Armand Berneman

Inhibition of Trypanosoma cruzi proline racemase affects host-parasite interactions and the outcome of in vitro infection

Proline racemase is an important enzyme of Trypanosoma cruzi and has been shown to be an effective mitogen for B cells, thus contributing to the parasites immune evasion and persistence in the human host. Recombinant epimastigote parasites overexpressing TcPRAC genes coding for proline racemase present an augmented ability to differentiate into metacyclic infective forms and subsequently penetrate host-cells in vitro. Here we demonstrate that both anti T. cruzi proline racemase antibodies and the specific proline racemase inhibitor pyrrole-2-carboxylic acid significantly affect parasite infection of Vero cells in vitro. This inhibitor also hampers T. cruzi intracellular differentiation.

Proline racemases: insights into Trypanosoma cruzi peptides containing D-proline

Trypanosoma cruzi proline racemases (TcPRAC) are homodimeric enzymes that interconvert the L and D-enantiomers of proline. At least two paralogous copies of proline racemase (PR) genes are present per parasite haploid genome and they are differentially expressed during T. cruzi development. Non-infective epimastigote forms that overexpress PR genes differentiate more readily into metacyclic infective forms that are more invasive to host cells, indicating that PR participates in mechanisms of virulence acquisition. Using a combination of biochemical and enzymatic methods, we show here that, in addition to free D-amino acids, non-infective epimastigote and infective metacyclic parasite extracts possess peptides composed notably of D-proline. The relative contribution of TcPRAC to D-proline availability and its further assembly into peptides was estimated through the use of wild-type parasites and parasites over-expressing TcPRAC genes. Our data suggest that D-proline-bearing peptides, similarly to the mucopeptide layer of bacterial cell walls, may be of benefit to T. cruzi by providing resistance against host proteolytic mechanisms.

Combined approaches for drug design points the way to novel proline racemase inhibitor candidates to fight Chagas' disease.

Chagas’ disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.

Salivary Natural Antibodies as a Basic Immune Barrier against Group A Streptococci

Immune protection against microbial pathogens in the secretions is primarily due to secretory immunoglobulin IgA (S-IgA) Abs acting as a first barrier against invasion.4, 6, 7, 9 The binding of pathogens with S-IgA favors their clearance in secretions by conveyance with the mucus stream. In most cases, these secretory antibodies (Abs) must be present before the antigenic stimulation because their role is to prevent the entry of the pathogen into the body. Indeed, the secretory immune response is delayed and its duration is usually short after antigen (Ag) clearing. According to these impairments, the protection by conventional S-IgA Abs is restricted to the cases of repeated antigenic challenges by chronic infection, asymptomatic portage, or vaccination with a living or long-lasting Ag. In contrast, natural Abs could be protective because these molecules, described in the serum,5 are present before the first contact with the pathogen, and thus bypass these limitations. Natural (preimmune) Abs are polyreactive and can recognize self and non-self Ags.1 We herein demonstrate the presence of natural Abs of the S-IgA isotype in human saliva and show that their polyreactivity to unrelated human and bacterial antigens allows binding with a wide variety of molecules at the surface of the streptococcal cells.

AN IMPROVED MICROCARRIER FOR MASS CULTURE OF HUMAN DIPLOID CELLS

Culture methods involving large volumes are necessary for mass production of eukaryotic cells. Cultures of such cells can be used for preparing useful compounds such as viral and parasitic antigens, hormones and enzymes. At the present time, only primary cultures or human diploid fibroblast cell strains (HDC) are accepted for the preparation of biological products.’ With the use of DEAE-Sephadex as envisaged by van Weze12 for growing anchorage-dependent cells, the ratio of culture surface to media volume can be increased. Moreover, this method makes possible the use of a unit process’ for cells which only grow in monolayers. However, the use of DEAE-Sephadex has been hampered by the cytotoxic effect due to excess cationic charge^.^,^ Levine’ improved the DEAE-Sephadex microcarrier system by preparing beads with a low charge density. In this paper, we report our preliminary results concerning the development of a new microcarrier which improves the adherence and the growth of HDC. Beads were prepared by grafting a DEAE anion exchange function onto the surface of Sephadex 50 (Pharmacia), then binding proteins to the DEAE according to 61