Characterization of secreted sphingosine-1-phosphate lyases required for virulence and intracellular survival of <i>Burkholderia pseudomallei</i>

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, plays a critical role in the orchestration of immune responses. S1P levels within the mammalian host are tightly regulated, in part through the activity of S1P lyase (S1PL) which catalyses its irreversible degradation. Herein we describe the identification and characterization of secreted S1PL orthologues encoded by the facultative intracellular bacteria Burkholderia pseudomallei and Burkholderia thailandensis. These bacterial orthologues exhibited S1PL enzymatic activity, functionally complemented an S1PL-deficient yeast strain, and conferred resistance to the antimicrobial sphingolipid D-erythro-sphingosine. We report that secretion of these bacterial S1PLs is pH-dependent, and is observed during intracellular infection. S1PL-deficient mutants displayed impaired intracellular replication in murine macrophages (associated with an inability to evade the maturing phagosome) and were significantly attenuated in murine and larval infection models. Furthermore, treatment of Burkholderia-infected macrophages with either S1P or a selective agonist of S1P receptor 1 enhanced bacterial colocalisation with LAMP-1 and reduced their intracellular survival. In summary, our studies confirm bacterial-encoded S1PL as a critical virulence determinant of B. pseudomallei and B. thailandensis, further highlighting the pivotal role of S1P in host-pathogen interactions. In addition, our data suggest that S1P pathway modulators have potential for the treatment of intracellular infection.We thank HL Ho & K Haynes (University of Exeter) for provision of strains and relevant vectors for yeast complementation studies. This work was supported by the Defence Science 26 and Technology Laboratory under contract DSTLX-1000060221 (WP1). CJM was funded by the EASTBIO Doctoral Training Partnership. The funders had no role in study design, data collection and analysis, or preparation of the manuscript

Structural, mechanistic and regulatory studies of serine palmitoyltransferase

SLs (sphingolipids) are composed of fatty acids and a polar head group derived from l-serine. SLs are essential components of all eukaryotic and many prokaryotic membranes but S1P (sphingosine 1-phosphate) is also a potent signalling molecule. Recent efforts have sought to inventory the large and chemically complex family of SLs (LIPID MAPS Consortium). Detailed understanding of SL metabolism may lead to therapeutic agents specifically directed at SL targets. We have studied the enzymes involved in SL biosynthesis; later stages are species-specific, but all core SLs are synthesized from the condensation of l-serine and a fatty acid thioester such as palmitoyl-CoA that is catalysed by SPT (serine palmitoyltransferase). SPT is a PLP (pyridoxal 5'-phosphate)-dependent enzyme that forms 3-KDS (3-ketodihydrosphingosine) through a decarboxylative Claisen-like condensation reaction. Eukaryotic SPTs are membrane-bound multi-subunit enzymes, whereas bacterial enzymes are cytoplasmic homodimers. We use bacterial SPTs (e. g. from Sphingomonas) to probe their structure and mechanism. Mutations in human SPT cause a neuropathy [HSAN1 (hereditary sensory and autonomic neuropathy type 1)], a rare SL metabolic disease. How these mutations perturb SPT activity is subtle and bacterial SPT mimics of HSAN1 mutants affect the enzyme activity and structure of the SPT dimer. We have also explored SPT inhibition using various inhibitors (e. g. cycloserine). A number of new subunits and regulatory proteins that have a direct impact on the activity of eukaryotic SPTs have recently been discovered. Knowledge gained from bacterial SPTs sheds some light on the more complex mammalian systems. In the present paper, we review historical aspects of the area and highlight recent key developments.</p

Carbon Nanotube/Polymer Nanocomposites Flexible Stress and Strain Sensors

Conformable stress and strain sensors are required for monitoring the integrity of airframe structures as well as for sensing the mechanical stimuli in prosthetic arms. For this purpose, we have developed a series of piezoresistive single-wall carbon nanotube (SWCNT)/polymer nanocomposites. The electromechanical coupling of pressure with resistance changes in these nanocomposites is exceptionally greater than that of metallic piezoresistive materials. In fact, the piezoresistive stress coefficient (pi) of a SWCNT/polymer nanocomposite is approximately two orders of magnitude higher than that of a typical metallic piezoresistive. The piezoresistive stress coefficient is a function of the nanotube concentration wherein the maximum value occurs at a concentration just above the percolation threshold concentration (phi approx. 0.05 %). This response appears to originate from a change in intrinsic resistivity under compression/tension. A systematic study of the effect of the modulus of the polymer matrix on piezoresistivity allowed us to make flexible and conformable sensors for biomedical applications. The prototype haptic sensors using these nanocomposites are demonstrated. The piezocapacitive properties of SWCNT/polymer are also characterized by monitoring the capacitance change under pressure

Role of a conserved arginine residue during catalysis in serine palmitoyltransferase

AbstractAll sphingolipid-producing organisms require the pyridoxal 5′-phosphate (PLP)-dependent serine palmitoyltransferase (SPT) to catalyse the first reaction on the de novo sphingolipid biosynthetic pathway. SPT is a member of the alpha oxoamine synthase (AOS) family that catalyses a Claisen-like condensation of palmitoyl-CoA and l-serine to form 3-ketodihydrosphingosine (KDS). Protein sequence alignment across various species reveals an arginine residue, not involved in PLP binding, to be strictly conserved in all prokaryotic SPTs, the lcb2 subunits of eukaryotic SPTs and all members of the AOS family. Here we use UV–vis spectroscopy and site-directed mutagenesis, in combination with a substrate analogue, to show that the equivalent residue (R370) in the SPT from Sphingomonas wittichii is required to form the key PLP:l-serine quinonoid intermediate that condenses with palmitoyl-CoA and thus plays an essential role enzyme catalysis.Structured summary of protein interactionsSPT binds to SPT by molecular sieving (View interaction

The chemical basis of serine palmitoyltransferase inhibition by myriocin

Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent, enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular-mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition.</p