Department of Microbiology
Honours Research Areas for 2008

Introduction

The Microbiology honours course consists of a one year supervised research project along with a relatively minor component of course work. Microbiological research is as varied in its nature as the microbial world itself and the research interests of the Department of Microbiology reflect this diversity.

The information in this page can also be downloaded in .pdf format.

Students wishing to apply may do so by registering their name with the Honours Coordinator (Prof. P. R. Fisher) along with a list of their three preferred supervisors. Students should also contact these prospective supervisors directly.

Fields of Research

Dr Christian Barth

E-mail: c.barth@latrobe.edu.au

Mitochondria originated as bacteria that have been engulfed by primitive eukaryotic cells. In the course of this endosymbiosis, most of the genes encoded by the mitochondrial genome have been transferred to the nucleus of the eukaryotic cell. However, a subset of essential mitochondrial genes is still retained in the mitochondrial genome, and the organelles carry out DNA replication, transcription and protein synthesis, processes that are different from the genetic processes in the nucleus. The size of the mitochondrial genome, its structure and gene organization, and the mode of gene expression and subsequent transcript processing are highly variable between the different species. Our studies focus on the following areas:

(1) Transcription and transcript processing in mitochondria of Dictyostelium discoideum

The mitochondrial genome of the eukaryotic microbe Dictyostelium discoideum has a size of approximately 55 kb, and is transcribed in a similar way as the human mitochondrial genome: large polycistronic transcripts are co-transcriptionally processed into smaller, mature RNA molecules (see Figure 1 for details; Barth et al., 1999; 2001). This makes Dictyostelium an attractive model for the study of the processes and the components involved in the expression of mitochondrial genes.

Our current research focuses on:

• the characterization of the mitochondrial RNA polymerase
• the identification of promoter sequences
• promoter recognition and regulation of mitochondrial gene expression
• the identification and characterization of other components of the transcription apparatus and
the transcript processing machinery

(2) Mitochondrial transcription and transcript processing in the human pathogen Acanthamoeba castellanii

Acanthamoeba castellanii is one of the most common protozoa in the soil and fresh water. The interest in the organism is based on its pathogenicity: it can invade the cornea, causing a painful and sight-threatening disease of the eye (amebic keratitis), but it can also spread to the central nervous system in which case it leads to the fatal disease amebic encephalitis. Acanthamoebae have also been associated with various secondary infections associated with immuno-compromised individuals such as AIDS patients and with several diseases in a variety of animals (Marciano-Cabral & Cabral, 2003). Moreover, Acanthamoebae can serve as a host for bacterial pathogens such as Staphylococcus aureus (an important and well known pathogen in hospitals due to its resistance to many antibiotics) or Legionella pneumophilia, the major cause of Legionnaire’s disease, a potentially fatal form of pneumonia.

The mitochondrial genomes of Dictyostelium and Acanthamoeba castellanii are strikingly similar in size, gene content and gene organisation. This implies that both organisms share the same mode of transcription and transcript processing. In our current research we make use of the knowledge we gained in the study of mitochondrial transcription in Dictyostelium to investigate these processes in Acanthamoeba. The complete understanding of these processes in the pathogen as well as the identification and characterisation of all components involved may reveal potential drug targets that aid in the treatment of Acanthamoeba infections.

(3) Replication and Maintenance of the Mitochondrial Genome in Dictyostelium discoideum

Mitochondrial DNA polymerase gamma (γ) is the sole enzyme found so far in a variety of organisms to be devoted to mitochondrial DNA replication. However, in other organisms such as Dictyostelium and in plants, the replicating enzyme has not been discovered yet. Recently, we have identified and cloned the gene for a mitochondrial DNA polymerase that has not been described in any other organism. The characterisation of this protein will allow further insight into the process of mitochondrial DNA replication and DNA repair.

