Actin microfilaments are one of the three major components of the cellular cytoskeleton. The cytoskeleton participates in maintaining adhesion to and communicating with the extracellular matrix, cell migration, division, and signaling. b-Actin (ACTB) mRNA was decreased in response to L. interrogans but increased in response to L. biflexa, both as compared to the uninfected control cells (Table 2) . Guanine nucleotide-binding protein alpha-13 subunit (GNA13) mediates the activation of the small GTPase RhoA [19] which when activated controls the assembly of focal adhesions and actin in the formation of stress fibers [20] . Although RhoA was not differentially regulated in response to the bacteria, Rho GTPase activating protein 5 (RhoGAP5) was differentially expressed following the same pattern as GNA13, in which both genes were downregulated in response to the pathogenic leptospires in comparison to the uninfected controls, and upregulated in response to the non-pathogen. The effect of decreased GNA13 may be to decrease stimulation of Rho, while The changes in expression of several additional genes are consistent with changes in cellular architecture as a result of leptospiral infection of these endothelial cells. For example, decreases in the mRNAs for radixin (RDX, a protein that links the actin cytoskeleton to the plasma), caveolins 1 and 2 (CAV1 and CAV2, which couple integrins to the Ras-ERK pathway, titin, the ECM component laminin b1, and integrin subunits a v and b 3 ( Table 2) , were seen in cells infected with L. interrogans Canicola as compared to the uninfected controls. In contrast, the L. biflexa Patoc caused increases in mRNA levels for the same genes in infected cells vs. uninfected controls (Table 2) . Together, all of these gene expression patterns are consistent with the hypothesis that one effect of L. interrogans serovar Canicola is to promote actin remodeling and detachment of the cells from the ECM. A fundamental stage in the pathogenesis of Leptospira infections is the ability of the bacteria to cross mucous membranes and underlying epithelial barriers, as well as endothelial cell barriers, and disseminate to different organs. Although Leptospira species are Ea.hy926 endothelial cells were plated in tissue culture treated glass chamber slides and allowed to reach near confluence (assessed visually). The bacteria were added at MOI = 10 and incubated with the endothelial cells for 1 or 3 hours at 37uC, then were washed and fixed. The slides were stained with phalloidin-FITC, which illuminates F actin, plus anti-Leptospira antibodies followed by TRITC-conjugated secondary antibody. Retraction of the cell bodies in response to L. interrogans Canicola, but not L. biflexa Patoc, is evident, particularly at 3 hr infection. The brighter staining of rounded and retracted cells with FITC-phalloidin may be due to disorganization of cellular architecture without complete depolymerization of the actin, which in the increased depth and decreased area of the cytoplasm would appear more concentrated and therefore brighter. Changes in endothelial cell morphology were most evident, and at earlier time points, in cells with which the L. interrogans bacteria were associated. One higher magnification micrograph of L. interrogans Canicola infected cells is included because the bacteria are small when viewing fields of endothelial cells that provide information on integrity of the monolayer. Micrographs are representative of multiple (.12) independent experiments. L. interrogans Copenhageni caused essentially the same changes in endothelial cell morphology as L. interrogans Canicola (data not shown). doi:10.1371/journal.pntd.0000918.g001 Endothelial Cell Responses to Leptospira www.plosntds.org extracellular bacteria apparently devoid of actin modifying exotoxins [21] [22] [23] , and devoid of the specialized secretion systems utilized by many bacterial pathogens to deliver toxins that disrupt the host cell cytoskeleton (as reviewed in [24] [25] [26] [27] [28] ), pathogenic leptospires might be indirectly targeting the cytoskeleton via cell surface attachment mechanisms that co-opt the host cell signaling to achieve the same result.