Sphingosine-1-phosphate lyases as novel therapeutic targets in intracellular bacteria
Custodio, Rafael Jose Da Silva
Thesis or dissertation
University of Exeter
A critical feature of the pathogenesis of B. pseudomallei is its ability to invade, survive and replicate in both phagocytic and non-phagocytic cells, hence a thorough understanding of this intracellular stage is essential in order to develop novel therapeutic strategies. Bioinformatics analysis on B. pseudomallei and B. thailandensis has revealed the presence of two distinct putative sphingosine-1-phosphate lyases (S1PLs). The human S1PL homolog is responsible for the irreversible degradation of sphingosine-1-phosphate (S1P), a critical regulator of immune responses. It was hypothesised whether Burkholderia-encoded S1PLs could represent a strategy employed by the bacteria to subvert the normal cellular functions of the host cells and contribute to B. pseudomallei pathogenesis. Using B. thailandensis as a model, we confirmed that both bacteria-encoded S1PLs orthologues are functionally equivalent to eukaryotic S1PL and are secreted in a pH-dependent manner, with secretion being observed during intracellular infection in macrophages. S1PL-deficient B. thailandensis mutants exhibited increased sensitivity to the antimicrobial sphingosine, attenuation in the Galleria mellonella infection model, impaired intracellular replication in macrophages, and inefficient phagosomal escape. Moreover, treatment of B. thailandensis-infected macrophages with S1P enhanced bacterial colocalisation with LAMP-1. Furthermore, either single or double deletion of S1PLs in B. pseudomallei displayed similar defective intracellular growth. Additionally, single deletion of S1PL displayed attenuation in BALB/c infection model. Macrophages infected with B. pseudomallei or B. thailandensis treated either with S1P or a specific agonist of S1P receptor 1 significantly enhanced phagocytic killing of intracellular bacteria. This study has highlighted the role of B. pseudomallei-encoded S1PL as a critical virulence determinant for intracellular survival, further establishing S1P-axis modulation as a strategy employed by Burkholderia to overcome the host immune response. In addition, herein has been demonstrated the potential therapeutic value of S1P pathway modulation for the treatment of intracellular infection.
PhD in Biological Sciences