Temporal changes to cellular bioenergetics, cholesterol metabolism, innate immune responses and microbiota during infection

Bacterial infections are major cause of morbidity and mortality in children under 5 years of age, especially in Low and Middle Income Countries (LMIC). In industrial countries, bacterial infections mainly affect the elderly (both in the community and in hospitals) and the immunocompromised (e.g. patients undergoing chemotherapy or infected with HIV).

While the ability to treat bacterial infections with antibiotics is arguably the most important achievement of modern medicine, the extensive misuse and overuse of antibiotics in human medicine and livestock has contributed to the rise and spread of antimicrobial resistance (AMR). The threat of antibiotic resistance is well reported, being sighted in many newspaper articles, high-level government reports and campaigns in recent years. Already responsible for 700,000 deaths per year, antibiotic-resistant bacteria are predicted to cause a staggering 10 million deaths by 2050. Whilst there has been a dramatic rise in the spread of multi-drug resistant bacteria, the discovery rate of new antibiotics has tumbled over the past decades, with only a few new approved drugs reaching the clinic. This highlights the urgent need to complement traditional drug discovery routes with new approaches to treat bacterial infections. Importantly, unintended targets of antibiotics include the normal, beneficial, gut bacteria (microbiota). Accordingly, we need to employ innovative approaches to treat bacterial infections, which minimise both selective pressure-promoting emergence of resistance and impact on the microbiota. The development of effective and novel control measures requires a systematic understanding of the biology of the disease, particularly the complex interactions between bacterial pathogens and their hosts in the context of the gut microbiota. By definition, this type of research relies on utilisation of robust and physiologically relevant animal models.

Citrobacter rodentium is mouse specific extracellular pathogen, which shares an infection strategy with human pathogenic E. coli strains (for example E. coli O157). Being a natural mouse pathogen, C. rodentium provides an ideal model to study infections with gut pathogens in the natural host and in the context of the gut microbiota. C. rodentium causes a self-limiting infection and triggers robust immune responses, proliferation of epithelial cells and displacement of the normal gut bacteria which mimic the characteristics observed during human infection with pathogenic E. coli. Recently, by applying state-of-the-art molecular methods to study the interaction of C. rodentium with cells that line the gut we found that the infection causes drastic changes to energy production and central metabolism in the host cells, in what seems to be an attempt to dampen inflammation. While previously the focus has been on immune cells, it is only now that we are starting to realise that controlling metabolism in epithelial cells is another key frontier in host-pathogen interactions.

In this project we aim to obtain an unprecedented molecular resolution of host-pathogen interactions over the duration of infection, in the context of a whole organism. Our proof-of-concept studies have already shown deregulation in the production and release of cholesterol during C. rodentium infection. In this project we will use wild type and mutant C. rodentium strains as well as wild type mice and mice deficient in a key controllers of cholesterol metabolism. We will study changes in metabolism over time, from infection to recovery, and match these changes to the presence of specific gut microbiota. The use of selected mutants, both bacterial and host, will allow us to unravel the molecular processes involved. We believe our pioneering approach would lead to conceptual shifts in understanding of host-bacterial infections and thus open new avenues for the development of novel treatment strategies for bacterial infection.

With only few exceptions, most studies of pathogen-host interactions report static observations of a single point in time. Here, we will use the Citrobacter rodentium model to investigate the temporal consequences of bacterial infection on metabolism and immune responses in intestinal epithelial cells (IECs), as well as dysbiosis. IECs will be isolated from infected mice at various days post infection; uninfected mice will be used as a control. Proteins will be extracted from IEC pellets, digested with trypsin, the resultant peptides labelled using the isobaric mass tag (TMT), pooled, fractionated offline and analysed by nanoscale online LC-MS. Multiple bioinformatics platforms and robust statistics will be used for data analysis. We will employ Q-RT-PCR, ELISA and WB, as well as metabolomics and lipidomics and functional assays, to cement the proteomics data and bioinformatics predications.

