Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells.

We previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ΔtetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its Δtet38, ΔtetR21, and ΔmgrA mutants, and a Δtet38 ΔtetR21 double mutant. After 2 h of contact, the Δtet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrA mutant. The growth patterns of RN6390 and the ΔmgrA and ΔtetR21 mutants within A549 cells were similar, while no growth was observed for the Δtet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells.

The alpha C protein mediates internalization of group B Streptococcus within human cervical epithelial cells.

Group B Streptococcus (GBS) is the leading cause of bacterial chorioamnionitis and neonatal pneumonia, sepsis, and meningitis. Deletion of the alpha C protein gene (bca) attenuates the virulence of GBS in an animal model; significant survival differences in the first 24 h of infection suggest a pathogenic role for the alpha C protein early in the infection process. We examined the role of alpha C protein in the association between GBS and mucosal surfaces using a human cervical epithelial cell line, ME180. Fluorescent and confocal microscopy and flow cytometry demonstrated that 9-repeat alpha C protein binds to the surface of ME180 cells. Isolated N-terminal region of this protein also binds to these cells and competitively inhibits binding of the full protein. Wild-type GBS strain A909 and the bca-null isogenic mutant JL2053 bound similarly to the surface of ME180 cells. However, A909 entered these cells threefold more. Internalization of A909 was inhibited with 2- and 9-repeat alpha C and with N-terminal region alone but not by repeat region-specific peptide. Translocation across polarized ME180 membranes was fivefold greater for A909 than for JL2053. These findings suggest a role for the alpha C protein in interaction with epithelial surfaces and initiation of infection.

Variability of outer membrane protein P1 and its evaluation as a vaccine candidate against experimental otitis media due to nontypeable Haemophilus influenzae: an unambiguous, multifaceted approach.

Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (