The ACM used in all experiments was a commercial product (BioXclude^®, Snoasis Medical, LLC, CO, USA) (Holtzclaw and Tofe, 2017). No human tissue samples were utilized in any of our experiments. The study has been approved by the UTHealth Institutional Biosafety Committee (IBC-22-066). The ACM used in all experiments is a commercial product. The amnion and chorion membranes are harvested from the placenta under sterile conditions with the donor’s consent, in accordance with the provisions of the Food and Drug Administration and the American Association of Tissue Banks. Harvested placental tissues are preserved while serological safety is confirmed. Following serological clearance, the amnion and chorion membranes are isolated from the placenta and processed via the proprietary Purion® process. The membranes are initially cleansed gently, followed by de-epithelialization of the amnion membrane. The de-epithelialized amnion membrane is then laminated onto the directly isolated chorion membrane. De-epithelialization enhances amnion-chorion membrane adhesion by exposing the basement membrane. Subsequently, the thicker (about 300μm) composite membrane undergoes chemical decontamination and dehydration under regulated conditions. This is followed by a terminal sterilization process utilizing gamma radiation and product packaging.

Streptococcus gordonii Challis, a gram-positive bacterium and one of the initial colonizers in the oral cavity, was used as a representative early colonizer (Socransky et al., 1998; Huang et al., 2011) in this study. S. gordonii was inoculated onto Todd Hewitt agar plates (comprised of 3 g of dehydrated Todd Hewitt Broth (THB) and 1.5 g of agar (Difco, Detroit, MI, USA) in 100 ml of H₂O) and cultured for 36 to 48 hours at 37°C in an anaerobic jar containing a bag of BD BBL CO₂ generator (Becton, Dickinson and Company, Sparks, MD, USA) to create a CO₂-enriched environment. The medium used for bacterial culture and experimental assays was THB.

S. gordonii colonies were transferred from THB agar plates to 1 ml of THB in an Eppendorf tube and grown overnight at 37°C to the stationary phase. S. gordonii overnight cultures were diluted in fresh 37°C prewarmed THB (1:4 dilution) and incubated for another hour at 37°C to log phase. The log phase S. gordonii was further diluted 100× in THB for the experimental assays.

HPLC is a type of chromatography, a laboratory technique for the separation of a mixture. MS is an analytical technique that identifies compounds in samples by measuring the mass/charge ratio of their constituent molecules. Pieces of samples (1 mm × 1 mm) from three ACMs were digested in the solvent, 200 ng of modified trypsin (Promega, Madison, WI, USA) and Rapigest (Waters Corporation, Milford, MA, USA), for 18 hours at 37°C. The resulting peptides were analyzed by HPLC-MS/MS coupled with an Orbitrap Fusion mass spectrometer (Thermo Scientific^TM, Waltham, MA, USA). Proteins and peptides in ACMs were identified by Mascot (v2.6, Matrix Science, London, UK) and Proteome Discoverer (v2.2, Thermo Scientific, San Francisco, US) and quantified with total ion count through Scaffold (v4, Proteome Software, Portland, US). The normalization algorithm applied in Scaffold normalizes spectral counting data of all data loaded in the experiment. The searches were performed with a precursor mass tolerance of 10 ppm and fragment ion mass tolerance of 0.8 Dalton using monoisotopic parent and fragment ion masses, allowing for up to two missed cleavages with trypsin digestion. The variable modifications were methionine sulfoxide and pyro-glutamate formation. The proteins and peptides were determined with a false discovery rate (FDR) <1.0% and were categorized by gene ontology (GO) terms.

