Department Of Microbiology 2021 Honours Programs In .

Professor Mariapia uTELEPHONE 61 3 9905 6162OFFICERoom 380, 15 Innovation Walk (Building pia-degli-espostiProfessor Mariapia Degli-EspostiDr Iona SchusterTWO VACANCIESDr Christopher AndoniouMCMV, Immunity and AgeingViral Infection and AutoimmunityProfessor Mariapia Degli-Esposti,Dr Christopher Andoniou and Dr Iona SchusterProfessor Mariapia Degli-Espostiand Dr Iona SchusterWith average life spans increasing we face novel challengesin managing age-associated health decline. A key factorin maintaining overall health is a well-functioning immunesystem. However, as we age the immune system becomesless functional with reduced production of T and B cells, aswell as changes in the quality and composition of respectivememory subsets. How immunological challenges such as viralinfections impact and shape the aging immune system is notwell understood. In this regard, we are particularly interestedin cytomegalovirus (CMV), a virus that is never fully clearedand remains with its host life-long. CMV infection causes thegradual expansion of certain CD8 T cell memory populations,a phenomenon that has been linked with both limiting andenhancing immune responses to other challenges. Usingthe well-established mouse model of murine CMV (MCMV)infection we aim to examine the impact of this in immunecompartments during ageing. Approaches will include highparameter multicolour flow cytometric analysis of immunecell subsets as well as bulk and single cell assays of immunefunctionality. The ultimate aim is to gain a better understandingof how CMV infection shapes the immune system over timeand how this affects the aging immune system.Viral infections have long been suspected to play a role inautoimmunity, with members of the herpes virus family suchas cytomegalovirus (CMV) specifically implicated. We use themodel of murine CMV, a natural pathogen of the mouse with highsimilarity to its human counterpart, to investigate the mechanismsunderlying the generation of protective antiviral responses andhow these correlate with the onset of autoreactive responses. Wehave shown that a strong anti-viral T cell response generated inthe absence of certain immune regulatory mechanisms improvesviral control. However, once the virus is controlled, this stronganti-viral response leads to increased generation of auto-specificimmune responses resulting in a loss of tissue function. Theautoimmune disease generated represents the best availablemodel of the second most common autoimmune disease ofman, Sjogren’s Syndrome, a condition that affects overall healthby severely compromising exocrine gland function. Experimentalapproaches will include in vitro and in vivo techniques usingwildtype as well as gene-targeted mouse strains. Techniquesinclude the preparation of different tissues for histological analysisof tissue pathology, characterization of infiltrating cell types, andassessment of changes in tissue architecture. Furthermore,we use flow cytometry to characterize and quantify immunecell populations isolated from different tissues at various timespost infection. The goal of this project is to further extend ourunderstanding of the processes and mechanisms underlying thegeneration of autoreactive immune populations in the contextof viral infection.2021 Microbiology Honours Projects 5

6 2021 ects

Dr Terry EPHONE 61 3 9902 9216OFFICERoom 231, level 2, 19 Innovation Walk (Building 76)Dr Terry Kwok-SchueleinTWO VACANCIESThe oncogenic type IV secretion system (T4SS) of H. pylori (left) is activated uponH. pylori-host interaction (right).The Molecular Mechanisms by WhichHelicobacter pylori Causes Stomach CancerHelicobacter pylori is a Gram-negative gastric bacterium thathas co-evolved with humans for more than 50,000 years. Itcolonises the stomach of over 50% of the world’s population,making it one of the most prevalent human pathogens. It is acausative agent of severe gastric diseases including chronicgastritis, peptic ulcer and stomach cancer. H. pylori has beenclassified as a Group I (high-risk) carcinogen.Highly virulent strains of H. pylori harbour a type IV secretionsystem (T4SS), a secretion machinery that functions as a“syringe” for injecting virulence proteins and peptidoglycaninto the host cell. We discovered that CagL, a specialisedadhesin present on the surface of the H. pylori T4SS, binds tothe human integrin α5β1 receptor on stomach lining cells.Thisbinding activates the T4SS and hence the secretion of virulencefactors including the highly immunogenic and oncogenic protein,CagA, into stomach cells. ‘Injected’ CagA then interacts withhost signalling molecules and triggers activation of a suiteof host responses. Interestingly, our recent findings suggestthat CagL can also directly modulate host cell functions. Theprecise mechanisms by which CagL functions both as ahost-activated sensor of the H. pylori T4SS and as a directactivator of aberrant host responses remain to be fullyunderstood.Our team uses multi-disciplinary state-of-the-art approachesto study the molecular mechanism of H. pylori type IV secretionand H. pylori-host interactions. We aim to understand themolecular basis of how H. pylori induces stomach cancer, withthe ultimate goal of providing knowledge for a better treatmentand/or prevention of H. pylori-associated stomach diseases.Projects are available to address the following key questions:i How does H. pylori trigger inflammation and carcinogenesisthrough the virulence functions of CagL and CagA?i Can cagL and cagA genotypes predict gastric cancer riskand therefore help pinpoint cancer-prone patients for earlytreatment?i How do CagL function as a host-activated sensor duringtype IV secretion?i How do CagL and CagA modulate host cell signallingduring chronic H. pylori infection?i Can we utilise the type IV secretion system of H. pylorifor delivery of therapeutic proteins?The available honours projects will enable one to gainexperience with the important techniques of molecularcloning and mutagenesis, bacterial culture, eukaryotic cellculture techniques, mouse infection models, CRISPR, RNAi,immunostaining, Western blotting, ELISA, confocal laserscanning microscopy, live cell imaging, etc. Someone who isenthusiastic in learning about the exciting secrets of bacteriahost interactions, infectious cancer biology and bacterialpathogenesis is strongly encouraged to apply.2021 Microbiology Honours Projects 7

