Streptococcus agalactiae is frequently the cause of bacterial sepsis and meningitis in neonates. In addition, it is a commensal bacterium that colonizes the mammalian gastrointestinal tract. During its commensal and pathogenic lifestyles, S. agalactiae colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present study, the serine-rich repeat protein Srr-1 from S. agalactiae was functionally investigated. Immunofluorescence microscopy showed that Srr-1 was localized on the surface of streptococcal cells. The Srr-1 protein was shown to interact with a 62-kDa protein in human saliva, which was identified by matrix-assisted laser desorption ionization-time-of-flight analysis as human keratin 4 (K4). Immunoblot and enzyme-linked immunosorbent assay experiments allowed us to narrow down the K4 binding domain in Srr-1 to a region of 157 amino acids (aa). Furthermore, the Srr-1 binding domain of K4 was identified in the C-terminal 255 aa of human K4. Deletion of the srr-1 gene in the genome of S. agalactiae revealed that this gene plays a role in bacterial binding to human K4 and that it is involved in adherence to epithelial HEp-2 cells. Binding to immobilized K4 and adherence to HEp-2 cells were restored by introducing the srr-1 gene on a shuttle plasmid into the srr-1 mutant. Furthermore, incubation of HEp-2 cells with the K4 binding domain of Srr-1 blocked S. agalactiae adherence to epithelial cells in a dose-dependent fashion. This is the first report describing the interaction of a bacterial protein with human K4.

To better understand the defense mechanism ofStreptococcus thermophilusagainst superoxide stress, molecular analysis of 10 menadione-sensitive mutants, obtained by insertional mutagenesis, was undertaken. This analysis allowed the identification of 10 genes that, with respect to their putative functions, were classified into five categories: (i) those involved in cell wall metabolism, (ii) those involved in exopolysaccharide translocation, (iii) those involved in RNA modification, (iv) those involved in iron homeostasis, and (v) those whose functions are still unknown. The behavior of the 10 menadione-sensitive mutants exposed to heat shock was investigated. Data from these experiments allowed us to distinguish genes whose action might be specific to oxidative stress defense (tgt,ossF, andossG) from those whose action may be generalized to other stressful conditions (mreD,rodA,pbp2b,cpsX, andiscU). Among the mutants, two harbored an independently inserted copy of pGh9:ISS1in two loci close to each other. More precisely, these two loci are homologous to thesufDandiscUgenes, which are involved in the biosynthesis of iron-sulfur clusters. This region, called thesufregion, was further characterized inS. thermophilusCNRZ368 by sequencing and by construction of ΔsufDandiscU₉₇nonpolar mutants. The streptonigrin sensitivity levels of both mutants suggest that these two genes are involved in iron metabolism.

Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents. We have determined the activity, dispersity, and oligomeric states of HuPON1 in solutions containing mild detergents using nondenaturing electrophoresis, size exclusion chromatography, and cross-linking. HuPON1 was active whatever its oligomeric state. In nonmicellar solutions, HuPON1 was polydisperse. In contrast, HuPON1 exhibited apparent homogeneity in micellar solutions, except with CHAPS. The enzyme apparent hydrodynamic radius varied with the type of detergent and protein concentration. In C₁₂E₈ micellar solutions, from sedimentation velocity, equilibrium analytical ultracentrifugation, and radioactive detergent binding, HuPON1 was described as monomers and dimers in equilibrium. A decrease of the detergent concentration shifted this equilibrium toward the formation of dimers. About 100 detergent molecules were associated per monomer and dimer. The assembly of amphiphilic molecules, phospholipids in vivo, in sufficiently large aggregates could be a prerequisite for anchoring of HuPON1 and then allowing stabilization of the enzyme activity. Changes of HDL size and shape could strongly affect the binding affinity and stability of HuPON1 and result in reduced antioxidative capacity of the lipoprotein.