Four commercially available proteins that were identified in the ACM analytes as described above were utilized in this assay to assess their antimicrobial efficacies, either individually or as mixtures of all 4 proteins. Each protein stock solution, lysozyme (100 mg/ml, Sigma Chemical Co., St. Louis, MO, USA), cathelicidin LL-37 (10 mg/ml, AnaSpec Inc., Fremont, CA, USA), lactoferrin (50 mg/ml, Athens Research & Technology Inc., Athens, GA, USA), or histone H2A/H2B dimer (20 µM, New England BioLabs, Ipswich, MA, USA), was mixed with log phase bacteria in Eppendorf tubes. The final concentrations in the individual-protein cultures were 10 mg/ml for lysozyme, 1 mg/ml for cathelicidin LL-37, 5 mg/ml for lactoferrin, and 2 µM for the histone H2A/H2B dimer. Each protein and 2 × 10⁵ S. gordonii cells in 55μl volume, were added to the wells of a glass-bottomed 96-well microtiter plate (Eppendorf North America, Hauppauge, NY, USA). For the synergistic assessment, a combination of lysozyme, cathelicidin LL-37, lactoferrin, and histone H2A/H2B dimer (in equal volumes) was mixed with the log phase bacteria at 1:10, 1:20, and 1:50 ratios. The mixtures were added to wells in glass-bottomed microtiter plates in volumes of 70 µl, 60 µl, and 54 µl, representing 100%, 50%, and 20% concentrations of each protein, respectively, relative to the individual protein experiments. The cell numbers per well were the same as those in the individual protein assays (2 × 10⁵ cells in each well). The negative controls were 2 × 10⁵ S. gordonii cells/well in THB without the addition of any protein. The positive controls were 6 mm circular pieces of ACM obtained using a biopsy punch (Miltex, Inc., York, PA, USA). The plates were incubated in a CO₂-enriched environment for 8 or 24 hours at 37°C in a box with a water compartment. At each time point, 40 µl of 800X-diluted (in saline) Live/Dead BacLight Bacterial Viability Kit Component B (Thermo Scientific^TM, Waltham, MA, USA) was added to each well and stained for 15 minutes at room temperature. All the experiments were performed in triplicate for each protein group, and the same experiments were repeated at least three times. The antimicrobial efficacy was determined using confocal microscopy and viable count recovery assays.

Confocal analysis was performed using a Nikon C2plus confocal microscope and NIS Elements AR software (Nikon Instruments Inc., Melville, NY, USA) for visual data collection. The filter set and laser settings utilized were 488 nm (green) for FITC and 561 nm (red) for TRITC. One or two representative images of each well were captured at different magnifications (100X or 200X) after the entire area of the bottom layer was viewed.

This assay was designed to quantify viable bacteria cultured with individual proteins or their combinations in varying dilutions. After confocal microscopy, the attached S. gordonii cells were scraped from the bottom of the glass well using a 1 ml pipet tip. The cells in the well were mixed well by pipetting and transferred to an Eppendorf tube. The bacteria were then sonicated five times at a setting of Output 3, Pulse, to break the bacterial chains (Sonic Dismembrator Model 100, Fisher Scientific, Hampton, NH, USA). Each sample was serially diluted in Hanks' balanced salt solution (HBSS, Thermo Scientific^TM, Waltham, MA, USA), and 15 μl of each dilution was subsequently plated onto Mitis Salivarius Agar plates (Difco, Detroit, MI, USA). The plate was incubated at 37°C for 24 to 48 hours in an anaerobic jar in a CO2-enriched environment, and colony-forming units (CFU) were counted manually under a stereo microscope.

Concentrations of 20 mg/ml for lysozyme, 2 mg/ml for cathelicidin LL-37, 10 mg/ml for lactoferrin, and 4 µM for the histone H2A/H2B dimer were utilized to determine the MIC for each protein. For the combination of the proteins, the same concentrations as in the glass-bottom well assay (10 mg/ml for lysozyme, 1 mg/ml for LL-37, 5 mg/ml for lactoferrin, and 2 µM for the histone H2A/H2B dimer) were utilized to assess the MIC. The mixtures containing the proteins that were serially diluted in THB and 2 x 10⁵ S. gordonii cells in 60 µl and 70 µl in total volume for the single protein and the protein combination, respectively, were cultured at 37°C for 16 hours. The bacterial growth control (negative control) was 2 × 10⁵ S. gordonii cells in 60 µl and 70 µl of THB for the individual protein control and the protein combination control, respectively. Serially diluted tetracycline (10, 2, 0.4, 0.08, 0.016, and 0.0032 µg/ml) was utilized as the positive control. After incubation, the cultures were mixed by pipetting and transferred to cuvettes. The optical density of the samples was then determined using a spectrophotometer (Eppendorf, Biophtometer, Eppendorf North America, Hauppauge, NY, USA) at a wavelength of 600 nm. The MIC of each protein or their combination was defined as the lowest concentration that completely inhibited bacterial growth.

For the viable bacterial cell count assessment, CFUs were compared among the control and protein groups using one-way analysis of variance (ANOVA) at 8-hour and 24-hour incubations, respectively. Comparisons between the means of individual proteins, combinations, and controls were analyzed by post hoc analysis (Tukey’s test). All values are expressed as the mean ± standard deviation. A p-value <0.05 was considered to indicate statistical significance.

