Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-chain Fatty Acyl-CoAs and a Novel CoA-induced Fit Mechanism

PaaI thioesterases are members of the TE13 thioesterase family that catalyze the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8-Å resolution, to reveal two double hotdog domains arranged in a back to back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle x-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn³⁷, Asp⁵², and Thr⁶⁸ are important for catalysis, and size exclusion chromatography analysis and x-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38 Å in the N terminus, and a loop region involving Tyr³⁸-Tyr³⁹. This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family.     

Ammonia enhanced dark respiration in Chlorella vulgaris is related to collapse of a transmembrane pH gradient

Abstract We obtained, by different methods, isogenic lipopolysaccharide (O antigen) and capsular polysaccharide (K antigen) mutants from Klebsiella pneumoniae strains able to induce experimental infections (cytitis and pyelonephritis) in rats. We compared the induction of experimental infections in rats by wild-type strains and the lipopolysaccharide and capsular polysaccharide mutants. The high-molecular mass lipopolysaccharide of K. pneumoniae is clearly implicated in the infection process of the rat urinary tract, whilst the capsular polysaccharide seems not to be involved to the same extent.     

Streptococcus pneumoniae promotes its own survival via choline-binding protein CbpC-mediated degradation of ATG14

ABSTRACT

Streptococcus pneumoniae

is an opportunistic bacterial pathogen that can promote severe infection by overcoming the epithelial and blood-brain barrier. Pneumococcal cell-surface virulence factors, including cell wall-anchored choline-binding proteins (Cbps) play pivotal roles in promoting invasive disease. We reported previously that intracellular pneumococci were detected by hierarchical macroautophagic/autophagic processes that ultimately lead to bacterial elimination. However, whether intracellular pneumococci can evade autophagy by deploying Cbps remains unclear. In this study, we explore the biological functions of Cbps and reveal their roles in manipulating the autophagic process. Specifically, we found that CbpC-activated autophagy takes place via its interactions with ATG14 (autophagy related 14) and SQSTM1/p62 (sequestosome1). Importantly, CbpC dampens host autophagy by promoting ATG14 degradation via the ATG14-CbpC-SQSTM1/p62 axis. CbpC-induced reductions in ATG14 levels result in impaired ATG14-STX17 complex formation. In pneumococcal-infected cells, ATG14 levels are dramatically reduced in a CbpC-dependent manner that results in suppression of autophagy-mediated degradation and enhanced bacterial survival. Taken together, our results reveal a novel mechanism via which pneumococci can manipulate host autophagy responses, in this case, by employing CbpC as a trap to promote ATG14 depletion. Our findings highlight a novel and sophisticated tactic used by S. pneumoniae that serves to promote intracellular survival.