s aureus strains baa 1717 usa300  (ATCC)

Journal: bioRxiv

Article Title: A chimeric, half-life extended lysin with a unique mode of action

doi: 10.64898/2026.05.13.724763

Figure Lengend Snippet: (A) Efficacy of Fc-CHAP-LSN constructs against in vitro formed biofilms. Bacterial quantification of 24 h-cultivated biofilms of S. aureus ATCC 43300 after 24 h exposure to SOC antibiotics or lysins. Concentrations indicated in µg/mL. Statistical analysis was performed using Graphpad’s one-way ANOVA with Dunnett’s correction for multiple comparisons; ****: P<0.0001; ***: P=0.001; **: P<0.01 ns: not significant (B) Graphical representation of the tested lysins in this assay. Cli: clindamycin; Dapto: daptomycin; LOQ: limit of quantification at 500 CFU/mL; Rif: rifampicin; Untr.: untreated control; Van: vancomycin;

Article Snippet: The following strains were used for this study: Staphylococcus aureus ATCC 43300 (ATCC), BAA-1717 (ATCC), NCTC 8178 (NCTC), Staphylococcus epidermidis DSM3269 (DSMZ), S. epidermidis RP62A (ATCC), Staphylococcus lugdunensis DSM4804 (DSMZ), Staphylococcus haemolyticus DSM20263 (DSMZ) and Staphylococcus haemolyticus DSM20265 (DSMZ).

Techniques: Construct, In Vitro, Control

Journal: bioRxiv

Article Title: A chimeric, half-life extended lysin with a unique mode of action

doi: 10.64898/2026.05.13.724763

Figure Lengend Snippet: Efficacy of Fc-CHAP-LSN against in vivo formed biofilms and in sepsis model. (A) Catheters with in vivo formed biofilms of S. aureus ATCC 43300 were explanted from CD1 mice 3 dpi and treated with either 50 µg/mL or 100 µg/mL Fc-L1-CHAP opt -LSN HEKa for 24 h at 37 °C. Rifampicin (10 µg/mL) was used as comparator. (B) Datapoints represent the biofilm bacterial burden in each catheter (n=6). Horizontal bars indicate the median burden for each group. When burden is below LOQ, half a value of LOQ is assigned for graphical and statistical purposes. Brown Forsythe ANOVA with Welch’s correction (GraphPad Prism) was used to calculate statistical significance against vehicle. dpi: days post infection; LOQ: limit of quantification at 10 CFU/catheter. (C) Lysin efficacy in mouse IV sepsis: Immunocompetent male CD1 mice were challenged with 3.23 × 10 7 CFU/mouse of S. aureus NCTC 8178, IV. Lysins were administered IV, twice (1 hpi and 25 hpi) at dose of either 100 µg/mouse or 500 µg/mouse. Vancomycin (Van) was dosed at 25 mg/kg, IV, q12 h, starting from 1 hpi. (D) Points on the graph represent CFU counts in individual mice (n=6), with horizontal lines indicating median CFU burden and interquartile range. Open circles indicate animals that needed to be terminated before planned endpoint due to clinical symptoms. Brown-Forsythe ANOVA with Welch’s correction was used to calculate the significance compared to vehicle group on log transformed data, with P values indicated in the graph. hpi: hours post infection; CFU: colony forming units; LOQ: limit of quantification at 54 CFU/g; For graphical and statistical purposes, values under LOQ were assigned half of the LOQ value.

Article Snippet: The following strains were used for this study: Staphylococcus aureus ATCC 43300 (ATCC), BAA-1717 (ATCC), NCTC 8178 (NCTC), Staphylococcus epidermidis DSM3269 (DSMZ), S. epidermidis RP62A (ATCC), Staphylococcus lugdunensis DSM4804 (DSMZ), Staphylococcus haemolyticus DSM20263 (DSMZ) and Staphylococcus haemolyticus DSM20265 (DSMZ).

