Carbon based nutrition of Staphylococcus aureus and the role of sugar phosphate transporters in intracellular bacterial replication

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.