Persistent Salmonella infection: characterization of non-replicating bacteria and Toxin-Antitoxin module function

Lead Research Organisation: Imperial College London
Department Name: Infectious Disease

Abstract

Bacterial pathogens cause many diseases in humans and are frequently well controlled by treatment with antibiotics. However, antibiotics are increasingly becoming inefficient. In addition to the well-documented cases of antibiotic resistance, persistence, characterised by relapsing infections following antibiotic treatment, is a major problem. It has been discovered recently that for many bacterial species, a proportion of bacterial cells grown in laboratory medium can enter a dormant-like state in which they are not affected by antibiotics. These bacteria are called persisters and little is known about how they arise. Salmonella is the causative agent of various diseases, ranging from gastro-enteritis to typhoid fever. We have visualised Salmonella persisters in infected tissues for the first time, and we have identified signals that lead to their formation. We showed that Salmonella forms large numbers of non-replicating persisters after being engulfed by immune cells called macrophages. By adopting this non-replicating mode, Salmonella survives antibiotic treatment and lingers in the host, accounting for its ability to cause recurrent infections. We have identified a family of genes (toxin/antitoxin modules) as the molecular pathways that lead to persister formation during infection. This project aims at deciphering how toxin/antitoxin modules work collectively and are regulated during infection. We will also carry out an in-depth analysis of the differences in the genetic program of persisters compared to proliferating bacteria to understand how non-growing bacteria can survive in the host on the long-term. Understanding better how persisters are formed and maintained in the host they infect is likely to provide valuable information for designing new drugs to coax them out of this state so that they become re-sensitised to antibiotics.

Technical Summary

Many bacterial pathogens cause persistent infection despite exposure to multiple courses of antibiotics, which are thought to contribute to the emergence of stable antibiotic resistance. Persisters are multidrug-tolerant bacteria that could account for the relapse of infections. Serovars of Salmonella enterica are important intracellular bacterial pathogens that cause a variety of human diseases, including gastroenteritis and typhoid fever. We developed a method allowing tracking of single cells and discovered that upon macrophage phagocytosis, a significant proportion of the Salmonella population forms non-replicating persisters. Then, I provided the first direct evidence that non-replicating persisters are also formed in infected mice where they survive extended periods of antibiotic treatment. I also showed that a family of genes, named class II Toxin/Antitoxin (TA) modules, is involved in the formation of Salmonella persisters. Class II TA operons encode a non-secreted toxin, which is an inhibitor of an essential cellular function and an antitoxin that neutralizes the toxin. The antitoxin is degraded under various stress conditions leading to growth arrest of the bacterial cell. How growth-arrested bacteria can establish a long-term viable but non-replicative state in host cells is unknown. The overall aim of the project is to characterize Salmonella persisters through 3 specific objectives. First, by undertaking an in-depth characterization of Toxin-Antitoxin modules I intend to understand how these proteins function collectively to govern entry into the persister state. Second, I will study the role of TA modules in the context of natural infection of mice. Third, I will investigate the basis for maintenance of a non-replicating viable state within host cells. The knowledge gained has great potential to offer opportunities for targeting persistent bacterial infections to improve antibiotic efficacy and counteract the generation of antibiotic resistance.

Planned Impact

Academic beneficiaries are described in another section. Other beneficiaries could include the private sector companies who are interested in developing drugs targeting persisters. Such drugs could potentiate antibiotics efficacy thereby diminishing the likelihood of recurrence of infections. Fifteen % of the apparently successfully treated typhoid patients suffer from relapse weeks, months or years after the first episode. Among women with an acute urinary tract infection, 20-30% will have a recurrent infection within 3-4 months, which leads medical practitioners to prescribe 6-months long antibiotic treatments. Recurrence is also often observed after Streptococcus and Staphylococcus infections. Moreover, the tuberculosis persistence is also thought to rely on the presence of persisters. Persistence, characterized by relapsing infections, is a major problem to human health as such infections frequently require multiple courses of antibiotics, which in turn are thought to contribute to the emergence of stable antibiotic resistance. With no new class of antibiotics developed since 1987, the antibiotic pipeline is drying up and unless appropriate drugs are found, we are facing a return to the pre-antibiotic era. Thus, fighting antimicrobial resistance is a national priority, as Chief Medical Officer Dame Sally Davies recently stressed, and targeting bacterial persisters would lead to making the most out of the available antibiotics while ensuring fewer are prescribed.
My work has the potential to improve human health and can help achieving long and healthy lives as I expect that it will have applications in targeting persistence. When bacterial persisters linger in their hosts silently, sometimes for decades, a drop in the host immunity can allow successful reactivation of an ancient infection. The numbers of immuno-compromised patients increase in both developing and developed parts of the world with HIV-infected patients, organ transplant recipients, cancer patients and other patients taking immunosuppressive drugs for disorders such as rheumatoid arthritis or inflammatory bowel disease; therefore, bacterial persistence is increasingly going to be a major threat.
It is not possible to predict with certainty if valuable intellectual property will emerge from the planned work, but if commercially exploitable discoveries are made, I and Imperial College's technology transfer company Innovations will ensure that it is protected properly by patents.

Publications

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Fisher RA (2017) Persistent bacterial infections and persister cells. in Nature reviews. Microbiology

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Hall AM (2017) Toxin-antitoxin systems: reversible toxicity. in Current opinion in microbiology

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Fisher RA (2016) Analysis of Macrophage-Induced Salmonella Persisters. in Methods in molecular biology (Clifton, N.J.)

 
Description RNAseq of Salmonella during infection of macrophages 
Organisation University of Wurzburg
Country Germany 
Sector Academic/University 
PI Contribution We collaborated with the Vogel group to generate RNA seq data of Salmonella during infection in macrophages. We designed the tools to sort the salmonella populations, set up the tools in their institute, prepared and sent samples, contributed to analysis, follow up experiments and wrote the manuscript(s)
Collaborator Contribution Our partners carried out the sequencing and the analysis of the data.
Impact PMID: 27841856
Start Year 2015
 
Description Structure/function analysis of Salmonella toxins 
Organisation Imperial College London
Department Department of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Elucidating mode of action of a toxin important for Salmonella persisters.
Collaborator Contribution Our collaborators characterized the crystal structure of a toxin.
Impact PMID: 27264868
Start Year 2015