Exploring the link between RseP and susceptibility to the bacteriocin H1 in Staphylococcus aureus

Abstract

Antimicrobial resistance is an escalating concern in today's interconnected world, with Staphylococcus aureus emerging as one of the primary causative pathogens. S. aureus is a common opportunistic pathogen found within the human skin microbiota, able to cause a wide range of infections with varying degrees of severity. With increasing numbers of deaths linked to antimicrobial resistance, the need for finding novel drugs and treatment alternatives is crucial to preventing a resurgence of historically high numbers of deaths related to infections. Bacteriocins have been investigated as an alternative to antibiotics. Bacteriocins are a diverse group of peptides ribosomally produced by bacteria that exhibit antimicrobial effects on mostly closely related species. They differ in complexity, inhibition spectra, and size. A particularly interesting family of bacteriocins is called the LsbB-family, consisting of four sequence-related native members and some synthetically engineered ones. H1 is a hybrid LsbB-family bacteriocin built up by the N-terminal half of enterocin K1 and the C terminal half of enterocin EJ97. All the members share the same receptor protein, RseP. RseP is a site-2 metalloprotease involved in regulating the σE factor in Escherichia coli. It has a conserved structure and is located in the cell membrane, with domains inside and outside the membrane. RseP homologs can be found across bacterial phyla and even in humans, making it a very conserved protein. This thesis aims to investigate the prevalence of H1 susceptibility in a collection of S. aureus isolates and to test whether differences in susceptibility can be linked to sequence variations in rseP. The collection comprised 129 isolates from the Laboratory of Microbial Gene Technology (LMGT). After the initial susceptibility testing, the rseP gene of 34 isolates was sequenced, and clustering at 100% identity on residue level was performed to identify the number of unique RseP sequences. A correlation analysis between H1 susceptibility and RseP sequence was conducted to determine the residues most significantly associated with susceptibility. The two most significant were positions 304 and 308, with E304 and K308 positively correlated with susceptibility, while K304 and I308 were negatively associated. To investigate their importance, rseP from a susceptible S. aureus strain was cloned and expressed in a non-susceptible ΔrseP strain L. lactis. After confirming the sensitivity of the new L. lactis mutant towards H1, E304 and K308 were substituted for K304 and I308. However, these substitutions alone were insufficient to confer nonsusceptibility, as this clone was still susceptible to H1. This indicates that more research is needed to identify all factors affecting the susceptibility of S. aureus towards H1.