We have recently found that EspO impacts the expression of IL-22-regulated proteins 8 days post infection (e.g. iNOS, Lcn2, Reg3, S100). We will investigate how EspO, and its partner protein Hax-1, modulate signalling downstream of the IL-22 receptor (as well as dysbiosis). We will infect WT mice with an espO mutant and use commercially produced IEC-specific hax-1 KO mice to investigate the role Hax-1 plays in C. rodentium infection and host inflammation.

We will investigate the role cholesterol biogenesis and efflux plays in pathogen-host interactions and inflammation using small molecules that impact on the activity of the transcription factors Srebp2 (regulating expression of genes involved in cholesterol biogenesis) and LXRs (regulating expression of gene involved in cholesterol efflux). We will use transgenic mice that either constitutively express LXR_alpha, or LXR_alpha/beta KO, specifically in IECs (using in-house bred control mice). Using the above approaches we will determine changes in protein abundance and link this to infection, inflammation and dysbiosis.

Bacterial infections (e.g. E. coli, Shigella and Salmonella) are a major cause of morbidity and mortality in children less than five years of age, especially in Low and Middle Income Countries (LMIC). In industrial countries, infections with enterohemorrhagic E. coli (EHEC, e.g. E. coli O157), Campylobacter and C. difficile are of major public heath concern. No vaccines are currently available to protect high-risk populations from these infections. Moreover, dramatic environmental and demographical changes together with movement of people and food across continents are coupled with the return of old pathogens and the emergence of new opportunistic infections. Together with these processes there has been a surge in multi-drug resistant bacteria and yet the discovery rate of new antibiotics has tumbled over the past decades, with only a few new approved drugs reaching the clinic.

As the human population ages, non-communicable chronic diseases (e.g. cardiovascular, cancer and diabetes) are becoming a dominant global health concern. Recently, it has become apparent that keeping the gut microbiota healthy is essential for human health. Importantly, unintended targets of antibiotics include commensal bacteria (microbiota). Faced with the spread of antibacterial resistance (ABR) there is an urgent need to complement the traditional antibiotic discovery routes with the development of innovative approaches to treat bacterial infections, which minimizes both selective pressure that promotes resistance and the impact on the microbiota. The development of effective and novel control measures requires a systematic understanding of the biology of the disease, particularly the complex interactions between bacterial pathogens and their hosts in the context of the microbiota. By definition, this type of research relies on utilisation of ROBUST and PHYSIOLOGICALLY RELEVANT animal models.

Citrobacter rodentium is an extracellular, mouse-specific pathogen that is frequently used to model infections with the human pathogens EHEC and enteropathogenic E. coli. C. rodentium reflects physiological host-pathogen interactions in immunocompetent mice and in the presence of the endogenous microbiota. My lab has pioneered the use of C. rodentium to study bacterial pathogenesis (PMID: 8945583) and host-pathogen immune interactions in the gut (PMID: 10338516). Over the last 10 years the use of this model has expanded exponentially and globally and has been utilized for the study of basic processes of mucosal immune responses, leading to fundamental discoveries and high profile publications (e.g. PMID: 22002608, PMID: 24581500, PMID: 28146477). Importantly, despite the growing appreciation of the role cellular metabolism plays during host-pathogen interactions, our understanding of changes to the metabolic networks of host cells during infection, particularly in intestinal epithelial cells, is incomplete. In this proposal we aim to use C. rodentium as a model to fill this fundamental gap in knowledge.

In this project we will use a robust and physiological animal model to obtain a holistic understanding and dynamic vision of pathogen-host interactions on mucosal surfaces, with emphasis on host metabolism, inflammation and the microbiota. As such, the project is perfectly aligned with the main IIB's remit: Basic and translational research into pathogens implicated in human infectious diseases; Research unravelling the complexities of the human immune system in health and disease; and Global health research that addresses the inequalities in health that arise particularly in poorly resourced countries. In particular, the project would benefit those interested in infection, immune responses, ABR, and the microbiome.

The project offers an important added value as C. rodentium infection impacts on processes frequently affected in chronic diseases and cancer (e.g. cholesterol metabolism and cell proliferation).