Engineering of bacteriophage-derived endolysins to treat streptococcal mammary gland infections

Bacteriophages, commonly known as phages, are specialized viruses that infect bacteria. Bacteriophage infection reprograms the bacterial host, leading to the substantial production of new viral particles. In the final stage of the infection cycle, the phage releases endolysins, initiating ‘lysis-from-within’, a process that degrades the bacterial cell wall and releases newly assembled viral particles. These endolysins, functioning as peptidoglycan hydrolases, maintain their effectiveness when applied externally to Gram-positive bacteria, a phenomenon known as ‘lysis-from-without’. This process induces bacterial lysis due to a high internal osmotic pressure inside the bacteria, comparable to a balloon bursting upon needle puncture. Consequently, endolysins emerge as highly promising antimicrobials with a unique mode of action, rendering them highly effective against antibiotic-resistant strains.

Endolysins derived from Gram-positive bacteriophages showcase a modular structure (for example CHAP and SH3_5 as shown in the figure), which allows the creation of engineered endolysins through genetic engineering. This approach leads to the development of new ‘domain swapped’ endolysin variants, exhibiting improved properties compared to the original wildtypes. Dr. Vander Elst’s research, conducted at Ghent University and KULeuven, focused on engineering domain-swapped endolysins targeting bovine mastitis-causing streptococci. Utilizing a high-throughput DNA assembly platform, the research team generated a library comprising tens of thousands of engineered variants. This library underwent screening for antibacterial activity in milk against streptococci isolated from mastitis-affected cows, which identified a superior endolysin with high bacterial killing that even demonstrated efficacy in eradicating S. uberis biofilm. The research team strategically engineered the superior endolysin to exhibit intracellular activity, enabling targeted action against streptococci inside the mammary epithelium. Notably, the superior endolysin also had a potentiation effect on cloxacillin treatment in raw cow’s milk. To validate the application potential of the superior endolysin, infected mice were intramammarily treated with cloxacillin combined with the superior endolysin (as shown in the figure), which showed a higher reduction in bacterial load, neutrophil infiltration and pro-inflammatory mediators in 60% of the mice compared to conventional antibiotic treatment. The results of the research team have been published in the scientific journals ‘Microbial Biotechnology’ and ‘Applied Microbiology and Biotechnology’.

Overall, these findings underscore the potential of engineered endolysins in improving the current treatment of bovine mastitis. The endolysin’s capacity to target the evolved virulence factors of mastitis-causing streptococci – being antibiotic resistance, biofilm formation, and intracellular survival – highlights the technology’s potential in overcoming the shortcomings of conventional antibiotic treatments. Simultaneously, engineered endolysins can enhance the effectiveness of existing antibiotic treatments – here demonstrated for cloxacillin – offering a complementary and promising approach to combating bovine mastitis caused by streptococci in the near and far future.

Niels Vander Elst was awarded a fellowship from the Belgian American Educational Foundation to start his research project at the Institute for Bioscience and Biotechnology Research, associated to the University of Maryland, in continuation of his veterinary medicine studies. He focused on investigating bacteriophage-derived enzymes (endolysins) as novel antimicrobials for bovine mastitis. Upon completion, Niels received additional funding from Research Foundation Flanders (FWO Vlaanderen) to finish his interdisciplinary PhD project as an interuniversity collaboration between Ghent University and KULeuven. After his PhD, Niels proceeded with his academic career at the Karolinska Institute in Stockholm, Sweden, where he continues to engineer endolysins, now with a focus on human health.

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