Untangling gene regulatory networks controlling host-pathogen interactions of the antimicrobial-resistant human pathogen Klebsiella pneumoniae
Lead Research Organisation:
University of Cambridge
Department Name: Veterinary Medicine
Abstract
The last decades have seen a rise in infections due to multi-drug-resistant bacterial pathogens, and the emergence of antibiotic resistant bacteria is one of the major challenges of our time. This serious threat to human health leads to worrying limitations of treatment options, especially against the so-called ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) and we risk an "apocalyptic" post-antibiotic era if urgent action is not taken.
The bacterium Klebsiella pneumoniae is an important cause of hospital- and community-acquired infections, causing for example pneumonia, skin/wound infections and catheter-associated urinary tract infections in the elderly and immunocompromised. Treatment of K. pneumoniae infections is hindered by the global spread of multidrug-resistant and hypervirulent strains, and carbapenem-resistant K. pneumoniae are classified by the WHO as a critical priority for new drug development. Of special concern are carbapenem-resistant isolates of the globally spreading strain called ST258 which frequently cause hospital-associated outbreaks and are a major contributor to the spread of carbapenem-resistance genes as they carry these on a piece of mobile DNA that can be easily spread to other bacteria. Clinical isolates of K. pneumoniae can be classified as classical or hypervirulent strains; while hypervirulent strains are a serious public health threat, the majority of Klebsiella disease burden is currently associated with classical strains. K. pneumoniae protects itself from the host immune response using many different virulence factors including a protective capsule, other surface structures and proteins that let it scavenge iron and stick to host cells. The most important of these virulence factors are currently limited to hypervirulent strains, and absent from the majority of classical K. pneumoniae isolates.
Although we know a lot about the genomes of K. pneumoniae, our understanding of the mechanisms by which K. pneumoniae causes disease is still limited. This is due to limited models of infection, outside the human, and the fact that clinically important lineages are highly diverse. In order to better manage and treat K. pneumoniae infections, a deeper understanding of its ability to cause disease in humans is urgently needed. Others have recently shown how Salmonella Typhimurium (a relative of Klebsiella) is able to evade killing by antibiotics by "hiding" inside host immune cells called macrophages, but K. pneumoniae was largely believed to lack this ability. However, we have recently shown that unexpectedly a K. pneumoniae ST258 strain can actively replicate inside macrophages, and we plan to try to understand this process in order to provide the basis for better treatments of K. pneumoniae in the future.
In this project, we will investigate how this strain responds to surviving inside macrophages by changing how it regulates its metabolism and growth, and how the host cell responds to the invading bacteria. We will try to reconstruct the complex control networks that regulates this ability in the bacterium, and we will compare this response to that of other related bacteria that also survive inside macrophages. Finally, we will look at other strains of K. pneumoniae from the environment, people in the community and hospital patients to see how widespread the ability is to survive inside host cells, and see if we can identify any genes or gene variants that might explain this. We believe that understanding all of these aspects will accelerate efforts to produce treatments for K. pneumoniae infection.
The bacterium Klebsiella pneumoniae is an important cause of hospital- and community-acquired infections, causing for example pneumonia, skin/wound infections and catheter-associated urinary tract infections in the elderly and immunocompromised. Treatment of K. pneumoniae infections is hindered by the global spread of multidrug-resistant and hypervirulent strains, and carbapenem-resistant K. pneumoniae are classified by the WHO as a critical priority for new drug development. Of special concern are carbapenem-resistant isolates of the globally spreading strain called ST258 which frequently cause hospital-associated outbreaks and are a major contributor to the spread of carbapenem-resistance genes as they carry these on a piece of mobile DNA that can be easily spread to other bacteria. Clinical isolates of K. pneumoniae can be classified as classical or hypervirulent strains; while hypervirulent strains are a serious public health threat, the majority of Klebsiella disease burden is currently associated with classical strains. K. pneumoniae protects itself from the host immune response using many different virulence factors including a protective capsule, other surface structures and proteins that let it scavenge iron and stick to host cells. The most important of these virulence factors are currently limited to hypervirulent strains, and absent from the majority of classical K. pneumoniae isolates.
Although we know a lot about the genomes of K. pneumoniae, our understanding of the mechanisms by which K. pneumoniae causes disease is still limited. This is due to limited models of infection, outside the human, and the fact that clinically important lineages are highly diverse. In order to better manage and treat K. pneumoniae infections, a deeper understanding of its ability to cause disease in humans is urgently needed. Others have recently shown how Salmonella Typhimurium (a relative of Klebsiella) is able to evade killing by antibiotics by "hiding" inside host immune cells called macrophages, but K. pneumoniae was largely believed to lack this ability. However, we have recently shown that unexpectedly a K. pneumoniae ST258 strain can actively replicate inside macrophages, and we plan to try to understand this process in order to provide the basis for better treatments of K. pneumoniae in the future.
In this project, we will investigate how this strain responds to surviving inside macrophages by changing how it regulates its metabolism and growth, and how the host cell responds to the invading bacteria. We will try to reconstruct the complex control networks that regulates this ability in the bacterium, and we will compare this response to that of other related bacteria that also survive inside macrophages. Finally, we will look at other strains of K. pneumoniae from the environment, people in the community and hospital patients to see how widespread the ability is to survive inside host cells, and see if we can identify any genes or gene variants that might explain this. We believe that understanding all of these aspects will accelerate efforts to produce treatments for K. pneumoniae infection.
Technical Summary
Klebsiella pneumoniae (Kp) is an important cause of nosocomial and community-acquired infections. Treatment is hindered by the global spread of multidrug-resistant strains, and of special concern are carbapenem-resistant isolates of sequence type ST258. Although Kp is well-characterised from a genomic perspective, our knowledge of the molecular mechanisms underpinning its pathogenicity is still limited.
