Sortase a mediated biomolecular engineering studies A potential strategy to tackle gram positive bacterial infections

Abstract

quotGram-positive bacterial infections are considered one of the vital public health issues newlinedue to increased antibiotic resistance cases, leading to various infections in immunecompromised patients. Among all Gram-positive bacteria, Staphylococcus aureus is one of the leading causes of severe risk to critically ill patients. The formation of biofilm newlineposes a significant treatment challenge for Staphylococcus aureus. The bacterium newlinedevelops a high level of resistance to various antimicrobial agents that are typically newlineeffective against their planktonic forms. These issues provide scope for developing new newlinemolecules or therapies for treating these bacterial infections. Therefore, we explored a newlinenew approach to target Staphylococcus aureus infection using its enzyme, sortase A. newlineSortase A is a transpeptidase enzyme present in many Gram-positive bacteria. It recruits newlinecell surface proteins onto the bacterial cell wall and plays a significant role in bacterial newlinevirulence. It recognizes its substrate using the LPXTG motif that is present at the Cterminal of the peptide and recruits its substrate onto the pentaglycine present on the newlinepeptidoglycan layer. Over the last two decades, it s become an important biomolecular newlinetool as it has the ability to perform in-vitro ligation. Sortase A provides a platform for newlinerobust, specific and highly efficient ligation. In the present study, we are trying to newlineexplore the potential use of sortase A present on the cell wall and the purified enzyme newlineagainst bacterial infections. newlineThe sortase A mediated ligation (SML) was used for the cell surface modification of newlineGram-positive bacterial cell wall and for developing antimicrobial surfaces. In Chapter newline2, we designed sortase A substrate mimics (peptides) having an LPETG motif at the Cterminal. It has been predicted that sortase A substrate mimics were stable, have no newlinehaemolytic activity and do not have their own standalone biofilm activity by Expasy newlineProtParam, HAPPENN and dPABBs software, respectively. In chapter 3, we have newlinedirectly installed the peptides on the sortase A of the bacterial cell wall and looked the newlineeffect of the peptides on biofilm formation. The incorporation of peptides on the cell newlinewall was measured through ELISA and Fluorescence Spectrophotometer. We have also newlineobserved a significant reduction in the biofilm formation property of modified bacterial cells. Further in Chapter 3, we studied other virulence parameters of the Gram-positive newlinebacterial cells, especially S. aureus. We have observed a significant reduction in the newlinevirulence of the bacteria in the presence of the peptide in terms of its IgG binding newlineproperty, fibronectin-binding and phagocytosis. We have also demonstrated bacterial newlinecolonization and biofilm formation inhibition on a silicon-coated latex catheter and an newlineex-vivo skin burn model. This work demonstrated the inability of surface-modified newlineGram-positive bacterial cells to establish infection, thereby presenting a novel strategy newlinefor attenuating its virulence. Further, purified sortase A was successfully used to attach newlineantimicrobial peptides on the synthetic surfaces, which provides a new opportunity for newlinethe medical industry to use SML to develop different bioactive surfaces to prevent newlinemicrobial growth.quot newline newline