Purification and crystallization of the hydroxylase component of the methanesulfonate monooxygenase from Methylosulfonomonas methylovora strain M2

The aim of the work described in this paper was two-fold: (1) the purification of the hydroxylase component of the MSAMO to electrophoretic homogeneity using a four-step chromatographic strategy and (2) the crystallization of the two-component hydroxylase of the MSAMO in order to enhance our understanding of the precise three-dimensional structure of the MSAMO, thus yielding an insight into the nature of the active site of this enzyme. Optimised crystallization conditions were identified allowing growth of crystals of the hydroxylase component of the MSAMO within five days. Crystals exhibited a brown colour suggesting the presence on an intact Rieske-iron sulfur centre and diffracted to 7.0 Å when a few degrees of data were evaluated on a beam line X11. © 2006 Elsevier Inc. All rights reserved.

Spray drying lactoferrin produces inhalable antimicrobial particles

The use of antimicrobial peptides and proteins as potential therapeutic agents in the management of multi-drug resistant infections is considered an attractive concept especially since such compounds should theoretically have low immunogenicity, high bioavailability with negligible toxicity. In this study we investigated the potential of developing a dry powder inhaler formulation of lactoferrin (a multifunctional iron binding protein). To achieve this, the protein was spray dried from a water only feedstock with suitably adjusted spray drying parameters. The particle size, degree of crystallinity, moisture content and yield of the spray dried powders along with the minimum bactericidal concentration (MBC) against Pseudomonas aeruginosa strain PAO1, were assessed. Dry powder inhaler formulations were prepared, and in vitro assessment studies using the multistage impinger were carried out to assess the aerosolisation performance of the formulations. Data obtained indicate that spray dried lactoferrin retains activity against biofilms and may be successfully employed in the treatment of chronic airway infections.

Molecular regulation of iron uptake in pseudomonas aeruginosa

The microbial demand for iron is often met by the elaboration of siderophores into the surrounding medium and expression of cognate outer membrane receptors for the ferric siderophore complexes. Conditions of iron limitation, such as those encountered in vivo, cause Pseudomonas aeruginosa to express two high-affinity iron-uptake systems based on pyoverdin and pyochelin. These systems will operate both in the organism's natural habitat, soil and water, where the solubility of iron at neutral pH is extremely low, and in the human host where the availability of free iron is too low to sustain bacterial growth due to the iron-binding glycoproteins transferrin and lactoferrin. Cross-feeding and radiolabelled iron uptake experiments demonstrated that pyoverdin biosynthesis and uptake were highly heterogeneous amongst P.aeruginosa strains, that growth either in the presence of pyoverdin or pyochelin resulted in induction of specific IROMPs, and that induction of iron uptake is siderophore-specific. The P.aeruginosa Tn5 mutant PH1 is deficient in ferripyoverdin uptake and resistant to pyocin Sa, suggesting that the site of interaction of pyocin Sa is a ferripyoverdin receptor. Additional Tn5 mutants appeared to exploit different strategies to achieve pyocin Sa-resistance, involving modifications in expression of pyoverdin-mediated iron uptake, indicating that complex regulatory systems exist to enable these organisms to compete effectively for iron. Modulation of expression of IROMPs prompted a study of the mechanism of uptake of a semi-synthetic C(7) α-formamido substituted cephalosporin BRL 41897A. Sensitivity to this agent correlated with expression of the 75 kDa ferri-pyochelin receptor and demonstrated the potential of high-affinity iron uptake systems for targeting of novel antibiotics. Studies with ferri-pyoverdin uptake-deficient mutant PH1 indicated that expression of outer membrane protein G (OprG), which is usually expressed under iron-rich conditions and repressed under iron-deficient conditions, was perturbed. Attempts were made to clone the oprG gene using a degenerate probe based on the N-terminal amino acid sequence. A strongly hybridising HindIll restriction fragment was cloned and sequenced, but failed to reveal an open reading frame correspondmg to OprG. However, there appears to be good evidence that a part of the gene codmg for the hydrophilic membrane-associated ATP-binding component of a hitherto uncharacterised periplasmic- binding-protein-dependent transport system has been isolated. The full organisation and sequence of the operon, and substrate for this putative transport system, are yet: to be elucidated,

Influence of iron on bacterial infections in leukaemia

The influence of iron metabolism, both on the invading bacterial pathogen and in the host is widespread and often appears to be crucial in determining the outcome of an infection. This study involved the investigation of leukaemia, a clinical disease where abnormal availability of iron may play a part in predisposing patients to bacterial infection. The iron status throughout a Gram-negative septicaemia and in 20 random, newly diagnosed leukaemic patients was assessed. The results revealed that the majority of the patients exhibited high serum iron levels and serum transferrin saturation often at 100%, with an inability to reduce the latter to within normal values during an infection episode. The antibody response to P.aeruginosa, E.coli and K.pneumoniae outer membrane protein (OMP) antigens were investigated by immunoblotting with sequential serum samples during infection in the leukaemic host. Antibodies to all the major OMPs, were observed, although recognition of iron-regulated membrane proteins (IRMPs) was in many cases weak. Results from the enzyme-linked immunosorbent assay indicated that in all patients antibody titre in response to infection was poor. Sub-MICs of mitomycin C significantly altered the surface characteristics of P.aeruginosa. The silver-stained SDS-PAGE gels of proteinase K digested whole cell lysates of strains PAO1, 6750, M7 and PAJ indicated that core LPS was affected in the presence of mitomycin C. In contrast, the rough strain AK1012 showed no observable differences. Results obtained using quantitative gas-liquid chromatographic analysis showed the amount of LPS fatty acids to be unaffected, however, the KDO and carbohydrate content in strains PAO1, 6750 and M7 under Fe+ and Fe- growth conditions were decreased by up to 4-fold in the presence of mitomycin C, indicating perturbed expression of LPS. The cell surface became significantly more hydrophobic in the P.aeruginosa strains, except AK1012 which was comparatively unaffected. The induction of protein G (OprG) in P.aeruginosa was found to be a sensitive indicator of media iron. The data indicated that expression of OprG can be modulated by growth rate/phase, availability of iron and by the presence of ciprofloxacin in the growth medium.