Fig 1.
Conditional in vitro treatments of B. pahangi adult worms.
A. The wsp copy number per worm in adult males after a 6-day treatment was measured by qPCR. The copy number reflects the abundance of bacteria in treated and control worms (***–p<0.001). B. Relative changes in the concentration of pyruvate within the worms measured after 1 and 6 days of treatment. Amount of pyruvate is expressed as μg per ml per worm. ***–p<0.001 as compared to relevant control (day 1 or day 6). C. Relative number of Mf released from treated females during the last 2 days of the 6-day treatment. Number of Mf released from control was assigned as 100%. ***–p<0.001 as compared to control and xxx–p<0.001 as compared to M+3BrPyr group. D. Viability of females treated for 6 days. The viability of control worms was taken as 100%. ***–p<0.001 as compared to control and xxx–p<0.001 as compared to M+3BrPyr group. E. Relative gene expression of wBm0209 and wBm0207 genes in treated worms. The analysis showed that treatment with sodium pyruvate induced the expression of bacterial genes as compared to control (***–p<0.001). M–medium control; M+NaPyr–treatment with sodium pyruvate; M+3BrPyr–treatment with 3BromoPyruvate; M+3BrPyr+NaPyr–treatment with 3BromoPyruvate and sodium pyruvate.
Fig 2.
Effect of treatment with a pyruvate kinase inhibitor on the fitness of both Wolbachia and the worms.
A. The wsp copy number per worm in adult males after a 6-day treatment was measured by qPCR (***–p<0.001). B. Relative changes in the concentration of pyruvate within the worms assigning 100% for control samples on day 1 of the treatment (***–p<0.001). C. Relative number of Mf released from female worms during the last 2 days of the 6-day treatment. Number of Mf released from control was assigned as 100%. ***–p<0.001 as compared to control and xxx–p<0.001 as compared to M+PKI III group. M–medium control; M+NaPyr–treatment with sodium pyruvate; M+PKI III–treatment with pyruvate kinase inhibitor III; M+PKI III+NaPyr–treatment with PKI III and sodium pyruvate; M+PKA–treatment with PK activator.
Fig 3.
Association of Bm-LDH and wBm0432 of Wolbachia.
A. Western blot of the pull-down assay confirming the formation of complexes between wBm0432-HIS and Bm-LDH-GST recombinant proteins. Lane 1, wBm0432-HIS immobilized on Ni-NTA beads (wBm0432-HIS-beads) was incubated with bacterial control extract (bacteria that did not express Bm-LDH-GST). Lane 2, free beads (no proteins were immobilized on the Ni-NTA beads) were incubated with bacterial control extract (bacteria did not express Bm-LDH-GST). Lane 3, wBm0432-HIS-beads were incubated with Bm-LDH-GST within the bacterial extract (bacteria expressing Bm-LDH-GST). Lane 4, free beads were incubated with Bm-LDH-GST within the bacterial extract (bacteria expressed Bm-LDH-GST). Western blot was treated with anti-GST (A’) or with anti-HIS (A”) antibodies. B, B’, B”. Confocal image showing the co-localization of wBm (red, B) and LDH (green, B’) in the cytoplasm of the parasite’s lateral chord. B”. Merged red and green channels. Colocalization of red and green signals merged as yellow. Magnification 63x. Negative control is presented in S2 Fig.
Fig 4.
Effect of treatment with LDH inhibitor on the Wolbachia-parasite interaction.
A. The wsp copy number per worm in adult males after a 6-day treatment was measured by qPCR (***–p<0.001). B. Relative gene expression analysis of Bm-LDH in samples treated with pyruvate (M+Pyr, grey, triangle), 3BromoPyruvate, inhibitor of glycolysis (M+3BrPyr, Blue, diamond) and control (M, gray, square). The analysis showed that pyruvate treatment induced expression of Bm-LDH after the first day of treatment. Inhibition of glycolysis only activated the expression of Bm-LDH in worms after 3 days of treatment. C. Schematic presentation of the role of Bm-LDH in pyruvate distribution between bacteria and conversion into lactate in parasite cells.
Fig 5.
Analysis of transcriptomic data obtained from B. malayi L3, L4 and adult worms.
A. Gene expression of PyrK (Bm3590) responsible for the final step in glycolysis (conversion of P-PEP to pyruvate). B. Gene expression of LDH (Bm3339) responsible for the conversion between lactate and pyruvate. C. Gene expression of HIF-1 (Bm3907), transcriptional factor that initiates the expression of glycolytic enzymes. D, E, F. Gene expression of PyrDH (Bm9000 (D), Bm7953 (E), Bm3652 (F)) responsible for the conversion of pyruvate to Acetyl-CoA. ***–p<0.001 as compared to L3 samples.