Sleeping with the Enemy: How Intracellular Pathogens Cope with a Macrophage Lifestyle

Sleeping with the Enemy: How Intracellular Pathogens Cope with a Macrophage Lifestyl

Novel insights into leishmania biology : the role of parasite and host-derived small non-coding RNAs

Infection with leishmania parasites causes severe chronic and potentially fatal illness in millions of people annually. Nevertheless, leishmania-host interactions remain understudied, and available treatments are sub-optimal. Pivotal to the establishment of infection, parasite replication and development of clinical disease is the subversion of microbicidal activities of host macrophages by leishmania. The overall aim of this thesis was to enhance our understanding of the modus operandi of macrophage subversion and explore the involvement of parasite- and host-derived small non-coding RNAs in this process. My first objective was to investigate whether leishmania exosomes act as shuttle vehicles to export and deliver leishmania RNAs to host macrophages, where they may contribute to pathogenesis. We used high-throughput sequencing to characterize the transcriptome of leishmania exosomes and found that leishmania exosomes are selectively and specifically enriched in small RNAs derived almost exclusively from non-coding RNAs such as rRNAs and tRNAs. In depth analysis revealed the presence of tRNA-derived small RNAs, a novel RNA type with suspected regulatory functions. Exosomes protected their RNA cargo from degradation and were competent to deliver RNAs to macrophages. Furthermore, our results demonstrated a remarkably high degree of congruence in exosomal small non-coding RNA content between two distinct leishmania species, which argues for a conserved mechanism for exosomal RNA packaging in leishmania. My second objective was to investigate whether macrophage miRNA expression is modulated during leishmania infection. Here, I was interested to know whether targeting of the host RNAi machinery is a potential novel mechanism of pathogenesis used by leishmania to control macrophage phenotype and promote chronic infection. I profiled miRNA expression in human macrophages at later stages of infection using two independent technologies. The data showed that leishmania infection induced an overall down-regulation of miRNA expression in macrophages. This down-regulation was not caused through effects on synthesis or stability of Drosha and Dicer, two essential enzymes involved in miRNA maturation. Taken together, my findings suggest that both leishmania- and host-derived small non-coding RNAs may contribute to pathogenesis. They open up new avenues of research on small RNA pathways in leishmania infection biology, which may identify novel therapeutic approaches.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Class IA phosphatidylinositol 3-kinase p110α regulates phagosome maturation.

Of the various phosphatidylinositol 3- kinases (PI3Ks), only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110α, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110α and contained PI3P and PI(3,4,5)P3; however, p110α and PI(3,4,5)P3 levels in phagosomes from p110α knockdown cells were decreased. Phagosomes from p110α knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, β-galactosidase. Phagosomes from p110α deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110α deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP) and homotypic vacuole fusion and protein sorting (HOPs) components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K

Phagosomes contain the class IA PI3K p110α, and display PI(3,4,5)P₃.

<p>Phagosomes from p110αknockdown cells show decreased p110α recruitment and PI(3,4,5)₃ levels. <b>A)</b><i>Phagosomes isolated from PMA-differentiated THP-1 cells contain the class IA PI3K p110α, and this is decreased in knockdown cells.</i> Late stage 4 h bead phagosomes were isolated by magnetic pulldown, lysed, and immunoblotted for p110α. Actin was done as a loading control. Densitometry of four independent experiments indicates a significant decrease in phagosomal p110α in phagosomes isolated from knockdown cells. Empty bars  =  control cell phagosomes, solid bars  =  phagosomes from p110α knockdown cells. p<0.05 <b>B)</b><i>Phagosomes from p110α knockdown cells contain less PI(3,4,5)P3, but similar amounts of PI3P as control cells.</i> Bead phagosomes were isolated by magnetic pulldown, fixed, stained for PI3P and PI(3,4,5)P3, and flow organellometry was done. A representative dot plot of SSC vs FL4 fluorescence channel depicting how the phagosomes were gated is shown. The data shows PI(3,4,5)P3 staining of 4 h phagosomes isolated from control and p110α knockdown cells from one experiment. Empty bars  =  control cell phagosomes, solid bars  =  phagosomes from p110α knockdown cells Data are means +/− s.e.m., from four independent experiments. The levels of PI(3,4,5)P3 were found to be significantly decreased in phagosomes isolated from p110α deficient cells. p<0.05. No significant differences were observed for PI3P levels. <b>C)</b><i>Kinetics of PI3P acquisition onto phagosomes.</i> Phagosomes were isolated at 30 min and 2 h and stained for PI3P. The graph shows geometric means normalized to isotype control. Open bars  =  phagosomes isolated from control cells, Solid bars  =  phagosomes isolated from p110α knockdown cells. No significant differences were observed, and PI3P levels appeared to remain consistent throughout phagosome maturation.</p

Knockdown of PI3K p110α does not impair phagocytosis of prey less than 3 µm in diameter.

