208 research outputs found
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
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
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
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
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
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
Salmonella vaccines for use in humans: present and future perspectives.
In recent years there has been significant progress in the development of attenuated Salmonella enterica serovar Typhi strains as candidate typhoid fever vaccines. In clinical trials these vaccines have been shown to be well tolerated and immunogenic. For example, the attenuated S. enterica var. Typhi strains CVD 908-htrA (aroC aroD htrA), Ty800 (phoP phoQ) and chi4073 (cya crp cdt) are all promising candidate typhoid vaccines. In addition, clinical trials have demonstrated that S. enterica var. Typhi vaccines expressing heterologous antigens, such as the tetanus toxin fragment C, can induce immunity to the expressed antigens in human volunteers. In many cases, the problems associated with expression of antigens in Salmonella have been successfully addressed and the future of Salmonella vaccine development is very promising
DNA vaccines: improving expression of antigens
Copyright © 2003 Garmory et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.DNA vaccination is a relatively recent development in vaccine methodology. It is now possible to undertake a rational step-by-step approach to DNA vaccine design. Strategies may include the incorporation of immunostimulatory sequences in the backbone of the plasmid, co-expression of stimulatory molecules, utilisation of localisation/secretory signals, and utilisation of the appropriate delivery system, for example. However, another important consideration is the utilisation of methods designed to optimise transgene expression. In this review we discuss the importance of regulatory elements, kozak sequences and codon optimisation in transgene expression
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