Figure 1.
Microscopy of M. hominis-infected Hela cells.
HeLa cells were analysed by scanning electron microscopy (A) and confocal laser microscopy (B) at 4 h, 24 h and 2 weeks (perm) post infection. Actin filaments were stained green (Alexa 488 phalloidin), nuclei blue (DAPI), surface-attached mycoplasmas magenta (B 4 h) or red (B 48 h and B perm) (mAb BG11 without fixation) and internalized mycoplasmas red (mAb BG11 after fixation and permeabilisation).
Figure 2.
Distribution of differentially regulated HeLa cell genes.
Numbers of up- and down-regulated (> 2 fold) HeLa cell genes at 4 h (t4) and 48 h (t48) after infection with M. hominis compared to uninfected HeLa cells. A chronically infected HeLa cell line (perm) was compared to HeLa cells infected with M. hominis at time point 0 h. A, numbers of up- and down-regulated genes; B, Venn diagram of upregulated genes; C, Venn diagram of down-regulated genes.
Table 1.
Genes highly up- or downregulated in HeLa cells at different stages of infection with M. hominis.
Figure 3.
Confocal microscopy of M. hominis invasion into, and bleb formation for evasion of HeLa cells.
A, HeLa cells analysed at 48 h post infection with M. hominis at an MOI of 500 by confocal laser microscopy. Actin filaments were stained green (Alexa 488 phalloidin), nuclei blue (DAPI), surface-attached mycoplasmas magenta (mAb BG11 without fixation) and internalized mycoplasma cells red (mAb BG11 after fixation and permeabilisation). The four-coloured image (A1) is also shown in four single coloured pictures (A2) for a better discrimination of intra- and extra-cellular mycoplasma cells and demonstration of actin rearrangement at the location of intracellular mycoplasmas. B, HeLa cells chronically infected with M. hominis. Actin filaments are stained green (Alexa 488 phalloidin), nuclei blue (DAPI), M. hominis cells red (mAb BG11). A M. hominis-filled protrusion of the HeLa cell membrane bordered by actin (green) is marked by an arrowhead. Inset: SEM of a chronically infected HeLa cell with a prominent protrusion. C, HeLa cells chronically infected with M. hominis. LAMP3 proteins are stained green (mAb MEM-259), M. hominis cells red (mAb BG11), membranes cyan (Alexa Fluor 594 Wheat Germ Agglutinin) and nuclei blue (DAPI). Co-localisation of LAMP3 and M. hominis appears yellow in a three colour overlay (omitting stain of the membranes).
Figure 4.
Comparison of microarray and RT-qPCR results.
Different preparations of total RNA of HeLa cells (04/11, 11/11 and 05/12) infected with M. hominis for 4 h, 48 h or 2 weeks (perm) at an MOI of 100 were subjected to microarray or RT-qPCR analyses and the fold change in expression levels of the named genes, with respect to that in uninfected HeLa cells, quantified as described in the Method section.
Table 2.
Number of pathway-assigned HeLa cell genes regulated at the different stages of infection with M. hominis.
Figure 5.
Network systems’ dependent transcript regulation.
Regulated HeLa cell genes at 4 h (t4), 48 h (t48) and 2 weeks (perm) after infection with M. hominis were sorted to KEGG- pathway maps and integrated into those belonging to the Metabolism, Genetic or Environmental Information Processing, Cellular Processes and Organismal Systems groups. The proportions of pathway genes that were regulated are shown.
Figure 6.
Model of M. hominis infection of HeLa cells.
Based on the chronology of the host response to a M. hominis infection, a model of infection-stage dependent colonization with M. hominis cell (blue sphere) was developed. Infection begins at t0 with focal adhesion, followed by opsonin-receptor-mediated phagocytosis and different stages of phago-lysosome maturation, ending at t∞, either with a protrusion of the host cell membrane as a prerequisite for mycoplasmal exocytosis or with a postulated escape into the cytoplasm of the host cell (?) or in destruction in the lysosome (light blue sphere). Infection-stage dependent genes of the immune system are upregulated, affecting Jak-STAT, MAPK and NOD-like receptor signalling and apoptosis. Upregulated genes are coloured light-red and down-regulated genes light-green. Fold changes in expression of genes were calculated from microarray results and represents the mean of quadruplicate assays.