Virginia Research Day 2021

. Peptide Nucleic Acids for the Prevention/Treatment of Persistent Infections Caused by Staphylococcus aureus Biofilms Nihan Akguc Col 1 *, Jayasimha Rao 2 , Govindarajan Rajagopalan 3 and Nammalwar Sriranganathan 1 1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 2 Department of Medicine, Division of Infectious Diseases, Carilion Clinic, Virginia Tech Carilion School of Medicine, 3 Department of Internal Medicine, Yale School of Medicine, Yale University.

Results

Methods

Staphylococcus aureus readily forms biofilms, which is one of the common mechanisms of antibiotic resistance and plays a crucial role in progression of infections in humans and animals. It is a challenge to treat such biofilm infections, often leads to antibiotic resistance, thereby causing persistent infection of wounds, heart valves and other living tissue as well as various indwelling devices such as catheters, cardiac implants and prosthetic joints, causing enormous economic and clinical burden. Hence, novel approaches to prevent/treat biofilm formation by this pathogen are urgently needed. In this context, inhibition of bacterial gene expression using peptide nucleic acids (PNAs) is an effective mean to either kill bacteria or to prevent biofilm formation. Therefore, we envisioned that a potential strategy for the prevention of S. aureus biofilm infections would be to use PNAs to down-regulate or abolish the expression of specific genes associated with biofilm formation. Introduction and Study Aim

Design and synthesis of P-PNAs Antisense PNAs linked with cell penetrating peptides (CPP) designated as P-PNAs targeting S. aureus -essential genes as well as genes important for biofilm formation were designed, and commercially synthesized along with scrambled PNAs (control) with CPP. The biofilm inhibitory activities of the synthesized P- PNAs targeting the three essential genes ( fmhB , yycF & yycG ), and four genes relevant to biofilm formation ( icaA , sarA , saeS , rot ) were tested against S. aureus P231 strain by a standard microtiter biofilm assay. The functions and targets DNA sequences of designed P-PNAs.

➢ S. aureus P231 strain was precultured in TSB- 1% glucose overnight at 37 °C with 5% CO 2 and diluted with fresh 2x concentration of TSB- 1% glucose to obtain a bacterial culture equivalent to 10 5 CFU/ml. Fifty microliters of this dilution were added to appropriate wells of a low binding 96-well microtiter plate. ➢ The indicated P- PNA concentrations were then added to bacterial diluents in two replicates. Appropriate amount of molecular grade water was added to the wells for each P-PNA concentrations. ➢ After 48 hours of incubation at 37 °C with 5% CO 2 , biofilm formation assay was performed.

Image of S. aureus P231 strain biofilm treatment with P-PNAs in microtiter plate after removing the liquid culture of the wells at 48 h.

Inhibitory effect of antisense PNA

Percentage biofilm inhibition in S. aureus by CPP-linked peptide nucleic acids (PNAs). Crystal violet staining was performed to assess biofilm formation on 96-well low binding microtiter plate at 48 hours.

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Conclusions and Future work

Acknowledgment The research was supported by VCOM:CVM One Health Research fund and foundation account. The first author was also supported by a TÜBİTAK -2219 international post doctoral fellowship research program during her Post doctoral studies.

• Our results showed that anti- sarA P-PNA at 50 μM prevented the biofilm formation on the surface of low binding microtiter plate. • CPP and scrambled PNAs had no inhibitory effects on bacterial growth as well as biofilm formation. • Our findings have the potential as an alternative strategy to prevent/treat biofilm formation by S. aureus and hence has clinical utility.

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