Day Group

Supervisors: Prof Victoria Korolik & Dr Christopher J Day

Molecular Microbiology

The natural habitat of campylobacters is the intestine of warm-blooded animals, and therefore chemotactic motility is an important mechanism involved in the colonisation and pathogenicity of this microorganism. Bacterial motility is subject to sensory control mechanisms that introduce a bias into the swimming direction of the organism towards beneficial environments and away from unfavourable conditions. Although chemotaxis has been demonstrated for Campylobacter the chemical substrates, mechanisms involved in the sensory control of motility and the role of chemotaxis in disease, are poorly understood. We, therefore, hypothesise that the chemosensory receptor proteins play a key role in chemotaxis and are involved in the pathogenicity of this organism as the first line of bacterial – host interaction and thus provide rational targets for the design of novel antimicrobial agents.

This project involves characterisation of interactions of the signaling domain of one of the chemoreceptors of C. jejuni, named Tlp1 with CheW and CheV chemotaxis proteins.  The major aim of this project is to identify which amino acids in the signaling domain of Tlp1 are responsible for binding with CheW and CheV though systematic site-specific mutagenesis followed by analysis of the mutated proteins using yeast 2-hybrid protein-protein interaction system.
This project will further the studies to elucidate the role of Tlp1 chemoreceptor in chemotaxis and pathogenicity of C. jejuni, which can potentially provide a tremendous insight into the mechanisms of chemotaxis of this organism.

Supervisors: Dr Ian Peak, Dr Christopher J Day & Prof Michael Jennings

Molecular Microbiology, Molecular Biology

The immune system responds to infections after it has recognised infectious agents. All bacteria secrete products, and some of these have profound effects on the host immune system, either acting as recognition molecules for immune attack, or by modifying the immune response to assist the microbe to survive. We are investigating how secreted molecules from pathogenic bacteria are detected. We are characterising host receptors for these secreted products, which will help understand diseases such as cholera, legionnaire’s disease, as well as infections caused by Pseudomonas in burned, and cystic fibrosis patients.

Techniques: Molecular genetics techniques, immunofluorescence microscopy, protein expression and purification, FACS analysis, cell culture and in vitro infections, in vivo infections using mouse models of disease, analyzing immune markers such as cytokine and chemokine responses of the host cell, small molecule purification and analysis by Mass Spectrometry and other techniques

Supervisors: Dr Christopher J Day & Prof Michael Jennings

Microbiology; cell assays; array technology; affinity and kinetics measurements

Recently we showed that pathogenic bacteria interact with host cell through direct contact of the carbohydrates expressed by both organisms (Day et al 2015 PNAS 112:E7266). Previously only three glycan-glycan interactions had been described (sea sponges, Lewis antigens and gangliosides) while our study extended this to over 60 new interactions. The role of glycan-glycan interactions in pathobiology and more widely throughout nature has not been fully elucidated. This project will investigate a wide range of bacterial polysaccharides for glycan binding and try to determine the minimal and sufficient structure required for these novel interactions. This study will utilise the glycomics arrays that we produce within the Institute for Glycomics as well as studies of affinity and kinetics using surface plamon resonance (GE Biacore T100) and micro isothermal calorimetry (TA Instruments nanoITC). Cell assays for bacterial adherence will also be performed.

Supervisors: Dr Christopher J Day & Prof Michael Jennings

Cell assays; array technology; affinity and kinetics measurements

Recently we showed that pathogenic bacteria interact with host cell through direct contact of the carbohydrates expressed by both organisms (Day et al 2015 PNAS 112:E7266). Previously only three glycan-glycan interactions had been described (sea sponges, Lewis antigens and gangliosides) while our study extended this to over 60 new interactions. The role of glycan-glycan interactions in pathobiology and more widely throughout nature has not been fully elucidated. In our studies we noted that bacteria that mimic human glycan structures were still capable of binding human glycan structures indicating that direct interactions between eukaryotic glycans are likely to occur. This project will investigate a range of eukaryotic glycans for their ability to recognise other eukaryotic glycans. This study will utilise the glycomics arrays that we produce within the Institute for Glycomics as well as studies of affinity and kinetics using surface plamon resonance (GE Biacore T100) and micro isothermal calorimetry (TA Instruments nanoITC). Cell assays to observe the binding of labelled glycans to appropriately glycosylated cells.

