Prophylaxis of disease caused by bacterial pathogens of man

This thesis reports research undertaken which will lead to improved pretreatments and therapies for disease caused by Clostridium perfringens, Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. C. perfringens is thought to be the most widely distributed bacterial pathogen and is the most important Clostridial species associated with enteric disease in domesticated animals. During warfare C. perfringens has been a significant causes of mortality. Between 1 and 10% of wounded personnel developed gas gangrene during the 1st and 2nd world wars. The ability of the bacterium to cause a range of diseases is due largely to the differential production of toxins. The first reported cloning and nucleotide sequencing of three of the four major toxins (α, β and ε-toxins) is documented in this thesis. The regulation of expression of α-toxin in C. perfringens has been investigated and methods for the expression of recombinant proteins in E. coli have been devised This information has been used to develop improved PCR-based diagnostic tests, and to investigate structure-function relationships. A high resolution crystal structure of a-toxin (phospholipase C) is reported. Using molecular and biophysical techniques, the functions of the two domains of the protein have been determined. Residues that play roles in the interaction of the toxin with host cell membranes have been identified using site-directed mutagenesis. This work has also provided a major insight into the structures and functions of related phospholipases C (the zincmetallophospholipases C) from other bacterial pathogens. This pioneering work with α-toxin is recognised by invitations to write reviews and book chapters on this subject and on bacterial phospholipases C. C. perfringens β-toxin has been shown to be related to pore forming toxins such as Staphylococcus aureus α-toxin. This finding suggests, for the first time, the mode of action of β-toxin. The interaction of C. perfringens ε-toxin with host cells has been investigated and progress made in identifying the cell-surface receptor for the toxin. Genetically engineered toxoids have been devised which induce high-level protection against α and ε-toxins. These vaccines are currently being developed by industry for veterinary use. Similar approaches have been used to devise a recombinant vaccine against Clostridium botulinum toxin F. The wider applications of toxins as therapeutics have also been investigated, and a novel cancer drug delivery system based on targeted lysis of drug-containing liposomes by α-toxin has been devised and patented. F. tularensis is the etiological agent of tularemia, a disease of man that is found in most countries in the Northern hemisphere and most frequently in Scandinavia, N. America, Japan and N. Russia. In this thesis the efficacy of antibiotics for the prevention and treatment of experimental tularemia is documented. Two surface antigens (lipopolysaccharide and FopA) have been evaluated as sub-unit vaccines. Of these, lipopolysaccharide shows potential as a protective antigen. However, because of the paucity of information available on this bacterium, a wider approach to vaccine development, involving the determination of the genome sequence of a fully virulent strain of F. tularensis has been undertaken. A preliminary analysis of the genome sequence is reported here, which has allowed the identification of targets for the development of a rationally attenuated mutant for use as a live vaccine. Y. pestis is generally recognised to have caused three major pandemics of disease, and credible estimates indicate that together these resulted in 200 million deaths. WHO figures indicate that there is a continuing public health problem from plague, especially in Africa, Asia and South America. In this thesis existing vaccines and antibiotics have been evaluated for the prevention and treatment of plague and found to have limitations. A number of approaches to the development of an improved vaccine have been investigated including rationally attenuated strains of the bacterium and isolated surface antigens. A sub-unit vaccine against plague has been devised based on recombinant forms of the F1- and V-antigens. This vaccine provides high level protection against both bubonic and pneumonic plague. This recombinant sub-unit vaccine has been patented and is currently in phase I clinical trials in man. This vaccine has been formulated for single oral or intranasal delivery, using microencapsulated or Salmonella-based delivery systems. Methods for enhancing the stability and efficacy of these vaccines have been investigated. Reviews on plague and plague vaccines have been written, confirming the status of the author as a world leader in this field. The work to devise an improved vaccine has also provided insight into the molecular basis of pathogencity of Y. pestis. A phoP / phoQ regulatory system has been discovered in the bacterium, which plays a key role in survival of the bacterium within macrophages. The V-antigen has been shown to be surface located to play a key role in the translocation of effector proteins into host cells. The biogenesis of the F1-capsular antigen has been investigated at a genetic and biophysical level. In order to underpin future work with this pathogen, the genome sequence is currently being determined. This work has already provided major new insights into the evolution of this pathogen. B. pseudomallei (formerly Pseudomonas pseudomallei) is found primarily in S. E. Asia, N. Australia and other tropical areas of the world. Melioidosis has recently appeared in temperate zones, including mainland France and the UK possible as a consequence of increased international travel. Acute disease can be treated with antibiotics but the bacterium can persist in the host and subsequent disease episodes can occur. In this thesis ciprofloxacin and doxycyline have been are evaluated and shown to have significant limitations for the treatment of melioidodis. In the longer term there is a requirement for an effective vaccine against melioidosis, and work is reported here to devise the genetic tools which will be necessary for the genetic manipulation of the bacterium, with a view towards the identification of virulence determinants

