Department of Microbiology
Honours Research Areas for 2011
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 (12.4 MB).
Students wishing to apply may do so by registering their name with the Honours Coordinator (Dr Naomi Bishop) along with a list of their three preferred supervisors. An application form (212 KB) 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
Mitochondria originated from bacteria and contain their own genome. Although most of the original genes
have been transferred to the nucleus of the eukaryotic host, a subset of essential genes is still
retained in the mitochondrial genome, and the organelles carry out DNA replication, transcription,
transcript processing, as well as protein synthesis. These genetic processes are of interest to us, as
errors occurring during these processes as well as mutations in the mitochondrial genome lead to
mitochondrial dysfunction, resulting in a variety of diseases, which mainly affect tissues of high
energy demand, such as the central nervous system and muscles. Our studies focus on the following areas:
(1) Mitochondrial transcription and transcript processing
- the characterisation of the mitochondrial RNA polymerase
- the identification and characterisation of mitochondrial 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
The human mitochondrial genome is only 16.5 kb in size, coding for only 13 proteins. Transcription occurs from a single control region, generating a single, large polycistronic transcript. It is likely that, as in bacteria, the co-transcription of genes is used for the coordinate regulation of genes. However, in contrast to the bacterial transcripts, the mitochondrial transcripts undergo varying degrees of processing. The processing mechanisms and the signals that dictate these events are currently under investigation by research groups from various disciplines, including those examining the role of mitochondria in apoptosis, ageing and disease.
Transcription from single promoters is common to the mitochondria of all vertebrata. In contrast, less derived organisms tend to have larger mitochondrial genomes, which, due to their size, require multiple if not many promoters for the expression of the more complex genomes. This is the case in fungi, for example, but multiple promoters are also required in plant mitochondria. In contrast to this, we recently discovered that in the simple protist Dictyostelium discoideum, the relatively large mitochondrial genome of 56 kb in size is transcribed from only a single promoter. Similar to the transcription processes in human mitochondria, the polycistronic transcript generated from the D. discoideum mitochondrial genome is processed into smaller, mature RNA molecules (Figure 1; Barth et al., 1999; 2001, 2007; Le et al., 2009). This makes D. discoideum an attractive model for the study of mitochondrial transcription and RNA maturation, as well as for the identification and characterization of the components involved in these processes.
Our current research focuses on:
(2) Mitochondrial transcription and transcript processing in the human pathogens 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 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. Acanthamoeba has 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. Moreover, Acanthamoeba serves as hosts for bacterial pathogens such as Staphylococcus aureus (a well known and important pathogen in hospitals due to its resistance to various antibiotics) or Legionella pneumophilia, which causes of Legionnaire’s disease, a potentially fatal form of pneumonia.
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 of the mitochondrial genome
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 D. discoideum and 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 characterization 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 D. discoideum, 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
- Ashley Harman, Honours students
- Luke Kennedy, PhD student
- Alkouni Makhlouf, PhD student
- Sam Manna, PhD student
- Maggie Mokbel, PhD student
- Michael Smith, PhD student
Further reading
- Barth, C., Greferath, U., Kotsifas, M. and Fisher, P.R. (1999). Polycistronic transcription and editing of the mitochondrial small subunit (SSU) ribosomal RNA in Dictyostelium discoideum. Current Genetics 36, 55–61.
- Barth, C., Greferath, U., Kotsifas, M., Tanaka, Y., Alexander, S., Alexander, H. and Fisher, P.R. (2001). Transcript mapping and processing of mitochondrial RNA in Dictyostelium discoideum. Current Genetics 39, 355–364.
- Barth, C., Le, P. and Fisher, P. (2007). Mitochondrial biology and disease in Dictyostelium. International Review of Cytology 263, 207-252.
- Kotsifas, M., Barth, C., de Lozanne, A., Lay, S. T. and Fisher, P.R. (2002). Chaperonin 60 and mitochondrial disease in Dictyostelium. Journal of Muscle Research and Cell Motility 23, 839–852.
- Le, P., Fisher, P.R. and Barth, C. (2009) Transcriptipn of the Dictyostelium discoideum mitochondrial genome occurs from a single initiation site. RNA 15, 2321-2330.
Dr Naomi Bishop
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 2011:
(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.
-
These projects will involve either carrying out a detailed analysis of a group of proteins involved in
endocytosis, autophagy, a genetic disorder of cellular trafficking, or focus on a single gene product
and its conservation across the Eukaryota.
-
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:
Is there a role for autophagy when 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. These virus-induced changes in cells obviously have detrimental effects but must also trigger "in-house" survival mechanisms – one of which is autophagy. Thus we aim to determine whether autophagy is initiated in infected cells to the benefit of either host or virus.
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.
Molecular dissection of the MNV-1 infectious clone
Currently we have a cDNA expression plasmid that encodes for the entire MNV-1 genome. This construct allows us to generate infectious MNV-1 viral RNA from a cDNA template. This is an exceptionally useful molecular tool and we aim to exploit it to help us understand the MNV-1 replication cycle. There are many aspects that can be explored with this clone, the primary one being the construction of a MNV-1 subgenomic replicon i.e. a self-replicating RNA molecule that does not encode for the MNV-1 structural proteins. Inclusion of reporter genes such as β-galactosidase or GFP enables us to directly visualize and quantitate virus replication.
Current members of the Molecular and Cellular Virology Laboratory:
- Andrea Mikulasova, Post doctoral scientist
- Leah Gillespie, Research assistant
- Rebecca Ambrose, PhD student
- Ben Cotton, PhD student
- Mary Khouri, Honours student
- Niomi Singh, Honours student
- Eden Whitlock, 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 Agrobacterim and on the importance of curdlan
production in the life of Agrobacterim. 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) Mobile elements in Agrobacterium
-
Our studies on plasmid biology and Agrobacterim EPS have intersected with the observation that
one of the "dead" transposons studied by Steve has a DNA segment that may have originated from
a member of the Rhizobiaceae. The segment contains an unusual opine-degradation-like gene
together with a kanamycin (Km)-resistance gene. Ferdiye, in her study of local Agrobacteria, was
surprised to find strains that were Km-resistant at high levels. We wish to determine the genetic
basis of such resistance, whether it is associated with a mobile element (Tn, In or plasmid) and/or
with opine-like genes that are either "orphan" genes or part of an opine-degradation operon.
- a thesis (65%)
- one literature review and an essay (22%)
- seminar and lecturette (7%)
- laboratory skills and general performance (6%)
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.
This year, two potential Honours projects are being offered for a Honours student with Dr Carl Kirkwood of the Enteric Virus Group at the Murdoch Childrens Research Institute. Dr Kirkwood is also an Honorary lecturer with the Microbiology Department and lectures as part of the MIC1IEP: Infections & Epidemics unit. Currently, Dr Kirkwood is supervising three PhD students at the MCRI enrolled via the Microbiology Department.
Projects will also be offered by the Burnet Institute at one of their centres of excellence – Virology, Immunology, Population, Health and International Health. In 2010, Burnet Institute Projects were offered in all of these areas as Honours projects to eligible students from Microbiology Departments at La Trobe, Monash, and Melbourne Universities. One Honours student enrolled through the Microbiology Department to carry out research based at the Burnet Institute in 2010.
For more information about the projects being offerred by the Murdoch Childrens Research Institute or Burnet Institute please contact Dr Naomi Bishop (03 9479 2232).
Assessment
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.