Supervisors: Dr Jessica Poole, Dr Christopher J Day & Prof Michael Jennings

Array technology; affinity and kinetics measurements

Carbohydrate binding proteins (also known as lectins) are a broad range of proteins with a wide specificity for carbohydrate structures. Recently we have found that a large number of bacterial and eukaryotic proteins have the ability to bind to glycans that had not previously been appreciated. This project will investigate a range of proteins from bacterial and eukaryotic sources for their ability to interact with glycans. This study will utilise the glycomics arrays that we produce within the Institute for Glycomics as well as studies of affinity and kinetics using surface plamon resonance (GE Biacore T100) and micro isothermal calorimetry (TA Instruments nanoITC).

Supervisors: Dr Yaramah Zalucki, Dr Christopher Day, Assoc Prof Thomas Haselhorst & Prof Michael Jennings

Structural biology, enzyme assays, antibiotic resistance, microbiology

Signal peptidase I is an essential enzyme in bacteria that cleaves signal peptides as the final step of protein export to the periplasm. We have discovered a novel secreted protein from Bacillus subtilils, TasA, whose signal peptide can bind signal peptidase I of E. coli (LepB), but is very inefficiently cleaved. In B. subtilis, the TasA signal peptide is removed a dedicated signal peptidase I called SipW, whose protein sequence is more similar to archaeal signal peptidase I. However, it is unknown why the TasA signal peptide is inefficiently cleaved by LepB, and requires an archaeal, not bacterial signal peptidase for its efficient removal. To answer this question, the student will purify SipW, and develop an enzyme assay to measure its cleavage of signal peptides. Subsequently both SipW and LepB will be compared in enzyme assays using the same substrates (based off the TasA signal peptide sequence). The project will develop insights into how archaeal and bacterial signal peptidases differ in their ability to process signal peptides.

The techniques involved in this study include enzyme assays, protein purification and protein analysis (coomassie gels and Western blots), cloning of genes (PCR, DNA manipulation) and other general molecular biology techniques.

Supervisors: Dr Lucy Shewell, Dr Christopher Day & Prof Michael Jennings

Glycobiology, Biochemistry, Biophysics, Cancer Biology

Approximately half or more of all human proteins carry a carbohydrate moiety through the process of glycosylation and it is well established that one of the universal features of cancer cells is aberrant glycosylation. The changes in glycosylation that occur in cancer cells include loss of expression or excessive expression of certain glycans (carbohydrates attached to proteins or lipids), increased expression of incomplete or truncated glycans, and the appearance of novel glycans. Glycoproteins, therefore, make ideal cancer biomarkers because these molecules are secreted or shed into the circulation from tissues or blood cells allowing them to be detected in the serum. Glycans terminating with the sialic acidNeu5Gc are not expressed at significant levels on healthy human tissues, because humans express an inactive cytidine monophosphate N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH) enzyme, and thus cannot synthesize Neu5Gc. Nevertheless, Neu5Gc-containing glycans are found in human tumour tissues, tumour cells and tumour secretions, and have been proposed as a tumour biomarker.

The Shiga toxigenic Escherichia coli (STEC) Subtilase cytotoxin (SubAB) recognizes α2-3 linked Neu5Gc via its pentameric B-subunit SubB. We purpose-engineered the SubB protein to increase specificity and selectivity for Neu5Gc containing glycans and have demonstrated that this mutant protein, termed SubB2M, recognizes Neu5Gc glycans exclusively and is able to detect Neu5Gc-enriched serum glycoproteins. We showed that SubB2M can detect elevated levels of Neu5Gc in serum samples from patients at all stages of ovarian cancer using only very small volumes of serum (~1μl) via surface plasmon resonance (SPR). SPR is a biophysical technique for measuring the binding of molecules in real-time without the use of labels. This project will investigate whether serum Neu5Gc levels are elevated in patients with other types of cancers compared to normal controls using a SubB2M-SPR assay. This project will also attempt to discover and characterize Neu5Gc-containing cancer biomarkers.