Production and properties of extracellular factors from Aeromonas salmonicida

The production of extracellular products by Aeromonas salmonicida, in vitro, has been investigated. The results indicated that the bacterium produces at least two haemolytic activities in vitro. Unshaken cultural conditions favoured the production of a haemolysin with a broad spectrum of activity against various erythrocyte types (H-lysin), whilst shaken cultural conditions favoured the production of a haemolysin active against trout erythrocytes (T-lysin). The effects of growth medium type and culture conditions on the production of these haemolytic activities has been investigated. The activity of the T-lysin appeared to be attributable to the combined effects of an activity which caused incomplete lysis of the erythrocytes (T1 activity) and caseinase. The T1 activity appears to be found in culture supernate associated with fragments of the bacterial cell wall or membrane resulting in apparent molecular heterogeneity. H-lysin activity appeared to be due to a single protein, which did not require a divalent cation for the expression of activity. The haemolysin was synthesised by the bacterium as an inactive precursor molecule (pro-H-lysin) which was cleaved by the bacterial protease to give the active haemolysin; other commercially available proteases were also able to effect this activation. An unidentified component of a variety of animal sera was also able to effect conversion of the pro-H-lysin to the active form, however, this conversion only occurred after the serum component had entered the bacterial cell. The H-lysin was purified 1770 fold using freeze fractionation, salt fractionation, ion exchange chromatography and gel filtration chromatography. The partially purified protein possessed erythrocyte lysing and glycerophospholipid:cholesterol acyltransferase activities, however it was not clear whether these activities were attributable to the same molecule. Investigation of the kinetics of erythrocyte lysis by the partially purified H-lysin suggested that the haemolysin possessed an enzymatic mode of action. In vitro the haemolysin was active against both rainbow trout leucocytes and tissue culture cells. However, in vivo the haemolysin had no obvious effect on rainbow trout.Ministry of Agriculture, Fisheries and Food, Weymout

Monoclonal antibodies against Yersinia pestis lipopolysaccharide detect bacteria cultured at 28 degrees C or 37 degrees C.

Four monoclonal antibodies were generated against Yersinia pestis lipopolysaccharide by immunising mice with a cell surface preparation from Y. pestis strain 1255. In an ELISA the monoclonal antibodies reacted with live whole cells of Y. pestis strain GB cultured at 28 degrees C or 37 degrees C. The lowest detection threshold for Y. pestis strain GB cultured at 28 degrees C was 4 x 10(5) cfu ml(-1) and for bacteria cultured at 37 degrees C was 1 x 10(4) cfu ml(-1). The monoclonal antibodies did not cross react with other pathogenic Yersinia in an ELISA, but showed some cross reactivity in an immuno-blot. The monoclonal antibodies could be used for the detection of Y. pestis cultured at different temperatures and with varying plasmid profiles as the lipopolysaccharide molecule is not temperature regulated and the genes encoding its biosynthesis are located on the bacterial chromosome

Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens

For decades, low doses of antibiotics have been used widely in animal production to promote growth. However, there is a trend to reduce this use of antibiotics in feedstuffs, and legislation is now in place in Europe to prohibit their use in this way. As a consequence, economically important diseases, such as necrotic enteritis (NE) of chickens, that are caused by Clostridium perfringens have become more prevalent. Recent research is creating a paradigm shift in our understanding of the pathogenesis of NE and is now providing information that will be necessary to monitor and control the incidence of NE in poultry

Functional Analysis of the Role of Toxin-Antitoxin (TA) Loci in Bacterial Persistence.

