Active site mutants of human secreted Group IIA Phospholipase A(2) lacking hydrolytic activity retain their bactericidal effect

The Human Secreted Group IIA Phospholipase A(2) (hsPIA2GIIA) presents potent bactericidal activity, and is considered to contribute to the acute-phase immune response. Hydrolysis of inner membrane phospholipids is suggested to underlie the bactericidal activity, and we have evaluated this proposal by comparing catalytic activity with bactericidal and liposome membrane damaging effects of the G30S, H48Q and D49K h5PLA2GIIA mutants. All mutants showed severely impaired hydrolytic activities against mixed DOPC:DOPG liposome membranes, however the bactericidal effect against Micrococcus luteus was less affected, with 50% killing at concentrations of 1, 3, 7 and 9 mu g/mL for the wild-type, D49K, H48Q and G30S mutants respectively. Furthermore, all proteins showed Ca2+-independent damaging activity against Liposome membranes demonstrating that in addition to the hydrolysis-dependent membrane damage, the hsPLA2GIIA presents a mechanism for permeabilization of phospholipid bilayers that is independent of catalytic activity, which may play a role in the bactericidal function of the protein (C) 2011 Elsevier Masson SAS. All rights reserved.

Conformation analysis of a surface loop that controls active site access in the GH11 xylanase A from Bacillus subtilis

Xylanases (EC 3.2.1.8 endo-1,4-glycosyl hydrolase) catalyze the hydrolysis of xylan, an abundant hemicellulose of plant cell walls. Access to the catalytic site of GH11 xylanases is regulated by movement of a short beta-hairpin, the so-called thumb region, which can adopt open or closed conformations. A crystallographic study has shown that the D11F/R122D mutant of the GH11 xylanase A from Bacillus subtilis (BsXA) displays a stable "open" conformation, and here we report a molecular dynamics simulation study comparing this mutant with the native enzyme over a range of temperatures. The mutant open conformation was stable at 300 and 328 K, however it showed a transition to the closed state at 338 K. Analysis of dihedral angles identified thumb region residues Y113 and T123 as key hinge points which determine the open-closed transition at 338 K. Although the D11F/R122D mutations result in a reduction in local inter-intramolecular hydrogen bonding, the global energies of the open and closed conformations in the native enzyme are equivalent, suggesting that the two conformations are equally accessible. These results indicate that the thumb region shows a broader degree of energetically permissible conformations which regulate the access to the active site region. The R122D mutation contributes to the stability of the open conformation, but is not essential for thumb dynamics, i.e., the wild type enzyme can also adapt to the open conformation.

The presence of a booster phenomenon among contacts of active pulmonary tuberculosis cases: a retrospective cohort

Abstract Background Assuming a higher risk of latent tuberculosis (TB) infection in the population of Rio de Janeiro, Brazil, in October of 1998 the TB Control Program of Clementino Fraga Filho Hospital (CFFH) routinely started to recommend a two-step tuberculin skin test (TST) in contacts of pulmonary TB cases in order to distinguish a boosting reaction due to a recall of delayed hypersensitivity previously established by infection with Mycobacterium tuberculosis (M.tb) or BCG vaccination from a tuberculin conversion. The aim of this study was to assess the prevalence of boosted tuberculin skin tests among contacts of individuals with active pulmonary tuberculosis (TB). Methods Retrospective cohort of TB contacts ≥ 12 years old who were evaluated between October 1st, 1998 and October 31st 2001. Contacts with an initial TST ≤ 4 mm were considered negative and had a second TST applied after 7–14 days. Boosting reaction was defined as a second TST ≥ 10 mm with an increase in induration ≥ 6 mm related to the first TST. All contacts with either a positive initial or repeat TST had a chest x-ray to rule out active TB disease, and initially positive contacts were offered isoniazid preventive therapy. Contacts that boosted did not receive treatment for latent TB infection and were followed for 24 months to monitor the development of TB. Statistical analysis of dichotomous variables was performed using Chi-square test. Differences were considered significant at a p < 0.05. Results Fifty four percent (572/1060) of contacts had an initial negative TST and 79% of them (455/572) had a second TST. Boosting was identified in 6% (28/455). The mean age of contacts with a boosting reaction was 42.3 ± 21.1 and with no boosting was 28.7 ± 21.7 (p = 0.01). Fifty percent (14/28) of individuals whose test boosted met criteria for TST conversion on the second TST (increase in induration ≥ 10 mm). None of the 28 contacts whose reaction boosted developed TB disease within two years following the TST. Conclusion The low number of contacts with boosting and the difficulty in distinguishing boosting from TST conversion in the second TST suggests that the strategy of two-step TST testing among contacts of active TB cases may not be useful. However, this conclusion must be taken with caution because of the small number of subjects followed.