Antibiotic Resistance → A Historical Perspective of Endolysins

Kevin Zhu
8 min readFeb 21, 2021

Written in collaboration with the LyseDevice Team

- New Scientist

Bacterial resistance to antibiotics has emerged in recent years as bacteria evolve to develop protective defense measures such as biofilms against antibacterial drugs [1, 2, 3]. Bacterial infections have become an imminent concern; causing 35,000 deaths yearly in the United States [4]. The World Health Organization estimates that antibiotic resistance will be the cause of 10 million deaths per year by 2050 if no suitable alternatives are found [5]. A prospective solution to the antimicrobial crisis is bacteriophages as they bind to protein receptors on bacterial walls and inject their genetic material, leading the host cell to die as phage copies burst through the membrane [6]. However, because phages can only bind to the surface receptors of a single strain of species of bacteria, they are highly vulnerable to the development of resistant bacterial mutants [7–8]. Phage cocktails containing a mixture of many bacteriophages have been produced to increase the robustness of treatments to mutations and target a broader range of potential bacterial strains, but cocktails are costly and time-consuming to test and produce.

Thus, another proposed alternative to antibiotics being presently investigated is phage endolysins. Typically, during the lysogenic reproductive cycle of a bacteriophage, lysin enzymes are produced by the phage within the host cell and degrade the cell wall via either hydrolysis or glycosidic bond cleaving, lysing the cell from the inside [9]. Against Gram-positive bacteria, lysins are highly effective bactericides when applied exogenously; similarly, in recent studies, lysins have been genetically modified with permeabilizing agents to degrade the membranes of Gram-negative bacteria, demonstrating a high potential for therapeutic use [10].

Similar to whole phages, lysins are effective at removing bacterial biofilms and are highly specific so they exhibit low toxicity to humans whole bacteriophages and antibiotics, lysins are resistant to all forms of bacterial resistance as they have evolved to uniquely target a molecule in the cell wall that is essential for the survival of the bacteria [11]. Current technology enables lysins to be produced in large quantities through recombinant DNA, while whole phages must be produced through the culturing of their host bacteria in liquid mediums. Automated flow chemistry has recently been developed to rapidly produce proteins de novo [12], however, it has not yet been applied to synthesize phage enzymes due to the unpredictable folding patterns of lysin loop regions between protein domains [13–14].

Due to the high specificity of lysin-based therapies, extensive experimental testing is required to create new therapies for individual species of bacteria, which is costly and time-consuming [15]. Recently, deep learning methods such as long short-term memory neural network (LSTM) have been proposed to process genomic sequences to computationally determine whether a species of bacteriophage lysin can target a particular bacteria based on the genome of the bacteria and the protein sequence of the lysin. This new advancement in technology has allowed for an efficient and highly accurate method to identify the proper lysin-based treatment for any bacterial infection [16]. Still, there are over 18,000 species of phages indexed in databases, and thousands of phages are being discovered yearly; likewise, a similarly large number of distinct lysins exist, with many capable of targeting only a specific bacterial strain [17]. As each species of lysin utilized in treatments must be produced in large quantities and stored locally for distribution to patients, it is currently infeasible for widespread therapeutic use for infections across a large number of pathogenic bacterial species [18–19]. Thus, new technology must be developed for the efficient production and distribution of novel lysin-based therapeutics to treat infections resulting from any strain of bacteria.

To obtain bacterial information, Oxford Nanopore Technologies’ MinION device allows for real-time analysis of nucleic acids. As an enzyme is passed through a protein nanopore, electrical signals are monitored to produce a resulting DNA sequence through the process of base calling [20–22]. The MinION’s ability to process long reads, portability, low-cost and hand-held features demonstrate the device as a breakthrough in sequencing processes [23–24].

Synthesized lysins can be delivered in a variety of methods to the site of infection, including intravenous and subcutaneous injections for immediate treatment in severe cases. Additionally, prescription pills, topical creams, and powders are commercializable forms of delivery that can be conveniently administered to the patient for mild bacterial infections. [25–26]

History

Antibiotics have been utilized since the early 20th century to treat a broad range of bacterial infections [27]. In 1928, Alexander Fleming discovered penicillin and in 1940, a bacterial penicillinase was identified. Since the development of the first effective antimicrobials in 1937, the evolution of resistance mechanisms through the misuse of antibiotics has limited their remedial use.

In the 1900s, following Twort and d’Herelle’s discovery of bacteriophages as agents capable of killing bacteria in association with diarrheal illnesses [28–30], Clark and Clark identified phage B563 from a Milwaukee sewage treatment plant which demonstrated a lytic activity on strains of streptococci, whereas the original phages were unable to infect the bacterium, and entitled the phenomenon “nascent lysis.” Winston Maxted further investigated the B563 phage’s lytic activity revealing its ability to kill streptococci sans the active phage [31]. The B563 phage was then renamed as C1, due to its activity on group C streptococci, who was the first to relatively refine the lysin. In the 1960s, Vincent Fischetti began working as a technical assistant on streptococcal-associated diseases including both scarlet and rheumatic fever.

A major difficulty in purifying the C1 lysin was a reduction in activity due to the irreversible binding of heavy metals and thiol-alkylation of the cysteine in the active site of the cysteine histidine-dependent amidohydrolase/peptidases (CHAP) domain [32]. Fischetti resolved this problem through the usage of sodium tetrathionate to reversibly obstruct and preserve the sulfhydryl group in the CHAP domain before purification; after purification, the lysin was reactivated by releasing the tetrathionate. Fischetti’s laboratory conducted experiments using the purified C1 lysin in decades to come as a mechanism for the extraction and identification of the streptococcal M protein and unravel the procedure where M protein and other surface proteins on Gram-positive bacteria were attached to the cell wall; his discoveries enabled the development of a treatment for the streptococcal pharyngeal infection. Lysins have since been applied to treat infections from many other bacterial genera.

Citations

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