Copper Homeostasis and virulence in Listeria monocytogenes

Lead Research Organisation: University of Manchester
Department Name: Medical and Human Sciences

Abstract

Listeria monocytogenes is a food borne bacterium that causes a number of serious infections in humans. Infection may be of the unborn/newly born infant where disease may manifest as listeric abortion, stillbirth or late-onset neonatal listeriosis. In adults, infection usually affects the central nervous system causing meningitis. In the USA L. monocytogenes accounts for 28% of all fatalities as a consequence of food borne infection and in cancer patients, L. monocytogenes is the most commonly encountered form of bacterial meningitis. The aims of this project are to understand copper homeostasis in L. monocytogenes and to establish its importance in infection.
In order to survive bacteria have to be able to adapt to changes in their local environment and maintain homeostasis. The ability to respond to changes in the levels of transition elements such as copper provides a dilemma. Bacteria have to balance how to obtain sufficient copper, an essential cofactor for certain enzymes, with the toxicity of copper to the cell. In L. monocytogenes the problem of copper homeostasis is particularly acute. It inhabits environments including soil, effluents and food where it will be exposed to fluctuating copper levels. The use of copper as an anti-microbial in animal feeds, as a disinfectant in factory-based farming and as a biocide in fruit production means L. monocytogenes will have to combat potentially toxic levels of copper during food production and processing.
Adapting to fluctuations in the level of copper is important during infections. L. monocytogenes replicates in a number of host tissues that will offer different challenges with regard to copper availability. Early in infection L. monocytogenes grows in the gall bladder, where there are high levels of copper. In contrast, in the liver and spleen it replicates in an environment containing little free copper and where the acquisition of sufficient copper for growth is essential. Adapting to these different niches within the host offers a challenge for a successful infection.
In terms of improvements to human health, there are a number of potential benefits. First, an understanding copper homeostasis in this important food borne pathogen will inform the current copper based treatments used in food production and lead to effective regimes to reduce L. monocytogenes contamination. Secondly by establishing the essential role for copper homeostasis in infection it may be possible in the longer term to develop novel non-antibiotic based approaches for treatment of infections based on disruption of copper homeostasis.

Technical Summary

The project aims to understand copper homeostasis in Listeria monocytogenes and the role of copper homeostasis in infection. Copper is essential for many bacteria being an important prosthetic group for certain enzymes. However at high levels copper is toxic in part due to its ability to redox cycle and catalyse the formation of oxygen derived free radicals. As such bacteria need to be able to maintain copper homeostasis.
In L. monocytogenes the problem of copper homeostasis is particularly acute. It inhabits a range of environments including soil, effluents and food where it will be exposed to fluctuating copper levels. The use of copper as an anti-microbial in animal feeds, as a disinfectant in factory-based farming and as a biocide in fruit production means L. monocytogenes will have to combat potentially toxic levels of copper during food production and processing.
Adapting to fluctuations in the level of copper is important during infections. L. monocytogenes replicates in a number of host tissues that will offer different challenges with regard to copper availability. Early in infection L. monocytogenes grows in the gall bladder, where there are high levels of copper, as such it will have to adapt to high levels of copper. In contrast, in the liver and spleen it replicates in an environment in which there will be little free copper and where the acquisition of sufficient copper for growth will be essential. Adapting to these different niches within the host offers a challenge in terms of maintaining a supply of copper for copper requiring enzymes whilst avoiding copper toxicity.
The proposal has five objectives.
1) To determine the role of the CopC/D protein in copper homeostasis.
2) To determine the mechanism of regulation of cueA and copC/D genes in response to copper.
3) To use micro-array analyses to show that a wider range of listerial genes are expressed during copper homeostasis and to identify these genes.
4) To test the hypothesis that adaptation to a low copper environment copper is essential for intracellular survival or replication in L. monocytogenes
5) To elucidate the role of copper homeostasis in the survival and growth of L. monocytogenes during infection in vivo: adaptation to both low and high copper levels in the liver, spleen and gall bladder.

Publications

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Title His-tagged RpoC (RNA polymerase) strain of Listeria monocytogenes 
Description This strain is very useful since it allows the rapid one step purification of RNA polymerase from Listeria monocytogenes to allow in vitro transcription experiments to be performed on Listeria monocytogenes promoters. 
Type Of Material Cell line 
Year Produced 2008 
Provided To Others? Yes  
Impact Currently being used by laboratories in the UK and USA but no published results as yet. 
 
Title pAULA-tRNA 
Description Integration vector to allow integration into the tRNA Arg locus in Listeria monocytogenes, thereby allowing single copy complementation to be achieved. 
Type Of Material Cell line 
Year Produced 2010 
Provided To Others? Yes  
Impact Currently being exploited in laboratories in Leicester UK to complement mutations prior to in vivo experiments. 
 
Title pUNK-1-LacZ 
Description The first purpose built lacZ transcriptional fusion vector for use in Listeria monocytogenes. 
Type Of Material Cell line 
Year Produced 2008 
Provided To Others? Yes  
Impact Currently being exploited in a number of laboratories world wide, no paper as yet. 
 
Description School Visits-Wilmslow, Manchester, ALtrincham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact In al cases approximately 80 sixth form students came to my presentation about Listeria and food borne infections. The presentation was interactive and followed by a Q&A session.

I informed via feedback that my talk was well received and formed the basis of a discussion group among the students the following week. In addition I was invited back the following year.
Year(s) Of Engagement Activity 2006,2007,2008,2009