Cellular crosstalk and disease pathogenesis

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  • Parasitic diseases are a constant threat to human health and survival. Understanding the complex cellular crosstalk between parasites and their hosts is crucial in developing methods for controlling and eradicating disease. Based at the University of California, Los Angeles, Professor Patricia Johnson is at the forefront of elucidating the molecular and cellular mechanisms that allow unicellular parasite Trichomonas vaginalis to establish and maintain infection. Professor Johnson’s research will enable new methods of diagnosis and therapy of trichomoniasis.

    Trichomoniasis is a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. This is the most common non-viral sexually transmitted disease with a reported 275 million people being affected worldwide according to The World Health Organization. Though an individual may be infected by T. vaginalis, they may be asymptomatic (no symptoms) or symptoms may not be noticeable, which means that reported cases may be drastically underestimated. A lag time in the development of distinct symptoms delays diagnosis and treatment; this delay may provide time for these microbes to exchange virulence factors and increase the spread of the parasite from host to host. In order for parasitic pathogens to propagate themselves successfully, they must possess molecular and cellular mechanisms that allow effective communication with regard to parasite–parasite and parasite–host interactions. Professor Johnson focuses her research on identifying these unique mechanisms of communication and interaction.

    Key mediator of communication
    Eukaryotic cells are defined as containing a membrane bound nucleus (the information centre containing its genetic make-up) and complex internal structures. Organisms comprised of eukaryotic cells include protozoa, fungi, plants and animals including humans. One of the ways in which eukaryotic cells communicate is via the exchange of information using mobile packaged cargo called extracellular vesicles (EVs). EVs promote cell–cell communication by passaging protein and genetic components from one cell to another. Once bound to their target cell, they can either remain at the surface of the membrane, detach, fuse with it or become internalised. EVs can contain proteins, lipids, sugar structures and RNA that can be used to modify cellular processes. It is now the general consensus that these cellular vehicles are important mediators of communication present within most, if not all, eukaryotic cells. Recent studies have highlighted the importance of EVs in parasite–parasite and parasite–host interactions through their ability to transfer virulence factors and drug resistant markers, modify host cell gene expression, promote parasite adherence and ability to establish infection and host cell proliferation.

    T. vaginalis (green) bound to a human epitheiall cell (pink). An unbound T. vaginalis cell is shown to the right. Note how the T. vaginalis cell spreads to increase its surface contact with the host cell. Image taken by Antonio Pereira-Neves and Marlene Benchimol, Rio de Janeiro, Brazil.

    Until recently, little was known about the pathogenesis of trichomoniasis and the mechanisms of virulence exhibited by T. vaginalis. For establishment of T. vaginalis infection, one key component of parasite cell–cell communication is the potential use of EVs termed exosomes. Professor Johnson led a previous study, along with her trainee Olivia Twu MD/PhD, which investigated whether T. vaginalis secreted exosomes and if so, what function they performed regarding parasite virulence. T. vaginalis vesicles were isolated and found to exhibit size, density and morphology similar to that of mammalian exosomes. Further analysis of T. vaginalis protein composition revealed extensive overlap with mammalian exosomal proteins. This high degree of conservation between T. vaginalis and mammalian exosome proteomes provided firm confirmation of exosome secretion by T. vaginalis. Further study involving the characterisation of the exosomal proteins of particularly virulent strains of T. vaginalis will be important in identifying those that affect pathogenesis.

    T. vaginalis viewed using electron microscopy. Note the four anterior flagella and a fifth one that is embedded in the outer membrane, running from the top (anterior end) down the side of the cell.

    Parasite exosome function
    The proteome refers to a set of proteins encoded by an organism’s genome. These proteins can be expressed differently depending on the type of cell and required function. During proteome analysis of T. vaginalis exosomes, surface proteins and proteases were identified which may have a role in pathogenesis through promoting parasite attachment to host ectocervical cells (Ects). Ects are the cells located on the surface of the host’s cervix, the lower region of the uterus. Having identified proteins within exosomes that could be responsible for pathogenesis, it was hypothesised that these exosomes may be used in initial infection of Ects. Indeed, it was found that T. vaginalis exosomes were able to fuse with and deliver their protein contents to Ect host cells.

    Understanding the nature of these factors will be crucial in elucidating how parasite exosome–host cell fusion occurs and how it can be controlled with appropriate targeted therapy to combat disease.