Bacterial Persistence: Methods and ProtocolsThe final publication is available at Springer via http://dx.doi.org/10.1007/978-1-4939-2854-5_11We have developed a method to analyze the functionality of putative TA loci by expressing them in Escherichia coli. Here, we describe the procedure for cloning recombinant TA genes into inducible plasmids and expressing these in E. coli. Following expression, toxicity, resuscitation of growth, and changes in persister cell formation are assayed. This can confirm whether predicted TA loci are active in E. coli and whether expression can affect persister cell formation

Vaccine delivery using nanoparticles

This is the final version of the article. Available from the publisher via the DOI in this record.Vaccination has had a major impact on the control of infectious diseases. However, there are still many infectious diseases for which the development of an effective vaccine has been elusive. In many cases the failure to devise vaccines is a consequence of the inability of vaccine candidates to evoke appropriate immune responses. This is especially true where cellular immunity is required for protective immunity and this problem is compounded by the move toward devising sub-unit vaccines. Over the past decade nanoscale size (<1000 nm) materials such as virus-like particles, liposomes, ISCOMs, polymeric, and non-degradable nanospheres have received attention as potential delivery vehicles for vaccine antigens which can both stabilize vaccine antigens and act as adjuvants. Importantly, some of these nanoparticles (NPs) are able to enter antigen-presenting cells by different pathways, thereby modulating the immune response to the antigen. This may be critical for the induction of protective Th1-type immune responses to intracellular pathogens. Their properties also make them suitable for the delivery of antigens at mucosal surfaces and for intradermal administration. In this review we compare the utilities of different NP systems for the delivery of sub-unit vaccines and evaluate the potential of these delivery systems for the development of new vaccines against a range of pathogens.This work was partly supported by grant number U54 AI057156 from the Western Regional Centre for Excellence, USA. The study performed in the laboratory of RWT was supported by NIH/NIAID grant U54 AI057156 from the Western Regional Center for Excellence. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIAID or NIH

Standardization of G. mellonella Larvae to Provide Reliable and Reproducible Results in the Study of Fungal Pathogens

This is the final version of the article. Freely available from MDPI via the DOI in this record.In the past decade, Galleria mellonella (wax moth) larvae have become widely used as a non-mammalian infection model. However, the full potential of this infection model has yet to be realised, limited by the variable quality of larvae used and the lack of standardised procedures. Here, we review larvae suitable for research, protocols for dosing larvae, and methods for scoring illness in larvae infected with fungal pathogens. The development of standardised protocols for carrying out our experimental work will allow high throughput screens to be developed, changing the way in which we evaluate panels of mutants and strains. It will also enable the in vivo screening of potential antimicrobials at an earlier stage in the research and development cycle

Variable protection against experimental broiler necrotic enteritis after immunisation with the C-terminal fragment of Clostridium perfringens alpha-toxin and a non-toxic NetB variant

Necrotic enteritis toxin B (NetB) is a pore-forming toxin produced by Clostridium perfringens and has been shown to play a key role in avian necrotic enteritis (NE), a disease causing significant costs to the poultry production industry worldwide. The aim of this work was to determine whether immunisation with a non-toxic variant of NetB (NetB W262A) and the C-terminal fragment of C. perfringens alpha-toxin (CPA247–370) would provide protection against experimental NE. Immunised animals with either antigen or a combination of antigens developed serum antibody levels against NetB and CPA. When CPA247–370 and NetB W262A were used in combination as immunogens, an increased protection was observed after oral challenge by individual dosing, but not after in-feed challenge

Will the enigma of Francisella tularensis virulence soon be solved?

Francisella tularensis is one of the most infectious bacterial pathogens known and is the causative agent of the zoonotic disease tularemia. In spite of the importance of this pathogen little is known about its virulence mechanisms. However, it is clear that the bacterium is an intracellular pathogen, replicating mainly in macrophages, with replication in amoebae also having been reported. The genome sequence of a high virulence strain of F. tularensis is close to completion and when available, will stimulate further research into virulence mechanisms

Oral immunization with a dam mutant of Yersinia pseudotuberculosis protects against plague.

Inactivation of the gene encoding DNA adenine methylase (dam) has been shown to attenuate some pathogens such as Salmonella enterica serovar Typhimurium and is a lethal mutation in others such as Yersinia pseudotuberculosis strain YPIII. In this study the dam methylase gene in Yersinia pseudotuberculosis strain IP32953 was inactivated. Unlike the wild-type, DNA isolated from the mutant could be digested with MboI, which is consistent with an altered pattern of DNA methylation. The mutant was sensitive to bile salts but not to 2-aminopurine. The effect of dam inactivation on gene expression was examined using a DNA microarray. In BALB/c mice inoculated orally or intravenously with the dam mutant, the median lethal dose (MLD) was at least 10(6)-fold higher than the MLD of the wild-type. BALB/c mice inoculated with the mutant were protected against a subcutaneous challenge with 100 MLDs of Yersinia pestis strain GB and an intravenous challenge with 300 MLDs of Y. pseudotuberculosis IP32953