Techniques: In Vivo, Infection, Transformation Assay

Journal: bioRxiv

Article Title: A chimeric, half-life extended lysin with a unique mode of action

doi: 10.64898/2026.05.13.724763

Figure Lengend Snippet: LSN-induced LSN-release from Fc-CHAP-LSN. (a) Purified LSN HEKa was incubated at increasing concentrations in 80% human serum and in presence of 10 8 CFU/mL S. aureus ATCC 43300 with (circles) or without (boxes) a fixed concentration of Fc-L1-CHAP opt -LSN HEK (1 µM) for 3h at 37°C. Samples were analyzed by SDS-PAGE followed by Western Blotting to quantify the amounts of free LSN in each reaction (x-axis). The remainder of the reaction was subjected to quantitative plating to determine the log10 CFU reduction relative to a growth control lacking lysins (y-axis). (B) Concentrations of spiked-in purified LSN are plotted against free LSN detected by Western blotting in the experiment described in panel (A).

Article Snippet: The following strains were used for this study: Staphylococcus aureus ATCC 43300 (ATCC), BAA-1717 (ATCC), NCTC 8178 (NCTC), Staphylococcus epidermidis DSM3269 (DSMZ), S. epidermidis RP62A (ATCC), Staphylococcus lugdunensis DSM4804 (DSMZ), Staphylococcus haemolyticus DSM20263 (DSMZ) and Staphylococcus haemolyticus DSM20265 (DSMZ).

Techniques: Purification, Incubation, Concentration Assay, SDS Page, Western Blot, Control

Journal: bioRxiv

Article Title: Multi-dimensional optimization of a lysin towards a ribolysin against life-threatening S. aureus infections: Fc-LysM-CHAP and its strong synergy with standard of care antibiotics

doi: 10.64898/2026.04.27.720530

Figure Lengend Snippet: a.) Summary of pharmacokinetic (PK)-enhancing modalities fused to LysM-CHAP. Indicated PK-enhancing domains were fused to L1 and tested for growth inhibition against Sa ATCC 43300 in supernatant screens. Architectures with detectable activity were further purified and their PK-enhancing effect tested in mice. Fc: human IgG1-Fc domain; mFc: monomeric Fc domain; FcRNbp: peptide binder to neonatal Fc receptor; HSA: human serum albumin; HSAdIII: domain III of HSA; IgGbp: peptide binder to IgG1; ABP: peptide binder to HSA. b.) Activity and PK of naked and Fc-fused LysM-CHAP variants. Indicated LysM-CHAP variants were purified as naked lysins or fused to N-terminal Fc-hel8, followed by characterization of MIC (in µg/mL) against Sa ATCC 43300. AUC at 4 h and 24 h timepoints (AUC 4h and AUC 24h ; in µg*h*mL -1 ) were determined from PK curves shown in c.). Note that AUC 24h was only determined for Fc-fused lysins. n.t.: not tested; MIC: minimal inhibitory concentration; AUC: