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La Trobe University
Department of Microbiology

Department of Microbiology
Honours Research Areas for 2010

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. An application form is attached to the Microbiology honours information booklet. Students should also contact these prospective supervisors directly.

Fields of Research

Dr Christian Barth

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


Picture of mitochondria 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 organisation, 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, 2007; Le et al., 2009). This makes Dictyostelium an attractive model for the study of the processes and components involved in the expression of mitochondrial genes.

    Our current research focuses on:

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

(2) Mitochondrial transcription and transcript processing in the human pathogen Acanthamoeba castellaniiand Naegleria fowleri

    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 sightthreatening 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 hosts 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

    Another organism studied in our laboratory is Naegleria fowleri, a facultative human pathogen typically found in the soil and in fresh water (lakes and rivers) but also in swimming pools and in drinking water. Naegleria is an amoeba-flagellate, so called because it can exist as an amoeba but also as swimming flagellates. The amoebae can enter the body through the olfactory neuroepithelium (usually during swimming) causing primary amebic meningoencephalitis, a fatal cerebral infection. Naegleria also infects other vertebrates as well as invertebrates. In addition, the amoebae can habour various bacteria and even viruses.

(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 as well as in Dictyostelium (Kotsifas et al., 2002; Barth et al., 2007). 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 characterization of homologous genes and their gene products in other organisms.

Current members of the Mitochondrial Genetics Laboratory:

  • Jessica Accari, PhD student
  • Leanne Bekhet, PhD student
  • Phuong Le. PhD student
  • Maggie Mokbel, PhD student
  • Luke Kennedy, PhD student
  • Michael Smith, PhD student
  • Eunice Le, Honours student
  • Sam Manna, Honours student

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, (i) 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). (ii) 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. (iii) Dysfunction in traffic through endoplasmic reticulum and secretory pathway can lead to diseases such as cystic fibrosis, diabetes mellitus, and a variety of cancers and neurological diseases.

In my laboratory we are investigating the molecular function of the endosomal, autophagic, and secretory pathways, and the role of specific host cell factors implicated in: autism spectrum disorder, lysosomal disorders, cancer, Huntington disease, amyotropic lateral sclerosis, and Fanconi anaemia. 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 2010:

(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 role of an un-characterized human cellular protein, or will study the manipulation of eukaryotic host cell trafficking pathways by a microbial 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
  • Pathogen growth and / or trafficking analysis

(2) In silico Analysis of Eukaryotic Trafficking Proteins

    There are two 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 can lead to bioinformatics-based or laboratory-based PhD projects, where the latter experimentally tests the in silico findings and predictions made
    Methods typically used in these types of projects include:
  • BLAST searches and sequence alignments
  • Secondary structure and coiled coil analyses
  • Analysis of chromosomal positions and synteny
  • Motif and domain searches
  • Characterising intron evolution and conservation
  • Detection of pseudogenes and paralogues
  • Phylogenetic tree creation

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, a lysosomal storage disorder called Batten Disease and Parkinson’s Disease. Honours projects will be offered in these broad areas. We have found that defects in these diseases arise from disturbed signaling pathways and we are investigating these pathways. Within this research area there are three main ongoing projects:

(1) Mitochondrial and neurodegenerative diseases

    We have discovered that the diverse cytopathologies of mitochondrial 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. The key signalling pathway that is affected involves an energy-sensing protein kinase called AMPK. Its activity is responsible for the “symptoms” of mitochondrial disease. Another signalling pathway, the TORC1 pathway is responsible for the “symptoms” of lysosomal disease. These two pathways and those for Parkinson’s Disease may overlap as some, but not all of the “symptoms” are similar. This is being investigated by creating disease in Dictyostelium via disruption or antisense inhibition of the expression of genes for selected 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, as well as the study of Ca2+ fluxes between the mitochondria and the cytosol.

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 co-immunoprecipitation, Western blotting, phosphorimaging analysis, standard and real time PCR/RT-PCR, high-speed and ultracentrifugation, pulsed-field and standard agarose gel electrophoresis, scintillation counting, radio-immunoassay, low level luminescence measurements, fluorometry, fluorescence microscopy, computer analysis of experimental results and of DNA sequences.

Dr Jason Mackenzie

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

Our overall objectives are to investigate and unravel the replication mechanism of two positivestranded RNA viruses (West Nile virus [WNV; a flavivirus] and Mouse Norovirus [MNV-1; a Norovirus]) that are highly pathogenic to humans and cause outbreaks of encephalitis and gastroenteritis, respectively. 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.

In particular we investigate:

How and where do flaviviruses and noroviruses replicate in cells?

Our research has focused on the roles of host and viral proteins in replicating the flavivirus and Norovirus genome. We have deduced all of the viral proteins that are required and also the site within cells where this replication occurs. We have found that there is a dramatic change in the organization and structure of cellular membranes that is associated with virus replication.

We know that these membranes are required for efficient virus replication and also help the virus to avoid immune detection. We have also shown that cholesterol plays a major role in helping the flavivirus replicate within the cell. All of these studies are providing us with a solid grasp of what is required for virus replication that integrates aspects of cell biology, biochemistry, molecular biology and immunology.

What determines viral protein localisation in cells?

