Table 1.
Toxicity of ciprofloxacin derivatives in mice.
Fig 1.
Effect of 7-day treatment with different doses of Cipro derivatives on the survival mice.
Swiss Webster mice were infected i.p. with 5x103 tachyzoites of T. gondii (RH strain) one day prior to the start of treatment. Results were evaluated by the Kaplan-Meier product limit method, and compared using the log-rank (Mantel-Cox) test. * P<0.05 vs. untreated controls. The numbers of treated mice in each group were: untreated, 10 (A), 14 (B and D) and 15 (C); 50 and 100 mg Cipro, 11 (three groups each); 150 mg Cipro, 8 (two groups); 50 and 100 mg Et-Cipro, 11 (three groups); 50 and 100 mg Ph-Cipro, 8 (two groups); 150 mg Ph-Cipro, 11 (three groups); 50 mg Adam-Cipro, 3 (one group); 100 mg Adam-Cipro, 12 (three groups).
Fig 2.
Early effects of ciprofloxacin (Cipro) derivatives in tachyzoite cell division inside host cells.
(A) Tachyzoites of T. gondii were treated with Cipro derivatives for 6 h, from 1–7 h post-infection, and the number of parasites per vacuole was analyzed by light microscopy. (B) After 6 h, the medium containing drugs was removed and replaced with fresh medium without drugs. Tachyzoites were allowed to proliferate for an additional 18 h, and the number of parasites per vacuole was counted. * P<0.05, results were analyzed by two-way ANOVA statistical test.
Fig 3.
Cipro derivatives did not affect parasite mitosis.
Parasites were labeled with anti-SAG1 (red, labeling the parasite’s cell surface) and Sytox green (to label the DNA). After 24 h of infection, untreated (control) parasites (top row) were typically arranged in ‘rosettes’, and displayed normal morphology, with one nucleus per cell. Many parasites were in the process of cell division, as evidenced by the presence of U-shaped mitotic nuclei (arrowheads). In cells treated with 5 μM of Et-Cipro (middle row) or Adam-Cipro (bottom row) for 24 h, tachyzoites were able to complete nuclear division, as evidenced by the presence of individualized nuclei, but could not complete cytokinesis, which led to the formation of multinucleated cells (arrows), and loss of the typical rosette organization. Images represent maximum projections of optical slices from confocal laser scanning microscopy.
Fig 4.
Cipro derivatives affected parasite daughter cells scission process.
Immunofluorescence microscopy of LLC-MK2 cells infected with tachyzoites of T. gondii and treated for 24 h with Cipro derivatives. Parasites were labeled with anti-HSP60 (recognizing the apicoplast, in green) and anti-IMC1 (recognizing the inner membrane complex or IMC, in red) antibodies. (A) Untreated (control) parasites were typically organized in ‘rosettes’ and had apicoplasts of normal shape and size. Tachyzoites treated with (B) 5 μM Et-Cipro, or (C) 1 or (D) 5 μM Adam-Cipro or (E) 10 μM Ph-Cipro, for 24 h, showed enlarged and abnormally-shaped apicoplasts (arrows), signs of cell division arrest (such as ‘tethered’ parasites, arrowheads), abnormally-shaped basal complexes (asterisks), and also missegregated apicoplasts (thick arrows) that were left outside daughter cell boundaries (marked by the anti-IMC1 labelling). Images represent optical slices (untreated) or maximum projections of optical slices (Et-Cipro and Adam-Cipro). (F) Quantification of vacuoles (n = 120) containing parasites presenting cell division arrest after treatment with Cipro derivatives for 24h, results are the mean of two independent experiments. (G) Apicoplast segregation (arrowhead) and parasite division defects were observed as a result of Cipro derivative treatment in the first event of tachyzoite division inside host cells. (H) Comparison of the distribution of basal IMC gap length in untreated control (n = 143), 1 μM Et-Cipro (n = 149), 5 μM Et-Cipro (n = 144), 1 μM Adam-Cipro (n = 150), 5 μM Adam-Cipro (n = 147) and 10 μM Ph-Cipro (n = 143) after 24 h of treatment. All treatment groups had P<0.05 compared to control (Kruskal-Wallis statistical test and Dunn’s Multiple Comparison Test).