area under curve. c.) PK profiles of naked and Fc-fused LysM-CHAP variants. Purified lysins were injected as a single slow bolus intravenous injection via a lateral tail vein into C57BL/6J mice at a dose of 500 µg/mouse. Blood samples were drawn at indicated timepoints and lysin levels quantified by ELISA. Points on the graph represent a mean measured blood concentration of n=3 with standard deviation. Note that we show a composite of two separate experiments, with naked and Fc-fused derivatives of L1, L1-1, L1-2 (experiment 1; last measured timepoint at 24 h, lower limit of quantification (LLOQ) 0.00625 µg/mL) having been measured separately to naked and Fc-fused derivatives of L1-3, L2-1, L2-2, L2-3, and L2-4 (experiment 2; last measured timepoint at 96 h, LLOQ 0.05 µg/mL)). d.) Efficacy of lysins against Sa ATCC 43300 in a neutropenic mouse thigh infection model. Neutropenia was induced in male C57BL/6J mice (n=5) with cyclophosphamide and animals were infected with Sa ATCC 43300 by intramuscular injection of 5×10 3 CFU/thigh in both thighs. Recombinant lysins, including exebacase, were applied at 1 hpi intravenously every 12 h (q12), at a dose of 500 µg/mouse. Vancomycin (VA) was used at dose of 100 mg/kg, q12 with treatment initiated 1 hpi. Data is depicted as log CFU/g of tissue in each thigh, harvested at 25 hpi (pre-treatment at 1 hpi). Each dot on the graph represents bacterial burden per gram of one thigh, with horizontal bars depicting median and interquartile range. Regular one-way ANOVA was used to calculate the significance compared to vehicle group with P values indicated in the graph. hpi: hours post infection; CFU: colony forming units; LOQ: limit of quantification (34 CFU/g). For graphical and statistical purposes, values under LOQ were assigned half of the LOQ value. e.) Bactericidal activity of different Fc-LysM-CHAP formats. Lytic activity of Fc-hel8-L1-3-variants with one or two LysM-CHAP arms, containing wild type Fc (brown) or Fc with RF mutations (to prevent Fc-binding to surface protein A; purple) and monomeric Fc variants thereof were tested in time kill assays in 80% human serum. Lysins were incubated at a final lysin concentration of 0.5 µM (refers to molar concentration of LysM-CHAP arms) with 10 6 CFU/mL Sa ATCC 43300 for 3 h at 37 °C. Shown are individual replicates, mean and SD. Median growth control (untreated) is indicated as a dashed horizontal line. Activity of parental Fc-hel8-L1-3 is shown in column 6. LOQ: limit of quantification (500 CFU/mL). For graphical purposes, values under LOQ were assigned a value of 400 CFU/mL.

