Binding and uptake of B3501 and B-4131 with human brain endothelial cell line, hCMEC/D3.

<p>HCMEC/D3 cells were exposed to <i>C. neoformans</i> serotype D wild type B3501 and its isogenic acapsular mutant B4131 for 2 hr and 4 hr at 37°C. Microscopic and live CFU counts were performed to determine the association and survival of cryptococci. (A) Association efficiency of B3501 and B4131 cryptococci with hCMEC/D3 cells. Like the H99-cap59 pair, live CFU counts showed a time dependent decrease in the number of associated cryptococci by 4 hr of incubation, ★ = P<0.01, ★★ = P<0.01 for both B3501 and B4131. There was no difference in association by encapsulated B3501 and acapsular B4131, P-value = >0.05 at 2 hr of incubation. However, B4131 was significantly more associated, P = 0.02 (Microscopy) and <0.01 (CFU) by 4 hr of incubation. (B) Internalization of encapsulated B3501 and its acapsular mutant derivative B4131 by hCMEC/D3 cells determined by fluorescence microscopy (Mic). The number of phagocytosed cryptococci increased significantly in a time dependent manner, P = 0.01 for both strains. However, there was no difference in internalization of B3501 and B4131, P = 0.5 and 0.2 at 2 hr and 4 hr respectively. Error bars are standard error of the mean, n = 6 repeats.</p

Binding and uptake of H99 and cap59 with human brain endothelial cell line, hCMEC/D3.

<p>Like bEnd3 cells, hCMEC/D3 cells were exposed to the <i>C. neoformans</i> serotype A isogenic strains, wild type H99 and acapsular derivative cap59 for 2 hr and 4 hr at 37°C. Microscopic (Mic.) and live CFU counts were performed to determine the association and survival of cryptococci. (A) Association efficiency of H99 and cap59 cryptococci with hCMEC/D3 cells. As opposed to microscopic counts, live CFU counts showed a time dependent decrease in the number of associated cryptococci by 4 hr of incubation, ★ = P<0.05 and ★★ = P<0.01 for H99 and cap59 respectively, suggesting a drop in viability of the hCMEC/D3 associated H99 and cap59 cryptococci. There was no difference in association of encapsulated H99 and acapsular cap59, P = 0.7 and 0.6 at 2 hr and 4 hr respectively. (B) Internalization of encapsulated H99 and acapsular cap59 cryptococci by human brain endothelial cell line, hCMEC/D3 cells. The internalized cryptococci and extracellular adherent were determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035455#pone-0035455-g001" target="_blank">Figure 1</a>. As with association, the number of phagocytosed cryptococci increased significantly in a time dependent manner, P = 0.01 and <0.01 for H99 and cap59 respectively. However, there was no difference in internalization of H99 and acapsular cap59 cryptococci, P = 0.5 and 0.8 at 2 hr and 4 hr respectively. Error bars are standard error of the mean, n = 6 repeats.</p

Binding and uptake of H99 and cap59 to the murine brain endothelial cell line, bEnd3.

<p>BEnd3 cells were exposed to wild type H99 and its acapsular derivative cap59 for 2 hr and 4 hr at 37°C. (A) The rate of association (bound and internalized cryptococci) was determined both by microscopy and live CFU counts. The rate of association increased significantly in a time dependent manner, (P<0.01 for both strains). However, there was no difference in association of encapsulated H99 or acapsular cap59 cells (P = 0.1 and 0.5 at 2 hr and 4 hr respectively). (B) Internalization of the two strains, H99 and cap59 by bEnd3 cells as determined by fluorescence microscopy. Internalized cryptococci (pre-stained with FITC) were distinguished from extracellular adherent ones by counter labelling with calcofluor white after infection. Intracellular cryptococci retained the FITC green signal while extracellular cells acquired the blue signal from calcofluor. The number of phagocytosed cryptococci increased significantly in a time dependent manner, (P<0.01 for both strains). However, there was no difference in internalization of encapsulated H99 and acapsular cap59, (P = 0.5 and 0.8 at 2 hr and 4 hr respectively). Error bars are standard error of the mean, n = 5 repeats.</p

Effect of viability on binding and phagocytosis of H99 and cap59 strains by BMEC.

<p>Heating for 15 min at 65°C prior to infection killed H99 and cap59 cryptococci. Brain microvascular cells (BMEC), bEnd3 and or hCMEC/D3 cells were exposed in parallel to live (LV) and heat-killed (HK) cryptococci for 2 hr and 4 hr at 37°C and the number of bound and internalized cryptococci was determined by fluorescence microscopy. (A and C) Association efficiency of live and heat-killed H99 or cap59 cryptococci by bEnd3 and hCMEC/D3 cell respectively. There was no difference in association of viable H99 and cap59 cryptococci with hCMEC/D3 cells at either time point, P>0.05. However, viable cryptococci were more associated than non-viable ones by 4 hr of incubation in bEnd3 cells, P<0.01 for both H99 and cap59 respectively. (B and D) Internalization efficiency of LV and HK H99 and cap59 cryptococci by bEnd3 and hCMEC/D3 cells. Both live and heat-killed cryptococci showed a time dependent increase in phagocytosis by both bEnd3 and hCMEC/D3 cells, however the rate of phagocytosis did not vary between the viable and non-viable cryptococci in both cell-lines, P>0.05 at both 2 hr and 4 hr of incubation. Error bars are standard error of the mean, n = 5 repeats.</p

Effect of opsonisation on cryptococcal binding and uptake by bEnd3 cells.