    Another interesting finding involves parasite modulation of adhesion to host cells to promote rapid infection. Highly adherent strains of T. vaginalis are thought to be able to communicate with less adherent strains via the passage of exosomes. Incubation of exosomes from highly adherent strains with less adherent strains has been shown to increase adhesion to Ects. Professor Johnson’s current work is focusing on identifying the molecules present on the surface of T. vaginalis exosomes and host cells. Understanding the nature of these factors will be crucial in elucidating how parasite exosome–host cell fusion occurs and how it can be controlled with appropriate targeted therapy to combat disease.

    Immunomodulation
    T. vaginalis exosomes can modulate host cell immune responses by inducing a response in signalling molecules that aid communication between different types of immune cells. Neutrophils circulate within the blood and upon sensing a signal are the first cells to migrate to the site of infection to begin killing invading pathogens. Monocytes like neutrophils are part of the innate immune response and differ in that they can mature into macrophages. T. vaginalis exosomes can increase and decrease levels of signalling molecules IL-6 and IL-8 respectively. IL-8 is known to be involved in a long-term inflammatory process and is integral for neutrophil recruitment.

    Another study led by Professor Johnson and Olivia Twu MD/PhD, in collaboration with the research group of Pier Luigi Fiori (Università degli Studi di Sassari, Italy), unveiled the presence of an inhibitory factor produced within T. vaginalis exosomes that can modulate the migration of immune cells called macrophages. This inhibitory factor is known as TvMIF and it is similar in form and functionality to its human homolog. TvMIF modulates the host immune response by binding to a surface receptor on monocyte cells. This interaction stimulates downstream pathways associated with IL-8 secretion and so increased host cell inflammation and proliferation. The ability of TvMIF to induce inflammation and cell proliferation may be linked to the increased incidence of urogenital cancers in individuals who have been infected by T. vaginalis. Here, Professor Johnson’s work highlights the importance of understanding the dynamic between the host immune system and immunomodulation via parasite-secreted molecules.

    Human neutrophils kill T. vaginalis using trogocytosis. Six human neutrophils (magenta) in contact with a Trichomonas vaginalis cell (green). Super-resolution live confocal microscopic imaging was used to view neutrophil trogocytosis of the parasite. Note the “green bites” of the parasite inside the two neutrophils at the bottom of the image.

    Another one bites the dust
    Trichomoniasis has been recorded to involve the swarming of neutrophils to the area of infection of the vaginal mucosa. These immune defenders are at the frontline of defence for the host and so dampening the IL-8 response may be a critical strategy for host infection and colonisation. In a recent study, a research team led by Professor Johnson and Professor Frances Mercer were the first to discover a novel mechanism by which host neutrophils kill T. vaginalis. Neutrophils are thought to use three main modes of killing infectious organisms; whole cell engulfment by phagocytosis, degradation by toxic granules and casting of Neutrophil Extracellular Traps (NETosis). Professor Mercer and Professor Johnson were able to use 3D and 4D live imaging to identify a novel mechanism by which human neutrophils kill T. vaginalis cells called trogocytosis.

    Trogocytosis is the process by which “bites” are taken from one cell by another. In this way, target cell surface molecules can be extracted via the transfer of fragments of the plasma membrane. It was found that multiple “bites” were taken from T. vaginalis cells by neutrophils prior to parasite death. This discovery is unique in that it is the first demonstration of neutrophils killing a pathogen via trogocytosis, hence establishing a new method for pathogen killing by human immune cells. Professor Johnson believes that further study regarding the characterisation of the molecular determinants of this mechanism will be integral for generating effective vaccines and immunotherapies to help mitigate chronic inflammation and (cellular) damage associated with T. vaginalis infection.

    This discovery is unique in that it is the first demonstration of neutrophils killing a pathogen via trogocytosis, hence establishing a new method for pathogen killing by human immune cells.

    Professor Johnson and Professor Jane Carlton at New York University took the lead on a research project which obtained and annotated the first draft genome sequence of T. vaginalis. This initiative led to the discovery of highly repetitive elements, relics of past prokaryote-to-eukaryote gene transfer events and evidence of genome expansion. Notably, expansion of genes associated with parasite metabolism, host cell invasion and phagocytosis of bacteria and host cells were identified. In conjunction with this research, Professor Johnson has pioneered the use of CRISPR/Cas9-mediated mutation and gene repair, along with homologous recombination for gene deletion. This knowledge of the T. vaginalis genome and use of cutting-edge technology is a powerful combination. The complex communication network between parasite and host comprised of a vast assortment of protein and gene vocabulary can be broken down into its basic components. Gene editing by CRISPR/Cas-9 will allow researchers to understand the biological roles of specific genes and create targeted therapy against trichomoniasis. Professor Johnson’s leadership in research on T. vaginalis, from parasite–host dynamics, genome topology to fine scale genome editing is providing the scientific community with an invaluable store of knowledge from which further research can be conducted to help combat disease and save lives.