Professor Jian LiEMAILjian.li@monash.eduTELEPHONE 61 3 9903 9702OFFICERoom 220, 19 Innovation Walk (Building liProfessor Jian LiDr Mohammad AzadLaboratory of Antimicrobial SystemsPharmacologyMy lab focuses on antimicrobial discovery andpharmacology against Gram-negative ‘superbugs’(namely Pseudomonas aeruginosa, Acinetobacter baumannii,and Klebsiella pneumoniae). There has been a markeddecrease in the discovery of novel antibiotics over the lasttwo decades. As no novel class of antibiotics will be availableagainst Gram-negative ‘superbugs’ in the near future, itis crucial to optimise the clinical use of ‘old’ polymyxinsusing systems pharmacology and to develop novel, saferpolymyxins and innovative therapeutics.My major research programs include:(1) optimising clinical use of polymyxins and their combinationsusing K/PD/TD) and systems pharmacology;(2) elucidation of mechanisms of antibacterial activity, resistance,and toxicity of polymyxins; and(3) discovery of novel, safer polymyxins and innovativetherapeutics against multidrug-resistant (MDR) Gramnegative bacteria.My lab is funded by the US National Institutes of Health (NIH)and Australian NHMRC.8 2021 Microbiology Honours ProjectsTHREE VACANCIESDr Yan ZhuDr Sue C. NangDeciphering the Mechanisms ofPolymyxin Resistance in P. aeruginosaUsing Computational BiologyProfessor Jian Li and Dr Yan ZhuP. aeruginosa is a critical threat to human health worldwide.Polymyxins are a group of last-line antibiotics against Gramnegative ‘superbugs’, including MDR P. aeruginosa.We are integrating genomics, transcriptomics, proteomics,metabolomics, and lipidomics to systematically examinebacterial responses to polymyxins and their combinations.This project aims to:(1) construct a genome-scale model of metabolism and geneexpression (ME model) for P. aeruginosa using literature andour multi-omics data;(2) use the constructed ME model to simulate cellularresponses to polymyxins; and(3) predict key genes and pathways contributing to polymyxinresistance and validate their functions with our comprehensivemutant library.This multidisciplinary project will, for the first time,characterise the complex interplay of signaling, regulationand metabolic pathways involved in polymyxin resistance,thereby optimising polymyxin chemotherapy in patients.

