Investigating the molecular mechanisms underlying the differential virulence of Salmonella enterica serovars Typhimurium and Choleraesuis
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Date
08/12/2021Author
Johnston-Menzies, Imogen Rena
Metadata
Abstract
Salmonella enterica is a facultative intracellular pathogen which impacts
human and animal health globally. Livestock animals, as a source of nutrition
and environmental contamination, represent a significant reservoir for human
non-typhoidal salmonellosis. For Salmonella enterica serovars, systemic
dissemination to sites outwith the intestinal environment is a phenotypic
marker of bacterial host-adaptation, a trait which influences infection
outcome. Host-adapted Salmonella Choleraesuis, for example, disseminates
to the bloodstream, liver, and spleen of infected pigs, causing little intestinal
inflammation but greater mortality whilst host-generalist Salmonella
Typhimurium causes self-limiting enteritis.
S. enterica encodes two type III secretion systems (T3SSs) which have
critical roles in bacterial pathogenesis – the T3SS-1 and T3SS-2. Both
systems are molecular syringes that inject effector proteins into host cells.
These effectors manipulate host-cell processes to aid either invasion (T3SS1) or intracellular survival (T3SS-2). As significant evidence suggests that
type III secretion influences host adaptation, the first aim of this study was to
characterise and compare the secretome of host-generalist S. Typhimurium
and host-adapted serovar S. Choleraesuis under T3SS-1 inducing
conditions.
A label-free quantitative proteomics approach was used to characterise the
secretome of two strains of well-defined virulence in livestock animals – S.
Typhimurium ST4/74 and S. Choleraesuis SCSA50 – and identified key
differences between the strains. This was the first comprehensive and
quantitative comparison between the secretomes of two different nontyphoidal S. enterica serovars and demonstrated that ST4/74 secreted more
T3SS-1 effectors than host-adapted SCSA50, a result which could not have
been predicted from the genome sequences alone. In addition, the proteomic
approach also identified several hypothetical proteins which were
investigated for T3SS-dependent secretion.
The subsequent aim of this study was to unravel regulatory differences
between ST4/74 and SCSA50 at the transcriptional level using quantitative
reverse transcription PCR (RT-qPCR). Initial comparative bioinformatic
analysis revealed single nucleotide polymorphisms in important regions of
the promoters of several factors which control T3SS-1 expression including
major transcriptional regulators HilA and HilE. Consequent RT-qPCR
investigation uncovered small but biologically relevant increases in
transcriptional expression of T3SS-1 transcriptional activators by ST4/74
compared to SCSA50 which could explain the differences in effector protein
secretion.
The importance of the T3SS is, however, in the context of host cells. The last
aim of this study was therefore to examine the interactions of ST4/74 and
SCSA50 with porcine cell lines using assays to assess invasion, intracellular
survival, net replication, and intracellular cytosolic populations. Whilst the
strains were similarly invasive, their intracellular lifestyles differed – in the
intestinal cell line IPEC-J2, ST4/74 replicated faster and had a larger
cytosolic population than SCSA50, two bacterial intracellular strategies linked
to activity of T3SS-1.
Overall, this study revealed significant differences in the repertoire and
amount of secreted proteins by host-generalist ST4/74 and host-adapted
SCSA50. Although more mechanistic exploration is required, increased
transcriptional expression of T3SS-1 transcriptional activators by ST4/74
potentially influences this. Paired with the differences in intracellular lifestyle,
this investigation identified type III secretion as a major differentiating factor
between S. Typhimurium and S. Choleraesuis and this data could inform on
new strategies for crucial broad-spectrum vaccine design.