Treatment of multi-drug-resistant Gram-negative bacterial infections using capsule depolymerases

A group of bacteria have recently emerged as a major threat to global human health. These "Gram-negative bacteria (GNB)" exploit the decreased capacity of critically ill patients to ward off infections; they are particularly dangerous as they accumulate genes that confer resistance to front-line antibiotics. Some of these infections are untreatable and morbidity and mortality are common. These bacteria are also becoming more virulent and are beginning cause serious infections outside critical care facilities. They impose a particularly severe burden on healthcare in Thailand: for example, in 2010 in excess of 90,000 hospitalized patients acquired infections with drug resistant GNB, around one third died and these infections resulted in excess of three million days of hospitalization. Because they have evolved so quickly, we do not have sufficient means to control these infections and to treat critically ill patients.
Most of the bacteria that cause these infections are protected from the patients' already compromised immune defences by the presence of a sugar-containing capsule; we know that removal of this protective outermost layer from the bacterial cell by capsule-degrading enzymes will enable the host to eliminate the attenuated pathogen. For this proposal, we wish to investigate the capacity of such enzymes ("capsule depolymerases") to strip the capsule from drug resistant strains of two of these bacterial species, Klebsiella pneumoniae and Acinetobacter baumannii. To begin the project, we will collect recent Thai isolates of these bacteria from hospitals in Bangkok and elsewhere in the country and sequence their genomes to enable us to compare them with global populations of similar bacteria. This analysis will inform on the range of capsule types carried by these "local" strains. We will then isolate bacterial viruses ("bacteriophage") that carry capsule depolymerases to enable them to dock onto the surface of their bacterial hosts prior to killing them. Once we have characterised a range of these enzymes, we will purify them and use molecular genetic techniques to engineer proteins in sufficient bulk for experiments in animal models of infection. If we are able to demonstrate that dosing infected rats with engineered enzymes favourably alters the course of infection, we will promote these novel agents as potential therapeutics for the treatment of these devastating infections in the human host.

The global emergence of multi-drug-resistant clones of Gram-negative bacteria in intensive care units has narrowed treatment options for these life-threatening infections. The burden on healthcare in Thailand is particularly severe and is increasing. For example, in 2010, over 90,000 hospitalized patients acquired such infections and one third died, resulting three million days of hospitalization. In Thailand, as elsewhere, the two most prominent responsible species are Klebsiella pneumoniae and Acinetobacter baumannii; isolates of both species initially colonise the gastrointestinal (GI) tract or lung as a prelude to invasion of the systemic circulation and are usually protected from patients' immune defences by polysaccharide capsules that comprise the outermost layer of the bacterial cell. We have shown that parenteral administration of depolymerase directed against capsules of bacteria causing lethal neonatal sepsis or inhalation anthrax resolve experimental infections; cure rates approach 100%. We wish to apply this technology to emergent Thai strains of K. pneumoniae and A. baumannii. To identify enzymes active against Thai clones, we will sequence the genomes of one hundred recent clinical isolates of each species and compare them with the global phylogeny. Bacteriophage that infect the most frequently encountered clones will be examined for the presence of capsule depolymerase: phage infecting capsulated bacteria usually produce enzymes enabling penetration of the capsular polysaccharide to facilitate viral docking on to the surface. Depolymerases will be purified and characterised, and encoding genes cloned to obtain recombinant enzymes from suitable vectors. We will develop rat models of GI tract and lung colonisation and systemic invasion, infect animals with prominent Thai clones and examine the impact of systemic administration of enzymes, alone and in combination, on the course of infection.

Who will benefit? The primary purpose of this application is to identify and characterise a limited palette of enzymes with the capacity to alter, in favour of the host, the progression of potentially lethal infections caused by MDR GNB pathogens. If positive data is obtained in rat models of infection, the possibility of generating a new range of tailored therapeutics against currently problematical pathogens emerges. Thus beneficiaries will include large pharmaceutical concerns and SMEs with an interest in developing new agents for the treatment and control of bacterial infections due to multi-drug-resistant pathogens. Most importantly, these agents will be targeted at bacterial clades currently responsible for the large majority of AMR GNB infections in Thailand and the major benefit should accrue to SMEs and pharmaceutical concerns with an interest in the wellbeing of the Thai population. The UCL and LSHTM applicants represent the Bloomsbury Research Institute, a joint initiative between these two institutions to establish a centre for excellence in important pathogen research, and we are in advanced negotiations with a major USA-based pharmaceutical company with the intention of joint discovery and development of a new generation of antibacterial agents. Strong data from the proposed project should accelerate further investment in capsule depolymerases as agents for the treatment of globally problematical infections. The study will also provide fresh insights into the molecular pathogenesis of these emerging pathogens through the use of cutting edge techniques. The eventual introduction of a new class of antibiotics will have enormous societal benefits and the identification of lead compounds with therapeutic efficacy is a major long-term goal of our work.
How will they benefit? The development of a new generation of antibacterial agents is not a primary commercial goal for the majority of large pharmaceutical organisations and there are only a limited number of smaller players in this field. It is therefore likely that badly needed new drugs, particularly for the treatment of infections caused by drug-resistant bacterial pathogens, will emerge from public-private partnerships. The industry has the resources and the capacity to undertake high throughput target-based screening of large compound libraries that could not be readily conceived in academia, but academics have a role to play in the identification of new approaches to drug discovery through creative insight. The identification of new therapeutic modalities that have been properly validated in robust models of infection will encourage commercial players to form longstanding partnerships with groups involved in applied basic science, and we already see this happening. We will share our data with interested parties with appropriate legal and intellectual safeguards in place and enter into negotiations of mutual benefit.
As a consortium, our commitment to antibiotic drug development is long term, as predicated by the long lead-in times for antibiotic drug discovery. It is likely that tangible benefits from this research, in the shape of new treatment paradigms, will emerge only after decades but there is currently no business model that can deliver antibiotics from initiation of the discovery phase to market in less than fifteen years. The postdoctoral staff working on this project will acquire skills in cutting edge molecular technologies that will significantly enhance their employability as career scientists, in the academic sphere but particularly in the pharmaceutical industry. In particular, the Songkla-based PDRA will have the opportunity to visit and acquire skills from three world-class institutions that will be of long-term benefit to the Thai economy and science base.