Legionella pneumophila pathogenesis : establishment of a new insect infection model and characterisation of the effector protein LtpD
Author(s)
Harding, Clare R.
Type
Thesis
Abstract
Legionella pneumophila is the causative agent of Legionnaires’ disease,
severe pneumonia acquired from inhalation of contaminated water droplets. In the
lung, L. pneumophila infects alveolar macrophages and creates a compartment
named the Legionella containing vacuole (LCV), which avoids degradation and
recruits components of the secretory pathway. LCV creation depends on the action
of the Dot/Icm system that translocates over 275 effectors into the host cell. To
study the function of these effectors, models that approximate human disease are
required.
Here, I characterise the larvae of Galleria mellonella as an infection model for L.
pneumophila. Infection resulted in larval mortality and bacterial replication in a
strain- and Dot/Icm-dependent manner. Flagella expression was dispensable for
bacterial virulence, however secreted phospholipases and the Dot/Icm effector SdhA
were shown to be important in virulence. Deletion of SdhA resulted in disruption of
the LCV membrane and destruction of haemocytes. The importance of SdhA
expression was confirmed in a mammalian model, validating the utility of G.
mellonella.
In the second part of this study, the novel protein LtpD was characterised. LtpD
was translocated via the Dot/Icm secretion system and localised to the LCV. A series
of truncation mutants defined a C-terminal 153 amino acid domain as required for
LCV localisation. This region was shown to bind directly to the lipid
phosphoinositide 3-phosphate. Further analysis revealed that LtpD also interacted
with the enzyme inositol monophosphatase 1, however did not change the enzyme’s
activity in vitro. Deletion of LtpD resulted in a subtle growth defect in mammalian
macrophages at late time points during infection. This growth defect was also seen
the G. mellonella and mouse lungs, confirming that LtpD is a virulence factor of L.
pneumophila.
In summary, here I present an infection model to investigate L. pneumophila
virulence and further characterisation of the Dot/Icm effector LtpD.
severe pneumonia acquired from inhalation of contaminated water droplets. In the
lung, L. pneumophila infects alveolar macrophages and creates a compartment
named the Legionella containing vacuole (LCV), which avoids degradation and
recruits components of the secretory pathway. LCV creation depends on the action
of the Dot/Icm system that translocates over 275 effectors into the host cell. To
study the function of these effectors, models that approximate human disease are
required.
Here, I characterise the larvae of Galleria mellonella as an infection model for L.
pneumophila. Infection resulted in larval mortality and bacterial replication in a
strain- and Dot/Icm-dependent manner. Flagella expression was dispensable for
bacterial virulence, however secreted phospholipases and the Dot/Icm effector SdhA
were shown to be important in virulence. Deletion of SdhA resulted in disruption of
the LCV membrane and destruction of haemocytes. The importance of SdhA
expression was confirmed in a mammalian model, validating the utility of G.
mellonella.
In the second part of this study, the novel protein LtpD was characterised. LtpD
was translocated via the Dot/Icm secretion system and localised to the LCV. A series
of truncation mutants defined a C-terminal 153 amino acid domain as required for
LCV localisation. This region was shown to bind directly to the lipid
phosphoinositide 3-phosphate. Further analysis revealed that LtpD also interacted
with the enzyme inositol monophosphatase 1, however did not change the enzyme’s
activity in vitro. Deletion of LtpD resulted in a subtle growth defect in mammalian
macrophages at late time points during infection. This growth defect was also seen
the G. mellonella and mouse lungs, confirming that LtpD is a virulence factor of L.
pneumophila.
In summary, here I present an infection model to investigate L. pneumophila
virulence and further characterisation of the Dot/Icm effector LtpD.
Version
Open Access
Date Issued
2013-03
Date Awarded
2013-09
Advisor
Frankel, Gad
Sponsor
Wellcome Trust (London, England)
Publisher Department
Division of Cell and Molecular Biology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)