Macrophage-bacteria interactions in cystic fibrosis: the cellular microbiology of the novel opportunistic bacterium Achromobacter species

Chronic infection and persistent inflammation in people with cystic fibrosis (PWCF) lead to progressive lung damage. Despite novel therapies to correct the CFTR disfunction chronic antibiotic treatments, compounded with extended life expectancy of PWCF, challenge the ecology of the lung microbiome and gives rise to the emergence of potential new pathogens displaying multidrug antibiotic resistance. Of these, Achromobacter sp. are particularly concerning since they are often the dominant bacteria recovered from sputum samples in an increasing number of PWCF including those with end-stage lung disease and they are also commonly found in the airways’ microbiome. We understand little about the association of Achromobacter with lung disease and lack information on pathogenic traits of Achromobacter sp. and their ability to interact with innate immune cells (e.g. macrophages, neutrophils), as well as an overall lack of information on the biology of these bacteria. The overarching objective of this project is to understand the cellular microbiology of Achromobacter infections.

Our group has recently demonstrated that Achromobacter CF isolates can survive and replicate in human monocytic macrophages by surviving in a modified phagolysosome, and also that the intracellular infection results in macrophage pro-inflammatory cell by pyroptosis. The mechanistic details of how this occurs are unknow, and also unknown are the bacterial factors involved in promoting inflammation.

This project has 2 objectives:

(i) Identifying the bacterial properties associated with intracellular survival
(ii) elucidating the macrophage cellular responses upon Achromobacter infection, especially in terms of proinflammatory responses and in particular, in human CF-defective cells.

To address objective (i) the PhD student will identify the bacterial properties involved in survival by a two-pronged approach. First, a candidate gene-based strategy to mutagenize genes suspected to be involved in intracellular survival (Primary targets include genes of putative Type III, IV and VI systems, O antigen and capsule biosynthesis clusters and several haemolysin-like genes. Second, in parallel, the PhD student will use an unbiased approach based on high-throughput transposon mutagenesis employing various systems (e.g. plasposons, TnSeg, Tradis). Mutants will be tested for intracellular survival using macrophages seeded in microtiter plates and the phenotypes further validated by genetic complementation. These experiments will identify genes involved in cell entry and intracellular survival using fluorescent microscopy and biochemical analyses, as appropriate.

To address objective (ii), the student will characterize the inflammatory response in macrophages upon Achromobacter infection in normal and CFTR-defective human cells. These studies will employ a variety of approaches including but not limited to cytokine profiling, pathway inhibitors, and a new developed microscopic assay to image, detect, and quantify inflammasome complexes in cells, as well as a various cell biology approaches to precisely identify the properties of the Achromobacter-containing vacuoles in macrophages. These studies will be complemented with setting up a mouse model of Achromobacter infection in lungs.

PhD candidates will join a vibrant, world-class team engaged in interdisciplinary studies on microbial pathogenesis using molecular biology, structural, biochemistry, and cell biology approaches, and are strongly advised to consult the following links (http://publish.uwo.ca/~mvalvano/Advice-to-grads.html) and (http://publish.uwo.ca/~mvalvano/index.html) for additional information on what to expect in the Valvano lab.

ENTRY REQUIREMENTS

Candidates should have or expect to obtain a 2:1 or higher Honours degree or equivalent in a relevant biomedical or life sciences subject.

English Language

Candidates applying from countries where the first language is not English should produce evidence of their competence through a qualification such as IELTS or TOEFL score.

The minimum recommended score for the School of Medicine, Dentistry and Biomedical Science is:
• IELTS score of 6.0 with not less than 5.5 in each of the four component elements of listening, reading, speaking and writing taken within the last 2 years;
• TOEFL score of 80+ (internet basted test), taken within the last 2 years, with minimum component scores of; Listening 17, Reading 18, Speaking 20, Writing 17);
• A valid Certificate of Proficiency in English grade A or B;
• A valid Certificate of Advanced English grade A; or
• A first or upper second class honours degree from a university based in the UK, Republic of Ireland or other suitably quality assured location in a country deemed by the UK Border Agency to be majority English speaking.

For a list of English Language qualifications also accepted by the School and University please see the following link:
http://www.qub.ac.uk/International/International-students/Applying/English-language-requirements/#English