Effect of diabetic condition and estrogen treatment on fungal burden.

<p>To induce diabetes, mice were injected with STZ as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147969#pone.0147969.g001" target="_blank">Fig 1</a>. STZ-treated diabetic (closed circle) (n = 7 or 8) or non-diabetic (open circle) (n = 2 or 3) mice were estrogenized (0.1 mg in sesame oil subcutaneously 72 h prior to inoculation) and inoculated with 1x10⁷ CFUs of <i>C</i>. <i>glabrata</i><b>(A)</b>. STZ-treated diabetic mice (n = 4/group) were subcutaneously injected with 0.1 mg of estrogen in sesame oil (closed circle) or sesame oil alone (open circle) 72 h prior to inoculation with 1x10⁷<i>C</i>. <i>glabrata</i><b>(B)</b>. Fungal burden was assessed longitudinally on days 1, 3, 7, 14, and 21 in vaginal lavage fluids. Diabetic mice often succumb to uncontrolled diabetes that resulted in reduced numbers of mice/group for latter time points. Results are expressed as median CFU and are cumulative of two repeat experiments. Data were analyzed using the Mann-Whitney U test. Significance is denoted as *, <i>P</i><0.05; **, <i>P</i>>0.01. Abbreviation: STZ, streptozotocin; CFU, colony forming unit.</p

Lack of biofilm formation during <i>C</i>. <i>glabrata</i> vaginitis.

<p>Vaginal tissues from <i>C</i>. <i>glabrata</i>-inoculated mice (diabetic, estrogenized, 1x10⁷ CFUs) and <i>C</i>. <i>albicans</i>-inoculated mice (non-diabetic, estrogenized, 5x10⁶) were excised 7 days post-inoculation to assess <i>in vivo</i> biofilm formation. Vaginae were stained with calcofluor white (blue) to visualize <i>C</i>. <i>albicans</i> yeast and hyphae <b>(A)</b>, Con A-R to visualize <i>C</i>. <i>glabrata</i> yeast and ECM <b>(C)</b>, and To-Pro-3 iodide (white) to visualize epithelial cell nuclei. Z-stack images were rotated to visualize height of <i>C</i>. <i>albicans</i><b>(B)</b> and <i>C</i>. <i>glabrata</i><b>(D)</b> biofilm. Confocal images were captured using a 60X oil emersion objective. The figure shows representative images of areas of biofilm growth from two repeat experiments using groups of 3 mice each. Abbreviations: CFU, colony forming unit; ConA-R, Concavalin A-Rhodamine conjugate; ECM, extra-cellular matrix.</p

Effect of inocula size on vaginal fungal burden in type 1 diabetic estrogenized mice.

<p>Diabetes was induced in mice (n = 10/group) by two i.p. injections of STZ (150 mg/kg). Diabetic mice (n = 8/group) were estrogen treated (0.1 mg in sesame oil subcutaneously 72 h prior to inoculation) and inoculated with either 2x10⁶ (closed circle) or 1x10⁷ (open circle) CFUs of <i>C</i>. <i>glabrata</i> and followed longitudinally for 21 days to assess fungal burden in vaginal lavage fluids. Diabetic mice often succumb to uncontrolled diabetes that resulted in reduced numbers of mice/group for latter time points. Results are expressed as median CFU and are representative of no less than 3 experiments. Data were analyzed using the Mann-Whitney U test. Abbreviations: STZ, streptozotocin; CFU, colony forming units.</p

Effect of mouse strain on fungal burden in diabetic estrogenized mice.

<p>Diabetes was induced in C57BL/6 (H-2^b) and C3H/HeN (H-2^k) mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147969#pone.0147969.g001" target="_blank">Fig 1</a>. STZ-treated diabetic mice (n = 8/group) were estrogenized and inoculated with <i>C</i>. <i>glabrata</i>. <i>C</i>. <i>glabrata</i> CFUs were quantified longitudinally in C57BL/6 (closed circle) and C3H/HeN (open circle) mice from vaginal lavage fluids collected on days 1, 3, 7, 14, and 21 post-inoculation. Diabetic mice often succumb to uncontrolled diabetes resulting in reduced mice/group in latter time points. Results are expressed as median CFU and only one experiment was performed. Data was analyzed using the Mann-Whitney U test. Significance is denoted as *, <i>P</i><0.05. Abbreviation: STZ, streptozotocin; CFU, colony forming unit.</p

Lack of synergistic effects on virulence during <i>C</i>. <i>glabrata</i> and <i>C</i>. <i>albicans</i> co-infections.

