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Elias Torres
Elias Torres

Ami Slic Mod 163

to assess the contribution of slic to inhibition of lysozyme in the presence of acp, we conducted a direct competitive infection experiment in which the slic mutant and the slic/p::slic* complemented strain were competed with the wild type strain in parallel. an average competitive index of 0.6 was obtained from three independent coinfection experiments on day 1 post-infection, while day 5 post-infection yielded a competitive index of 1.2 ( fig 7g ). the relative number of cfus recovered from the vaginal swabs was 2.4, 2.2, and 2.2-fold lower for the slic mutant versus the wild type strain on days 1, 3, and 5 post-infection, respectively ( fig 7h ). the defect was fully restored for the slic/p::slic* strain, with cis of 1.0, 1.0, and 1.6 on days 1, 3, and 5, respectively ( fig 7h ).

Ami Slic Mod 163

we conducted complementary biochemical and genetic approaches to assess the contribution of slic to acp-mediated inhibition of c-type lysozyme from chicken egg white (hewl, sigma). assays were conducted using 100 volumes of 0.1 m sodium phosphate, 0.1 m nacl, ph 7.5 and 2 mm sodium azide as buffer and volume. experimental samples containing 2.5 m of hewl were incubated with increasing concentrations of slic (p) (0.05 m, 0.1 m, and 0.25 m). the control wells contained hewl alone. after incubation, the reaction was initiated by addition of the 50 g/ml dq lysozyme substrate.

for the immunolocalization experiments, slic and slic* were purified and concentrated as described above and labeled with the alexa fluor 488 protein labeling kit (life technologies). to visualize the localization of slic, n. gonorrhoeae cells were grown to mid-log phase (od 600 of 0.5) in gcdm and fixed with 4% paraformaldehyde for 1h at room temperature. cells were blocked and washed three times in pbs with 0.2% gelatin and 0.1% triton x-100. slic-alexa fluor 488 was diluted 1:50 in pbs, added to cells, and incubated for 1h at room temperature in the dark. after incubation, cells were washed three times in pbs and mounted on a slide with dapi-containing mounting medium. microscopy was performed using a leica tcs sp5 ii multiphoton laser scanning confocal microscope, and images were analyzed with leica las af lite version 2.6.0 software. immunofluorescence microscopy confirmed the surface localization of slic on n. gonorrhoeae cells ( fig 3a ). in contrast, both slic and slic* localized to the periplasmic side of the outer membrane in the complemented strain, slic/p::slic* ( fig 3b ), indicating that the protein is anchored to the membrane in a similar manner as bamd and sura, which are known to be periplasmic surface-exposed proteins. we also observed that the slic complemented strain, slic/p::slic*, exhibited a relatively weaker signal on the cell surface when compared to the wild type ( fig 3c ). these results suggest that the expression of slic may be down-regulated in the complemented strain. overall, these experiments confirmed that slic is exposed on the cell surface and is likely associated with the outer membrane. it is also noteworthy that the slic protein was detected in the cytoplasm of all cells, including the wild type, slic/p::slic, and slic/p::slic*, and that this signal was reduced in the complemented strain. slic may therefore be actively secreted from the cell, or slic and/or slic* may be degraded in this location. further studies will be needed to address these hypotheses.


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