Identification of ethyl-6-bromo-2((phenylthio)methyl)imidazo[1,2-a]pyridine-3-carboxylate as a narrow spectrum inhibitor of Streptococcus pneumoniae and its FtsZ.

Filamentous temperature-sensitive mutant Z (FtsZ) is a key cell-division protein recognized as an important target for anti-bacterial drug discovery, especially in the context of rising multi-drug resistance. A respiratory pathogen, Streptococcus pneumoniae, is rapidly evolving antibiotic resistance, thus posing a clinical risk in the developing world. Inhibiting the conserved protein FtsZ, leading to the arrest of cell division, is an attractive alternative strategy for inhibiting S. pneumoniae. Previously, Vitamin K3 was identified as an FtsZ-targeting agent against S. pneumoniae. In the present work, docking studies were used to identify potential anti-FtsZ agents that bind to the Vitamin K3-binding region of a homology model generated for S. pneumoniae FtsZ. Compounds with imidazo[1,2-a]pyridine-3-carboxylate core were synthesized and screened for their anti-proliferative activity against S. pneumoniae. Remarkably, the hit compound IP-01 showed anti-bacterial action against S. pneumoniae without any activity on other bacteria. In S. pneumoniae, IP-01 showed similar inhibitory action on FtsZ and cell division as Vitamin K3. Sequence alignment identified three unique residues within S. pneumoniae FtsZ that IP-01 binds to, providing a structural basis for the observed specificity. IP-01 is one of the first narrow-spectrum agents identified against S. pneumoniae that targets FtsZ, and we present it as a promising lead for the design of narrow-spectrum anti-FtsZ anti-pneumococcal compounds.

Vitamin K3 inhibits FtsZ assembly, disrupts the Z-ring in Streptococcus pneumoniae and displays anti-pneumococcal activity.

The respiratory pathogen, Streptococcus pneumoniae has acquired multiple-drug resistance over the years. An attractive strategy to combat pneumococcal infection is to target cell division to inhibit the proliferation of S. pneumoniae. This work presents Vitamin K3 as a potential anti-pneumococcal drug that targets FtsZ, the master coordinator of bacterial cell division. Vitamin K3 strongly inhibited S. pneumoniae proliferation with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 6 µg/ml. Vitamin K3 disrupted the Z-ring localization in both S. pneumoniae and Bacillus subtilis within 30 min of treatment, while the membrane integrity and nucleoid segregation remain unchanged. Several complementary experiments showed that Vitamin K3 inhibits the assembly of purified S. pneumoniae FtsZ (SpnFtsZ) and induces conformational changes in the protein. Interestingly, Vitamin K3 interfered with GTP binding onto FtsZ and increased the GTPase activity of FtsZ polymers. The intrinsic tryptophan fluorescence of SpnFtsZ revealed that Vitamin K3 delays the nucleation of FtsZ polymers and reduces the rate of polymerization. In the presence of a non-hydrolyzable analog of GTP, Vitamin K3 did not show inhibition of FtsZ polymerization. These results indicated that Vitamin K3 induces conformational changes in FtsZ that increase GTP hydrolysis and thereby, destabilize the FtsZ polymers. Together, our data provide evidence that Vitamin K3 derives its potent anti-pneumococcal activity by inhibiting FtsZ assembly.

Heterogeneity in pneumolysin expression governs the fate of Streptococcus pneumoniae during blood-brain barrier trafficking.

Outcome of host-pathogen encounter is determined by the complex interplay between protective bacterial and host defense strategies. This complexity further amplifies with the existence of cell-to-cell phenotypic heterogeneity in pathogens which remains largely unexplored. In this study, we illustrated that heterogeneous expression of pneumolysin (Ply), a pore-forming toxin of the meningeal pathogen, S. pneumoniae (SPN) gives rise to stochastically different bacterial subpopulations with variable fate during passage across blood-brain barrier (BBB). We demonstrate that Ply mediated damage to pneumococcus containing vacuolar (PCV) membrane leads to recruitment of cytosolic "eat-me" signals, galectin-8 and ubiquitin, targeting SPN for autophagic clearance. However, a majority of high Ply producing subset extensively damages autophagosomes leading to pneumococcal escape into cytosol and efficient clearance by host ubiquitination machinery. Interestingly, a low Ply producing subset halts autophagosomal maturation and evades all intracellular defense mechanisms, promoting its prolonged survival and successful transcytosis across BBB, both in vitro and in vivo. Ply therefore acts as both, sword and shield implying that its smart regulation ensures optimal disease manifestation. Our elucidation of heterogeneity in Ply expression leading to disparate infection outcomes attempts to resolve the dubious role of Ply in pneumococcal pathogenesis.