Phage-Antibiotic Therapyin the Postantibiotic EraPulmonary Toxicity of Novel PolymyxinCombination TherapiesProf Jian Li and Dr Sue C. NangDr Mohammad Azad and Professor Jian LiAntimicrobial resistance has become one of the greatestglobal threats to human health and pandrug-resistant (PDR)Klebsiella pneumoniae has been identified by the WHO asone of the 3 top-priority pathogens urgently requiring noveltherapeutics. These ‘superbugs’ cause life-threateninginfections, particularly in the critically ill, and polymyxinsare often used as the last option. Worryingly, increasingemergence of polymyxin resistance highlights the urgencyto develop novel therapeutics to treat PDR K. pneumoniae.Bacteriophage (i.e. phage) have recently attracted substantialattention as a potential alternative against PDR bacterialinfections; however, resistance to phage therapy (includingcocktails) in K. pneumoniae can rapidly develop. Fortunately,phage resistance may restore bacterial susceptibility to certainantibiotics and therefore, optimal phage-antibiotic combinationtherapy provides a superior approach to fight against thesesuperbugs. Contemporary antimicrobial pharmacology playsa critical role in optimizing antibiotic dosage regimens, butlacks systems and mechanistic information. Importantly,antibiotic dosing strategies cannot be easily extrapolatedinto phage therapy, mainly due to the complex disposition,host specificity and self-replication of phages. As the optimalphage-antibiotic combination and dosage regimens alsodepend on the dynamics of infection and host responses,innovative strategies incorporating systems pharmacologyand host-pathogen-phage-antibiotic interactions havea significant potential in optimising phage-antibioticcombinations. This project will employ cutting-edge systemspharmacology to generate urgently needed information forrationally optimising novel phage-antibiotic combinations inpatients.Current dosing recommendations of parenteral polymyxinsare suboptimal for treatment of respiratory tract infectionsdue to poor drug exposure at the infection site. Moreover,nephrotoxicity is the dose-limiting factor and can occur inup to 60% of patients. Pulmonary delivery of polymyxinsas monotherapy and in combination with other antibioticshas offered a great promise for bacterial eradication in therespiratory tract. However, we have shown that polymyxinslocalise in mitochondria of human lung epithelial cells andactivate multiple apoptotic pathways. This multi-disciplinaryproject aims to investigate the effect of polymyxins and theirsynergistic combinations with other key classes of antibiotics onhuman lung epithelial cells, using fluorescence activated cellsorting (FACS), metabolomics, proteomics, transcriptomicsand cutting-edge imaging techniques. This project will providethe much-needed pharmacological information for safer andmore efficacious use of polymyxin inhalation therapy againstlife-threatening lung infections.2021 Microbiology Honours Projects 9

Professor Trevor LithgowEMAILtrevor.lithgow@monash.eduTELEPHONE 61 3 9902 9217OFFICERoom 233; Room 252, 19 Innovation lithgow-lab/homeProfessor Trevor LithgowDr Rhys DunstanMapping Diversity of Bacteriophagewith Genomics and Structural BiologyDr Rhys Dunstan and Professor Trevor LithgowBacteriophages (phages) dominate numerous ecosystems,and are most often isolated from water sources. Recently,phage discovery has been accelerated using new generationsequencing strategies: (i) viral metagenomics, in which complexphage populations are harvested en masse from environmentalsources, and (ii) data mining archives of bacterial genomesequence information, to detect embedded prophage andphage-related sequences. As powerful as they are, thesebioinformatics-based approaches do not yield virions forwet-lab analyses. Understanding the diversity present inthe architecture and the biology of phages requires isolatingand characterizing active virions.10 2021 Microbiology Honours ProjectsTWO VACANCIESDr Christopher StubenrauchThis project aims to assess phage diversity through a classicalenvironmental microbiology approach: using water samplescollected from diverse locations around the world, the phagestherein will be concentrated and plated on a lawn of bacteria.Attention will be focused on phages that infect the pathogenKlebsiella pneumoniae or a closely related plant commensalKlebsiella pseudopneumoniae that looks set to emerge as animportant pathogen. Using a combination of electron microscopyto assess virion morphology, and bioinformatics for comparativegenomics and protein identification, the project wouldclassify, catalogue and compare the various phage isolated.Finally, a systematic assessment of cocktails of the variousphage will be undertaken to determine killing efficacy forfuture therapeutic work.

Understanding how bacteria piecetogether αβ-barrel proteinsDr Christopher Stubenrauch and Professor TrevorLithgowWithin crowded biological systems, like a bacterial cell,folding factors are essential for ensuring correct proteinassembly on a biologically relevant time scale. Traditionally, 3classes of outer membrane proteins have been recognisedthat each follow distinct and well-characterised assemblypathways: β-barrel proteins, lipoproteins, and secretins.While the majority of proteins fall clearly within the confines ofthese three protein groups, a complex protein class referredto as αβ-barrel proteins does not.While the importance of αβ-barrel proteins in AMR anddisease is widely appreciated, their mechanisms of assemblyare not. This project aims to determine which folding factorsassist in the assembly of αβ-barrel proteins that lead bacteriato becoming such successful pathogens and involvesthe use of a range of general molecular microbiologicaltechniques, MIC analyses, western immunoblotting, andpulse chase assembly assays.Members of the αβ-barrel protein family can readily befound in most Gram-negative bacteria and are one of themost important virulence factors that promote antimicrobialresistance (AMR) and bacterial pathogenesis. The modelbacterium we study, Escherichia coli, houses up to 6different αβ-barrel proteins, including the protein TolC – thepromiscuous outer membrane component of a range ofantimicrobial efflux pumps and type 1 secretion systems.2021 Microbiology Honours Projects 11

