Treatment of Systemic Infection by Modification of the Bacterial Phenotype

Lead Research Organisation: School of Pharmacy
Department Name: Pharmaceutics

Abstract

Meningitis and sepsis remain devastating diseases, particularly in the young. If patients survive, there are usually serious consequences that include mental retardation, seizures, cerebral palsy and hearing loss. Antibiotic therapy may not always be effective because the infection progresses very rapidly and antibiotic resistant bacteria are now more commonly found. There is widespread recognition that new approaches to the treatment of the disease are urgently needed. These serious infections in the newborn infant and in older children are caused by a relatively small number of bacterial types and they are almost all protected from the effects of the patient?s immune system by a coat, or capsule, comprising linked sugar molecules. There is ample evidence that the capsule is essential for the survival of the bacteria within the tissues of the patient, as without it they are no longer able to protect themselves from immune attack. We are looking for ways to rapidly remove this protective layer as a novel way of treating the infection ? our approach, which we call phenotypic modification, does not rely on directly killing the bacteria in the way that most antibiotics do, but aims to convert them to a ?less fit? form that cannot survive in the body. We have found an enzyme, derived from a bacterial virus or ?bacteriophage?, which quickly and selectively strips the capsule from the bacterial surface and sensitises the pathogenic organisms to the body?s defences. We have used the enzyme to treat experimental infections in newborn rats and we find that injection of very small amounts of material can very effectively cure the animals of what is invariably a fatal infection. This is the first demonstration that we know of which shows that ?phenotypic modification? can work in whole animals. We now which to achieve a greater understanding of the nature of the infection in the animal model by studying the distribution of the pathogen in animal tissues and organs and we would also like to establish that the positive therapeutic outcome that we observe is really due to removal of the protective capsule at the site of infection. Finally, we propose to use ?microarray technology? to establish whether key rat gene products are involved in the determination of the therapeutic outcome.

Technical Summary

Capsulate bacteria cause a range of severe systemic infections and there is a large body of evidence linking their invasive potential to expression of capsular polysaccharides on the bacterial surface. This relationship is particularly strong with Gram-negative bacteria causing sepsis and meningitis, and relatively few capsular serotypes are responsible for most of these infections. Escherichia coli K1 strains are a common cause of sepsis and meningitis in the newborn, and produce capsules composed of alpha-2,8-linked polysialic acid (PSA) that significantly enhances virulence. The difficulty of treating these infections is compounded by the steady increase in antibiotic resistance associated with these pathogens. We are developing an approach to the treatment of these infections based on modulation of capsule expression at the bacterial surface; removal or inhibition of biosynthesis of PSA may disarm these organisms and allow the patients? host defences to counter the infection. We have established that this principle (abrogation of infection by modification of the bacterial phenotype) is sound by treating E. coli K1-infected neonatal rats with endosialidase E (endoE), which specifically hydrolyses PSA. Intraperitoneal administration of small quantities of the protein (0.25mcg), given as a single dose early in the course of the infection, abolishes bacteraemia and prevents death.

We now wish to establish parameters for enzyme-mediated cure of systemic infection in the neonatal rat model and define the key events that determine the outcome of the infection and its resolution by endoE. We will produce sufficient quantities of highly pure recombinant endoE to allow structural elucidation of the enzyme. Structural data will enable us to improve the pharmacokinetic properties by removing non-essential domains from the protein and to design non-catalytic enzyme mutants to establish beyond doubt that in vivo catalysis of PSA is essential for endoE-mediated abrogation of infection. The whole body distribution of E. coli K1 in neonatal rats will be determined at appropriate time points after intestinal colonisation by orally administered bacteria using microbiological, histological and immunohistochemical techniques.In addition, we will evaluate the effect of enzyme treatment on this bioburden. Finally, we will investigate the effects of K1 infection, and its treatment with endoE, on the major organs of the neonatal rat using histopathological techniques and determine the effects of infection and treatment on gene expression in selected organs using DNA microarrays. The proposal represents a thorough programme of basic research to develop a novel therapy that may have wider therapeutic application
 
Description Innovation Award
Amount £65,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2007 
End 09/2010
 
Description K1 histochemistry 
Organisation University of Gothenburg
Department Mucin Biology Group
Country Sweden 
Sector Academic/University 
PI Contribution Providing tissue samples for determination of temporal and physiological parameters associated with E. coli K1 gastrointestinal colonization, including Tff2 composition of mucin.
Collaborator Contribution Determination of mucin barrier integrity and site of K1 translocation from gut to blood
Impact Manuscript based on mucin layer integrity in preparation; graduated PhD student from my laboratory has joined the mucin biology group at the University of Gothenburg led by Gunnar Hansson.
Start Year 2011
 
Description K1 histochemistry 
Organisation University of Gothenburg
Department Mucin Biology Group
Country Sweden 
Sector Academic/University 
PI Contribution Providing tissue samples for determination of temporal and physiological parameters associated with E. coli K1 gastrointestinal colonization, including Tff2 composition of mucin.
Collaborator Contribution Determination of mucin barrier integrity and site of K1 translocation from gut to blood
Impact Manuscript based on mucin layer integrity in preparation; graduated PhD student from my laboratory has joined the mucin biology group at the University of Gothenburg led by Gunnar Hansson.
Start Year 2011
 
Description K1-GFP fusion protein 
Organisation University of Turku
Country Finland 
Sector Academic/University 
PI Contribution We undertook histochemical characterisation of tisssues using this reagent
Collaborator Contribution Produced by recombination technology a GFP fusion protein as a key reagent for histological localisation obacteria in rat tissues
Impact We provide compelling evidence that the neonatal rat pup model, unlike almost all of alternatives, possesses key features of the infection in the human neonate: non-invasive establishment of infection following colonisation of the intestinal tract; a strong age dependency; temporal development along the gut-blood-brain transit; colonisation and penetration of the meninges after invasion of the cerebral spinal compartment. A192PP efficiently produced a systemic lethal infection in almost all pups, with considerable organ involvement. Nevertheless, small single doses of endoE resolved infection in the majority of animals and prevented death. As far as can be ascertained, resolution of the infection was due to in situ removal of the capsule from bacteria in the blood compartment.
Start Year 2006
 
Description radio interview - Broadcasting House 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I was interviewed about my research by this Radio 4 programme with respect to control of notifiable diseases and the work from thisd MRC grant was part of the feature

I was invited to participate in a NewsNight BBC2 programme on a related topic the following year
Year(s) Of Engagement Activity 2006