CLOSTNET PUBLICATIONS

1. Lipoproteins of bacterial pathogens.
Kovacs-Simon A, Titball RW, Michell SL.
Room 416, School of Biosciences, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter, Devon, United Kingdom EX4 4QD.
Abstract
Bacterial lipoproteins are a set of membrane proteins with many different functions. Due to this broad-ranging functionality, these proteins have a considerable significance in many phenomena, from cellular physiology through cell division and virulence. Here we give a general overview of lipoprotein biogenesis and highlight examples of the roles of lipoproteins in bacterial disease caused by a selection of medically relevant Gram-negative and Gram-positive pathogens: Mycobacterium tuberculosis, Streptococcus pneumoniae, Borrelia burgdorferi, and Neisseria meningitidis. Lipoproteins have been shown to play key roles in adhesion to host cells, modulation of inflammatory processes, and translocation of virulence factors into host cells. As such, a number of lipoproteins have been shown to be potential vaccines. This review provides a summary of some of the reported roles of lipoproteins and of how this knowledge has been exploited in some cases for the generation of novel countermeasures to bacterial diseases.

Infect Immun. 2011 Feb;79(2):548-61. Epub 2010 Oct 25
PMID: 20974828
PMCID: PMC3028857[Available on 2011/8/1]

2. Molecular basis of toxicity of Clostridium perfringens epsilon toxin.
Bokori-Brown M, Savva CG, Fernandes da Costa SP, Naylor CE, Basak AK, Titball RW.
Biosciences, College of Life and Environmental Sciences, University of Exeter, UK Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck College, London, UK.
Abstract
Clostridium perfringens ε-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C.perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of ε-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of ε-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, ε-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.

© 2011 The Authors Journal compilation © 2011 FEBS.

FEBS J. 2011 Apr 23. doi: 10.1111/j.1742-4658.2011.08140.x. [Epub ahead of print]
PMID: 21518257

3. Reconsidering the Sporulation Characteristics of Hypervirulent Clostridium difficile BI/NAP1/027
DA Burns*, D Heeg*, ST Cartman and NP Minton (joint first authorship)
Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom
Abstract
Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and a major burden to healthcare services worldwide. In recent years, C. difficile strains belonging to the BI/NAP1/027 type have become highly represented among clinical isolates. These so-called ‘hypervirulent’ strains are associated with outbreaks of increased disease severity, higher relapse rates and an expanded repertoire of antibiotic resistance. Spores, formed during sporulation, play a pivotal role in disease transmission and it has been suggested that BI/NAP1/027 strains are more prolific in terms of sporulation in vitro than ‘non-epidemic’ C. difficile types. Work in our laboratory has since provided credible evidence to the contrary suggesting that the strain-to-strain variation in C. difficile sporulation characteristics is not type-associated. However, the BI/NAP1/027 type is still widely stated to have an increased rate of sporulation. In this study, we analysed the sporulation rates of 53 C. difficile strains, the largest sample size used to-date in such a study, including 28 BI/NAP1/027 isolates. Our data confirm that significant variation exists in the rate at which different C. difficile strains form spores. However, we clearly show that the sporulation rate of the BI/NAP1/027 type was no higher than that of non-BI/NAP1/027 strains. In addition, we observed substantial variation in sporulation characteristics within the BI/NAP1/027 type. This work highlights the danger of assuming that all strains of one type behave similarly without studying adequate sample sizes. Furthermore, we stress the need for more rigorous experimental procedures in order to quantify C. difficile sporulation more accurately in the future.

Online at PLoS One 15 Sept 2011 http://dx.plos.org/10.1371/journal.pone.0024894

4. Spores of Clostridium difficile clinical isolates display a diverse germination response to bile salts.
D Heeg*, DA Burns*, ST Cartman and NP Minton
*joint first author
Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom
Abstract
Clostridium difficile spores play a pivotal role in the transmission of infectious diarrhoea, but in order to cause disease spores must complete germination and return to vegetative cell growth. While the mechanisms of spore germination are well understood in Bacillus, knowledge of C. difficile germination remains limited. Previous studies have shown that bile salts and amino acids play an important role in regulating the germination response of C. difficile spores. Taurocholate, in combination with glycine, can stimulate germination, whereas chenodeoxycholate has been shown to inhibit spore germination in a C. difficile clinical isolate. Our recent studies of C. difficile sporulation characteristics have since pointed to substantial diversity among different clinical isolates. Consequently, in this study we investigated how the germination characteristics of different C. difficile isolates vary in response to bile salts. By analysing 29 isolates, including 16 belonging to the BI/NAP1/027 type, we show that considerable diversity exists in both the rate and extent of C. difficile germination in response to rich medium containing both taurocholate and glycine. Strikingly, we also show that although a potent inhibitor of germination for some isolates, chenodeoxycholate does not inhibit the germination, or outgrowth, of all C. difficile strains. Finally, we provide evidence that components of rich media may induce the germination of C. difficile spores, even in the absence of taurocholate. Taken together, these data suggest that the mechanisms of C. difficile spore germination in response to bile salts are complex and require further study. Furthermore, we stress the importance of studying multiple isolates in the future when analysing the nutrients or chemicals that either stimulate or inhibit C. difficile spore germination.