    What drove you to pursue research on T. vaginalis?
    My interests in biology stem back to my early encounters with nature on my childhood farm in Virginia, USA. This upbringing encouraged an insatiable curiosity with how life is created and developed. As a young scientist-to-be, I was mystified by the ability of the farm animals to reproduce themselves, and accompanying my father to assist in the births of litters of pigs and single calves only furthered this fascination. At university, I chose to focus my studies on biology and biochemistry, and became fascinated with the molecular and sub-cellular biology of the simplest unit of life: the cell. During my postdoctoral studies, I was allured to the complex interactions between parasites and their human hosts, while studying a parasite that causes African sleeping sickness, Trypanosoma brucei. When deciding what parasite to research as an independent scientist, I was drawn to Trichomonas vaginalis based on its many intriguing, yet poorly studied, biological characteristics and its status as a neglected parasitic infection that primarily negatively affects the health of women.

    References

    • Twu O, de Miguel N, Lustig G, Stevens GC, Vashisht AA, et al. (2013) Trichomonas vaginalis Exosomes Deliver Cargo to Host Cells and Mediate Host:Parasite Interactions. PLoS Pathog 9(7): e1003482. doi:10.1371/journal.ppat.1003482
    • Marti M, Johnson PJ (2016) Emerging roles for extracellular vesicles in parasitic infections. Current Op in Micro 32:66-70
      Twu O, Johnson PJ (2014) Parasite Extracellular Vesicles: Mediators of Intercellular Communication. PLoS Pathog 10(8): e1004289. doi:10.1371/journal. ppat.1004289
    • Mercer F, Ng SH, Brown TM, Boatman G, Johnson PJ (2018) Neutrophils kill the parasite Trichomonas vaginalis using trogocytosis. PLoS Biol 16(2): e2003885. https://doi.org/10.1371/journal.pbio.2003885
    • Twu O, Dessí D, Vu A, Mercer F, Stevens GC, de Miguel N, … Johnson PJ (2014) Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses. Proceedings of the National Academy of Sciences of the United States of America, 111(22), 8179–8184. http://doi.org/10.1073/pnas.1321884111
    • Carlton, J.M et al (2007) Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 315: 207-212.
    • Janssen, B. D., Chen, Y.-P., Molgora, B. M., Wang, S. E., Simoes-Barbosa, A., & Johnson, P. J. (2018). CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis. Scientific Reports, 8, 270. http://doi.org/10.1038/s41598-017-18442-3
  • Research Objectives
    Professor Johnson’s cellular research focuses on host–parasite interactions from both the host and parasite side.

    Funding
    NIH

    Collaborators
    Two of Professor Johnson’s trainees were key players in this research:

    • Olivia Twu, MD/PhD (exosome & TvMIF work)
    • Frances Mercer, PhD (neutrophil trogocytosis work)

    The research discussed here also involved collaboration with the laboratories of Professor Pier Luigi Fiori (Università degli Studi di Sassari, Italy), Professor Jane Carlton (New York University) and Dr Natalia de Miguel (Instituto Tecnológico Chascomús, Buenos Aires, Argentina).

    Bio
    Professor Johnson earned her PhD in Biological Sciences at The University of Michigan, followed by postdoctoral research training with Professor Piet Borst, at The Netherlands Cancer Institute in Amsterdam. She was a research scientist at The Rockefeller University in New York City, working with Nobel Laureate Professor Christian de Duve, before establishing her own laboratory at the University of California, Los Angeles.

    Contact
    Patricia J. Johnson, PhD
    Professor, Department of Microbiology, Immunology and Molecular Genetics
    UCLA
    1602 Molecular Sciences Building
    609 Charles E. Young Drive, East
    Los Angeles, CA 90095, USA

    E: johnsonp@ucla.edu
    T: +1 310 825 4870
    W:

  • Cellular crosstalk and disease pathogenesis

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