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

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

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