Chemotransport contributes to the effect of oscillatory fluid flow on human bone marrow stromal cell proliferation.

Mechanical loads produce a diverse set of biophysical signals that may regulate bone cell activity, but accumulating evidence suggests that interstitial fluid flow is the primary signal that bone cells perceive. Because we previously demonstrated that oscillatory fluid flow increases human bone marrow stromal cell proliferation, we investigated the contribution of fluid shear stress and chemotransport, two stimuli induced by interstitial fluid flow. Alterations in flow rate at a constant peak shear stress were associated with decreases in oscillatory fluid flow-induced marrow stromal cell proliferation, while variations in peak fluid shear stress had no significant effect. Modulation of marrow stromal cell proliferation by flow rate may be attributed to changes in the release of ATP and intracellular calcium signaling. We found that if the flow rate is decreased while maintaining a constant peak fluid shear stress, marrow stromal cells release less ATP into the extracellular environment. Moreover, as the flow rate decreased fewer cells respond to fluid flow with an increase in intracellular calcium concentration. These data suggest that chemotransport is a prerequisite for marrow stromal cells to respond to interstitial fluid flow.

Epithelial cells are sensitive detectors of bacterial pore-forming toxins.

Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins (including pneumolysin from Streptococcus pneumoniae, alpha-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis) at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial poreforming toxins, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by the insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response, while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.

Deglycosylation of human glycoconjugates by the sequential activities of exoglycosidases expressed by Streptococcus pneumoniae.

Streptococcus pneumoniae produces three surface-associated exoglycosidases; a neuraminidase, NanA, a beta-galactosidase, BgaA, and a beta-N-acetylglucosaminidase, StrH. the proposed functions of NanA, which removes terminal sialic acid, include revealing receptors for adherence, affecting the function of glycosylated host clearance molecules, modifying the surface of other bacteria coinhabiting the same niche, and providing a nutrient source. However, it is unclear whether following desialylation S. pneumoniae can further deglycosylate human targets through the activity of BgaA or StrH. We demonstrate that NanA, BgaA and StrH act sequentially to remove sialic acid, galactose and N-acetylglucosamine and expose mannose on human glycoproteins that bind to the pneumococcus and protect the airway. In addition, both BgaA and NanA were shown to contribute to the adherence of unencapsulated pneumococci, to human epithelial cells. Despite these findings, triple exoglycosidase mutants colonized mice as well as their parental strains, suggesting that any effect of these genes on colonization and disease may be host species-specific. These studies highlight the importance of considering the complete ability of S. pneumoniae to deglycosylate human targets and suggest that in addition to NanA, BgaA and StrH also contribute to pneumococcal colonization and/or pathogenesis.

Phase variable desialylation of host proteins that bind to Streptococcus pneumoniae in vivo and protect the airway.

Most clinical isolates of Streptococcus pneumoniae consist of heterogeneous populations of at least two colony phenotypes, opaque and transparent, selected for in the bloodstream and nasopharynx, respectively. Microarray analysis revealed 24 orfs that demonstrated differences in expression greater than twofold between variants of independent strains. Twenty-one of these showed increased expression in the transparent variants, including 11 predicted to be involved in sugar metabolism. A single genomic region contains seven of these loci including the gene that encodes the neuraminidase, NanA. In contrast to previous studies, there was no contribution of NanA to adherence of S. pneumoniae to epithelial cells or colonization in an animal model. However, we observed NanA-dependent desialylation of human airway components that bind to the organism and may mediate bacterial clearance. Targets of desialylation included human lactoferrin, secretory component, and IgA2 that were shown to be present on the surface of the pneumococcus in vivo during pneumococcal pneumonia. The efficiency of desialylation was increased in the transparent variants and enhanced for host proteins binding to the surface of S. pneumoniae. Because deglycosylation affects the function of many host proteins, NanA may contribute to a protease-independent mechanism to modify bound targets and facilitate enhanced survival of the bacterium.