In the presence of GlcN-6P, SiaR bound the probe and GlcN-6P slightly increased the binding affinity. While the presence of GlcN-6P did not result in a major change in the binding affinity of SiaR, the change in the shift does suggest that GlcN-6P is interacting with SiaR and impacting its ability to bind to its operator. Other phosphosugars of the sialic acid catabolic pathway (sialic acid, ManNAc, and GlcNAc-6P) nor GlcN-1P altered SiaR-binding (unpublished data) [14]. Taken together with the expression data, this demonstrates that GlcN-6P interacts with SiaR

and has an effect on its DNA-binding properties. SiaR is not displaced from the DNA, but instead functions as an activator with GlcN-6P as a co-activator. As in our previous studies [14], the binding of SiaR to the EMSA probe resulted in the appearance of two shifted bands (Figure

GDC 0449 VX-689 6). This was even more apparent when lower concentrations of SiaR were present in the binding reaction. The double shift is possibly caused by the binding of multiple SiaR proteins to the probe. This is a likely explanation, considering that the region protected by SiaR is large (53 bp) [14]. Further work will be necessary to determine the exact cause for the double shift. GlcN-6P accumulates in a nagB mutant To confirm that Neu5Ac was transported and catabolized in the 2019ΔcyaA ΔnagB mutant strain, 31P NMR spectroscopy of intact cells was used. Cultures of wild-type 2019 and 2019ΔcyaA ΔnagB were grown to early exponential phase and cAMP and/or Neu5Ac were added and the 31P spectrum was obtained (Figure 7). A peak was detected near 5 ppm when cAMP was added to either strain. When Neu5Ac was added, a peak was detected near 7 ppm in the 2019ΔcyaA ΔnagB mutant that was absent in the wild-type strain. This peak was also absent in either strain when Neu5Ac was omitted. This indicated the accumulation of a significant amount of a phosphorylated compound in the mutant strain when exogenous Neu5Ac was

present. Since the Neu5Ac catabolic pathway is blocked at NagB in the mutant strain, Neu5Ac would be converted nearly to GlcN-6P, but not Fru-6P. Taken together with the interaction of GlcN-6P with purified SiaR, this indicates that GlcN-6P is accumulating in the 2019ΔcyaA ΔnagB mutant and is responsible for the activation of the nan operon. Figure 7 BIBF 1120 clinical trial Detection of intracellular GlcN-6P by 31 P NMR spectroscopy. 31P NMR spectra were obtained following the growth of cells in the presence of exogenous cAMP and/or Neu5Ac. A. 2019ΔcyaA ΔnagB with Neu5Ac and cAMP. B. 2019 wild-type with Neu5Ac and cAMP. C. 2019ΔcyaA ΔnagB with cAMP. D. 2019 wild-type with cAMP. E. 2019 wild-type without supplement. Discussion The importance of sialic acid in the protection of NTHi from the host immune response requires that most of the sialic acid transported into the cell is activated by SiaB and utilized for the decoration of the LOS and biofilm matrix.