Characterisation of nongrowing Salmonella during macrophage infection
File(s)
Author(s)
Fisher, Robbie Andrew
Type
Thesis
Abstract
Persisters are antibiotic tolerant cells thought to be responsible for the
recalcitrance and the recurrence of many bacterial infections. Salmonella has
been shown to form persisters during macrophage infection, the source of
which is a reservoir of nongrowing bacteria. This thesis describes the use of
dual RNA-seq, combined with fluorescence dilution and FACS, to analyse the
transcriptomes of nongrowing and growing Salmonella as well as the
transcriptomes of their respective host macrophages during infection.
Interestingly, nongrowing Salmonella were found to express the SPI-2 type
three secretion system (T3SS) and infected macrophages were found to be
polarised towards an M2 phenotype compared to uninfected bystander cells.
Further activity in nongrowing Salmonella was uncovered using fluorescence
dilution combined with DAPI-staining, which provided evidence of DNA
synthesis during infection. In addition, the toxin-antitoxin module yhfG-fic was
investigated in Salmonella after a deletion mutant in this module was found to
have a defect during infection, however this was discovered to be due to a
polar effect of the deletion on the expression of the downstream gene pabA.
Heterogeneity in the lag distributions of growing and nongrowing Salmonella
post-infection was also investigated in this study, using the ScanLag
technique. Deletion of TA modules, which have previously been linked to
persister formation, had a minimal effect of the lag distributions of nongrowing
Salmonella, however the lag times of growing bacteria were increased. The
SOS response was found to affect lag times and is important for Salmonella
recovery post-infection, regardless of growth state.
recalcitrance and the recurrence of many bacterial infections. Salmonella has
been shown to form persisters during macrophage infection, the source of
which is a reservoir of nongrowing bacteria. This thesis describes the use of
dual RNA-seq, combined with fluorescence dilution and FACS, to analyse the
transcriptomes of nongrowing and growing Salmonella as well as the
transcriptomes of their respective host macrophages during infection.
Interestingly, nongrowing Salmonella were found to express the SPI-2 type
three secretion system (T3SS) and infected macrophages were found to be
polarised towards an M2 phenotype compared to uninfected bystander cells.
Further activity in nongrowing Salmonella was uncovered using fluorescence
dilution combined with DAPI-staining, which provided evidence of DNA
synthesis during infection. In addition, the toxin-antitoxin module yhfG-fic was
investigated in Salmonella after a deletion mutant in this module was found to
have a defect during infection, however this was discovered to be due to a
polar effect of the deletion on the expression of the downstream gene pabA.
Heterogeneity in the lag distributions of growing and nongrowing Salmonella
post-infection was also investigated in this study, using the ScanLag
technique. Deletion of TA modules, which have previously been linked to
persister formation, had a minimal effect of the lag distributions of nongrowing
Salmonella, however the lag times of growing bacteria were increased. The
SOS response was found to affect lag times and is important for Salmonella
recovery post-infection, regardless of growth state.
Version
Open Access
Date Issued
2017-09
Date Awarded
2018-03
Advisor
Helaine, Sophie
Holden, David
Sponsor
Medical Research Council (Great Britain)
Grant Number
MR/J006874/1
Publisher Department
Department of Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)