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The activity of engineered, peptide-displaying enzymes is modulated by binding to specific anti-peptide antibodies. This new concept of a quantitative antibody detection system allows test kits to be set up for fast diagnosis of infectious diseases. To develop a quick and homogeneous assay for the detection of human immunodeficiency virus (HIV) infection, we have explored two acceptor sites of the bacterial Escherichia coli ␤-galactosidase for the accommodation of HIV antigenic peptides. Two overlapping epitopes (namely P1 and P2) from the gp41 envelope glycoprotein, contained in different sized peptides, were inserted in the vicinity of the enzyme active site to generate a set of hybrid, enzymatically active ␤-galactosidases. Regulable enzymes of different responsiveness to monoclonal antibody binding were generated with both acceptor sites tested. These biosensors were also sensitive to immune sera from HIVinfected patients. Modeling data provide insight into the structural modifications in the vicinity of the active site induced by peptide insertion that strongly affect the responsiveness of the engineered proteins through different parameters of their catalytic properties.Insertional fusion technologies are useful instruments for the analysis of membrane protein topology and structure-function relationship, as well as for the generation of randomized protein libraries and enzymatic biosensors (1). The ␤-galactosidase enzyme (EC 3.2.1.23), encoded by the Escherichia coli lacZ gene, hydrolyzes lactose into glucose and galactose (2) which are then metabolized for cell growth. In addition, this enzyme also hydrolyzes other substrates producing colored compounds useful to monitor a wide range of biological processes. The three-dimensional structure of ␤-galactosidase (3) permits the permissiveness of solvent-exposed loops to heterologously inserted peptides to be explored. In this context, we previously constructed some recombinant ␤-galactosidases displaying foot-and-mouth disease virus (FMDV) 1 B-cell epitope peptides accommodated in solvent-exposed surfaces (4, 5). The peptide insertion results in hybrid ␤-galactosidases with reduced enzymatic activity. However, in the presence of antipeptide monoclonal antibodies or polyclonal sera, these hybrid enzymes translate the antigen-antibody interaction into an easily measurable increase of the enzymatic activity (4 -6).Because the results obtained with these FMDV-based biosensor prototypes were very promising in the context of a fast and easy diagnosis of infectious diseases in a homogeneous assay, we were prompted to design new recombinant ␤-galactosidases as biosensors for anti-HIV human antibodies. The development of such a homogeneous colorimetric assay could be of great impact in human health and also be helpful in better understanding the mechanism of enzymatic regulation in biosensors by testing whether ␤-galactosidase enzymatic modulation is restricted to particular epitopes or specific peptide sequence, length, or conformation. Therefore, we have inserted i...

Production and release of heat-labile toxin (LT) by wild-type enterotoxigenic Escherichia coli (ETEC) strains, isolated from diarrheic and asymptomatic Brazilian children, was studied under in vitro and in vivo conditions. Based on a set of 26 genetically diverse LT(+) enterotoxigenic E. coli strains, cell-bound LT concentrations varied from 49.8 to 2415 ng mL(-1). The amounts of toxin released in culture supernatants ranged from 0% to 50% of the total synthesized toxin. The amount of LT associated with secreted membrane vesicles represented <5% of the total toxin detected in culture supernatants. ETEC strains secreting higher amounts of LT, but not those producing high intracellular levels of cell-bound toxin, elicited enhanced fluid accumulation in tied rabbit ileal loops, suggesting that the strain-specific differences in production and secretion of LT correlates with symptoms induced in vivo. However, no clear correlation was established between the ability to produce and secrete LT and the clinical symptoms of the infected individuals. The present results indicate that production and release of LT by wild-type human-derived ETEC strains are heterogeneous traits under both in vitro and in vivo growth conditions and may impact the clinical outcomes of infected individuals.

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Haemophilus influenzae type b (Hib) causes invasive infections in infants and young children. Vaccines consisting of Hib capsular polysaccharide (polymer of ribosylribitol phosphate [PRP]) conjugated to a protein are effective in the prevention of such infections. The production of capsular polysaccharide type b was studied in three cultivation conditions: single, glucose pulse, and repeated batch. Specific polysaccharide production (Yp/x) was calculated for all experiments, showing the following values: 67 (single-batch cultivation), 71 (glucose pulse), 75 (repeated-batch cultivation, first batch), and 87 mg of PRP/g of dry cell weight (DCW) (repeated-batch cultivation, second batch). Biomass concentration reached approximately 1.8 g of DCW/L, while polysaccharide concentration was about approximately 132 mg/L in the three fermentation runs. Polysaccharide synthesis is associated with cell growth in all studied conditions as established by Kono's analysis and Luedeking-Piret's model.