(4) Identification and characterisation of nuclear-encoded mitochondrial proteins in Dictyostelium, plants and mammals

Genes of both the mitochondrial and the nuclear genome control the main function of the mitochondria, the synthesis of ATP by oxidative phosphorylation (OXPHOS). Decreased OXPHOS capacities, caused by mutations in the mitochondrial genome or by impairment in the expression of nuclear genes involved in mitochondrial biogenesis can result in mitochondrial dysfunction. This can elicit disorders in mammals, plants, yeast and, as we recently discovered, in Dictyostelium (Wilczynska et al., 1997; Kotsifas et al., 2002). We make use of the ability to induce such mitochondrial disorders in Dictyostelium and the resulting changes in the phenotype of the organism in order to identify unknown nuclear-encoded mitochondrial proteins that are essential for mitochondrial function. The discovery of the genes encoding these proteins in the Dictyostelium genome will form the basis for the identification and characterisation of homologous genes and their gene products in other organisms.

Current members of the Mitochondrial Genetics Laboratory:

• Phuong Le. PhD student
• Maggie Mokbel, PhD student
• Luke Kennedy, PhD student
• Michael Smith, PhD student
• Jessica Accari, Honours student
• Daniela Basa, Honours student

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Dr Naomi Bishop

Email: n.bishop@latrobe.edu.au

All diseases, whether due to infectious agents or genetic causes, are due to disturbances at the cellular level. The field of cell biology is becoming increasingly important to the study of disease and, conversely, the study of the cellular basis of human disease has already produced some fundamental insights into the function of eukaryotic cells. Specifically, defects along the endosomal pathway are linked to disorders such as hypertension (defective endocytosis of sodium channels), many types of cancer (defective downregulation of growth factor receptors), pigmentation defects (defective maturation and transport of melanin granules in melanosomes in skin cells), familial hypercholesterolemia (defective cellular uptake of lipoproteins), and more than 40 specific lysosomal storage diseases, which typically cause neurological symptoms (due to defective transport of hydrolytic enzymes).

The autophagic pathway merges with the endocytic pathway, and has functions in maintenance of cell homeostasis, immune presentation, and in facilitating programmed cell death (type II cell death, where apopotosis is type I). Defects in autophagy are likewise linked to a range of diseases, including cancers, myopathies, and neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease.

Many microorganisms, including bacteria, fungi, and viruses manipulate endocytic and/or autophagic pathways when they infect eukaryotic host cells. Studying these processes provides us with fundamental information on the pathogenic mechanisms of these microorganisms and highlights potential therapeutic targets. The research also provides insights into the normal physiological role of these pathways in the host cell. Within these research areas there will be two types of projects available for Honours projects in 2008:

(1) Molecular and Cellular Pathogenesis

Two projects are offered in the area of molecular and cellular pathogenesis. These projects will be laboratory based and will study the manipulation of eukaryotic host cell endocytic and/or autophagic pathways by either a viral, bacterial, or fungal pathogen.

Methods typically used in these types of projects include:
• DNA manipulation, purification, restriction analysis and sequencing,
• Polymerase chain reaction (PCR)
• Protein expression
• Mammalian cell culture
• Transfection of mammalian cells
• Immunofluorescence
• Viral, bacterial or fungal growth analysis

(2) In silico Analysis of Eukaryotic Trafficking Proteins

There are several projects available in the area of eukaryotic genomics, which will be carried out in a "dry" laboratory. It is not anticipated that these projects will involve any "wet" experiments, and all aspects of the project will use computer-based methods. No experience in computer programming is required, nor will programming be involved in these projects. These projects may, however, lead to laboratory-base PhD projects that will experimentally test the in silico findings and predictions made.

These projects will involve either carrying out a detailed analysis of a group of proteins involved in endocytosis or autophagy, or focus on a single gene product and its conservation across the Eukaryota.

