Microbial pathogenesis and microbial biotechnology
Dr Simmonds’ has two current research interests:
The first regards the evolutionary and medical implications of the production of endopeptidase enzymes by streptococci (link 1).
The second regards the use of heterotrophic bacteria to bioconvert agricultural waste into useful product or industrial feedstock (link 2).
Applications for postgraduate study in Dr Simmonds’ lab are welcome. A major in microbiology or microbial biotechnology is essential, and university level papers in chemistry and mathematics are highly desirable. It is essential for students to hold a University scholarship (or equivalent) before they undertake project work. Applications for scholarships can be found at: http://www.otago.ac.nz/postgraduate/index.html
Dr Simmonds has recently hosted Professor Kelly Doran http://www.bio.sdsu.edu/faculty/doran.html on sabbatical from San Diego State University. Their collaborative project aims to investigate the role of a streptococcal endopeptidase on the pathogenesis of group B streptococci.
Dr Simmonds has also enjoyed a long standing collaboration with professors Gary Sloan http://bsc.ua.edu/about/faculty-directory/gary-l-sloan/ and Russell Timkovich http://chemistry.ua.edu/faculty_profiles/russell-timkovich/ of the University of Alabama at Tuscaloosa which has resulted in multiple joint publications and the exchange of PhD students between their respective laboratories.
Yang, S., R.S. Simmonds and E.J. Birch. 2014. Physiochemical characterization and thermal porperties of lipids from R. opacus PD630. Food and Public Health. 4:87-92.
Chen, Y., R.S. Simmonds and R. Timkovich. 2013. Proposed docking interface between peptidoglycan and the target recognition domain of zoocin A. Biochemical and Biophysical Research Communications. 441:297-300.
McLeod, F.S.A. and R.S. Simmonds. 2013. Penicillin facilitates the entry of antisense constructs into Streptococcus mutans. FEMS Microbiology Letters. 349:25-31.
Chen, Y., R.S. Simmonds, J.K. Young and R. Timkovich. 2013. Solution structure of the recombinant target recognition domain of zoocin A. Proteins: Structure, Function and Bioinformatics. 81:722-727.
Group B Streptococcus (GBS) infection in pregnancy can lead to serious medical outcomes including death. Vaginal colonization is unusual in childhood but becomes more common in late adolescence (Figure 1); approximately 10% to 30% of pregnant women are colonized asymptomatically with GBS. Urinary tract infections caused by GBS complicate 2-4% of pregnancies, additionally during pregnancy or the postpartum period, women can also develop amnionitis, endometritis, sepsis, or rarely, meningitis caused by GBS. Other manifestations included bacteremia (31%), endometritis (8%), chorioamnionitis (4%), pneumonia (2%), and puerperal sepsis (2%). GBS also causes severe invasive disease in young infants. Approximately 50-70% of vaginally delivered infants of GBS colonized mothers will become colonized, and 1-2% of colonized infants develop GBS invasive disease. The majority of newborn infections occur within the first week of life and have a mortality rate of up to 9%.
Figure 1. Interaction of GBS with human cervical epithelial cells.
Visualization of adherent GBS by SEM (A), and light microscopy followed by Gram staining (B)
Bacterial endopeptidases are peptidoglycan hydrolysing enzymes with a narrow host range, i.e. the enzyme will only bind to and lyse the cell wall of a few closely related strains of bacteria. Knocking out either the gene encoding a recently discovered GBS endopeptidase, or ciaR, a gene which regulates the expression of the GBS endopeptidase, yielded strains of GBS attenuated for persistence in a mouse model.
The GBS endopeptidase will be produced in a genetically modified Escherichia coli and used to determine the biochemical activity of the purified GBS endopeptidase against peptidoglycan derived from a variety of bacterial strains. This work will allow us to establish the binding site of the enzyme and the precise point of hydrolysis within the GBS peptidoglycan. We also wish to determine if the GBS endopeptidase may have potential for use as a prophylactic treatment to reduce GBS infections in pregnant women. Initially it will be tested to see if it can prevent the attachment to and invasion of human tissue culture cells by GBS. Later it will be tested to see if it can prevent the invasion of mice by GBS.
Early studies have shown that the expression level of the GBS endopeptidase gene are regulated at least in part by a GBS virulence regulator. This project will seek to examine what environmental signals the GBS is responding to and how this translates to expression of the GBS endopeptidase gene. This project requires the use of a qRTPCR assay to determine which virulence genes in GBS are expressed under conditions permissive for invasion of human tissue culture cells.
Rhodococcus opacus strain PD630 has been shown to accumulate triacylglycerol (TAG) to very high levels both in laboratory culture (87%) and at pilot-plant scale (38%). In most respects R. opacus PD630 is an admirable industrial producer strain, it is easy to grow and reaches very high densities in batch fermentations (Figure 2). Four enzymes are involved in the TAG biosynthesis pathway. These enzymes all draw on the cellular pool of acyl-CoA intermediates and sequentially attach the acyl units to the glycerol backbone. Diacylglycerol acyltransferase (DGAT) is the last enzyme in the pathway and its presence or absence is critical to the formation of TAG containing intracellular inclusion bodies. Accumulation of TAG in this strain (and others) seems to be a survival mechanism and occurs in response to growth under starvation conditions, characterized by low nitrogen levels in the presence of excess carbohydrate. The mechanism by which nitrogen level controls TAG accumulation in R. opacus PD630 is not well understood and it is the focus of Dr Simmonds’ current research to understand this process at the molecular level.
Figure 2. Growth of R. opacus PD630 on chemically defined medium.
This project seeks to determine if the expression of non-host fatty acid biosynthesis genes in R. opacus PD630 will lead to the accumulation of TAGs with novel FA compositions. This project requires use of molecular biology techniques to assemble five lipid biosynthesis genes into an operon and to clone these in Escherichia coli. These genes will then be subcloned into an E. coli – Rhodococcus shuttle vector and transformed into R. opacus PD630. Once in R. opacus PD630 the genes will be examined for expression and the lipid produced by the bacterium will be examined for presence of novel lipids.
The accumulation of TAG in R. opacus PD630 is highly regulated, but the mechanism by which this is achieved is unknown. This project seeks to understand the genetic mechanisms involved in the accumulation of TAG in R. opacus PD630. This project requires the use of a qRTPCR assay to determine which lipid biosynthesis genes in R. opacus PD630 are expressed under growth conditions either permissive or non-permissive of lipid accumulation.