Fig 5.
Transmission electron microscopy analysis of tachyzoites treated with Cipro derivatives.
(A) Untreated cells infected with parasites undergoing normal cell division by endodyogeny. IMC (arrowheads) scaffolds daughter cells while nucleus (N) is undergoing closed mitosis. Each daughter cell is inheriting one apicoplast (inset). (B) Tachyzoites of T. gondii treated with 1 μM Et-Cipro for 48 h. Treatment led to parasite cell division arrest. Arrowheads point at daughter cells delimited by the inner membrane complex and show ‘tethered’ daughter parasites. (C) T. gondii tachyzoites treated with 5 μM Adam-Cipro for 24 h. Treatment led to parasite cell division arrest, leading to the formation of ‘tethered’ daughter cells. This derivative also affected the formation of the inner-membrane complex (IMC), and one of the daughter cells is devoid of this structure (arrowhead and inset). (D) 5 μM Et-Cipro for 24 h. Treatment increased the IMC basal gap length and apicoplast positioning. Arrowhead points to the region of the parasite’s surface devoid of an IMC (gap). (E) Tachyzoites treated with 5 μM Et-Cipro for 48 h. Part of the IMC envelope is missing (arrowhead) in the apical region of a daughter cell budding (arrow points to IMC scaffolding the daughter cell bud). (F) Tachyzoites treated with 5 μM Et-Cipro for 48 h. Treatment caused apicoplast (inset) to be positioned outside the boundaries of the daughter cell IMC (arrowhead), which is likely to lead to apicoplast missegregation during parasite division. A-apicoplast; HC-host cell; M- mitochondrion; N-nucleus; PV-parasitophorous vacuole; Rp-ropthries. Scale bars: (A) 1 μm, 0.2 μm inset; (B) 2 μm (C) 0.5 μm, 0.2 μm inset; (D) 1 μm; (E) 1 μm; (F) 1 μm, 0.2 μm inset.
Fig 6.
Treatment of T. gondii with Cipro also leads to cell division arrest, apicoplast missegregation and IMC alteration.
Tachyzoites of T. gondii were treated with 20 μM of Cipro for 24, 48 and 72 h. (A) Quantification of vacuoles (n = 120) containing parasites presenting cell division arrest after treatment with 20 μM of Cipro for 24, 48 and 72 h, results are the mean of two independent experiments. (B-H) Immunofluorescence and TEM microscopy of LLC-MK2 cells infected with tachyzoites of T. gondii and treated for 48 and 72 h with 20 μM Cipro. For immunofluorescence parasites were labeled with anti-HSP60 (recognizing the apicoplast, in green), anti-IMC1 (recognizing the inner membrane complex or IMC, in red) antibodies and DAPI (to label the DNA). While untreated tachyzoites presented normal morphology (B), parasites treated for 48 and 72 h presented cell division arrest, such as ‘tethered’ parasites (C-E and G), abnormally-shaped basal complexes (C and F square brackets, and inset), missegregated (C’ arrow) and mispositioning apicoplasts (C’ thick arrows), parasites displaying regions of pellicle without IMC (asterisk in D and arrows in H) and large degenerated parasites showing abnormal nucleus size (E arrow).
Fig 7.
Cipro derivatives and Cipro affect MORN1 localization during parasite division.
In parasites presenting normal division process MORN1 localizes at the basal complex of the mother cell (arrow) and also caps the final end of the IMC in budding daughter cells (arrowheads) (A), a single strong point in the middle of the cell was also observed in normal non-dividing parasites (B rosette 2 curved arrow). Tethered daughter cells resulting from the treatment with 5 μM Et-Cipro (B-C) and Adam-Cipro (D-E) for 24h, and 20 μM Cipro for 72h (F) showed wide MORN1 basal caps (B and D large arrows) and lack or a weak deposition of MORN1, at the basal end of mother (arrow) and budding daughter cells (arrowheads) (B-F)
Table 2.
Effect of treatment with Cipro, Et-Cipro, Adam-Cipro and Ph-Cipro on the growth of Cryptosporidium parvum sporozoites in host cells.