Methods typically used in these types of projects include:
• BLAST searches
• Secondary structure and coiled coil analyses
• Analysis of chromosomal positions and synteny
• Motif and domain searches
• Multiple sequence alignments e.g. CLUSTAL W
• Characterising intron evolution
• Detection of pseudogenes and paralogues
• Phylogenetic tree creation (using maximum parsimony, maximum likelihood, neighbour-joining, and minimum evolution methods)

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Prof. Paul Fisher

Email: p.fisher@latrobe.edu.au

The activities of cells in unicellular and multicellular organisms, including humans, need to be and are regulated by extracellular signals. The proteins involved in sensing and responding to them have been conserved and elaborated upon during evolution. Not surprisingly, perturbation of the pathways that process (transduce) these signals contributes to a variety of diseases. We are using Dictyostelium discoideum as a model to study the cytopathologies associated with certain neurological and neuromuscular genetic disorders – namely mitochondrial diseases and a lysosomal storage disorder called Batten Disease. Honours projects will be offered in the area of mitochondrial disease. We have found that defects in mitochondrial disease arise from disturbed signaling pathways and we are investigating these pathways. Within this research area there are three main ongoing projects:

(1) Mitochondrial diseases

We have discovered that the diverse cytopathologies of mitocondrial disease are caused by altered intracellular signal transduction, not by insufficient ATP. In Dictyostelium the result is impaired phototaxis, thermotaxis, development and growth as well as increased susceptibility to Legionella infection. This is being investigated by creating mitochondrial disease in Dictyostelium via disruption or antisense inhibition of the expression of genes for mitochondrial proteins encoded either in the nuclear genome or the mitochondria.

(2) Signalling pathways for phototaxis

Genetic disorders that affect the mitochondria vary in their severity and as the underlying genetic defect becomes more severe, different cellular activities become impaired in turn. In Dictyostelium one of the first signs of mitochondrial disease is impaired phototaxis. We are therefore investigating the signalling pathways involved in phototaxis and how mitochondrial disease affects them. Proteins that have been identified as important for signal transduction in phototaxis include serpentine receptors, heterotrimeric and small GTP-binding proteins (RasD), cytoskeletal proteins (filamin, GRP125, myosin II), ErkB (a MAP kinase) and Protein kinase B (PKB). We have shown that several of these proteins belong to a signalling complex in which filamin acts as a scaffold for assembly of the complex.

(3) Ca²⁺ signalling

Mitochondrial diseases may affect Ca²⁺ signalling and this in turn could contribute to the cytopathology. Using a recombinant jellyfish protein that luminesces in a Ca²⁺-sensitive manner, we developed a method for accurately measuring intracellular Ca²⁺ concentrations in living Dictyostelium cells in real time. The cytosolic free Ca²⁺ levels in cells are a result of the balance between Ca²⁺ sequestration and release by Ca²⁺-binding proteins, as well as Ca²⁺ influx from and efflux to the extracellular medium and intracellular organelles. Current research involves molecular genetic study of the functions of the Ca²⁺ channels and pumps in the plasma and organellar membranes.

Techniques required in these research areas include DNA isolation and cloning in E. coli, transformation of Dictyostelium (same methods as for mammalian cells), DNA restriction analysis, protein isolation and purification, Southern and northern blotting using non-radioactive probes (colour, chemiluminescent, chemifluorescent), creation of recombinant clones for tagged protein expression in E. coli and antibody generation, 1D and 2D PAGE, protein coimmunoprecipitation, western blotting, phosphorimaging analysis, standard and real time PCR/RT-PCR, high-speed and ultracentrifugation, pulsed-field and standard agarose gel electrophoresis, scintillation counting, radioimmunoassay, low level luminescence measurements, fluorometry, fluorescence microscopy, computer analysis of experimental results and of DNA sequences.