<p>Live (LV) and heat-killed (HK) H99 cryptococci were opsonised with mouse derived anti-capsule IgG antibody, 18B7 and the rate of adherence and internalization was determined by fluorescence microscopy. (A) Association of opsonised (op) and non-opsonised (Nop) cryptococci with bEnd3 cells. Rate of association of opsonised and non-opsonised H99LV cryptococci was similar, P = 0.8 and 0.4 at 2 hr and 4 hr respectively. Similarly, there was no difference between opsonised and non-opsonised heat-killed H99 cryptococci, P = 0.9 and 0.5 at 2 hr and 4 hr respectively. (B) Rate of internalization of opsonised and non-opsonised cryptococci internalized by bEnd3 cells. The number of internalized H99LV cryptococci was similar, regardless of opsonisation status (P = 0.9 at both 2 hr and 4 hr). Similarly, there was no difference between opsonised and non-opsonised heat-killed H99 cryptococci, P = 0.5 and 0.4 at 2 hr and 4 hr respectively. Error bars are standard error of the mean, n = 5 repeats.</p

A sampling from the zebrafish toolbox.

<p>(A) Selective ablation of macrophages. Transgenic fish with macrophage-specific expression of Gal4 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002349#ppat.1002349-Ellett1" target="_blank">[2]</a> and Gal4-dependent expression of nitroreductase-mCherry fusion protein were incubated at 3 dpf with 5 mM metronidazole or vehicle for 24 hours. Neither transgenics nor controls exposed to metronidazole had any loss of viability or developmental defects. Ablation efficiency of macrophages is >90% (R. Gratacap and R. Wheeler,unpublished data). Scale bar = 100 µm. (B) OXYellow <i>Candida albicans</i> reports on oxidative stress in vivo. Zebrafish larvae were infected in the hindbrain with OXYellow <i>C. albicans</i> (expressing mCherry constitutively and EGFP under the oxidative stress-induced catalase promoter) and imaged at 24 hours post-infection. Green/red ratio quantifies oxidative stress (K. Brothers and R. Wheeler, unpublished dat). Scale bar = 10 µm. (C) <i>Cryptococcus neoformans</i> infects zebrafish embryos. Zebrafish were infected with EGFP-expressing <i>C. neoformans</i> and imaged. Clusters of fungi are seen in the tail (S. Johnston and R. Ma, unpublished data). Scale bar = 100 µm.</p

Advantages of embryonic zebrafish model for study of innate immune-pathogen interaction.

<p>Advantages of embryonic zebrafish model for study of innate immune-pathogen interaction.</p

RGS14, but not talin2, is involved in the regulation of αMβ2 binding activity in macrophages.

<p>J774.A1 macrophages were transfected using magnetofection with either GFP-tagged talin1 or 2 or Flag-tagged RGS14 and 48 hr later, challenged with C3bi-RBC, processed for immunofluorescence using antibodies against Flag for cells expressing Flag-RGS14 plasmid (GFP-tagged proteins remained unstained) as well as antibodies to detect the C3bi-RBC (by anti-rabbit antibodies against the opsonising rabbit antibody) and analysed by epifluorescent microscopy (<b>A</b>) as described in the Methods section. (<b>A</b>) Representative examples; (<b>B</b>) cells were scored for RBC association, and indices are the mean ± S.D. of at least three independent experiments. *, <i>p</i><0.001; **, <i>p</i><0.05.</p

RGS14 is the Rap1 effector essential for RBC binding during αMβ2 activation.

<p>(<b>A</b>, <b>B</b>) J774.A1 macrophages were transfected with 60pmol of individual or pooled RGS14 or Scrambled siRNA as indicated and 5 days later, cells were analysed for RGS14 expression (<b>A</b>,<b>B</b>) and binding of C3bi-RBC (<b>C</b>). (<b>A</b>) Lysates of siRNA transfected J774.A1 cells were analysed by western blotting for the presence of RGS14 and focal adhesion kinase (FAK) as indicated. (<b>B</b>) Relative band intensities were determined as described in the Methods section, with the ratio of Scrambled siRNA and FAK intensities set to 100% for the negative control. (<b>C</b>) siRNA transfected J774.A1 cells were challenged with C3bi-RBC, processed for immunofluorescence and scored for RBC association, as described in the Methods section. Relative association indices were related to the values obtained from the negative (scrambled) controls and they are the mean ± S.D. of at least three independent experiments. Not significant (ns), <i>p</i>>0.05; *, <i>p</i><0.05.</p

Variation in host responses to <i>C</i>. <i>neoformans</i> infections.

<p>(A) A measured intracellular proliferation rate (IPR) for each donor showing median of at least 2 biological repeats each (median = 0.53, mean = 0.6307, SD = 0.5275 and Coefficient of variation = 83.64%). (B) Variable rates of vomocytosis observed between and within donors showing median of at least 2 biological repeats each (median = 41.11%, mean = 41.55%, SD = 15.53 and Coefficient of variation = 37.37%). (C) Correlation between intracellular parasitism and non-lytic expulsion events (R square = 0.2501, P-value = 0.0005). Each point on the graph represents data from a single blood donation. Since not all participants were available to provide four repeat donations across the study period, the number of repeated samples varies from donor to donor.</p