The outcome of phagocytic cell division with infectious cargo depends on single phagosome formation

Abstract

Given that macrophages can proliferate and that certain microbes survive inside phagocytic cells, the question arises as to the post-mitotic distribution of microbial cargo. Using macrophage-like cells we evaluated the post-mitotic distribution of intracellular Cryptococcus yeasts and polystyrene beads by comparing experimental data to a stochastic model. For beads, the post-mitotic distribution was that expected from chance alone. However, for yeast cells the post-mitotic distribution was unequal, implying preferential sorting to one daughter cell. This mechanism for unequal distribution was phagosomal fusion, which effectively reduced the intracellular particle number. Hence, post-mitotic intracellular particle distribution is stochastic, unless microbial and/or host factors promote unequal distribution into daughter cells. In our system unequal cargo distribution appeared to benefit the microbe by promoting host cell exocytosis. Post-mitotic infectious cargo distribution is a new parameter to consider in the study of intracellular pathogens since it could potentially define the outcome of phagocytic-microbial interactions.

Figure 1. The possible post-mitotic outcomes of intracellular particle distribution and modeling of post-mitotic intracellular particle distribution.

(A) Schematic diagram of Outcome I: all the intracellular particles in the mother cell are distributed into one of the daughter cells with a clean daughter cell generated after cell division (all-or-none distribution). (B and C) Schematic diagram of Outcome II: intracellular yeast cells in the mother cell are distributed equally (B) or unequally (C) into both daughter cells after cell division (equal or unequal distribution). (D) Theoretical distribution index (DI_T) for various numbers of intracellular particles calculated from Equation (4), with the aid of a computer program, assuming that the intracellular particles were stochastically distributed into two daughter cells during cell division. DI_T for n = 1–100 were plotted in this Figure. (E) Probability of Outcome I, IIa and IIb (P_I, P_IIa and P_IIb) of various numbers of intracellular particles calculated from Equations (5)–(7), with the aid of a computer program, assuming that the intracellular particles were stochastically distributed into two daughter cells during cell division. P_I, P_IIa and P_IIb for n = 1–100 were plotted in this Figure.

Figure 2. Outcome I and II of post-mitotic intracellular particle distribution.

(A) Post-mitotic Outcome I of intracellular particle distribution. Images showing J774.16 cells infected with HK Cap67 undergoing cell division and sorting the intracellular yeasts into one of the two daughter cells. Frames are labeled according to the start of the imaging process, which is approximately 1 h after phagocytosis of yeasts was initiated. The thick arrow indicated single giant phagosome formation caused by phagosomal fusion. The thin arrow indicated macrophage-like cell division. Images were collected at 20×. Bar, 10 µm. (B) Post-mitotic Outcome II of intracellular particle distribution. Images showing J774.16 cells infected with C. neoformans strain 24067 undergoing cell division and sorting the intracellular in both daughter cells. Frames are labeled according to the start of the imaging process, which is approximately 1 h after phagocytosis of yeasts was initiated. The arrow indicated macrophage cell division. Images were collected at 20×. Bar, 10 µm.

Figure 3. Comparison of experimental and theoretical post-mitotic particle distribution.

(A) Post-mitotic DI of intracellular particle distribution. DI for C. neoformans strains 24067, H99, heat-killed H99 (HK H99), H99 phospolipase mutant (H99 PLB1), Cap67, heat-killed Cap67 (HK Cap67), Cap67 coated with polysaccharide (Cap67+PS), C. gattii or heat-killed C. gattii (HK C. gattii). The horizontal lines denoted the means of calculated DI, which were not immediately comparable between the groups as described in the Results. The data were collected from three or more independent experiments for every condition. In every experiment, movies were recorded for approximately 24 h with 300–400 cells in the microscopic field from which the macrophage-like cells underwent cell division with intracellular particles were selected for analysis. (B) Normalized post-mitotic DI by stochastic modeling of intracellular particle distribution. To eliminate the inaccuracy introduced by numerical variances of intracellular particles, the normalized DI was calculated as described in the Results. The distribution of intracellular particles was stochastic if the normalized DI equaled to one (dash line). While normalized DI were either larger or smaller than 1, the distribution of intracellular particles into daughter macrophage-like cells was non-stochastic and was skewed to unequal distribution or equal distribution, respectively. One-way ANOVA test revealed significant variances between the groups (p<0.0001). Newman-Keuls multiple comparison test revealed significant difference between H99 and H99 PLB1 (^# p<0.05), Cap67 and Cap67+PS (^## p<0.01). One sample t test revealed that post-mitotic distribution of most yeasts had significantly larger DI than 1 which denoted stochastic distribution (dash line) (**p<0.01, ***p<0.001).

Figure 4. Linear correlation of percentage of macrophage-like cells with single phagosome formation versus the normalized DI.

The percentages of cells demonstrating single phagosome formation in all the experiments were calculated and plotted versus the normalized DI. Statistical analysis revealed a strong linear correlation. The best-fit line intersected the X-axis near a normalized distribution index of 1, which denotes a stochastic distribution in situations where there is no phagosomal fusion.

Figure 5. Extrusion of intracellular yeasts during mitosis of macrophage-like cells and fusion of two daughter macrophage cells with intracellular yeasts post mitosis.

(A) Fusion of two daughter macrophage cells with intracellular yeasts post mitosis. Frames are labeled according to the start of the imaging process, which is approximately 1 h after phagocytosis of C. neoformans strain H99 was initiated. The thick arrow indicated the fusion of two daughter cells after division and the thin arrow indicated macrophage-like division. Images were collected at 20×. Bar, 10 µm. (B) Extrusion of intracellular yeasts during mitosis of macrophage-like cells. After phagocytosis of C. neoformans, some macrophage-like cells undergoing cell division extruded intracellular yeasts either before or after the cell round-up, a morphological change that indicates the initiation of cell division (time zero). Frames are labeled according to the start of the imaging process, which is approximately an hour after phagocytosis of C. gattii was initiated. The thick arrow indicated the extrusion of intracellular C. neoformans and the thin arrow indicated macrophage-like cell division. Images were collected at 20×. Bar, 10 µm.