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Dr Jason Mackenzie

E-mail: j.mackenzie@latrobe.edu.au

Our overall objectives are to investigate and unravel the replication mechanism of two positive-stranded RNA viruses (West Nile virus [a flavivirus] and Mouse Norovirus [a Norovirus]) that are highly pathogenic to humans and cause outbreaks of encephalitis and gastroenteritis. Our aims are to determine how and where these viruses replicate within infected cells and what host components/organelles are “used and abused” by the virus. We aim to correlate this abuse of host with the pathogenic outcomes associated with viral infection. In conjunction with these studies we are investigating how viruses can evade our immune system and in particular how viruses can bypass the antiviral activities of our first line of defence; the innate immune system

(1) Roles of Cellular Lipids in Flavivirus Replication

Our current research has sought to evaluate the role of cellular lipids in flavivirus RNA replication and membrane induction. We have observed that a host protein regulating cholesterol biosynthesis is upregulated during infection and redistributes to the sites of virus replication. Additionally compounds affecting the cells capacity to produce and recycle cholesterol have differing effects on virus replication, with drugs affecting cholesterol biosynthesis having the most profound effects. One consequence of the redistribution of cholesterol is the apparent dissociation of lipid raft molecules. This consequence has implications relating to cellular metabolic pathways including immune activation cascades.
In collaboration with Rob Parton (Institute of Molecular Biosciences)

(2) Visualization of Flavivirus-Replication in Live Cells

We have previously identified the protein composition and roles of unique cytoplasmic membrane structures that are induced upon flavivirus infection. These membrane structures appear crucial to the efficient replication of flaviviruses and are intimately linked to the exponential increase in virus production. These membranes can be easily identified with antibodies with both the light and electron microscopes, however these are static representations. Recently we have identified the viral protein responsible for these membrane changes and thus we can directly target this protein for analysis. We aim to utilize the green fluorescent protein and time-lapse epifluoresence to visualize the formation and proliferation of virus membranes over real-time in living cells. To this end we have constructed in-frame insertions of the GFP gene into the Kunjin virus infection clone and replicon. Analysis of these constructs after expression reveals that they are defective for replication. Fortunately though they can be rescued with a helper replicon to produce and express the GFP-fusion proteins. Currently we are investigating techniques to provide us with viable constructs in the future.
In collaboration with Gareth Griffiths (EMBL, Heidelberg, Germany)

(3) Flavivirus evasion of interferon-stimulated antiviral proteins

In response to infection by pathogens our cells and body produces proteins that fight and combat the invading pathogen. The production of such “anti-viral” proteins is tightly regulated though, primarily by chemicals known as interferons. One of these antiviral proteins is MxA. MxA has broad spectrum antiviral properties against many viruses, in particular viruses similar to influenza and measles viruses. One of our aims was to assess whether MxA could also impart these antiviral activities against flaviviruses. Therefore we observed whether over-expression of MxA, independently of intereferon, could protect cultured cells against flavivirus infection. Analyses revealed that either flavivirus RNA replication or virus production was hampered by MxA expression. This evasion does not appear to be due to a viral-encoded antagonist, although an unknown host protein does appear to specifically associate with MxA during infection. The role and identity of this protein is currently under investigation. Interestingly some of our data has indicated that the prolific membrane rearrangements and rapid flavivirus assembly process may “hide” the viral components from MxA and other host surveillance proteins thus preventing the host cells from stimulating protective mechanisms.

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Dr Vilma Stanisich

E-mail: v.stanisich@latrobe.edu.au

Projects In Plasmid Biology

(1) Mercury-resistance transposons of the Tn5053/502-family

Our interests include the epidemiology of these elements, their evolutionary relationships to each other and to Tn (or In) elements that contain related mer (HgII-resistance) and/or tni (transposition) modules, and the mechanistic basis of their unusual target-specificity and their ability (under some ircumstances) to transpose randomly (Steve)..

(2) Evidence of evolutionary divergence, and of conservation, in promiscuous (IncPβ) plasmids

A detailed study of several IncPβ plasmids has revealed that their conserved "backbone" has been disrupted at two locations by multiple insertions of Tn or In elements. The extant "nested transposons" are not self-mobile, but can be induced to relocate when an appropriate tnpA (transposase) in provided in trans. The varying molecular composition of the moveable elements highlights the fact that seemingly "dead" transposons can continue to contribute to bacterial diversity in unexpected ways (Steve).