Our aim is to define these mechanisms underpinning Kp infections of eukaryotic immune cells through four complementary work packages. WP 1 will simultaneously profile the transcriptome of ST258 isolate RH201207 during macrophage infection and of the host in response by dual RNA-seq. WP 2 will reconstruct the gene regulatory networks responsible for the transcriptional responses of Kp to macrophage invasion using the complementary functional genomics approaches of ChIP-seq and TraDISort. It will be possible to fully determine the upstream and downstream regulatory pathways of key regulators and reconstruct the complete transcriptional networks. WP 3 will identify the transcriptomic changes under infection-relevant conditions to compare RH201207 to other pathogens such as S. Typhimurium and E. coli as well as a previously published Kp strain. WP 4 will examine the diversity of host interaction within large collections of fully sequenced clinical Kp isolates by analysing their capacity to replicate inside macrophages using fluorescence dilution. This will allow us to perform exploratory genome wide association studies to test the association of mutations and the gene presence between each variant and the potential for intramacrophage replication.
The knowledge gained has great potential to establish broadly applicable technologies for studies of bacterial gene regulation networks, identify genes necessary to infect and manipulate host cells which could act as novel antibacterial and antivirulence drug targets and discover pathways of the immune response to Kp exposure
Our aim is to define these mechanisms underpinning Kp infections of eukaryotic immune cells through four complementary work packages. WP 1 will simultaneously profile the transcriptome of ST258 isolate RH201207 during macrophage infection and of the host in response by dual RNA-seq. WP 2 will reconstruct the gene regulatory networks responsible for the transcriptional responses of Kp to macrophage invasion using the complementary functional genomics approaches of ChIP-seq and TraDISort. It will be possible to fully determine the upstream and downstream regulatory pathways of key regulators and reconstruct the complete transcriptional networks. WP 3 will identify the transcriptomic changes under infection-relevant conditions to compare RH201207 to other pathogens such as S. Typhimurium and E. coli as well as a previously published Kp strain. WP 4 will examine the diversity of host interaction within large collections of fully sequenced clinical Kp isolates by analysing their capacity to replicate inside macrophages using fluorescence dilution. This will allow us to perform exploratory genome wide association studies to test the association of mutations and the gene presence between each variant and the potential for intramacrophage replication.
The knowledge gained has great potential to establish broadly applicable technologies for studies of bacterial gene regulation networks, identify genes necessary to infect and manipulate host cells which could act as novel antibacterial and antivirulence drug targets and discover pathways of the immune response to Kp exposure
Publications
Murray G
(2023)
The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs
in Proceedings of the National Academy of Sciences
Coll F
(2022)
PowerBacGWAS: a computational pipeline to perform power calculations for bacterial genome-wide association studies.
in Communications biology
Murray GGR
(2021)
Mutation rate dynamics reflect ecological change in an emerging zoonotic pathogen.
in PLoS genetics
Description | Research Visit Grant |
Amount | £2,880 (GBP) |
Funding ID | GA003920 |
Organisation | Microbiology Society |
Sector | Learned Society |
Country | United Kingdom |
Start | 03/2024 |
End | 05/2024 |
Description | Differential physiology of West Africa restricted Mycobacterium tuberculosis complex analysed by deep transcriptome sequencing |
Organisation | Noguchi Memorial Institute for Medical Research (NMRR) |
Country | Ghana |
Sector | Academic/University |
PI Contribution | Analysis of RNASeq data from M. tuberculosis |
Collaborator Contribution | Generation of RNASeq data from M. tuberculosis |
Impact | Analysis ongoing. Will lead to publications |
Start Year | 2023 |
Description | Gene fitness and essentiality of Klebsiella pneumoniae during macrophage infections |
Organisation | Macquarie University |
Country | Australia |
Sector | Academic/University |
PI Contribution | Follow-up of TraDIS analysis: generating and analysing mutations |
Collaborator Contribution | Dr. Francesca Short generated TraDIS libraries |
Impact | Ongoing analysis: will result in publications |
Start Year | 2022 |
Description | Identification of Burkholderia cenocepacia factors important for intramacrophage stages and development of acute infection in a zebrafish infection model |
Organisation | University of Montpellier |
Country | France |
Sector | Academic/University |
PI Contribution | Analysis of TraDIS libraries from Zebrafish infections using Burkholderia cenocepacia |
Collaborator Contribution | Dr Annette Vergunst generated TraDIS libraries |
Impact | Analysis in progress: will lead to publications |
Start Year | 2022 |
Description | Interaction of M. abscessus with human macrophages |
Organisation | University of Cambridge |
Department | Department of Medicine |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Analysis of data from Dual RNA-seq of Mycobacterium abscessus and human macrophages and Gene essentiality of M. abscessus by CRISPR-Cas9 |
Collaborator Contribution | Provision of data from Dual RNA-seq of Mycobacterium abscessus and human macrophages and Gene essentiality of M. abscessus by CRISPR-Cas9 |
Impact | Analysis of data in preparation for publication |
Start Year | 2023 |
Description | Persister formation in Klebsiella pneumoniae |
Organisation | Harvard University |
Department | Harvard Medical School |
Country | United States |
Sector | Academic/University |
PI Contribution | Collaboration on the use of our TraDIS libraries in the analysis of persister formation in Klebsiella |
Collaborator Contribution | Use of Prof. Helaine's models of persister formation |
Impact | Travel grant for Senior Research Associate to conduct research in Prof. Helaine's laboratory |
Start Year | 2024 |
Description | Cambridge Festival |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Hands on demonstration of DNA Extraction from fruits as part of the University-wide Cambridge Festival |
Year(s) Of Engagement Activity | 2023,2024 |
URL | https://www.festival.cam.ac.uk |