analysis of ESBL-producing E. coli strains (702: FIR (ESC_RA7174AA_AS_04180); 708: FIR (ESC_RA7666AA_AS_01170); 723: FhuA (ESC_ RA7193AA_AS_00052); 724: FhuA (ESC_RA7194AA_AS_03122); 728: FhuA (ESC_RA7198AA_AS_01051); 770: FIR (ESC_RA7701AA_AS_01976), using the protein sequence of E. coli BL21 FhuA (WP_000124402.1) as a reference. To further illustrate the conserved regions of the proteins (Fig. 7) we used the Jalview sequence alignment tool 55 .
Statistical analysis. Analysis of the data was conducted by using GraphPad Prism 7 software (Version 7.0d). For muralytic assays, the activity of each cleared lysate of cells expressing Innolysins was tested in triplicate and means of activity was compared with the average activity of the cleared lysates of cells carrying the empty vector. The significance of the difference in muralytic activity was assessed with Unpaired-Samples t-test using 95% confidence interval for the mean difference. The same software and also the same statistical test was used to analyze results from the growth inhibition assays, using the optical density (OD 655 ) of the bacterial growth after incubation with the cleared lysate of each protein compared to the optical density of cells grown after treatment with the cleared lysate of cells carrying the empty vector. For the antibacterial assay, all bacterial counts were converted to log-scale and means, and standard deviations were calculated afterwards. Antibacterial activity was tested in triplicate in three independent experiments. Decimal reductions of cells were calculated by the difference between the average logarithmic concentrations of cells treated with Innolysin and cells treated with 20 mM HEPES-NaOH (pH 7.4) as a negative control. The significance of the decimal reductions of cells was assessed with Paired-Samples t-test using 95% confidence interval percentage.