Professor Dena LyrasEMAILdena.lyras@monash.eduTELEPHONE 61 3 9902 9155OFFICERoom 152, 19 Innovation Walk (Building 76)Professor Dena LyrasDr Yogitha SrikhantaTWO VACANCIESDr Steven MiletoDr Milena AwadA/Professor Priscilla JohanesenUnderstanding the Host Repair Responseto Clostridioides difficile InfectionAnti-sporulation strategies targetingspore-forming pathogensProfessor Dena Lyras and Dr Steven MiletoProfessor Dena Lyras and Dr Yogitha SrikhantaGastrointestinal infections often induce epithelial damagethat must be repaired for optimal gut function. Whileintestinal stem cells are critical for this regeneration process,how they are impacted by enteric infections remains poorlydefined. We recently investigated infection-mediateddamage to the colonic stem cell compartment and howthis affects epithelial repair and recovery from infection,using the pathogen Clostridioides difficile, which induces aspectrum of diarrheal diseases mediated by two exotoxins,TcdA and TcdB. These toxins share sequence andstructural homology but may contribute to disease severityunequally. We have shown that infection disrupts mouseintestinal cellular organisation and integrity deep into theepithelium, and exposes the otherwise protected stem cellcompartment. This disruption of the gut epithelium occursprimarily through TcdB-mediated damage and altered stemcell signalling and function, resulting in a significant delayin recovery and repair of the intestinal epithelium. However,the mechanisms of stem cell intoxication, and the effectsof different TcdB variants on stem cell function, remainunknown. Animal models of infection will be used, togetherwith specific C. difficile mutants and strain variants, tostudy virulence factors and host interactions, allowing usto gain a mechanistic understanding of how these bacteriainteract with, and damage, the host gut. We will also usevarious tissue culture systems to examine the specificmolecular mechanisms that lead to TcdB-mediated stemcell dysfunction, including stem cell-derived organoids.Spore-forming bacteria include the devastating human andanimal pathogen Clostridioides difficile, the food spoilagepathogen Bacillus cereus and the agent for bioterrorismBacillus anthracis. Spores are the infectious particles ofthese pathogens and their resistant and unique structuremakes them difficult to eradicate. Their persistenceproperties enable the spread of disease, resulting infatalities and economic devastation in environments suchas health care settings, the food industry and publicspaces in the case of weaponised anthrax. Despite thesemajor problems, there are no strategies to prevent sporeproduction. C. difficile is of considerable medical interestdue to the high disease burden and global challenge ofmanaging the consequences of infection. C. difficile sporesare highly resistant to antibiotic treatment and disinfectantsand are responsible for facilitating disease transmissionand recurrent infection. Our published work has shownthat cephamycins, a group of beta-lactam antibiotics, caninhibit spore formation of C. difficile epidemic isolates byblocking the activity of spore-specific penicillin bindingproteins. Of clinical relevance, co-treatment of mice with thecephamycins and the standard-of-care C. difficile treatmentvancomycin, which is ineffective against spores, preventedrecurrent infection. This project will extend our C. difficilespore formation inhibition studies to other spore-formingbacteria, including both pathogens and commensals, towork towards better drug delivery strategies for treatmentof diseases caused by these pathogens.12 2021 Microbiology Honours Projects

Interrogating the effects of thehuman host protease plasminogen onClostridioides difficile infectionUnderstanding the Role of BacterialStructures in the Transfer of AntibioticResistance Genes During ConjugationProfessor Dena Lyras and Dr Milena AwadProfessor Dena Lyras, Dr Yogitha Srikhanta andA/Professor Priscilla JohanesenThe human and financial cost of Clostridioides difficile globalepidemics is substantial and alarming, with C. difficile listedas the number one antibiotic-resistant bacterial threat in theUSA. A key driver of C. difficile infection relates to the abilityof this bacterium to form spores, an inert and highly robustcell type, which allows survival of the bacterium in hostileenvironments. Spores initiate and transmit disease, andcontribute to disease relapse, whereas the vegetative cell formof C. difficile colonises the gut and produces potent toxinsthat cause disease. We have found that the host proteaseplasminogen migrates to the gut following toxin mediateddamage and that C. difficile spores, but not vegetative cells,recruit plasminogen to their

enrolled through Microbiology. Microbiology Coursework The coursework conducted within the Department of Microbiology consists of short courses termed colloquia, a statistics course and a seminar series. BSc students need to complete two colloquia, BBiomedSci students complete o