Carbon based nutrition of Staphylococcus aureus and the role of sugar phosphate transporters in intracellular bacterial replication
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Date
28/06/2014Author
Bell, John Alexander
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Abstract
The Gram positive bacterium Staphylococcus aureus is a major cause of human
disease in industrialized countries. This multifaceted pathogen is adapted to thrive
in a variety of host niches, including the intracellular compartment. S. aureus
rapidly develops antibiotic resistance, and infections due to resistant clones pose
a global threat, calling for novel therapeutic approaches. The ability to exploit
host nutrients and efficiently metabolize these resources for growth is paramount
for bacterial pathogenesis. Understanding the nutritional and metabolic
determinants that underpin bacterial virulence may lead to the identification of
novel antimicrobial targets. This thesis investigates carbon nutrition and
metabolism of community-acquired methicillin resistant S. aureus (CA-MRSA)
USA300, a widely spread, hyper virulent multi-resistant strain.
The dependence of S. aureus on carbohydrates for growth was considered first. In
vitro studies in supplemented chemically defined media showed that sugar
phosphates, such as hexose phosphates and glycerol phosphates, promote
staphylococcal growth more efficiently than glucose. Deletion mutations were
introduced to the two putative sugar phosphate transporter genes present in the
S. aureus genome, uhpT (hexose phosphate permease) and glpT (glycerol
phosphate permease). Phenotypic analysis of USA300 mutants and heterologous
expression of the transporters in a previously described Listeria monocytogenes
Δhpt mutant, totally unable to use sugar phosphates, confirmed that S. aureus
UhpT and GlpT have different substrate specificities. Whilst both can transport
glycerol monophosphate (excluding glycerol-2-phosphate) and the
organophosphate antibiotic fosfomycin, hexose monophosphates are only
imported via UhpT. Since sugar phosphates are only present in significant
amounts inside living tissues, particularly the intracellular compartment, the role
of S. aureus UhpT and GlpT in pathogenesis was investigated by constructing a
double deletion mutant. The ΔuhpTΔglpT USA300 mutant was used to infect
several relevant mammalian cell lines. In the conditions tested, it was found that
UhpT and GlpT played no role in the intracellular replication of S. aureus. By
contrast, Listeria exploits sugar phosphates from the host cell cytosol via the
homologous hexose phosphate transporter, Hpt, to maximise replication and
enhance virulence. The distinct requirement of sugar phosphates for intracellular
proliferation may reflect intrinsic differences in carbon nutrient dependence
between the two organisms. It was confirmed that S. aureus can efficiently use
other readily available carbon sources for growth, such as amino acids. In
contrast, Listeria is strictly dependent upon sugar-derived carbon for growth, due
to an incomplete tricarboxylic acid cycle. Whilst the double ∆uhpT∆glpT mutation
had no effect in S. aureus, expression of staphylococcal uhpT or glpT restored
wild-type intracellular growth in the L. monocytogenes ∆hpt mutant. Taken
together, the results illustrate that sugar phosphate permeases have a contextual
role in bacterial virulence, where the background in which the genes are
expressed determine their contribution as a virulence factor.
The intracellular dynamics of S. aureus was also explored using
immunofluorescence microscopy. It was observed that, during epithelial cell
infection, USA300 remains enclosed in a membrane-bound vacuole. This
localisation may form a barrier to cytosolic sugar phosphates and potentially
explain the absence of effect of the sugar phosphate permease deletions in
intracellular proliferation. Preliminary characterisation of the S. aureus containing
vacuole (SACV) was performed and it was found to be positive for the Rab7 late-endosomal
GTPase and for trans-Golgi markers. This suggests that SACVs
converge at the Golgi apparatus. Interestingly, a USA300 mutant lacking the global
regulatory system agr was unable to proliferate intracellularly and did not acquire
Rab7 or Golgi markers. Since the Δagr mutation did not cause any impairment in
carbon source dependent growth, these preliminary data suggest that
modification of the SACV by Agr-regulated effectors may play a key role in
modulating cellular processes that control staphylococcal intracellular survival
and/or replication.
Evidence presented in this thesis provides a platform for further exploration of S.
aureus host cell nutrient dependence and the mechanisms that drive replication.