Projects In Agrobacterium Biology

(3) The molecular biology of EPS (extracellular polysaccharide) production

Our studies have dealt mainly with the production of curdlan by a high-yielding Agrobacterium strain like those used in commercial curdlan production. This water-insoluble EPS is produced under N-depleted conditions, is structurally simple (a linear, (1-->3)-β-glucan) and its production involves three structural genes (crdASC) whose precise roles and regulation are under investigation (Ferdiye).

A hitherto "cryptic" and water-soluble EPS (named EPS-X) is elicited by elevated MnII levels and is co-produced with curdlan. A putative EPS-X synthesis/secretion region has been identified whose size (ca. 17 kb) suggests that EPS-X is a heterpolysaccharide. The aim of the work is to identify the essential epx genes, determine whether EPS-X is novel (i.e. its sub-unit composition, structure and pathway of synthesis) and whether there is "cross-talk" between the crd and epx production systems (Danielle).

(4) The regulatory cascade leading to EPS production

We hope to determine the regulatory cascade leading to curdlan (and EPS-X) production by assessing the roles of various global regulators, namely: a two-component system (NtrBC) that senses intracellular N-status (Sanja), the "alarmone" (RelA) that initiates the bacterial stringent response to environmental stress (Ferdiye) and the metalloprotease/chaperone (FtsH) that is essential for survival in stationary phase (Danielle). The role of additional, positive-acting, functions that may be curdlan-specific (the CrdR regulatory protein) (Sanja and Ferdiye) or EPS-X-specific (an orphan sensor kinase) (Danielle) are also being studied.

(5) The biological role(s) of EPS (horses for courses?)

EPS production is a complex process initiated by an interplay of various environmental factors (physical and nutritional) and cellular physiological triggers. The EPS armoury of Agrobacterium is impressive – it has the capacity to produce at least six though not simultaneously, suggesting one or more combinations of specific elicitors for each EPS. The success of Agrobacterium as a soil saprophyte may be underscored by its EPS versatility. We are studying the epidemiology of curdlan production by agrobacteria from local soils (Ferdiye) and comparing the roles of cellulose and curdlan in natural contexts (e.g. attachment to, and survival on, plant tissues; resistance to soil predators and physical stressors e.g. temperature, dessication, toxic agents) (Sanja and Prof Matthysse).

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External projects

External projects are carried out with a supervisor working externally to La Trobe University. Much of the research for these projects will be carried out off-site from the Bundoora campus, with some assessment and other tasks completed at the Department of Microbiology. Students undertaking external projects are also assigned an internal supervisor.

Dr Carl Kirkwood from the Enteric Virus Group, a research group in the Murdoch Childrens Research Institute (the research arm of the Royal Children's Hospital), and Dr Johnson Mak from the Virology Program at Macfarlane Burnet Institute for Medical Research are offering honours projects in 2008.

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Dr Carl Kirkwood
Murdoch Childrens Research Institute

E-mail: carl.kirkwood@mcri.edu.au

phone: 03 8341 6439

The Enteric Virus Research Group is part of the Murdoch Childrens Research Institute, which is the research arm of the Royal Children's Hospital. Dr. Carl Kirkwood is a group leader in the Enteric Virus Group and has the following Honours project available:

Characterisation and Comparison of Rotavirus G9P[8] Strains during Outbreaks in the Northern Territory.

Rotavirus is the major cause of acute gastroenteritis in children in developed and developing countries. In Australia over 10,000 children are admitted to hospital each year. The National Rotavirus Reference Centre undertakes surveillance and serotype characterisation of rotavirus strains causing severe gastroenteritis in young children throughout Australia.

