Jennings Group

Supervisors: Dr Freda Jen and Prof Michael Jennings

National Health and Medical Research Council (NHMRC) funded research project

This project aims to develop a vaccine to protect against bacterial infection cause by non-typeable haemophilus influenzae and Neisseria gonorrhoeae. The bacteria that cause gonorrhoea (Neisseria gonorrhoeae), middle ear infections and exacerbations of chronic obstructive lung disease (non-typeable haemophilus influenzae) have become multi-drug resistant. These diseases are a major health and economic burden. In the absence of new drugs, a vaccine to prevent these diseases has emerged as a major unmet need in human health. We aim to develop a new vaccine that targets a bacterial-specific sugar that we have discovered is the Achilles heel of these bacteria.

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 John Atack and Prof Michael Jennings

Many host-adapted bacterial pathogens contain phase variable methyltransferases, which control expression of multiple genes, and known as phasevarions (phase variable regulons).

Our studies will investigate the specificity, and the mode of gene regulation through differential methylation by these phase-variable methyltransferases. We will clone and over-express newly identified methyltransferases to determine their recognition sequences. We have identified a number of genes in both human and animal pathogens that are differentially expressed in several phasevarions: we will investigate how methylation alters regulation of these genes. We will investigate if these genes are regulated directly or indirectly, and determine the effect of removing any recognition sequences from the promoters of these genes. This project will use protein over-expression and purification methods to allow us to study these methyltransferases in vitro. Surface plasmon resonance will be used to conduct kinetic measurements, and gel-shift assays (EMSA) will be used to study binding affinity and ability.  Reporter constructs will be made to observe the effect of methyltransferase phase-variation on the level of expression from individual promoters.

Supervisors: Dr John Atack, Prof Michael Jennings

Non-typeable Haemophilus influenzae is a major human adapted pathogen and causes a number of acute and chronic diseases of the human respiratory tract, including middle ear disease, otitis media (OM) in children, exacerbations in chronic obstructive pulmonary disease (COPD) in the elderly, and pneumoniae. Invasive disease (meningitis and septicaemia) caused by NTHi is increasing annually and is a particular problem in infants under 1 year of age, where the mortality is close to 20%. Antibiotic resistance is increasing each year, resulting in NTHi being on the World Health Organisations list of priority pathogens. There is no currently licensed vaccine available for NTHi. Vaccine design is a problem for NTHi as individual strains show high genetic diversity, and many antigenic proteins are phase-variable – their expression is randomly and reversibly switched on or off. If a vaccine target is able to randomly turn off, the vaccine would lose effectiveness.

This project will: 1) determine the best possible combination of conserved protein antigens to include in a universal NTHi vaccine from both current and putative vaccine candidates; 2) study the role and regulation of a number of uncharacterised NTHi proteins that show high sequence and strain conservation; and 3) determine if know proteins that have been discounted from use in vaccines as they are phase-variable can be used in vaccines as their expression is critical for certain disease stages or colonisation of particular host niches.

Supervisors: Dr Freda Jen & Prof Michael Jennings

Molecular Biology, Molecular Microbiology

Many pathogenic bacteria modify proteins after translation. Some of these modification are on proteins on the surface of the bacteria that are key in understanding host:pathogen interactions and in developing vaccines.Recent advances in Neisseria meningitidis have identified post-translation modification of virulence factors with glycans and phosphorylcholine. Some key post-translation modification pathway components have also been identified, but the picture is incomplete. The aim of this project will be to conduct transposon mutagenesis and screen for loss of key post-translation modifications. In this way novel post-translation modification pathway components will be identified and investigated.

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 Yaramah Zalucki & Prof Michael Jennings

Microbiology, protein purification and protein structure analysis

Codon usage is biased at the 5’ end of secretory genes, with the highest percentage of non-optimal codons found compared to any region of the genome. The exact role for the observed bias is unknown. We have strong evidence that changing signal sequence non-optimal codons to the most optimal codon in the synonymous codon family results in structural changes in the mature region of the protein. However, detailed analysis on the exact nature of the structural change has not been done. In this project, we will alter the codon usage in the signal sequence of two small proteins (>20 kDa), purify them and determine any structural differences by NMR and other techniques. Determining any structural change from altering signal sequence codon usage will be a novel find, and important in the field structural biology and how proteins are targeted for protein export. The techniques used in this project will involve cloning, PCR, protein purification, NMR analysis, protein analysis (Western and coomassie staining techniques, DNA sequencing and phenotypic analysis of any mutants made.

Supervisors: Dr Yaramah Zalucki & Prof Michael Jennings

Microbiology, molecular genetics and antibiotic resistance

Antibiotic resistance is in N. gonorrhoeae is a major public health concern. One of the major determinants of resistance is the MtrCDE efflux pump, which exports compounds from the inner membrane to the extracellular milieu. Expression of the efflux pump is controlled by a repressor, MtrR, and a conditional activator, MtrA. We have identified a number of novel promoter mutations in the mtrCDE, whose role in increasing expression of the efflux pump has not been characterised. In this project, we will place these mutations individually, and in conjunction with known promoter mutations, to measure their effect on antibiotic resistance in N. gonorrhoeae. These mutations will also be placed in the context of a promoter-less lacZ fusion, to measure their effect on the strength of the promoter. We will also look at how these mutations influence the binding of the two known regulators of the efflux pump, MtrA and MtrR to the promoter region. The techniques used in the project will involve cloning, PCR, MIC assays, RNA extraction and other general microbiology and 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 Freda Jen, Prof Ifor Beachman, Prof Michael Jennings

Research area: Molecular biology

Project details: Guinea pig serum was serendipitously discovered to be an effective anti-tumour agent over 65 years ago with respect to transplantable lymphomas. It was found that the agent responsible for this remarkable anti-tumour property was L-asparaginase in the sera. There is much evidence that the anti-tumour properties of L-asparaginases are due to depletion of exogenous L-asparagine on which the tumour cells depend for growth, being essentially auxotrophic for L-asparagine. However, the identification of the guinea pig serum L-asparaginase has been problematical since its discovery. L-asparaginase was only found in guinea pig serum and not in the sera of other species investigated, including mouse and rat. The aim of this project is to determine the genetic and biochemical origins of the liver and serum enzymes in guinea pig and in those related species which also have both isozymes.

Techniques: Molecular biology

Supervisors: Dr Freda Jen, Prof Michael Jennings

Research area: Molecular biology

Project details: Neisseria gonorrhoeae is a host adapted bacterial pathogen that infects male urethral and female cervical and causes sexually transmitted disease gonorrhoea. Lipooligosaccharides (LOS) is one of the major virulence factors of N. gonorrhoeae and is composed of multiple possible glycoforms due to the phase variation (high frequency of ON/OFF switching of gene expression) of the genes involved in LOS biosynthesis. Gonococcal LOS structure is caped with a N-acetyl-5-neuraminic acid (Neu5Ac). However, N. gonorrhoeae cannot synthesize the CMP-Neu5Ac required for LOS biosynthesis and must acquire it from the host. In most of the LOS biosynthetic pathway, the core-oligosaccharide is assembled in the cytoplasm. Previously, the alpha-2,3-sialyltransferase, Lst of N. gonorrhoeae was proposed to be a surface exposed outer membrane protein. In our unpublished study, we investigated the cellular location of Lst and all our results indicated that Lst is located inside of the cell suggesting that there must be a transport system or a trans-sialidase transport CMP-Neu5Ac from the host to inside of cells. The aim of this project is to discover a novel CMP-Neu5Ac transporter in pathogenic Neisseria.

Techniques: Molecular biology