PLoS One. 2012;7(2):e32381. Epub 2012 Feb 22
PMID: 22384234
PMCID: PMC3285209 Free PMC Article

5. Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production.
ST Cartman, ML Kelly, D Heeg, JT Heap, and NP Minton
Clostridia Research Group, School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom
Abstract
Clostridium difficile causes a potentially fatal diarrheal disease through the production of its principal virulence factors, toxin A and toxin B. The tcdC gene is thought to encode a negative regulator of toxin production. Therefore, increased toxin production, and hence increased virulence, is often inferred in strains with an aberrant tcdC genotype. This report describes the first allele exchange system for precise genetic manipulation of C. difficile, using the codA gene of Escherichia coli as a heterologous counterselection marker. It was used to systematically restore the Δ117 frameshift mutation and the 18-nucleotide deletion that occur naturally in the tcdC gene of C. difficile R20291 (PCR ribotype 027). In addition, the naturally intact tcdC gene of C. difficile 630 (PCR ribotype 012) was deleted and then subsequently restored with a silent nucleotide substitution, or "watermark," so the resulting strain was distinguishable from the wild type. Intriguingly, there was no association between the tcdC genotype and toxin production in either C. difficile R20291 or C. difficile 630. Therefore, an aberrant tcdC genotype does not provide a broadly applicable rationale for the perceived notion that PCR ribotype 027 strains are "high-level" toxin producers. This may well explain why several studies have reported that an aberrant tcdC gene does not predict increased toxin production or, indeed, increased virulence.

Appl Environ Microbiol. 2012 Jul;78(13):4683-90. Epub 2012 Apr 20
PMID: 22522680
PMCID: PMC3370502 [Available on 2013/1/1]

6. Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation.
Kirk DG, Dahlsten E, Zhang Z, Korkeala H, Lindström M.
Department of Food Hygiene and Environmental Health, Centre of Excellence in Microbial Food Safety Research, Faculty of Veterinary Medicine, University of Helsinki, Finland.
Abstract
A key survival mechanism of Clostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organism Bacillus subtilis, nothing is known about these mechanisms in C. botulinum. Using the ClosTron gene-knockout tool, sigK, encoding late-stage (stage IV) sporulation sigma factor K in B. subtilis, was disrupted in C. botulinum ATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast, sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis of sigK in the parent strain revealed expression at the late log growth phase in the parent strain. Analysis of spo0A, encoding the sporulation master switch, in the sigK mutant and the parent showed significantly reduced relative levels of spo0A expression in the sigK mutant compared to the parent strain. Similarly, sigF showed significantly lower relative transcription levels in the sigK mutant than the parent strain, suggesting that the sporulation pathway was blocked in the sigK mutant at an early stage. We conclude that σ(K) is essential for early-stage sporulation in C. botulinum ATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organism B. subtilis. Understanding the sporulation mechanism of C. botulinum provides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

Appl Environ Microbiol. 2012 Jul;78(13):4590-6. Epub 2012 Apr 27.
PMID: 22544236
PMCID: PMC3370484 [Available on 2013/1/1]

7. Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum.
Vasileva D, Janssen H, Hönicke D, Ehrenreich A, Bahl H.
Abteilung Mikrobiologie, Institut für Biowissenschaften, Universität Rostock, Albert-Einstein-Str. 3, D-18051 Rostock, Germany.
Abstract
Iron is a nutrient of critical importance for the strict anaerobe Clostridium acetobutylicum, as it is involved in numerous basic cellular functions and metabolic pathways. A gene encoding a putative ferric uptake regulator (Fur) has been identified in the genome of C. acetobutylicum. In this work, we inactivated the fur gene by using insertional mutagenesis. The resultant mutant showed a slow-growing phenotype and enhanced sensitivity to oxidative stress, but essentially no dramatic change in its fermentation pattern. A unique feature of its physiology was the overflowing production of riboflavin. To gain further insights into the role of the Fur protein and the mechanisms for establishment of iron balance in C. acetobutylicum, we characterized and compared the gene-expression profile of the fur mutant and the iron-limitation stimulon of the parental strain. Not surprisingly, a repertoire of iron-transport systems was upregulated in both microarray datasets, suggesting that they are regulated by Fur according to the availability of iron. In addition, iron limitation and inactivation of fur affected the expression of several genes involved in energy metabolism. Among them, two genes, encoding a lactate dehydrogenase and a flavodoxin, were highly induced. In order to support the function of the latter, the ribDBAH operon responsible for riboflavin biosynthesis was also upregulated significantly. Furthermore, the iron-starvation response of C. acetobutylicum involved transcriptional modifications that were not detected in the fur mutant, suggesting that there exist additional mechanisms for adaptation to low-iron environments. Collectively, these results demonstrate that the strict anaerobe C. acetobutylicum senses and responds to availability of iron on multiple levels using a sophisticated system, and that Fur plays an important role in this process.

Microbiology, 2012 Jul;158(Pt 7):1918-29. Epub 2012 May 3.
PMID: 22556358

8. Analysis of metronidazole susceptibility in different Clostridium difficile PCR-ribotypes
Moura I, Spigaglia P, Barbanti F, Mastrantonio P
ISS Rome, Italy
J Antimicrob Chemother (Submitted 2012)

9. A two-component system controls the expression of genes encoding CBM6-containing proteins in response to straw in Clostridium cellulolyticum.
Hamza Celik
Laboratoire de Chimie Bactérienne, IMM-CNRS-UMR7283, University of Aix-Marseille, Marseille, France

Soon to be Submitted