Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Biochemistry & Biophysics
Non Technical Summary
Bacteria are a growing threat in human and veterinary medicine, as well as in agriculture, where valuable crops are increasingly subject to microbial disease, and industry, where microbial corrosion and fouling have long been a major problem. We are losing ground against bacteria in all these areas. Traditional antibiotics are rapidly losing their effectiveness against bacterial disease in humans and are being withdrawn from applications in veterinary clinic and animal husbandry. Chemical biocides for use in agriculture and industry are being disqualified because of their toxicity to the environment.It is with these trends in mind that I propose to continue our efforts to advance the basic biology of bacteriophage, the viruses of bacteria, as a natural alternative for combating bacteria in health, agriculture and industry.Bacteria inhabit every niche in the biosphere, at an estimated population of -1029, or more than ten million trillion for each human on earth (Hendrix et al., 1999). Bacterial infections cause most of the important diseases of humans, animals and plants. Moreover, bacteria are major sources of contamination, degradation and corrosion in many critical economic and industrial sectors, especially in fuel pipelines and storage facilities. To combat bacteria, the world has relied on mass-produced antibiotics for therapeutics and on chemical "biocide" treatments in agriculture and industry. Both strategies are now near or at the stage of collapse, for several reasons. First, bacteria are a particularly insidious and resourceful enemy. They are very flexible genetically, exchanging DNA with their colleagues, both related and unrelated, at enormous rates and mutating extremely rapidly, so antibiotic-resistant bacteria are constantly evolving.Bacteriophages, or "phage", are simply viruses that grow on bacteria; they have been successfully doing this for billions of years, and as a consequence of their successful evolution, now exist in unimaginable diversity in every ecosystem where bacteria are found. Typically, in the natural environment, 10 -100 phage particles can be found for every bacterial cell, leading to estimates exceeding 1031 particles in the biosphere. It has been known since early in the 20th century, when phage were discovered, that phages kill bacteria, and can do so effectively in the context of the infected human, animal or plant. Bacteriophage biology thus offers an inexhaustible reservoir of natural biological tools and knowledge to combat bacterial pathogenesis.The phage infection cycle involves (1) adsorption to the target bacterial cell; (20) penetration of the phage genomic nucleic acid into the host cytoplasm; (3) vegetative growth, involving replication of the genomic nucleic acid and viral morphogenesis; (4) lysis of the host, n which the cell envelope is disrupted, releasing the progeny virions. Our group has led the world in the study of the lysis process at the molecular level(Cahill and Young, 2019; Chamakura and Young, 2019). Besides its fundamental nature, as the most common cytocidal event in the biosphere, phage lysis is now relevant in its key role in the phage therapeutics for human medicine, crop protectional and animal husbandry.Over the past four decades, our group has established that there are two strategies for phage lysis: Multi-Gene Lysis (MGL), for the dsDNA Caudovirales , which have up to -700 genes; and Single Gene Lysis for the ubiquitous ssRNA Leviviridae (3 -4 genes; 3-4 kb) and ssDNA Microviridae (8 -10 genes; 5 kb). In MGL systems, lysis, and thus the length and fecundity of the infection cycle, is actively and precisely scheduled by a small membrane protein, the holin, through control of a muralytic enzyme, the endolysin and in collaboration with other phage proteins from a still-growing menu of at least 11 more functional and diverse types. In SGL systems, lysis is not precisely timed; instead, a single protein (Sgl) promotes dysfunction of cell wall biosynthesis and/or homeostasis sufficiently severe to evoke, eventually, host autolysis(Chamakura and Young, 2019).As noted above, the holins constitute the master control of MGL systems. We have shown that there are two general types of holins: canonical holins and pinholins, which, as the names imply, differ in the size of hole formed in the membrane (micron-scale and nm-scale, respectively). There are two fundamental types of endolysins: canonical and SAR; the former are globular cytoplasmic enzymes that are released to attack the cell wall through the large micron-scale holes formed by canonical holins. In contrast, SAR endolysins pass through the membrane using the host secretion system and accumulate at the cell wall in inactive form; when the pinholins act to permeabilize the cytoplasmic membrane with multiple nm-scale holes, the SAR endolysins are activated and attack the cell wall. In addition, in infections of Gram negative bacteria, we have shown that the outer membrane must be actively disrupted even after the cell wall is destroyed by the endolysin activity. Most phages encode spanins, which disrupt the OM by fusing it with the IM, after the cell wall has been degraded. Moreover, there are two types of spanins: two-component spanins and unimolecular spanins. Finally, there are specific antiholins that negatively regulate holins and pinholins. So far we have identified membrane, periplasmic and cytoplasmic antiholins and membrane antipinholins. Recently, we have also found that about 10% of phages of Gram-negative hosts use small polypeptides that we call disruptins to disrupt the OM. Finally, we have also found that some phages encode proteins we have designated as releasins that are required for activation of SAR endolysins. This brings the current total of lysis protein classes for MGL systems of Gram-negative hosts to 13, most of which contain a great diversity of protein sequences. Our group discovered all of these functional classes, except for the canonical endolysins.In small lytic phages, where genomic space is extremely limited, lysis is accomplished by Sgl proteins, which, in