Da Costa Ramos, Juliana; (2019) The algal chloroplast as a platform for synthesis of lytic enzymes targeting Gram-negative pathogens. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The rapid rise of antibiotic resistance and shortage of new antibiotics in the drug development pipeline is reaching crisis point, and the scientific response must include a supporting infrastructure for delivery and production. A primary threat to human health is Gram-negative bacteria, which are naturally more resistant to antimicrobial drugs due to the presence of an outer membrane acting as an additional protective barrier. A growing body of research promotes the use of endolysins as novel antibiotics. In their native context, these enzymes lyse the peptidoglycan layer of the bacterial cell wall to release phage progeny from within the host bacterium at the end of the infection cycle. It has been shown that they can also lyse bacteria “from without”, holding great promise as next-generation antibiotics by targeting molecules essential for bacterial viability. This work demonstrates that the eukaryotic microalgae Chlamydomonas reinhardtii can be modified to produce endolysins that target Gram-negative bacteria, offering a new photosynthetic platform with limitless potential for sustainable production. The C. reinhardtii chloroplast presents an excellent compartment for the expression of endolysins as it has a prokaryotic origin, mimicking the environment in which endolysins are naturally produced. Furthermore, it does not contain PG, so the accumulation of endolysin proteins does not pose a deleterious effect on cell viability due to the lack of substrate. Transgenic lines of C. reinhardtii expressing the globular endolysin LysAB2 targeting Acinetobacter baumannii were produced by chloroplast transformation, and recombinant protein accumulation and antimicrobial activity were demonstrated. Enzymatic and antibacterial assays showed clear endolysin activity in algal crude extract against reference strains and multidrug resistant clinical isolates of A. baumannii. Synthetic enzymes known as Artilysins – endolysins fused with antimicrobial peptides so that they act more effectively against Gram-negative pathogens – were also expressed in the C. reinhardtii chloroplast.

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