Periodic Reporting for period 1 - WolAntiS (Using antisense inhibition to understand Wolbachia symbiosis and antiviral protection)
Reporting period: 2019-03-01 to 2021-02-28
Animals are almost universally associated with microbes, interacting with them in intricate ways. Wolbachia - a maternally transmitted intracellular bacterium - is the most widespread animal symbiont, infecting 50% of known insect species. This symbiont induces a range of phenotypes, including reproductive manipulations, facilitating its own spread within host populations, and antiviral protection. Wolbachia blocks the replication of viruses and in mosquitoes it can prevent virus transmission to humans. Wolbachia is therefore a WHO-recommended strategy for the control of most vector-borne viral diseases – including dengue and Zika – and has been deployed in experimental field releases. To date, however, we lack a mechanistic understanding of Wolbachia-induced antiviral protection. This is unfortunate, as it impedes wider exploitation of the Wolbachia-insect symbioses, e.g. for disease control in species that are poor Wolbachia hosts or in designing novel treatments for viral infections.
This proposal aimed at addressing key technological and knowledge deficits. The first objective was to develop an antisense strategy to target the function of Wolbachia genes. Second, to determine which Wolbachia genes are involved in animal-microbe symbiosis and antiviral protection.
This work is important due to the prevalence of Wolbachia in nature and its interaction with insects (eg. pests, pollinators) and pathogenic filarial nematodes causing human disease. Understanding Wolbachia-host interaction will allow us to understand insects and nematodes better, improve current efforts to control vector-borne and filarial-caused diseases.
This proposal aimed at addressing key technological and knowledge deficits. The first objective was to develop an antisense strategy to target the function of Wolbachia genes. Second, to determine which Wolbachia genes are involved in animal-microbe symbiosis and antiviral protection.
This work is important due to the prevalence of Wolbachia in nature and its interaction with insects (eg. pests, pollinators) and pathogenic filarial nematodes causing human disease. Understanding Wolbachia-host interaction will allow us to understand insects and nematodes better, improve current efforts to control vector-borne and filarial-caused diseases.
I have tested a panel of cell-penetrating peptides known to target other intracellular bacteria for their ability to co-localize with Wolbachia within insect cells. Fluorescently-labelled peptides were applied onto Wolbachia-infected cells, their localization observed using a confocal microscope, and co-localization with Wolbachia measured using custom ImageJ macros.
Subsequently, 12 bp synthetic antisense molecules, spanning the ATG and ribosome-binging sites, were designed for two Wolbachia genes: wsp and ftsZ. These genes were selected, as antibodies against them were readily available, and we confirmed their robustness in the silencing protocol. We conjugated the antisense molecules with the cell-penetrating peptides targeting Wolbachia, and applied the onto Wolbachia-infected cells. The knock-down of selected Wolbachia genes was tested with the Western blot using Drosophila tubulin as a loading control. This work, together with the similar protocol using antisense molecules conjugated to cholesterol analogues, has led to the development of the first antisense inhibition protocol for selective and specific targeting of Wolbachia genes.
Although the results of the project were not deployed yet, they were disseminated to both, academic and non-academic audiences in a series of activities. During the project, I participated in the following conferences and scientific events:
1) The British Microbiology Society Annual Conference 2019, Belfast, UK, 08 – 11/04/2019
2) Royal Entomological Society – Special Interest Group: Insects: Infection & Immunity, Online Conference, 24-25 September 2020
3) Virtual Symbiosis Seminar, organized by prof. Greg Hurst and Prof. Nicole Gerardo (https://docs.google.com/spreadsheets/d/1DHkn2CjRXz9QbJPcSDuibYmn6e1khhgd2xPIxXjXVMY/edit#gid=943392522) 7th July 2020
The results of the project have been written up in the following publications/preprints:
- Chrostek E§, Martins NE§, Marialva MS, Teixeira L (2020) Wolbachia-conferred antiviral protection is determined by developmental temperature. bioRxiv, doi: 10.1101/2020.06.24.169169. (citations: 3)
§ Contributed equally
- Corbin C, Jones JE, Chrostek E, Fenton A, Hurst GDD (2020) Thermal sensitivity of the Spiroplasma-Drosophila hydei protective symbiosis: The best of climes, the worst of climes. Molecular Ecology doi: 10.1111/mec.15799.
The approaches and results of the action were disseminated further to non-scientific audience by the fellow invited to act as:
- An Invited author of the article: “Wolbachia – basic science and applications of an antiviral bacterium” for Microbiology Today (https://bit.ly/3eyZ03T) a magazine by the Microbiology Society with articles for all readers, including parliamentarians and policy-makers (06/08/2019).
- An Expert interviewed by “The Scientist Magazine”, a professional magazine providing coverage of recently published scientific papers (19/07/2019.
