The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD + -or NADP + -dependent reversible conversion of α-ketoglutarate (AKG) to L-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP + -dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate, and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general.

25 26 KEY WORDS: Streptococcus pneumoniae, infection tolerance, autophagy, pneumolysin, pore-27 forming toxin, cholesterol dependent cytolysin, cation-π interaction 28 2 ABSTRACT 29 The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic 30 colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host 31 compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore 32 forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although 33 pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the 34 primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a 35 bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. 36 Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of 37 pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and 38 T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel 39 inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) 40 in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this 41 interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, 42 inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and 43 autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that 44 was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue 45 damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. 46 Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal 47 157 pneumolysin (Ply-H; 5CR6 and 4QQA) (16, 22). Superposition of Ply-NH structure with the 158 recently reported structures of Ply-H (5CR6 and 4QQA) (16, 22) produced root mean square 159 deviation (r.m.s.d.) of 2.4 and 1.2 Å, respectively, over 471 Cα atoms. Superposition of specific 160 domains, D1-3 of Ply-NH and Ply-H (5CR6) yielded r.m.s.d. of 0.75 Å and alignment of only D4 161

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD+- or NADP+-dependent reversible conversion of α-ketoglutarate (AKG) to L-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP+-dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general.