We have cloned and expressed all of the different MNV- 1 open-reading frame 1 proteins from cDNA expression plasmids. Localisation studies have revealed that the different viral proteins display unique localisation patterns within transfected cells and are targeted separately to distinct organelles; many of which are utilized by the virus during replication. Thus one of our aims is to identify the targeting signals embedded within the separate proteins that could potentially direct virus replication to distinct sites within infected cells. Identified motifs can then be mutated in cDNA infectious clones of the virus to determine whether such mutations dramatically alter the capability of the virus to replicate.

Can we visualize virus 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 timelapse epi-fluoresence to visualize the formation and proliferation of virus membranes over real-time in living cells. We aim to combine this analysis with fluorescently labeled RNA molecules in attempts to observe virus replication, assembly and maturation in living cells in real-time.

Role of apoptosis in Mouse Norovirus infection

Recently we have observed that infection of cells with Mouse Norovirus (MNV-1) induces apoptosis. This has been readily observed by a destruction of host cell nuclei. We have also observed a similar trait when cells were transfected with a plasmid encoding for one of the MNV-1 nonstructural proteins. As apoptosis is an end-point for cells, we would like to address whether this is something the virus would like to occur or whether it is the host cell responding to infection. Thus we aim to determine whether MNV-1 is affecting induction of apoptosis. This project will be in collaboration with the laboratory of Dr John Silke in Biochemistry.

Flavivirus evasion of interferonstimulated 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.

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.

Current members of the Molecular and Cellular Virology Laboratory:

  • Andrea Mikulasova, Post doctoral scientist
  • Leah Gillespie, Research assistant
  • Jennifer Hyde, PhD student
  • Rebecca Ambrose, PhD student
  • Ben Cotton, PhD student
  • Jacinta Ortega, Honours student

Dr Vilma Stanisich

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

Projects in Plasmid Biology

(1) Mercury-resistance transposons of the Tn5053-family
    The Tn5053/Tn402 family of elements includes fully functional transposons (Tn) as well as a subgroup comprised of classic Class I integrons (In). Our interests concern the distribution and evolutionary relationships of these elements as well as the mechanistic basis of transposition ability. These transposons exhibit unusual target-specificity as well the ability (under some circumstances) to transpose to random target sites. We are interested in the transposon-encoded and target-encoded properties that are responsible for the two different pathways of transposition.
(2) Pseudomonas resistance plasmids
    Dr Steve Petrovski, a graduate from the laboratory, analysed the structure of IncPβ broad host range plasmids and showed that their evolutionary divergence was due to multiple insertions of Tn or In elements into a conserved "backbone". He also showed that seemingly "dead" transposons could generate bacterial diversity in unexpected ways. Steve and several honours students, including Damien, studied a novel "nested" transposon located in a new type of mobilisable Pseudomonas plasmid. The nested element is fully functional and includes a Tn5053/Tn402 family transposon. Although the host Pseudomonas strain was a clinical isolate, we suspect that both the plasmid and the base element of the transposon were originally from an environmental source. We wish to explore this possibility.

Projects In Agrobacterium Biology

(3) The molecular biology of EPS (extracellular polysaccharide) production
    We are interested in the production of curdlan by a high yielding Agrobacterium strain similar to those used in commercial curdlan production (Fig. 1). This structurally simple EPS [a linear, (1-->3)-β-glucan] is produced under N-depleted conditions by a three-gene synthesis locus (crdASC). In contrast, a hitherto “cryptic” EPS (named EPS-X) is elicited by elevated MnII levels and is co-produced with curdlan. Danielle has identified a large gene cluster (ca. 20 kb) that is probably the main EPS-X synthesis locus.
    We also have evidence that the regulatory cascade leading to production of curdlan and EPS-X involves global regulators, namely, the "alarmone" (RelA) that initiates the bacterial stringent response to environmental stress (Ferdiye) and the two-component systems (NtrBC and NtrYX) that sense cellular N-status (Sanja). Sanja has also shown that another regulatory protein, CrdR, has a key role in "cross-talk" between curdlan production and EPS-X production.
(4) 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 (six EPS at least) and versatile (not all EPS are produced concurrently) and may contribute to success of the bacterium both as a pathogen and as a soil saprophyte. Ferdiye is studying the incidence of curdlan production by agrobacteria from local soils and other sources. She is interested in whether curdlan production is a feature of all three biovars (biovars I, II and III) and the extent to which it has been conserved. Answers to these questions provide information on the evolution of Agrobacterium and on the importance of curdlan production in the life of Agrobacterium. Sanja and Danielle are assessing the functional role(s) of curdlan and EPS-X in events that are likely to occur in nature (e.g. cell-to-cell aggregation; attachment to, and survival on, plant tissues; resistance to soil predators e.g. amoebae; resistance to physical stressors e.g. temperature, desiccation, toxic agents).
(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 (six EPS at least) and versatile (not all EPS are produced concurrently) and may contribute to success of the bacterium both as a pathogen and as a soil saprophyte. Ferdiye is studying the incidence of curdlan production by agrobacteria from local soils and other sources. She is interested in whether curdlan production is a feature of all three biovars (biovars I, II and III) and the extent to which it has been conserved. Answers to these questions provide information on the evolution of Agrobacterium and on the importance of curdlan production in the life of Agrobacterium. Sanja and Danielle are assessing the functional role(s) of curdlan and EPS-X in events that are likely to occur in nature (e.g. cell-to-cell aggregation; attachment to, and survival on, plant tissues; resistance to soil predators e.g. amoebae; resistance to physical stressors e.g. temperature, desiccation, toxic agents).

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.

Assessment

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

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.