Subsequently, 12 bp synthetic antisense molecules, spanning the ATG and ribosome-binging sites, were designed for two Wolbachia genes: wsp and ftsZ. These genes were selected, as antibodies against them were readily available, and we confirmed their robustness in the silencing protocol. We conjugated the antisense molecules with the cell-penetrating peptides targeting Wolbachia, and applied the onto Wolbachia-infected cells. The knock-down of selected Wolbachia genes was tested with the Western blot using Drosophila tubulin as a loading control. This work, together with the similar protocol using antisense molecules conjugated to cholesterol analogues, has led to the development of the first antisense inhibition protocol for selective and specific targeting of Wolbachia genes.
Although the results of the project were not deployed yet, they were disseminated to both, academic and non-academic audiences in a series of activities. During the project, I participated in the following conferences and scientific events:
1) The British Microbiology Society Annual Conference 2019, Belfast, UK, 08 – 11/04/2019
2) Royal Entomological Society – Special Interest Group: Insects: Infection & Immunity, Online Conference, 24-25 September 2020
3) Virtual Symbiosis Seminar, organized by prof. Greg Hurst and Prof. Nicole Gerardo (https://docs.google.com/spreadsheets/d/1DHkn2CjRXz9QbJPcSDuibYmn6e1khhgd2xPIxXjXVMY/edit#gid=943392522) 7th July 2020
The results of the project have been written up in the following publications/preprints:
- Chrostek E§, Martins NE§, Marialva MS, Teixeira L (2020) Wolbachia-conferred antiviral protection is determined by developmental temperature. bioRxiv, doi: 10.1101/2020.06.24.169169. (citations: 3)
§ Contributed equally
- Corbin C, Jones JE, Chrostek E, Fenton A, Hurst GDD (2020) Thermal sensitivity of the Spiroplasma-Drosophila hydei protective symbiosis: The best of climes, the worst of climes. Molecular Ecology doi: 10.1111/mec.15799.
The approaches and results of the action were disseminated further to non-scientific audience by the fellow invited to act as:
- An Invited author of the article: “Wolbachia – basic science and applications of an antiviral bacterium” for Microbiology Today (https://bit.ly/3eyZ03T) a magazine by the Microbiology Society with articles for all readers, including parliamentarians and policy-makers (06/08/2019).
- An Expert interviewed by “The Scientist Magazine”, a professional magazine providing coverage of recently published scientific papers (19/07/2019.
This work has led to the development of the first direct protocol to study Wolbachia gene functions within the native system inside the insect cells. This is a significant step beyond the state-of-the-art in unculturable insect symbionts, where genotype-phenotype links have been established in very few cases. So far, most successful approaches were technically challenging, laborious, and focused on small numbers of candidate genes.
This project has many potential interdisciplinary impacts, which include: (i) Host-microbe interactions: Identification of mechanisms deployed by Wolbachia to interact with the hosts. This will enable design of new drugs and treatments for bacterial and viral diseases. (ii) Ecology: Dissecting consequences of the mechanisms of symbiosis and antiviral protection on insect population structure and infected individuals spread. This will allow to predict more accurately the outcomes of the current efforts using Wolbachia to limit the spread of dengue and Zika. (iii) Cell biology: Understanding of Wolbachia integration into host cells physiology and its engagement of host machinery. Wolbachia’s inconspicuousness within the host cell may uncover new biological pathways and phenomena. (iv) Vector control: Improvement of current efforts to control vector-borne diseases through understanding of the mechanism of virus-blocking. This will allow generation of new transgenic insects refractory to pathogens for the management of diseases in humans, livestock, and plants. (v) Biomedical sciences: Identification of novel Wolbachia drug targets for the treatment of filarial diseases and design of novel antifilarial treatments. (vi) Agriculture: Antiviral protection of beneficial insects and elimination of pest insects and worms.
This project has many potential interdisciplinary impacts, which include: (i) Host-microbe interactions: Identification of mechanisms deployed by Wolbachia to interact with the hosts. This will enable design of new drugs and treatments for bacterial and viral diseases. (ii) Ecology: Dissecting consequences of the mechanisms of symbiosis and antiviral protection on insect population structure and infected individuals spread. This will allow to predict more accurately the outcomes of the current efforts using Wolbachia to limit the spread of dengue and Zika. (iii) Cell biology: Understanding of Wolbachia integration into host cells physiology and its engagement of host machinery. Wolbachia’s inconspicuousness within the host cell may uncover new biological pathways and phenomena. (iv) Vector control: Improvement of current efforts to control vector-borne diseases through understanding of the mechanism of virus-blocking. This will allow generation of new transgenic insects refractory to pathogens for the management of diseases in humans, livestock, and plants. (v) Biomedical sciences: Identification of novel Wolbachia drug targets for the treatment of filarial diseases and design of novel antifilarial treatments. (vi) Agriculture: Antiviral protection of beneficial insects and elimination of pest insects and worms.