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

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

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

Identification of type II toxin-antitoxin modules in Burkholderia pseudomallei

© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reservedThis is an open access article that is freely available in ORE or from the publisher's web site. Please cite the published version.Type II toxin-antitoxin (TA) systems are believed to be widely distributed amongst bacteria although their biological functions are not clear. We have identified eight candidate TA systems in the genome of the human pathogen Burkholderia pseudomallei. Five of these were located in genome islands. Of the candidate toxins, BPSL0175 (RelE1) or BPSS1060 (RelE2) caused growth to cease when expressed in Escherichia coli, whereas expression of BPSS0390 (HicA) or BPSS1584 (HipA) (in an E. coli ΔhipBA background) caused a reduction in the number of culturable bacteria. The cognate antitoxins could restore growth and culturability of cells.Wellcome Trus

Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis

addresses: Biosciences, College of Life and Environmental Sciences, Geoffrey Pope Building, University of Exeter, Stocker Road, Exeter, Devon, EX4 4QD, UK.notes: PMCID: PMC3025829types: Journal Article; Research Support, Non-U.S. Gov't© 2011 Wand et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Burkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella) infection models

ATP-binding cassette systems in Burkholderia pseudomallei and Burkholderia mallei

BACKGROUND: ATP binding cassette (ABC) systems are responsible for the import and export of a wide variety of molecules across cell membranes and comprise one of largest protein superfamilies found in prokarya, eukarya and archea. ABC systems play important roles in bacterial lifestyle, virulence and survival. In this study, an inventory of the ABC systems of Burkholderia pseudomallei strain K96243 and Burkholderia mallei strain ATCC 23344 has been compiled using bioinformatic techniques. RESULTS: The ABC systems in the genomes of B. pseudomallei and B. mallei have been reannotated and subsequently compared. Differences in the number and types of encoded ABC systems in belonging to these organisms have been identified. For example, ABC systems involved in iron acquisition appear to be correlated with differences in genome size and lifestyles between these two closely related organisms. CONCLUSION: The availability of complete inventories of the ABC systems in B. pseudomallei and B. mallei has enabled a more detailed comparison of the encoded proteins in this family. This has resulted in the identification of ABC systems which may play key roles in the different lifestyles and pathogenic properties of these two bacteria. This information has the potential to be exploited for improved clinical identification of these organisms as well as in the development of new vaccines and therapeutics targeted against the diseases caused by these organisms

Development of Vaccines Against Burkholderia Pseudomallei

Burkholderia pseudomallei is a Gram-negative bacterium which is the causative agent of melioidosis, a disease which carries a high mortality and morbidity rate in endemic areas of South East Asia and Northern Australia. At present there is no available human vaccine that protects against B. pseudomallei, and with the current limitations of antibiotic treatment, the development of new preventative and therapeutic interventions is crucial. This review considers the multiple elements of melioidosis vaccine research including: (i) the immune responses required for protective immunity, (ii) animal models available for preclinical testing of potential candidates, (iii) the different experimental vaccine strategies which are being pursued, and (iv) the obstacles and opportunities for eventual registration of a licensed vaccine in humans