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Understanding physiological changes of Escherichia coli in response to endolysin LNT113 treatment

  • 송혜민 (아주대학교 일반대학원)

초록/요약

In response to the alarming rise in multidrug-resistant (MDR) Gram-negative pathogens and the lack of effective antibiotics, there is an urgent need for alternative antibacterial strategies with novel mechanisms of action. Bacteriophage endolysins are emerging as promising candidates, as they enzymatically degrade the bacterial cell wall with high specificity and a low likelihood of resistance development. LNT113 is a recombinant endolysin engineered by fusing EC340 from Escherichia coli phage PBEC131 with cecropin A, a natural antimicrobial peptide from Hyalophora cecropia. In order to extend the potential of LNT113 as a promising alternative to conventional antibiotics, the probability of bacteria to develop resistance to LNT113 should be addressed. Therefore, this study explored the physiological and transcriptomic responses of E. coli to subinhibitory LNT113 stress and identified genetic elements required for bacteria to adapt to and tolerate subinhibitory LNT113. Transcriptome analysis identified 552 differentially expressed genes (DEGs) following subinhibitory LNT113 treatments. To functionally evaluate bacterial responses, thirteen DEGs related to stress response and envelope maintenance were deleted respectively, producing thirteen mutant strains. Among them, Δ(prmB–yfcL) and ΔfabB were attenuated in their viability under subinhibitory LNT113 treatments, indicating their susceptibility to LNT113. With regard to the prmB–yfcL operon, individual genes were deleted respectively to define the critical gene required for bacterial tolerance. Consequently, it was found that ΔaroC and ΔmepA were more vulnerable to LNT113 treatments. NPN uptake assays demonstrated that the outer membranes of ΔfabB, ΔaroC, and ΔmepA were more permeable to LNT113, and introduction of pWSK129::fabB, pWSK129::aroC, and pWSK129::mepA into each mutant strain restored the membrane integrity. These findings suggest that fabB- mediated unsaturated fatty acid biosynthesis and mepA-dependent peptidoglycan remodeling are critical for maintaining envelope integrity during endolysin stress, while aroC may contribute to bacterial tolerance to LNT113 indirectly through metabolic adaptation. This work provides insights into bacterial defense responses and may guide the development of more effective endolysin-based therapies.

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목차

1. Introduction 1

2. Materials and Methods 4
2.1 Bacterial strains and growth conditions 4
2.2 Expression and purification of endolysin LNT113 8
2.3 RNA extraction 8
2.4 RNA sequencing and analysis 9
2.5 Quantitative real-time PCR 9
2.6 Bacterial turbidity reduction assay 19
2.7 1-N-phenylnaphthylamine (NPN) uptake assay 19

3. Results 20
3.1 Transcriptomic profiling of E. coli MG1655 in response to endolysin LNT113 treatment 20
3.2 Experimental validation of differentially expressed genes 26
3.3 Construction of mutants lacking candidate resistance genes against LNT113 31
3.4 Susceptibility test of the 13 mutant strains to LNT113 treatments using turbidity reduction assay 34
3.5 Susceptibility test of selected mutant strains to LNT113 treatments using NPN-based membrane permeability assay 37

4. Discussion 44

5. Reference 48

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