<p>Cells were either fed Alexafluor 633-SE labelled BSA-coupled 3 µm magnetic beads or infected with Alexafluor 633-SE labelled <i>M. smegmatis.</i> Flow cytometric analysis was done to determine uptake. Solid circles  =  control cells, empty squares  =  p110α knockdown cells. Data are means +/− s.e.m., from three independent experiments. No significant differences were observed.</p

Phagosomes from p110α knockdown cells show marked decreases in LAMP-1 acquisition and phagolysosome fusion.

<p><b>A)</b> Confocal microscopy of control and p110α knockdown cells shows reduced phagosomal LAMP-1 acquisition. The graph to the right shows counts of phagosomes colocalized with LAMP-1 staining. At least 100 phagosomes in each of three independent experiments were counted. Open bar  =  Control cells, Solid bar  =  p110α knockdown cells. Means +/− s.e.m. are shown. **p<0.01. <b>B)</b> Confocal microscopy of control and p110α knockdown cells shows reduced phagosome - lysosome fusion. Cells were loaded with Texas Red dextran, which was then chased into lysosomes. The graph to the right shows the numbers of phagosomes that were positive for a dextran signal. At least 100 phagosomes in each of three independent experiments were counted. Open bar  =  control cells, Solid bar  =  p110α knockdown cells. Means +/− s.e.m. are shown. *p<0.05.</p

Magnetic bead phagosomes from p110α knockdown cells show defective acquisition of the lysosomal proteins LAMP-1 and LAMP-2.

<p>Kinetic analysis of phagosome maturation by flow organellometry shows <b>A)</b> normal acquisition of the early endosomal marker Rab5 and EEA-1, and <b>B)</b> marked defects in the acquisition of the lysosomal membrane proteins LAMP-1 and LAMP-2 by p110α knockdown cells. Solid circles  =  control cells, empty squares  =  p110α knockdown cells. Timepoints were taken at 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours post bead treatment. Data are means +/− s.e.m., from five independent experiments. Ratios of geometric mean over isotype control of 1 or less are interpreted as negligible signal. *p<0.05. <b>C)</b> Whole cell expression of phagosome markers are at similar levels in control and p110α knockdown cells. Staining of permeabilized cells and flow cytometric analysis of various phagosome markers in control and p110α knockdown cells showed no significant differences in cellular levels. Empty bars  =  control cells, solid bars  =  p110α knockdown cells. Data are means +/− s.e.m., from three independent experiments.</p

Delivery of the lysosomal enzyme β- galactosidase is impaired in p110α knockdown cells.

<p><b>A)</b> Knockdown of p110α results in decreased acquisition of the lysosomal enzyme β- galactosidase by phagosomes containing either BSA coated beads or M. smegmatis. Cells were fed prey that had been previously surface labelled with Alexafluor 633-SE and the fluorescent β-galactosidase substrate, C₁₂RG. Cleavage of C₁₂RG releases fluorescence which is read in the FL2 channel of the flow cytometer. This was then normalized to the Alexafluor 633 signal (FL4) for phagocytic uptake. Solid circles  =  control cells, empty squares  =  p110α knockdown cells. Timepoints were taken at 1 hour, 2 hours, 4 hours, and 6 hours post bead treatment or infection. *p<0.05, **p<0.001, **p<0.0005. Data are means +/− s.e.m., from at least four independent experiments. <b>B)</b> Inducible p110α knockdown via a distinct shRNA sequence also showed defects in β-galactosidase acquisition. Knockdown was induced by doxycycline treatment prior to experimentation, which also induces expression of TurboRFP which is linked to shRNA production. The histogram on the left shows induction of red fluorescence when cells were given doxycycline. Cells were also fed the green fluorescent β-galactosidase substrate, C₁₂FDG, which is read in the FL1 channel. Empty bars  =  Control cells (no doxycycline induction), Solid bars  =  Cells induced for p110α knockdown. *p<0.05, **p<0.001. Data are means +/− s.e.m. from five independent experiments.</p