Article Snippet: Overnight cultures of Sa ATCC 43300 and BAA-1717 were grown in tryptic soy broth (TSB) at 37 °C and shaking at 220 rpm, diluted 1:20 into fresh TSB to an optical density at 600 nm (OD) of ∼0.05, and further grown until reaching mid-log phase (OD ∼1.0).

Techniques: Inhibition, Activity Assay, Purification, Concentration Assay, Injection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Infection, Recombinant, Binding Assay, Incubation, Control

Journal: bioRxiv

Article Title: Multi-dimensional optimization of a lysin towards a ribolysin against life-threatening S. aureus infections: Fc-LysM-CHAP and its strong synergy with standard of care antibiotics

doi: 10.64898/2026.04.27.720530

Figure Lengend Snippet: a.) X-ray structure of LysM-CHAP L1-3 bound to a stem-peptide analogue (PDB: 11CI). Shown is a space-filling model of LysM (yellow) and CHAP (blue). The stem peptide analogue (orange) is shown as a ball-and-stick model. The modeled NAG-NAM chain from PDB:4UZ3 is shown as a transparent ball-and-stick model. The continuous substrate binding groove is indicated in grey. The inlet shows a zoomed-in view of the contacts between LysM and the stem-peptide analogue. b.) Interactions between stem peptide and the L1 CHAP active site (PDB: 11CH). The stem peptide is shown as a ball-and-stick model. Stem peptide amino acids are labeled in orange font. Amino acids from the CHAP domain in contact with the stem peptide are labeled in black font and shown as ball-and-stick models. Polar interactions between active site residues and the stem-peptide analogue are indicated with dotted lines. The top inlet shows the interactions between the stem peptide L-Lys residue and Phe94/Tyr159 in the CHAP domain (see Supplemental Discussion). The bottom inlet shows electron densities of the stem peptide and the catalytic Cys98 sulfur atom forming a 1.8 Å long covalent bond (indicated in green). The red arrow indicates the C α -atom of D-Ala in the stem peptide. c.) Correlation between Sa binding and lysin potency. Single alanine substitutions were introduced into the substrate binding pocket of the LysM domain in catalytically active Fc-hel14-L1-4 and catalytically inactive Fc-hel14-L1-4* (* denotes a Cys98Ser mutation in L1-4). Variants were purified from Expi293 supernatants. Catalytic rates of Sa (ATCC 43300) cell binding (k on ) and dissociation (k off ) of Fc-hel14-L1-4* derivatives were quantified using biolayer interferometry. ODRRs of catalytically active Fc-hel14-L1-4 counterparts were determined at 0.2 µM final enzyme concentration. k off (left) and k on (right) are plotted against ODRR of corresponding alanine mutants. Pearson coefficients (r) and P-values (P) are indicated for the correlation between k off and ODRR and k on and ODRR in the top left of each graph. d.) Differential impact of LysM mutations on the activity of CHAP variants. ODRRs of a set of 33 combinatorial motif engraftment (CME) designs of Fc-hel14-L1-4 containing either a wild type (wt) LysM domain or LysM domains harboring a Ser18Ala mutation (LysM(18A)) or Ser18Ala plus Ile45Ala mutations (LysM(18A,45A)) were determined in supernatants and normalized to the parental Fc-hel14-L1-4 construct. Rates of LysM(wt) variants are plotted against the corresponding LysM(18A) (blue) and LysM(18A,45A) (red) derivatives. e.) High throughput screen for active site mutants of Fc-hel14-L1-4(18A) with enhanced ODRRs. Active site variants of Fc-hel14-L1-4(18A) were generated using CME (blue) or inverse folding (orange) design strategies and transfected into Expi293 cells. Secretion levels in supernatants were determined by biolayer interferometry and lysin activities were determined by OD reduction assays. ODRRs were normalized to the parental Fc-hel14-L1-4 construct. Secretion levels were normalized to the original Fc-hel8-L1-3 construct. Secretion and ODRR were further expressed as fold changes relative to the parental Fc-hel8-L1-3 reference. Secretion and relative ODRRs of Fc-hel14-L1-5 and Fc-hel14-L1-6 are indicated. f.) Summary of engineering steps leading to Fc-hel14-L1-5 and Fc-hel14-L1-6. Substitutions in LysM, linker-2 and CHAP domain relative to Fc-hel8-L1-3 are indicated. g.) Dose-response behavior of lysin leads in OD reduction assays. Supernatants of Fc-hel8-L1-3, Fc-hel14-L1-4, Fc-hel14-L1-5 and Fc-hel14-L1-6 were serially diluted and measured for activity in OD reduction assays. Final assay lysin concentration (as determined by biolayer interferometry) is plotted against ODRR of each variant. h.) Summary of normalized ODRRs of lysin leads. ODRRs were recorded in supernatants from independent transfections and normalized relative to Fc-hel14-L1-4. ODRR were then expressed as fold changes relative to the Fc-hel8-L1-3 reference. Individual ODRRs are shown, horizontal lines indicate mean ODRRs.

Article Snippet: Overnight cultures of Sa ATCC 43300 and BAA-1717 were grown in tryptic soy broth (TSB) at 37 °C and shaking at 220 rpm, diluted 1:20 into fresh TSB to an optical density at 600 nm (OD) of ∼0.05, and further grown until reaching mid-log phase (OD ∼1.0).

Techniques: Binding Assay, Labeling, Residue, Mutagenesis, Purification, Concentration Assay, Activity Assay, Construct, High Throughput Screening Assay, Generated, Transfection, Variant Assay

