The cellular mechanisms underpinning the host restriction of Salmonella Typhi

The bacterial pathogen Salmonella Typhi causes a severe systemic disease in humans called typhoid fever, which is of major global importance and results in over 27 million cases of disease and 200,000 deaths each year. S.Typhi is a host-adapted pathogen that is exclusively restricted to humans but the disease mechanisms governing this host specificity are unknown.

S.Typhi initiates typhoid fever by injecting virulence proteins into human host cells to direct uptake and replication within an intracellular membrane-bound compartment called the Salmonella-containing vacuole (SCV). S.Typhi is incapable of establishing infection in mouse cells where the mammalian Rab32 GTPase localises to the SCV and directs the pathogen for degradation. When Rab32 is eliminated from cells using genetic engineering S.Typhi survives within a mouse. This shows that Rab32 is critical for the pathogen's strict host-specificity. The Rab GTPase family (~60 members) control cellular communication pathways by recruiting specialised 'effector' proteins to membrane-bound compartments. How mouse Rab32 destroys S.Typhi, and through which effectors, is unknown. Strikingly, human Rab32 localises to SCVs in infected human cells where the pathogen survives and establishes infection. This demonstrates a critical difference in the mouse and human Rab32 pathways. How S.Typhi survives the action of human Rab32 and its cognate effectors is not understood.

Fundamental to Rab function is the membrane to which they are anchored but studying this presents formidable challenges. I propose to build a new experimental reconstitution approach that tackles this very important problem by focusing on the key relationship between Rabs and their membrane. I will engineer membrane-bound particles displaying host-specific Rab32 that will mimic SCVs in mouse and human cell-free extracts. In this way, I will capture and identify the mystery Rab32 effectors, and understand how they operate fundamentally in the physiological membrane environment. SCV proteomics and infection-based screens will form complementary approaches for identifying the pivotal Rab32 effectors. The role and regulation of the Rab32-effectors and their interaction with intracellular S.Typhi will be determined during infection of mouse and human cells.

Resolving the cellular mechanisms of S.Typhi's host restriction will reveal pivotal cell biology underlying typhoid, and has the potential to speed the development of our anti-infectives arsenal and broaden the scope for therapeutic intervention. Furthermore, the powerful reconstitution system would be applicable to any Rab GTPase combination making it an effective tool for multiple research avenues in the future addressing human diseases.

The human-restricted bacterial pathogen Salmonella Typhi causes a severe systemic disease of global importance called typhoid fever. S Typhi initiates infections by directing intracellular replication within Salmonella-containing vacuoles (SCVs). S.Typhi is incapable of establishing infection in animal cells where the pathogen is killed. I want to understand the cellular mechanisms behind S.Typhi's destruction in animal cells and the pathogen's human host restriction.

The small GTPase Rab32 localises to SCVs where it drives S.Typhi degradation in mouse but not human macrophages. This shows Rab32 determines host-specificity and reveals a vital difference in Rab32 pathways. Rabs recruit cognate mammalian effector proteins to direct membrane transport but how mouse Rab32 destroys S.Typhi, and through which effectors, is unknown, as is the reason why human Rab32 is ineffective. Understanding Rab32 would unveil a new pathogen killing mechanism and provide a molecular key for combating S.Typhi in human cells.

Fundamental to Rab function is their cooperation with the membrane to which they are anchored but studying this presents formidable challenges. I aim to build a new approach that focuses on the key relationship between Rabs and their membrane to unveil disease mechanisms. By immobilising phospholipid bilayers on silica microspheres I will engineer membrane platforms anchored with host-specific Rab32 that mimic SCVs. Rab32 platforms will enable me to reconstitute the operation of Rab32 in mouse and human cell extracts. I will capture and identify the pivotal Rab32-machinery, characterise its regulation and cooperation with lipids, and reconstitute processes underlying S.Typhi degradation. In parallel, Rab32 effectors and pathway components will be identified via SCV proteomics and infection-based screens. Cell biology studies will dissect the Rab32 network, and resolve cellular and virulence mechanisms that are decisive in the host restriction of S.Typhi.

Impact on Research Staff
MRC NIRG support will give me the resources to launch my independent career, recruit research staff, and generate an experimental foundation that will enable me to publish in peer-review journals, obtain future grant support and expand my new group. Building this dynamic and creative research environment will require the training of outstanding microbiologists and cell biologists at Cambridge University that will augment the expertise in my group and supply the principle investigators of the future.

Impact on Society
My research will benefit society by addressing the bacterial pathogen S.Typhi, which represents a considerable global health burden that causes ~27,000,000 infections per year and 200,000 deaths worldwide. This threat is exacerbated by the inexorable rise in multidrug resistance, and the stalled development of antimicrobials and vaccines, which impede current prevention strategies. Society is still absolutely reliant on antibacterial drugs discovered decades ago, a remarkable and increasingly ominous situation. To identify novel therapeutic strategies it is vital that cutting edge combinatorial biology is applied to uncover and understand the cell components and pathways, the hijack mechanisms, that underlie bacterial pathogenesis. Studying S.Typhi has remained challenging and has perhaps been neglected due its human host specificity so developments like the recent generation of engineered S.Typhi animal models promises to capitalise on new cell biology breakthroughs and speed the development of new medicines. Part of combating disease is generating public awareness. By learning about my research the public become aware of the science behind the human health threats posed by S.Typhi. Such an understanding is important in seeking to augment research support and foster future scientists.

Rab32 is implicated in human genetic diseases, e.g. Hermansky-Pudlak syndrome, and advances in this field will have an impact on therapeutic avenues that will benefit the health of society.

Impact on Academia
The research will have broad appeal to small GTPase biologists (~150 Ras family GTPases), infection biologists and scientists studying genetic diseases. My research will benefit academics by providing mechanistic insight that could be exploited in their own research. This is exemplified by the proposed Rab32 project that aims to identify new host factors and mechanisms that are crucial to S.Typhi degradation in non-permissive mouse macrophages, which will be of great interest to those studying typhoid fever, macrophages and genetic diseases. Moreover, the reconstitution methodology I am developing will represent a substantial technological advance for the Rab field and will be widely available for the scientific community and is suitable for studying any Rab.

Impact on Translational Research and Industry
Studying S.Typhi remains a challenge due its strict human adaptation. The majority of our current understanding on Salmonella pathogenesis derives from studies on S.Typhimurium in mice but murine typhoid and human typhoid differ in a number of important respects (e.g. S.Typhi-specific virulence factors). Identifying crucial cellular factors that determine host-restriction could be exploited in future studies by generating engineered animals that are susceptible to S.Typhi, which would expand our understanding of typhoid pathogenesis and speed the generation of new therapeutics. Rabs are prime drug targets due to their central roles in disease and findings from my research have the potential to benefit translational research and industry. New Rab-effector interactions could highlight potential targets for new therapeutic interventions and form the basis of protein structure-based drug design or high-throughput drug screens.