HmuY Haemophore and Gingipain Proteases Constitute a Unique Syntrophic System of Haem Acquisition by Porphyromonas gingivalis

Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin

Unique Structure and Stability of HmuY, a Novel Heme-Binding Protein of Porphyromonas gingivalis

Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-β fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection

Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies In Vitro and In Vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo

Aspirin induces cell death and caspase-dependent phosphatidylserine externalization in HT-29 human colon adenocarcinoma cells

The induction of cell death by aspirin was analysed in HT-29 colon carcinoma cells. Aspirin induced two hallmarks of apoptosis: nuclear chromatin condensation and increase in phosphatidylserine externalization. However, aspirin did not induce either oligonucleosomal fragmentation of DNA, decrease in DNA content or nuclear fragmentation. The effect of aspirin on Annexin V binding was inhibited by the caspase inhibitor Z-VAD.fmk, indicating the involvement of caspases in the apoptotic action of aspirin. However, aspirin did not induce proteolysis of PARP, suggesting that aspirin does not increase nuclear caspase 3-like activity in HT-29 cells. This finding may be related with the ‘atypical’ features of aspirin-induced apoptosis in HT-29 cells. © 1999 Cancer Research Campaig

Mixed model approaches for the identification of QTLs within a maize hybrid breeding program

Two outlines for mixed model based approaches to quantitative trait locus (QTL) mapping in existing maize hybrid selection programs are presented: a restricted maximum likelihood (REML) and a Bayesian Markov Chain Monte Carlo (MCMC) approach. The methods use the in-silico-mapping procedure developed by Parisseaux and Bernardo (2004) as a starting point. The original single-point approach is extended to a multi-point approach that facilitates interval mapping procedures. For computational and conceptual reasons, we partition the full set of relationships from founders to parents of hybrids into two types of relations by defining so-called intermediate founders. QTL effects are defined in terms of those intermediate founders. Marker based identity by descent relationships between intermediate founders define structuring matrices for the QTL effects that change along the genome. The dimension of the vector of QTL effects is reduced by the fact that there are fewer intermediate founders than parents. Furthermore, additional reduction in the number of QTL effects follows from the identification of founder groups by various algorithms. As a result, we obtain a powerful mixed model based statistical framework to identify QTLs in genetic backgrounds relevant to the elite germplasm of a commercial breeding program. The identification of such QTLs will provide the foundation for effective marker assisted and genome wide selection strategies. Analyses of an example data set show that QTLs are primarily identified in different heterotic groups and point to complementation of additive QTL effects as an important factor in hybrid performance

Fragile x syndrome and autism: from disease model to therapeutic targets

Autism is an umbrella diagnosis with several different etiologies. Fragile X syndrome (FXS), one of the first identified and leading causes of autism, has been modeled in mice using molecular genetic manipulation. These Fmr1 knockout mice have recently been used to identify a new putative therapeutic target, the metabotropic glutamate receptor 5 (mGluR5), for the treatment of FXS. Moreover, mGluR5 signaling cascades interact with a number of synaptic proteins, many of which have been implicated in autism, raising the possibility that therapeutic targets identified for FXS may have efficacy in treating multiple other causes of autism

Involvement of the Wbp pathway in the biosynthesis of Porphyromonas gingivalis lipopolysaccharide with anionic polysaccharide

The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharide (LPS) molecules, O-LPS and A-LPS. We have recently shown that P. gingivalis strain HG66 lacks A-LPS. Here, we found that introduction of a wild-type wbpB gene into strain HG66 restored formation of A-LPS. Sequencing of the wbpB gene from strain HG66 revealed the presence of a nonsense mutation in the gene. The wbpB gene product is a member of the Wbp pathway, which plays a role in the synthesis of UDP-ManNAc(3NAc)A in Pseudomonas aeruginosa; UDP-ManNAc(3NAc)A is sequentially synthesized by the WbpA, WbpB, WbpE, WbpD and WbpI proteins. We then determined the effect of the PGN-0002 gene, a wbpD homolog, on the biosynthesis of A-LPS. A PGN-0002-deficient mutant demonstrated an A-LPS biosynthesis deficiency. Taken together with previous studies, the present results suggest that the final product synthesized by the Wbp pathway is one of the sugar substrates necessary for the biosynthesis of A-LPS

Thermodynamics of irreversible adsorption of water vapours by montmorillonite

The adsorption–desorption of water vapours on swelling layer silicate is considered in the framework of the Brunauer-Emmett-Teller (BET) and Frenkel-Halsey-Hill (FHH) formalisms. The theoretical dependencies calculated with reasonable and consistent values of model parameters are shown to agree quite satisfactorily with the experimental data. Based on these equations, the description of the adsorption hysteresis related to Type III adsorption isotherms is proposed, which also yields a good reproduction of the experimentally observed values