National surveillance has identified that serotype G9 has emerged as a significant cause of severe gastroenteritis in children in Australia since it has been first identified in 1999, and worldwide during the past 5 years. During this period we have seen two large outbreaks of gastroenteritis occur in the Northern territory, 2001 and 2007.

The project will characterize serotype G9 strains obtained during the second outbreak in 2007 in the NT, and will compare them to the previous G9 outbreak in 2001. Analysis undertaken will include molecular biology methods such as RT-PCR, sequence analysis and Northern hybridisation. Understanding the changes that have occurred in the re-emergence of the G9 serotype is important for implementation strategies of rotavirus vaccines.

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Dr Johnson Mak
Macfarlane Burnet Institute for Medical Research

E-mail: johnson.mak@med.monash.edu.au or mak@burnet.edu.au

phone: 03 9282 2217

The HIV Assembly Group aims to better define the replication processes of HIV-1 and its closely related viruses through basic research, and to identify critical features that can be exploited for translation to novel antiviral strategies for clinical applications. The successful candidate should be enthusiastic about HIV research, and a wide range of molecular virology techniques will be taught in these projects.

(1) Codon Usage in HIV-1

Synonymous codons are different codon sequences that encode for the same amino acid. Selected groups of viruses, including HIV-1, have evolved to use AU-rich codons for protein synthesis. Interestingly, the natural hosts of these viruses rely on a completely different subset of codons (GC-rich codons) and transfer RNAs (tRNAs) for the synthesised peptide chains. As the codon utilised by these viruses are distinct from their natural host cells, the reliance of rare AU-rich codon in viral genomes implies an important but unexplored biological regulation process. The objective of this project is to define the precise mechanism of codon bias in viral replication.

(2) Retroviral recombination

Retroviral recombination is an important route to generate multiple drug resistant- (MDR) and immune escaped-HIV-1. We have developed a novel system to assess the process of retroviral recombination in HIV-1 genomes, and we are exploring the potential to suppress the process of retroviral recombination using small molecules inhibitors to regulate the recombination process. Results from this work will help to develop new tools to regulate the production of MDR and immune escape HIV-1.

(3) Host Cell Factors and HIV-1 Replication in Primary Cells

Our data show that requirement of host cell factors in HIV-1 replication could be drastically different between cell line system and natural target cells of HIV-1, and we are currently dissecting how some of these host cell factors support the replication of HIV-1 and defining their true contribution to HIV biology.

(4) Virion lipids in HIV-1

The lipid rafts hypothesis remains one of the highly debate topic in modern biology. Our novel observation has led us to realize that viral pathogens are nature's ideal model system to examine the lipid rafts hypothesis. Envelope viruses such as HIV-1 (nature's specific type of rafts) can be readily isolated and lipid-modified. This project will provide prove of concept data showing lipid enriched pathogens can be used to examine and to help end the debate of the lipid rafts hypothesis.

(5) Biochemical and Structural Analysis of HIV-1 Proteins

During HIV-1 assembly, viral proteins and host cell factors undergo a series of 'cooperative' interaction and 'coordination' which lead to the formation of virion particles. The objective of this project is to define some of these events at the atomic levels through structural biology study, with the aim to develope novel small olecule inhibitors to suppress the production of infectious HIV-1.

(6) Tracking the Movement of HIV-1 in Living Cells

The movement of viral genetic materials and proteins in an infected cell is not well defined. We have established Australia’s first level 3 pathogen imaging facility at the Burnet Institute, and we are currently using this technology to define the trafficking process of viral RNAs and proteins. Through tracking of the movement of viral factors, we will be able to define the parameters that regulate virus movement in the cell, and to develop novel strategy to suppress the virus propagation.

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Assessment

• a thesis (65%)
• one literature review and an essay (22%)
• seminar and lecturette (7%)
• laboratory skills and general performance (6%)

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Further Information

Further information about Honours in Microbiology can be obtained from the Undergraduate Handbook; or contact the Department of Microbiology.

Faculty of Science, Technology and Engineering course information is also available online.