general, are small polypeptides encoded by genes embedded in a distinct reading frame inside important viral genes. Our group has shown that there are at least two classes of Sgls: protein-antibiotics (PA) and L-like. PAs are proteins that block cell wall synthesis, just like classic antibiotics, leading to lysis when the infected cell divides. We have discovered three PA-type Sgls that block three different steps in the universally conserved cell wall biosynthesis pathway. The L-like Sgls cause lysis by a still-undiscovered mechanism that does not involve inhibition of cell wall biosynthesis, leading to random lesions in the cell envelope. We have conducted in-depth genetic analysis of the prototype L Sgl and identified its key residues and dependencies on host protein folding mechanisms.I am the Director of the Center for Phage Technology (CPT), established by the Board of Regents of the TAMU System in 2010 with the explicit mandate to promulgate and advance the translational applications of phage biology in agriculture, industry and human health. Until recently, the CPT has been focused on agricultural applications of phage therapy(Das et al., 2015). However, in 2016, the CPT was involved in the famous "Tom Patterson case", in which a prominent academic scientist was saved from a life-threatening bacterial infection by treatment with phage preparations. Phages isolated and purified at the CPT constituted the first phage cocktail administered in modern medical history(Schooley et al., 2017). The impact of this successful phage-based intervention, as well as a number following, has completely changed the environment surrounding phage therapy and its potential practitioners. Under my direction, the CPT is now heavily committed to advancing phage therapy in both agricultural and clinical directions.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
The over-arching basic research objective is to determine and characterize the "Lys-ome", which is the complete set of proteins used by phages to effect lysis of the host bacterial cell. In the process, we will learn how these proteins work and what specific aspects of the bacterial cell are targeted. Ultimately the long-term goal is to understand the molecular systems by which bacteria synthesize and maintain the cell wall and how we can interfere with these systems for the development of antibiotics useful in health and agriculture. Moreover, understanding these systems may lead to the development of more effective phage-based therapeutics by mutating and engineering the lysis genes. Ultimately this knowledge will benefit the fight against bacterial disease in agriculture, animal husbandry and in veterinary and human medicine.
Project Methods
Determination and Characterization of the MGL "Lys-ome"We will continue to employ the combination of genetics, and biochemistry, along with molecular, cellular and structural biology approaches that have been successful for us over the past 41 years at Texas A&M. Our general approach is to start with a thorough genetic characterization of the system, thus providing a foundation of biological relevance and operational definitions, as well as generating the necessary components for positive and negative controls. Although gene discovery can alter our immediate experimental plans, our current priorities in the MGL area for the next five years are:Spanin mechanism and functionIn the next five years, the focus will be on the remarkable spanins, which fuse membranes during lysis. The two types of spanins resemble both in terms of domain structure and function the two main classes of membrane-fusion proteins used by enveloped viruses to infect mammalian cells. Since the spanins can be studied with phage genetics, they represent a major opportunity to study these still mysterious membrane-fusion events that are central to viral pathogenesis in major diseases like HIV. We have developed a fusion assay using bacterial spheroplasts and will exploit this with our large collection of spanin mutants and synthetic polypeptides. We will also attempt to obtain crystal structures and/or solution structures (using NMR) for the prototype two-component and unimolecular spanin systems, by supplying our collaborators with mutant strains and purified proteins.New MGL protein classesWe will also focus on the two new classes of MGL proteins: releasins and disruptins. Releasins are unique in licensing dynamic membrane topology of endolysins. Disruptins are small, amphipathic proteins that are used to weaken the outer membrane; surprisingly, when purified and used in vitro, they function as phage encoded versions of the cationic antimicrobial peptides (CAMPs) produced by mammalian cells. The unique power of phage genetics will be used to determine the mechanisms of both these new MGL proteins. Our biophysical and structural collaborators will be supplied with mutants, phenotypes and constructs to be used in characterizing lysis at both the atomic level and in the context of the infected single cell using single-cell and single-molecule super-resolution fluorescence microscopy.Finding and characterizing new SGLsIn the SGL area, there has been a recent hyper-expansion in the number of small lytic phage genomes, particularly of the ssRNA phages. Previously, there were only 10 unique ssRNA phage genomes, of which we had shown three to encode PA-type Sgls and 7 to encode L-like Sgls. Recent meta-genomes and meta-transcriptomes have increased the number to >200. We have already started identifying the likely Sgl proteins using bioinformatics, and synthesizing the genes encoding these proteins. We will use classic bacterial genetics as well as whole-genome sequencing to identify the targets of these diverse Sgl proteins, none of which have any homologues in the databases. The hypothesis is that ssRNA phage Sgl proteins have evolved to attack every step in host cell wall synthesis and homeostasis. This is an exciting horizon which could lead to the development of many new antibiotics. Also, we will test a new model that the L-like Sgls act by binding the universal cell wall precursor, Lipid II, using biochemistry, genetics and single-cell fluorescence microscopy.