Back to microbes

With all the excitement over the human genome and its potential for increasing understanding of human disease, microbial disease research has slipped out of the limelight.

But the study of microbial pathogenesis, or the mechanisms of causing disease, is alive and well in Australia, judging by the NHMRC grants awarded last week to microbiologists around the country.

A variety of microbes are under investigation by researchers, who are interested in teasing out the basic pathways used by bacteria and other pathogens to take advantage of their environment, causing disease. With the explosion of information available from genomic studies of pathogenic microbes, the opportunity is now there to really look specifically at regulation of metabolic pathways and production of toxins.

Prof Julian Rood, head of the Microbiology Department at Monash University, says that getting a basic understanding of the fundamental mechanisms at work in microbial pathogenesis is the goal of the research on which his group is working.

"It's only when you understand the disease mechanism properly that you can design appropriate drugs and interventions," he explains.

He received two NHMRC grants last week to pursue his studies on anaerobic bacterium Clostridium perfringens and its close relative Clostridium septicum. These two bacteria cause gas gangrene and Rood is interested in precisely how that occurs.

Rood plans to look at the regulation of the Clostridium toxin genes to determine how they are controlled. His team already knows there is a two-component signal transduction system, which is key to the regulation of these genes.

"We've developed the ability to make mutants in Clostridium by homologous recombination. We can then study the role of the gene that has been knocked out," he explains. Using a variety of methods, the researchers will reintroduce genes and look at the regulatory processes under different environment conditions.

With the second grant, Rood and lab member Dr Dena Lyras are taking a slightly different approach, using reverse genetics to look at the effect on the gas gangrene disease process itself. In collaboration with Prof Richard Boyd and Dr John Emmins of the Department of Pathology and Immunology at Monash, the researchers want to know how the host responds to the infection, and why the normal inflammatory response to bacterial infection is down-regulated.

Virulence genes

Another Monash laboratory, headed by Assoc Prof John Davies, is investigating the regulatory networks controlling virulence genes in the bacterial species Neisseria gonorrhoeae and Neisseria meningitidis, which as their names suggest, cause gonorrhoea and meningitis respectively.

Davies and Dr Charlene Kahler plan to use a microarray that covers every single gene in the genome of each organism, including those from N. meningitidis serogroups A and B. The array is made at Monash and is the result of collaboration between Davies' lab and six other groups around the world. Armed with this valuable tool, the researchers will look at mutants with the regulatory genes knocked out.

"Using these we can monitor the expression of genes in the mutants in response to changing environmental conditions," explains Davies. "The aim is to understand as completely as we can how virulence is controlled by bacteria."

Davies says that while the project is basic science, the end results could lead to new candidates for vaccines and drug targets.

Prof Ben Adler, also at Monash, is interested in a "pathogenicity island" in Shigella, the species that causes bacillary dysentery. This is a specific region of DNA that is not a regular part of the bacterial genome, which contains virulence genes and other sequences involved in the pathogenic mechanism.

"These are genomic regions that bacteria have acquired laterally," he says, and explains that he wants to know exactly how they acquire the pathogenic sequences.

According to Adler, the islands can be quite large. In the case of Shigella's 'she' island, it is 43 kilobases long and contains in excess of 30 genes. Some of these genes are clearly involved in pathogenesis, he says, while not much is known about others. One gene in particular is of interest, a cytotoxic protease, and Adler would like to know how it binds and gets into epithelial cells, and how it manages to kill the cell.

Unlike a plasmid, however, pathogenicity islands are not capable of self-replication. They integrate into the genome and are often lost from the bacteria via a very precise excision method. Adler suggests that there are a couple of mechanisms which might be involved in transferring the island from one bacterium to another, including via a bacteriophage, or bacterium-infecting virus, or by some form of self-mobilisation.

With Dr Harry Sakellaris and Dr Martin Scanlon, Adler plans to achieve transfer between populations of Shigella under artificial conditions in the lab. "No one has shown this yet, but it clearly happens in nature," he says. Adler also received another grant for a separate project, to characterise outer membrane proteins in Leptospira species, which are collectively responsible for a zoonotic, or animal-borne human disease known as leptospirosis.

While the main antigen for immunity to Leptospira is lipopolysaccharide (LPS), there is increasing attention being paid to identifying possible vaccine candidates from the outer membrane proteins, says Adler.

He plans to systematically examine these proteins to determine whether they are involved in the immune response, a process that requires identification and cloning of the relevant genes, purification of the protein in a biologically correct form and animal studies to look at the immunogenicity of the protein.

Metabolic pathways

Up at the University of Queensland, Assoc Prof Alastair McEwan and Assoc Prof Michael Jennings are studying a variety of metabolic pathways in bacteria, particularly in relation to uptake of metal ions, oxidative stress and pathogenicity.

For a start, McEwan and co-workers have found a novel mechanism for iron uptake in Pseudomonas aeruginosa, which they intend to investigate.

"Iron uptake is a central element of pathogenicity in many microorganisms, and a variety of mechanisms have evolved," he says. In the long term, McEwan says that the development of antibacterial compounds to block iron uptake pathways are a possibility.

A separate project, headed by Jennings along with McEwan and Prof James Paton at the University of Adelaide, will look at the importance of manganese as a biological antioxidant, and its uptake by Streptococcus pneumoniae.

"Mutants with a loss of manganese uptake are hypersensitive to oxidative stress. The pathway seems to be critical for growth and survival," explains McEwan.

The fundamental aim of the projects, he says, is to work out the genetics, with a view to eventually being able to block the pathways. But for now, the goal is to get a more complete idea of the function of the pathways and their relationship to mechanisms of pathogenicity.