Microbial NTPDases and their contribution to infection

© 2012 Dr. Patrice RiedmaierLegionella pneumophila is an intracellular bacterium that has adapted to infect human hosts. Infection of susceptible individuals can result in the onset of severe atypical pneumonia commonly referred to as Legionnaires’ disease. Genome sequence analysis of L. pneumophila has revealed the presence of numerous eukaryotic-like proteins and proteins with eukaryotic domains. These include two apyrases annotated lpg0971 and lpg1905, which are homologues of human CD39/NTPDase1. Lpg0971 and Lpg1905 belong to the GDA1_CD39 superfamily that are characterised by their ability to hydrolyse terminal phosphoanhydride bonds of extracellular nucleoside di- and tri-phosphates, such as ATP and ADP. Prior to the commencement of this study, the importance of Lpg1905 to bacterial infection has been established. In this study, the contribution of both NTPDases to Legionella infection was investigated, in particular the role of Lpg0971 during infection and further structural and functional characterisation of Lpg1905. A biochemical assay that measures the release of inorganic phosphate was used to determine the functional characteristics of recombinant, refolded Lpg0971. Highest enzymatic function was observed in the presence of manganese (II) ions. Additionally, Lpg0971 displayed affinity for ATP hydrolysis only. To ascertain whether Lpg0971 contributed to vacuolar replication within host cells, bacterial replication was tested in human alveolar macrophages and amoebae. The lpg0971 mutant was unable to replicate over 72 h in both cell lines; however, complementation of the mutant restored replication. Moreover, the two NTPDases appeared to be functionally redundant during replication in macrophages. The elucidation of the crystal structure of Lpg1905 during the course of this study provided insight into the residues surrounding the substrate in the active site. Three residues were selected for their proximity to the substrate binding pocket and targeted site-directed mutagenesis was performed. These studies revealed the importance of Arg56 for binding the substrate during hydrolysis and Try346 for positioning the substrate during hydrolysis. The conservative mutation R56K resulted in a mutant derivative with NTPase activity only (35% of wild type activity), whereas the nonconservative mutation Y346A produced a mutant derivative with higher NDPase activity (35% of wild type activity) than NTPase activity (20% of wild type activity). Comparison of the ability of these mutant derivatives to complement lpg1905 mutant during in vitro replication assays within THP-1 macrophages demonstrated the importance of NTPase activity rather than NDPase activity of Lpg1905 to bacterial replication. This was supported by a chimeric mutant derivative in which the ACR1 motif of Lpg1905 was mutated from TGSR to SHTS that exhibited only NTPase activity (40% of wild type activity) but was also able to replicate within THP-1 macrophages. Lastly, enzyme kinetic comparisons between wild type and chimeric Lpg1905 revealed differences in both the substrate binding affinity and the action of various NTPDase inhibitors. Of the various inhibitors tested, gadolinium (III) chloride was the most potent. Overall this study established the role of both Lpg0971 and Lpg1905 in Legionella infection and highlighted structural and functional characteristics of both L. pneumophila NTPDases

A type III effector antagonizes death receptor signalling during bacterial gut infection.

Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response