Relevant Degree Programs
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2020)
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.
Phagosome maturation is a key innate immune response involving interactions of phagosomes with the endosomal system. These interactions result in the creation of a destructive, antimicrobial phagolysosome compartment. How this process is regulated is not entirely known, and intracellular pathogens such as Mycobacterium tuberculosis (Mtb) inhibit phagosome maturation as a survival strategy. We examined phagosome maturation from the perspective of two factors, one host- and one pathogen-derived. First, we determined whether the class IA phosphatidylinositol 3-kinase (PI3K), p110α, contributes to maturation regulation. Of the various PI3Ks, only hVps34 is known to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in p110α, we discovered that this PI3K isoform controls maturation processes beyond Rab7 acquisition, leading to delivery of lysosomal markers. Phagosomes from p110α knockdown cells were markedly deficient in LAMP-1, LAMP-2 and β-galactosidase, and could not fuse with lysosomes. Despite lacking lysosomal components, p110α deficient phagosomes recruited Rab7 and its effectors RILP and HOPS components Vps16 and Vps41, suggesting that in addition to Rab7, p110α is required for phagolysosome formation. We also examined how Mtb mediates phagosome maturation arrest by screening an Mtb genomic library for factors able to disrupt yeast vacuolar protein sorting (VPS). Since VPS is homologous to mammalian endosomal trafficking, factors that inhibit VPS might also arrest phagosome maturation. Four proteins able to disrupt yeast VPS were identified in this screen, two hypothetical proteins, Rv0900 and Rv1268c, the P-type ATPase Rv0425c, and PE-PGRS62. To study its effects on macrophage function, we generated M. smegmatis able to express PE-PGRS62. Murine macrophages infected with this construct had arrested phagosome maturation, displaying decreased Rab7 and LAMP-1 recruitment. Infected macrophages also expressed 2-3 fold less iNOS protein when compared to cells infected with control bacteria. Loss of PE-PGRS62 expression in M. marinum resulted in greater iNOS levels, and complementation of the mutant with PE-PGRS62 restored the ability to inhibit iNOS expression. Marked differences in colony morphology were also seen in M. smegmatis expressing PE-PGRS62 and in the M. marinum PE-PGRS62 transposon mutant. Our results suggest that PE-PGRS62 supports mycobacterial virulence via inhibition of phagosome maturation and iNOS expression.
Human infection with protozoa of the genus Leishmania results in a spectrum of disease manifestations collectively termed the leishmaniases. These diseases involve a chronic infection of macrophages which display a deactivated phenotype. Various proteins secreted by leishmania are known to interact with host signaling molecules, bringing about activation of negative feedback loops. Some of these have been shown to block interferon-γ signaling and to inhibit macrophage microbicidal functions. Unlike the well characterized secretion mechanisms used by bacterial pathogens, the mechanism(s) by which leishmania and other eukaryotic pathogens secrete proteins into host cells has remained elusive. The overall aim of this project was to gain a more thorough understanding of host immune-modulation by leishmania, based on the hypothesis that much of these effects are mediated by secreted proteins. The project goals were to: 1) comprehensively identify the proteins secreted by leishmania, 2) determine the mechanism by which proteins are secreted from leishmania into host cells, and 3) determine the functional properties of leishmania secreted compounds. To achieve these goals, a global proteomic analysis of leishmania secreted proteins was carried out using quantitative mass spectrometry. This identified 358 bona fide leishmania secreted proteins many of which were orthologs of proteins considered to be markers of mammalian exosomes. Subsequent experiments confirmed that leishmania secrete exosomes into conditioned media. Comparative proteomics showed that exosomes account for at least 50% of protein secretion by leishmania. Furthermore, the results showed that the cargo profile of leishmania exosomes is influenced by changes in temperature and pH, similar to those experienced by promastigotes after host invasion. Microscopy of leishmania infected cells confirmed the novel finding that leishmania use exosomes to deliver proteins into host cells. Additional studies demonstrated that leishmania exosomes have immunosuppressive properties which, in a cargo dependent manner, modulate the responses of monocytes, dendritic cells, and T lymphocytes. These findings suggest that leishmania utilize exosomes in long-range cellular communication and immune-modulation. In conclusion, this research has significantly advanced the current knowledge of leishmania biology, through the identification of novel secreted molecules, discovery of a secretion system, and description of the immune-modulating effects of leishmania exosomes.