Pathology of Tnf-deficient mice infected with Plasmodium chabaudi adami 408XZ.

Tumor necrosis factor alpha (Tnf) plays a pleiotropic role in murine malaria. Some investigations have correlated Tnf with hypothermia, hyperlactatemia, hypoglycemia, and a suppression of the erythropoietic response, although others have not. In this study, we have evaluated parasitemia, survival rate and several pathological features in C57BL/6JTnf(-/-) and C57BL/6JTnf(+/+) mice after infection with Plasmodium chabaudi adami 408XZ. Compared to the C57BL/6JTnf(+/+) mice, C57BL/6JTnf(-/-) mice showed increased parasitemia and decreased survival rate, whereas blood glucose, blood lactate and body weight were not significantly different. However, C57BL/6JTnf(-/-) mice suffered significantly more from severe anemia and hypothermia than C57BL/6JTnf(+/+) mice. These results suggest that Tnf is an important mediator of parasite control, but not of anemia development. We hypothesize that the high mortality observed in the Tnf knock-out mice is due to increased anemia and pathology as a direct result of increased levels of parasitemia.

TNF as a malaria candidate gene: polymorphism-screening and family-based association analysis of mild malaria attack and parasitemia in Burkina Faso.

We have previously obtained strong evidence for linkage of mild malaria attack to the MHC region, with a peak close to the tumor necrosis factor (TNF) gene. We screened, for polymorphisms, the entire TNF gene in the same sample of 34 families comprising 197 individuals living in a Plasmodium falciparum endemic area and we found 17 polymorphisms. In a longitudinal study, we investigated whether the 11 most frequent and informative polymorphisms were associated with mild malaria attack and maximum parasitemia, which was the highest parasitemia in each individual over 2 years. Mild malaria attack and maximum parasitemia were positively correlated. Transmission disequilibrium tests showed nominal evidence for association between TNF-1031, TNF-308, TNF851 and TNF1304 polymorphisms, and mild malaria attack on the one hand, and between TNF-238, TNF851 and TNF1304 polymorphisms, and maximum parasitemia on the other hand. After accounting for multiple tests, we confirmed the association of TNF-238 with maximum parasitemia and the association of TNF1304 and TNF851 with maximum parasitemia and mild malaria attack. The association tests with mild malaria attack suggest a moderate effect of TNF-308 polymorphism. In conclusion, our study suggests that several TNF variants may be part of the genetic determinants for maximum parasitemia and/or mild malaria attack.

Comparison of pathology in susceptible A/J and resistant C57BL/6J mice after infection with different sub-strains of Plasmodium chabaudi.

Susceptible A/J and more resistant C57BL/6J mice were infected with Plasmodium chabaudi chabaudi 54X, P.c. chabaudi AS and Plasmodium chabaudi adami 408XZ. As expected, most C57BL/6J mice survived the infections with the different isolates. But in contrast to previous observations, not all A/J mice succumbed to infection: just over 50% of A/J mice survived infections with P.c. chabaudi 54X, while 80% survived P.c. chabaudi AS. The more virulent parasite, P.c. adami 408XZ, was able to kill all A/J mice and 20% of C57BL/6J mice after an intravenous infection with 10(5) pRBC. A detailed study of four parameters of pathology (body weight, body temperature, blood glucose and RBC counts) in both mouse strains after a P.c. adami 408XZ infection showed similar patterns to those previously reported after infection with P.c. chabaudi AS. These data suggest that environmental factors as well as parasite polymorphisms might influence the severity of malaria between susceptible and resistant mice.

Kinetic and spectroscopic characterization of native and metal-substituted beta-lactamase from Aeromonas hydrophila AE036.

Two metal ion binding sites are conserved in metallo-beta-lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur-to-metal charge transfer bands are observed for all mono- and di-metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non-competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.

Mono- and binuclear Zn2+-beta-lactamase. Role of the conserved cysteine in the catalytic mechanism.

When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistance to most beta-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn2+-beta-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn2+-beta-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn2+ (mononuclear) and di-Zn2+ (binuclear) Zn2+-beta-lactamases are catalytically active but with different kinetic properties. The mono-Zn2+-beta-lactamase requires the conserved cysteine residue for hydrolysis of the beta-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.

Mono- and binuclear Zn-beta-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions.

The Bacteroides fragilis Zn-beta-lactamase is active with a mono- and a binuclear zinc site. The apoenzyme produced by removal of both Zn ions does not recover full activity upon readdition of Zn2+ in contrast to an active mono-Zn form prepared at pH 6.0. Differences in k(cat) values observed are substrate-dependent implying distinct mechanisms for the mono- and binuclear species. The substrate profile of a Zn,Cd hybrid obtained by selective exchange of one zinc ion is different from that of the Zn2 enzyme with a remarkable 15-fold increased activity with cefoxitin as substrate.

Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-beta-lactamase activity and stability.

Two Zn2+ binding sites were found in the Aeromonas hydrophila AE036 metallo-beta-lactamase. The affinity of the first binding site for Zn2+ ions is so high that the dissociation constant could not be determined, but it is significantly lower than 20 nM. The mono-Zn2+ form of the enzyme exhibits a maximum activity against its carbapenem substrates. The presence of a Zn2+ ion in the second lower affinity binding site results in a loss of enzymatic activity with a Ki value of 46 microM at pH 6.5. The kinetic analysis is in agreement with a noncompetitive inhibition mechanism. The Zn content of the A. hydrophila enzyme is also strongly pH-dependent. With an external Zn2+ ion concentration of 0.4 microM, occupancy of the higher affinity site by metal ions is lower than 10% at pH 5 and 10. The affinity for the second binding site seems to increase from pH 6 to 7.5. Fluorescence emission and circular dichroism spectra revealed slight conformational changes upon titration of the apoenzyme by Zn2+ ions, resulting in the successive saturation of the first and second binding sites. Differential scanning calorimetry transitions and intrinsic fluorescence emission spectra in the presence of increasing concentrations of urea demonstrate that the catalytic zinc strongly stabilizes the conformation of the enzyme whereas the di-Zn enzyme is even more resistant to thermal and urea denaturation than the mono-Zn enzyme. The Zn2+ dependency of the activity of this metallo-beta-lactamase thus appears to be very different from that of the homologous Bacteroides fragilis enzyme for which the presence of two Zn2+ ions per molecule of protein appears to result in maximum activity.