<p>Diabetes was induced in C57BL/6 mice (n = 20) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147969#pone.0147969.g001" target="_blank">Fig 1</a>. STZ-treated diabetic mice were estrogenized and inoculated with 1x10⁷<i>C</i>. <i>glabrata</i> (n = 5), 5x10⁴<i>C</i>. <i>albicans</i> (n = 6), or both (n = 6). Mice were lavaged on days 1, 3, and 7 post-inoculation to measure fungal burden (<i>C</i>. <i>glabrata</i>, open circle; <i>C</i>. <i>albicans</i>, closed circle) <b>(A)</b> and PMN infiltration (dashed line denotes high-responder cutoff, further described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147969#pone.0147969.ref005" target="_blank">5</a>]) <b>(B)</b>. LDH was quantified in lavage fluids from day 7 post-inoculation <b>(C)</b>. Vaginal tissues were excised from co-inoculated mice on day 10, and <i>C</i>. <i>glabrata</i>-specific (red) and <i>C</i>. <i>albicans</i>-specific (green) PNA-FISH probes were used to stain fungal cells <b>(D)</b>. Fluorescent images of vaginal epithelium (E) and lumen (L) were captured using a 20X objective (insert) and 4X digital zoom (expanded insert 80X). Results are expressed as median CFU and PMN counts and mean LDH ± SEM. Shown are representative data from two experiments. Fungal burden and PMN data were analyzed using the Mann-Whitney U test, and LDH concentrations were analyzed with the unpaired Student’s <i>t</i> test. Significance is denoted as follows, *, <i>P</i><0.05; **, <i>P</i><0.01; n.s., not significant. Abbreviations: STZ, streptozotocin; LDH, lactate dehydrogenase; PNA-FISH, protein nucleic acid-fluorescent in situ hybridization; E, epithelium; L, lumen; SEM, standard error of the mean.</p

Lack of immunopathology during <i>C</i>. <i>glabrata</i> vaginitis.

<p>Diabetes was induced in mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147969#pone.0147969.g001" target="_blank">Fig 1</a>. STZ-treated diabetic mice (n = 8) were estrogenized, inoculated with <i>C</i>. <i>glabrata</i>, and underwent vaginal lavage on days 1, 3, 7, 14, and 21 to assess PMN infiltration into the vaginal lumen (the dashed line denotes the high-responder cutoff, further described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147969#pone.0147969.ref005" target="_blank">5</a>], and the gray box is representative of typical PMN migration observed in 75% of <i>C</i>. <i>albicans</i> vaginal infections) <b>(A)</b>. Lavage fluids from day 7 post-inoculation were collected from <i>C</i>. <i>glabrata</i>-infected mice (diabetic, estrogenized, 1x10⁷ CFUs) and uninoculated mice (diabetic, estrogenized) and analyzed for IL-1β <b>(B)</b> and S100A8 <b>(C)</b> protein levels by ELISA. IL-1β and S100A8 were also evaluated in banked specimens from <i>C</i>. <i>albicans</i> inoculated (5x10⁶ CFUs) estrogenized non-diabetic mice on day 7 post-inoculation. The results are expressed as median PMN counts and mean protein concentration ± SEM and cumulative of two repeat experiments. For IL-1β quantification, pooled lavage fluids from <i>C</i>. <i>glabrata</i>-inoculated mice (n = 15), <i>C</i>. <i>albicans</i>-inoculated mice (n = 8), and uninoculated diabetic mice (n = 4) were analyzed. For S100A8 quantification, individual lavage fluids from <i>C</i>. <i>glabrata</i>-inoculated mice (n = 10), <i>C</i>. <i>albicans</i>-inoculated mice (n = 10), and uninoculated diabetic mice (n = 4) were analyzed. Data were analyzed using the unpaired Student’s <i>t</i> test. Significance is denoted as *, <i>P<</i>0.05; n.s., not significant. Abbreviations: STZ, streptozotocin; PMN, polymorphonuclear leukocyte; CFU, colony forming units; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean; IL-1β, interleukin-1β.</p