Structure of 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major cause of opportunistic infection and is resistant to most antibiotics. As part of efforts to generate much-needed new antibiotics, structural studies of enzymes that are critical for the virulence of P. aeruginosa but are absent in mammals have been initiated. 2-Keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8Ps), also known as 2-dehydro-3-deoxyphosphooctonate aldolase, is vital for the survival and virulence of P. aeruginosa. This enzyme catalyzes a key step in the synthesis of the lipopolysaccharide (LPS) of most Gram-negative bacteria: the condensation reaction between phosphoenolpyruvate (PEP) and arabinose 5-phosphate to produce 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P). This step is vital for the proper synthesis and assembly of LPS and the survival of P. aeruginosa. Here, the recombinant expression, purification and crystal structure of KDO8Ps from P. aeruginosa are presented. Orthorhombic crystals were obtained by vapor diffusion in sitting drops in the presence of 1 mM phosphoenlpyruvate. The structure reveals the prototypical α/ß TIM-barrel structure expected from this family of enzymes and contains a tetramer in the asymmetric unit.

Immunomodulatory proteins from hookworms reduce cardiac inflammation and modulate regulatory responses in a mouse model of chronic Trypanosoma cruzi infection.

Introduction: Hookworms are parasitic helminths that secrete a variety of proteins that induce anti-inflammatory immune responses, stimulating increased CD4 + Foxp3+ regulatory T cells and IL-10 production. Hookworm-derived recombinant proteins AIP-1 and AIP-2 have been shown to reduce inflammation in mouse models of inflammatory bowel disease and inflammatory airway disease by inducing CD4+Foxp3+ cells and IL-10 production. In contrast, chronic infection with the protozoal parasite Trypanosoma cruzi, the causative agent of Chagas disease, leads to chronic inflammation in tissues. Persistence of the parasites in tissues drives chronic low-grade inflammation, with increased infiltration of inflammatory cells into the heart, accompanied by increased production of inflammatory cytokines. There are no current antiparasitic drugs that effectively reduce or prevent chronic myocarditis caused by the onset of Chagas disease, thus new therapies are urgently needed. Therefore, the impact of AIP-1 and AIP-2 on myocarditis was investigated in a mouse model of chronic T. cruzi infection. Methods: Female BALB/c mice infected with bioluminescent T. cruzi H1 strain trypomastigotes for 70 days were treated once daily for 7 days with 1mg/kg AIP-1 or AIP-2 protein by intraperitoneal injection. Control mice were left untreated or treated once daily for 14 days with 25mg/kg aspirin in drinking water. At 84 days of infection, splenocytes, cardiac tissue and serum were collected for evaluation. Results: Treatment with both AIP-1 and AIP-2 proteins significantly reduced cardiac cellular infiltration, and reduced cardiac levels of IFNγ, IL-6 and IL-2. AIP-2 treatment reduced cardiac expression of COX-2. Further, while incubation with AIP-1 and AIP-2 proteins did not induce a significant upregulation of an immunoregulatory phenotype in dendritic cells (DC), there was a modest upregulation of CD11c +CD11b+MHCII+SIRPα+ expression, suggesting a regulatory phenotype. Ex-vivo stimulation of splenocytes from the treatment groups with AIP-1 loaded DC induced reduced levels of cytotoxic and pro-inflammatory T cells, stimulation with AIP-2 loaded DC specifically induced enhanced levels of CD4+CD25+Foxp3+ regulatory T cells among treatment groups. Discussion: All in vivo and in vitro results demonstrate that hookworm-derived AIP-1 and AIP-2 proteins reduce T. cruzi induced cardiac inflammation, possibly through multiple anti-inflammatory mechanisms.

Structure of type II dehydroquinase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes opportunistic infections and is resistant to most antibiotics. Ongoing efforts to generate much-needed new antibiotics include targeting enzymes that are vital for P. aeruginosa but are absent in mammals. One such enzyme, type II dehydroquinase (DHQase), catalyzes the interconversion of 3-dehydroquinate and 3-dehydroshikimate, a necessary step in the shikimate pathway. This step is vital for the proper synthesis of phenylalanine, tryptophan, tyrosine and other aromatic metabolites. The recombinant expression, purification and crystal structure of catalytically active DHQase from P. aeruginosa (PaDHQase) are presented. Cubic crystals belonging to space group F23, with unit-cell parameters a=b=c=125.39 Å, were obtained by vapor diffusion in sitting drops and the structure was refined to an R factor of 16% at 1.74 Šresolution. PaDHQase is a prototypical type II DHQase with the classical flavodoxin-like α/ß topology.