The morphogenic protein CopD controls the spatio-temporal dynamics of PBP1a and PBP2b in Streptococcus pneumoniae

International audiencePenicillin-binding proteins (PBPs) are key to the assembly of peptidoglycan, the major component of the bacterial cell wall. Although several PBP-specific regulatory proteins have been identified in different species, little is known about how the activity of PBPs is controlled and coordinated during the cell cycle. In this study, we characterize the unknown function protein Spr1400 and demonstrate its regulatory function on two PBPs in Streptococcus pneumoniae. For that, we use a combination of technics ranging from bacterial genetics and protein biochemistry to microscopy imaging. First, we show that pneumococcal Spr1400 localizes late to the cell division septum. Furthermore, deletion of spr1400 results in wider cells. Using co-immunoprecipitation and bacterial two hybrid (B2H), we observe that Spr1400 interacts with two PBPs, the class A PBP PBP1a and the class B PBP PBP2b, which are required for cell elongation. Microscale thermophoresis combined with B2H further reveals that these interactions occur through their transmembrane domains. We also show that Spr1400 co-localizes with PBP1a and PBP2b throughout the cell cycle. Strikingly, deletion of spr1400 alters the dynamics of PBP1a and PBP2b. Indeed, the two PBPs persist longer at the division site and localize later at the division site of daughter cells. Collectively, these data demonstrate that Spr1400, thus named CopD for coordinator of PBP1a and 2b dynamics, is a spatio-temporal regulator of PBP1a and PBP2b required for pneumococcal morphogenesis

The variant hERG/R148W associated with LQTS is a mutation that reduces current density on co-expression with the WT

International audienceBackground: A variant of the ether-à-go-go related channel (hERG), p.Arg148Trp (R148W) was found at heterozygous state in two infants who died from sudden infant death syndrome (SIDS), one with documented prolonged QTc and Torsade de Pointes (TdP), and in an adult woman with QTc > 500 ms, atrio-ventricular block and TdP. This variant was previously reported in cases of severe ventricular arrhythmia but very rarely in control subjects. Its classification as mutation or polymorphism awaited electrophysiological characterization. Methods: The properties of this N-terminal, proximal domain, hERG variant were explored in Xenopus oocytes injected with the same amount of RNA encoding for either hERG/WT or hERG/R148W or their equimolar mixture. The human ventricular cell (TNNP) model was used to test the effects of changes in hERG current.Results: R148W alone produced a current similar to the WT (369±76 nA (mean±SEM), n=13 versus 342±55 nA in WT, n=13), while the coexpression of 1/2 WT + 1/2 R148W lowered the current by 29 % versus WT (243±35 nA, n=13, p<0.05). The voltage dependencies of steady-state activation and inactivation were not changed in the variant alone or in coexpression with the WT. The time constants of fast recovery from inactivation and of fast and slow deactivation analyzed between –120 and +20 mV were not changed. The voltage-dependent distribution of the current amplitudes among fast-, slow- and non-deactivating fractions was unaltered. A 6.6 % increase in APD90 from 323.5 ms to 345 ms was observed using the human cardiac ventricular myocyte model.Conclusions: Such a decrease in hERG current as evidenced here when co-expressing the hERG/R148W variant with the WT may have predisposed to the observed long QT syndrome and associated TdP. Therefore, the heterozygous carriers of hERG/R148W may be at risk of cardiac sudden death