Design and Selection of Engineered Lytic Proteins With Staphylococcus aureus Decolonizing Activity

Abstract

Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15–0.6 μM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 μM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 μg/cm2). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.

Cellular and Enzymatic Determinants Impacting the Exolytic Action of an Anti-Staphylococcal Enzybiotic

“…Our results agree with a previous study reporting that Lys11 constructs bearing either the endopeptidase or amidase domain directly fused to the CBD failed to induce visible lysis against heat-killed S. aureus [ 46 ]. However, in certain experimental conditions, structurally related staphylococcal endolysins were shown to display normal, or even superior, lytic activity, after the deletion of the amidase CD [ 45 , 47 ]. Therefore, for related endolysins, the outcome of combining or deleting functional domains can vary significantly, probably depending on the intrinsic features of the individual domains.…”

Biology and Regulation of Staphylococcal Biofilm

“…Dozens of recent papers described different attempts to reduce biofilm formation or to destroy an already-formed biofilm in order to avoid the removal or replacement of an implanted material. Enzymes, antimicrobial peptides, bacteriophages, and natural compounds from vegetal origins have been used, mainly in vitro, with variable results [ 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 ]. Interesting observations were reported by Caballero Gomez in the field of meat-chain production using natural compounds extracted from essential oils alone or in combination with EDTA [ 133 ].…”

“…Gutierrez and colleagues obtained very interesting results by using an engineered endolysin treatment in vitro and in vivo in a skin model of infection. The authors obtained a complete disinfection of the contaminated sites after treatment [ 138 ]. Similar results were envisioned by using lytic bacteriophages on already-formed biofilms.…”

Endolysin, a Promising Solution against Antimicrobial Resistance

“…Furthermore, continuous administration of ClyR for 40 days obviously decreased the severity of caries in the rat model infected with a single or a mixed bacteria of S. mutans and S. sobrinus [ 131 ]. The engineered phage endolysin LysRODIΔAmi prevented biofilm formation at low protein concentrations of 0.15–0.6 μM in S. aureus and had no toxicity toward human keratinocytes, even at high concentrations of 22.1 μM [ 132 ].…”