Techniques: SPR, affinity purification, protein gel electrophoresis, western blotting

Supervisors: Dr Lucy Shewell, Dr Christopher Day & Prof Michael Jennings

Molecular Microbiology, Glycobiology

The cholesterol-dependent cytolysins (CDCs) are a family of toxins produced by a number of Gram-positive human pathogens including Streptococcus, Clostridium, Listeria, Bacillus and Gardnerella. These toxins form pores in cholesterol-containing membranes, hence it was thought that cholesterol was the cellular receptor. We have found that the CDCs bind with high-affinity to glycan targets and that these glycans serve as cellular receptors. This project aims to further investigate the glycan binding of several of the CDCs by using molecular modeling to identify key residues involved in binding to the glycan targets. Site-directed mutants of these residues will be generated and analysed using a range of techniques, including surface plasmon resonance (SPR) and cell-based assays, to confirm their role in glycan binding. Identifying key residues of the CDCs involved in glycan binding will provide insight into the function and tropism of these toxins and may assist in the development of inhibitors of these toxins.

Techniques: molecular biology, SPR, affinity and kinetics measurements; cell assays

Supervisors: Dr Chris Day, Prof Joe Tiralongo, Dr Alison Peel & Assoc Prof Daniel Kolarich

Glycomics, glycoproteomics, infection, evolution, zoonotic disease

Animals such as bats are considered prime hosts for zoonotic diseases before they "jump" to humans. Viruses such as influenza or COVID-19 are known to infect different host species, where they can gain novel functions and increase their virulence.

The cell surface of mucosal barriers in the respiratory tract plays a fundamental role in this interplay. This cell surface, but also any body fluid proteins are extensively modified with species specific sugars called glycans. These glycans build a universal language used by cells but also abused by pathogens. Though eukaryotic organisms share one alphabet, evolution made them speak multiple different languages and dialects. We have developed glycomics & glycoproteomics tools to translate these languages and uncover how pathogens learned to speak and interpret glyco-languages between different species. Understanding this relationship is crucial as viral, bacterial and parasite pathogens have developed elaborate strategies to jump between hosts – and many of these strategies involve cell surface glycans. The influenza virus is just one fairly well understood example that uses this strategy.

As part of this project several student projects are available that include 1.) characterisation of the plasma/serum glycome across several vertebrae species; 2.) investigation and understanding cross-species recognition of pathogen adhesins; 3.) novel, cutting-edge science to understand the role of glycosylation and infection in flying foxes;

4.) working in and with an interdisciplinary and international team delivering first-hand knowledge and skills in a variety of biochemical and immunological skills and techniques.

The outcomes of these highly collaborative (across Griffith, national and international partners) projects will provide novel clues how infectious diseases can spread and uncover novel targets to stop their distribution and uncover novel biology in animal species of primary interest for the distribution of zoonotic pathogens. Students will be introduced to biochemistry and immunology laboratory workflows that include (but are not limited to) SDS-PAGE, Western blotting, Proteomics and Glycomics sample preparation, acquisition and data analyses and gain general knowledge in Biochemistry, Glycobiology and Immunology.

Techniques: mass spectrometry, glycomics, proteomics, microarray, Immunoglobulins, protein purification, immunological techniques.

Supervisors: Associate Professor Thomas Haselhorst and Dr Chris Day

Structural Biology, Computational Chemistry, NMR spectroscopy, Microbiology

Project details: The decade long overuse of antibiotics in poultry agriculture and consequently the transferral of antibiotic resistance to humans and the associated health problems underlines the urgent need for novel antibiotic-independent strategies, such as feed supplements (prebiotics) that augment commercial poultry performance and provide food safety. This PhD project aims to develop prebiotic treatment options to reduce the colonisation of Campylobacter jejuni in the chicken intestinal tract. Structural and biophysical investigations of glycan-glycan interactions followed by monitoring the bacterial load in chickens and potential cross-contamination into chicken will form the main part of the thesis. Expected outcomes will be the development of a potentially commercially viable non-antibiotic treatment option for poultry farmers in Australia.

Techniques: Structural investigations on Glycans in solution with NMR spectroscopy, biophysical methods and molecular modelling, developing of a virtual glycan array approach, monitoring Campylobacter bacterial strains.