Listeria monocytogenes

Quicklinks: | Cell-Cell Interactions | Copper Homeostasis | 

Introduction 

Listeria monocytogenes is a significant food-borne intracellular pathogen that is capable of establishing intracellular infections in immuno-compromised individuals. This bacterial pathogen is capable of infection of both phagocytic and non-phagocytic mammalian cells.

Cell-cell Interactions between Listeria monocytogenes and protozoan hosts

Project Staff: Connor Thomas (Supervisor), Rethish Nadhanan, Malaysia (PhD Student). 

Past Students: Alisha Akya, Iran (PhD Student)

The primary ecological niche of L. monocytogenes is assumed to be soil and water. It is not yet known whether this organism, like some other intracellular pathogens (eg. Legionella), is maintained in the environment by association with simple animal cells such as protozoans.

One aim of my research group is to examine the ability of L. monocytogenes to establish an intracellular lifestyle with single celled animals common in soil and water. This work has significant implications for establishment of reservoirs of these bacterial pathogens in the environment. For example, we wish to determine whether L. monocytogenes can initiate intracellular infections of simple single celled animals, such as protozoa (including amoebae eg. Acanthamoeba polyphaga, and ciliated protozoans eg. Tetrahymena). We hypothesise that the intracellular infection of these animals allows L. monocytogenes to survive in the environment. Furthermore, we believe that this association provides a vehicle for persistent contamination of food processing environments and ultimately the contamination of foods. Since food-borne transmission is the primary mechanism of infection of susceptible individuals, this hypothesis has significant implication for the food industry at large. Given recent fatal outbreaks of Listeria infections in South Australia, this project has significance for the local food processing industry. Future work on this research topic will build on work already developed by a PhD student. The project will involve co-culture experiments between L. monocytogenes and suitable protozoans cell models. The outcome of co-culture experiments conducted under differing environmental conditions will be evaluated by fluorescence microscopy, electron microscopy and basic bacteriology. The influence of defined mutations within known virulence genes in L. monocytogenes on the interaction will also be examined.

Copper homeostasis in Listeria monocytogenes

Project Staff: Connor Thomas (Supervisor), Andrew Pointon (Co-supervisor), Francesca Bell (PhD Student), Mei Mei Hii (MSc Student), Caroline Yeoh (Honours Student) 

A second aspect of our work on L. monocytogenes involves the role of copper homeostasis in persistence of L. monocytogenes infections in the livers and spleens of infected mice. Mutations in a copper transporting P-type ATPase (CtpA) significantly reduce persistent infection (Francis, M.S. and Thomas, C.J. (1997) Microbial Pathogenesis. 22:67-78). Inflammation induced by infection apparently decreases copper levels in the liver and increases levels in the spleen. Under these conditions, mutants are apparently unable to maintain copper homeostasis within macrophages and consequently are eliminated from infected tissue. Furthermore, in the absence of copper homeostasis, copper ions can catalyse production of superoxide and hydroxyl radicals that lead to damage and death of the bacterial cell.

The distribution of ctpA seems to be restricted to plasmid DNA carried by environmental and food and isolates of L. monocytogenes. All isolates of L. monocytogenes also carry DNA encoding a putative copper transport associated P-type ATPase (lctA). The role of lctA in copper transport is unknown, however mutations in ctpA are known to significantly affect ability to grow in the presence of sub-toxic concentrations of copper and under copper starvation conditions. Double mutants (lctA and ctpA) are super sensitive to extracellular copper and most interestingly, also to other divalent cations such as cadmium. This indicates that ctpA, and the plasmids that carry this gene, may be of prime importance in enabling establishment and maintenance of clonal populations of L. monocytogenes in environmental niches.

Sequence analysis of DNA flanking ctpA has identified at least 4 genes are involved in copper homeostasis and there is evidence that expression is regulated by a two component regulatory system located close to the copper transport P-type ATPase gene (ctpA) (see GenBank Accession Number U15554). We have created a number of mutations in the identified genes and are in the process of analysing the in vitro effects on copper tolerance. The following simple diagram shows the genetic arrangement of the plasmid encoded genes identified.

The amino acid sequence of OrfA has homology to a regulatory protein. We have cloned and over-expressed orfA and purified protein. We have used DNA gel shift assays to establish the role of the protein in regulation.

orfC may encode a cytoplasmic copper binding protein that interacts with the protein encoded by ctpA. We do not know what the function of orfB is, but it may encode an extracellular protein that binds copper ions and presents them to CtpA or a cytoplasmic protein that acts as a cytoplasmic shuttle for the copper ions transported by CtpA. Immedialely downstream of these genes are ORFs encoding a two component regulatory system, multicopper oxidases and another copper transporting ATPase.  Together, these genes comprise a copper homeostasis islet.

There are several possible projects stemming from this work, all of which extend work conducted by several PhD students. Each involve an investigation of the role of the unidentified accessory copper transport genes (orfB and orfC) or the role of the two component regulatory system, on copper homeostasis using in vitro experimental systems. Projects involving this work will give students experience in a variety of molecular cloning methods, production of specific mutants, protein expression systems, and methods for study of relevant aspects of microbial physiology.