HPLC-MS/MS identified 1007 proteins and peptides among the three ACMs, with over half of the proteins and peptides being shared by the three membranes. There is variability among the proteins and peptides in the membranes because the results are a semi-quantification where the presence of proteins or peptides is indicated by the isotopic composition of the elements in a molecule. The proteins with reported antimicrobial ability among these 1007 proteins and peptides are lactoferrin, histone H2A, histone H2B, cathelicidin LL-37, and lysozyme ( Figure 1 ). Since these antibacterial molecules consist of more than 100 amino acids, they are mentioned as proteins here for consistency. However, some of these compounds have been described as peptides in the literature (Ramuta et al., 2021).

The spectral counts of proteins or peptides with antimicrobial properties in ACM were determined based on the fragments resulting from ionization and their signal intensity ( Figure 2 ). Histone H2A, histone H2B, and lysozyme were present in all three membranes. Cathelicidin LL-37 was present in one membrane, and lactoferrin was present in two membranes. Generally, histone H2A and histone H2B had a higher count and more homogenous presence than others.

S. gordonii typically presents as long chains of cocci ( Figure 3 , Control). For individual proteins, lactoferrin treatment resulted in fewer bacterial cells relative to the control, with the majority of them alive ( Figure 3 , Lactoferrin), indicating that lactoferrin mainly inhibited bacterial growth at both time points. Cathelicidin LL-37, on the other hand, exhibited bactericidal ability, killing the majority of the cells ( Figure 3 , Cathelicidin LL-37) at 8 hours and even more at 24 hours. Lysozyme disrupted the long chains and killed most bacteria. In contrast to the other three proteins, S. gordonii thrived in the presence of the histone H2A/H2B dimer at 8 and 24 hours, indicating minimal antimicrobial activity against S. gordonii.

The combinations of the four proteins, even at 1/5 of the concentrations used for the single protein, demonstrated significant bactericidal activity. Similar to the protein combinations, the intact ACM also resulted in the complete absence of live bacteria ( Figure 4 ). The large round green dots in Figure 4 of the ACM were from the staining solution, based on the shapes and large diameters. The results of glass-bottom well assays were further confirmed with bacterial viability cell count assays (see below).

To confirm the confocal analysis results and quantify the living S. gordonii cells, the bacterial viability was determined. Lactoferrin, cathelicidin LL-37, and lysozyme at the indicated concentrations significantly reduced the survival of S. gordonii at both time points relative to that of the controls and histones, via one-way ANOVA analysis or the subsequent post hoc test (p <0.001) ( Table 1 ).

The enhanced bactericidal effect of the protein combination was evident ( Table 1 ). The protein combination (at the same concentrations as those of the individual proteins) and its 2X dilution completely eliminated S. gordonii in the cultures at both incubation time points. Even its 5X dilution killed comparable amounts of individual lactoferrin, cathelicidin LL-37, and lysozyme ( Table 1 ). In addition, ACM also resulted in 0% survival of S. gordonii in the cultures at both incubation time points ( Table 1 ).

The MIC test establishes the lowest concentration of an antimicrobial agent that prevents the visible growth of a microorganism. The MIC values for each protein group are shown in Table 2 . Histone H2A/H2B dimers, even at a concentration of 4 µM, were not able to inhibit S. gordonii growth and, therefore, did not reach the MIC. This finding is in agreement with the results of the confocal analysis and viable cell counts. S. gordonii formed precipitates immediately upon mixing with cathelicidin LL-37, even at the lowest concentration (0.06 mg/ml) after serial 2X dilutions. This prevented us from obtaining a reliable OD value. The MICs for lactoferrin and lysozyme were 10 mg/ml and 20 mg/ml, respectively. For the protein combination group, the presence of cathelicidin LL-37 in combination did not cause any precipitation of the S. gordonii cells, even at the highest concentrations. A much lower concentration of each protein was required for the combination MIC relative to the individual protein assays ( Table 2 ).

Since the histone H2A/H2B dimer did not exhibit obvious antimicrobial activity against S. gordonii in any of the assays (confocal analysis, viable cell count, and MIC test), the MIC of the three-protein combination without the presence of histones was further assessed. The MIC for the three-protein combination was approximately three times greater than that for the four-protein combination ( Table 2 ), indicating the synergistic effect of the histone H2A/H2B dimer in the protein combination in eliminating S. gordonii.