Journal: bioRxiv

Article Title: Multi-dimensional optimization of a lysin towards a ribolysin against life-threatening S. aureus infections: Fc-LysM-CHAP and its strong synergy with standard of care antibiotics

doi: 10.64898/2026.04.27.720530

Figure Lengend Snippet: a.) Principle of the BLI assay for measuring binding between lysins and surface immobilized Sa cells. Biotinylated Sa cells are immobilized on streptavidin (SA) biosensors (step 1), followed by blocking with biocytin (step 2). Biosensors are then exposed to solutions containing different Fc-LysM-CHAP variants at increasing concentrations, followed by measurement of association rates (k on ; step 3). After binding, the sensors are transferred to buffer solutions, followed by measurement of lysin dissociation rates (k off ; step 4). b.) Impact of LysM mutations on ODRRs of active site variants of Fc-hel14-L1-4. ODRRs of a set of 33 combinatorial motif engraftment (CME) active site derivatives of Fc-hel14-L1-4 containing either the wild type LysM domain (LysM(wt)), or LysM domains harboring the S18A (LysM(18A)) or S18A/I45A (LysM(18A,45A)) mutations were determined in supernatants and normalized to parental Fc-hel14-L1-4. Shown are normalized ODRRs of individual CME variants. Horizontal lines indicate means of all CME variants in the respective LysM background. c.) Impact of LysM mutations on MICs of active site mutants of Fc-hel14-L1-4. Supernatants described under b.) were subjected to growth inhibition assays against Sa strains BAA-1717 (left) and ATCC 43300 (right). MICs were normalized relative to the Fc-hel8-L1-3 construct using the formula MIC Fc-hel8-L1-3 /MIC variant . Shown are values for individual constructs and means of all CME variants in each LysM background. d.) Effect of LysM mutations on MICs of active site mutants of Fc-hel14-L1-4. MIC data from panel c.), but with fold Fc-hel8-L1-3 MIC changes of LysM(wt) variants plotted against the corresponding LysM(18A) (blue) and LysM(18A,45A) (red). MIC data against Sa BAA-1717 (left) and ATCC 43300 (right) are shown. e.) High throughput screen for active site mutants of Fc-hel14-L1-4(18A) with improved MICs. Supernatants described in were subjected to growth inhibition assays against Sa BAA-1717 (X-axis) and ATCC 43300 (Y-axis). Determined MICs were normalized relative to the Fc-hel8-L1-3 construct using the formula MIC Fc-hel8-L1-3 /MIC variant . CME variants are shown in blue, inverse folding variants are shown in orange. Lead variants Fc-hel14-L1-5 and Fc-hel14-L1-6 are highlighted. f.) Trade-off between secretion and ODRR for variants designed by CME and inverse folding approaches. Same as .), but only showing secretion and activities of the most active CME (blue) and inverse folding (orange) variants at each secretion bin. g.) Summary of MICs of Fc-hel8-L1-3, Fc-hel14-L1-4, Fc-hel14-L1-5 and Fc-hel14-L1-6 as determined from supernatants. Shown are individual MIC values determined for Sa BAA-1717 from independent transfections. Median MICs are indicated with horizontal lines.

Article Snippet: Overnight cultures of Sa ATCC 43300 and BAA-1717 were grown in tryptic soy broth (TSB) at 37 °C and shaking at 220 rpm, diluted 1:20 into fresh TSB to an optical density at 600 nm (OD) of ∼0.05, and further grown until reaching mid-log phase (OD ∼1.0).

Techniques: Binding Assay, Blocking Assay, Inhibition, Construct, Variant Assay, High Throughput Screening Assay, Transfection

Journal: bioRxiv

Article Title: Multi-dimensional optimization of a lysin towards a ribolysin against life-threatening S. aureus infections: Fc-LysM-CHAP and its strong synergy with standard of care antibiotics

doi: 10.64898/2026.04.27.720530

Figure Lengend Snippet:

Article Snippet: Overnight cultures of Sa ATCC 43300 and BAA-1717 were grown in tryptic soy broth (TSB) at 37 °C and shaking at 220 rpm, diluted 1:20 into fresh TSB to an optical density at 600 nm (OD) of ∼0.05, and further grown until reaching mid-log phase (OD ∼1.0).

Techniques: Purification, Concentration Assay, Variant Assay, Activity Assay