Because Leptospira interrogans has been shown to bind to proteoglycans on the mammalian cell surface [17] , we tested a proteoglycan synthesis inhibitor, b-xyloside, for the ability to decrease damage to endothelial cell layers caused by L. interrogans Canicola. b-xyloside inhibits transfer of glycosaminoglycan chains to protein cores; a control sugar analog, a-galactoside, was tested in parallel. As shown in Figure 4 , inhibition of proteoglycan synthesis did not fully prevent the damage to the endothelial cell layers caused by L. interrogans. The inhibition of glycosaminoglycan chain attachment does not significantly affect the formation of holes in the cell layer caused by L. interrogans Canicola as assessed visually and by measurement of L. interrogans penetration of the cell layers (data not shown). b-xyloside does cause a reduction of L. interrogans Canicola and Copenhageni attachment to these cells ( [17] and data not shown), but does not abolish bacterial attachment, consistent with the hypothesis that additional nonproteoglycan molecules serve as substrates for L. interrogans attachment to cells. Direct bacterial attachment to the cells does appear to be required for the damage to the endothelial cell layers, as supernatants harvested from infected cell layers (infection times of 1-24 hr) and sterilized by centrifugation and filtration through 0.1 mm filters did not affect endothelial cell layer integrity (data not shown). Therefore, non-proteoglycan cell surface receptors are likely to be those primarily involved in the responses of the endothelial cells to L. interrogans attachment, and efforts to identify both the host cell and the bacterial cell molecules involved in these interactions are underway. As noted in the publication reporting the sequence of two L. biflexa Patoc strains [29] , there are a number of proteins predicted in the published L. interrogans genomes that are not present in the L. biflexa Patoc genome, including some that are postulated to have potential adhesin activities. These include proteins containing leucine-rich repeats, which are involved in many protein-protein interactions [29] . As stated in the publication of the L. biflexa genome, it is intriguing Ea.hy926 cell layers were treated with the proteoglycan synthesis inhibitor b-xyloside, or the control a-galactoside, as described in [17] prior to infection with L. interrogans sv. Copenhageni. After 3 hr, the cell layers were washed and fixed, then stained with phalloidin-FITC. Neither reagent significantly reduced disruption of the cell layers by L. interrogans, as alterations in cell morphology and significant gaps between cells were seen when L. interrogans was present, and trans-endothelial cell layer migration was not significantly affected (data not shown). Consistent with this result, chondroitin sulfates B and A, which do and do not inhibit L. interrogans attachment to mammalian cells, respectively [17] , also had no effect (not shown). doi:10.1371/journal.pntd.0000918.g004