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Vector-borne Trypanosoma brucei parasites develop in artificial human skin and persist as skin tissue forms

Christian Reuter, Fabian Imdahl, Laura Hauf, Ehsan Vafadarnejad, Philipp Fey, Tamara Finger, Heike Walles, Antoine-Emmanuel Saliba, Florian Groeber-Becker, Markus Engstler

Preprint posted on May 13, 2021 https://www.biorxiv.org/content/10.1101/2021.05.13.443986v1

Human skin infection model successfully recapitulates transmission of African trypanosomes

Selected by Isabella Maudlin

Background

African trypanosomes are unicellular, flagellated parasites transmitted by the bite of the tsetse fly, causing devastating disease in humans and livestock in Sub-Saharan Africa. African trypanosomes cycle between their mammalian hosts and insect vectors in a complex manner. In the mammalian host, trypanosomes multiply extracellularly in the blood and lymphatic systems as proliferative ‘slender’ forms. As parasite density increases, quorum sensing (a form of cell-cell communication that triggers gene expression changes when population density is high) results in the differentiation from ‘slender’ to ‘stumpy’ forms that are growth-arrested and thought to be pre-adapted to survival in the tsetse fly vector.

Once ingested by the tsetse fly during a bloodmeal, African trypanosomes differentiate into ‘procyclic’ forms that multiply in the tsetse midgut and migrate to either the salivary glands or the proboscis, depending on the species. After this migration, trypanosomes differentiate into ‘metacyclic’ forms that are capable of infecting a new host during the next bloodmeal of the tsetse fly. The most important change, unique to metacyclic and bloodstream form trypanosomes, is the expression of a protective surface coat called the variant surface glycoprotein (VSG) that allows the trypanosome population to evade the mammalian immune system. In addition to the bloodstream, trypanosomes have more recently been found to proliferate in the skin of humans and animals. These skin populations are thought to be a major reservoir for long-term infections and could be particularly important for infections in asymptomatic individuals.

A key to studying infection of any parasite is to develop an infection model. In the case of studying trypanosomes, this is usually limited to mouse models. In this preprint, Reuter et al. developed a human skin infection model and successfully recapitulated transmission of T. brucei by tsetse flies, allowing for the first time the in vivo study of trypanosome infection of human skin and furthering our understanding of the development of infection in humans. This could help to fight trypanosome infections and improve the lives of many people in Sub-Saharan Africa.

Key Findings

1. Human skin equivalents resemble native human skin

An advanced primary human skin equivalent (a high-density skin equivalent) was developed as a model for infection. High-density dermal equivalents were comprised of normal human epidermal keratinocytes, and histology showed that after 15 days of culture the skin equivalents resembled native human skin with a fully differentiated 4-layer epidermis. Confirming differentiation, immunohistochemical staining showed that at day 23 the proper physiological skin markers were expressed in different layers of the skin. To determine how heterogeneous the primary human skin equivalents were, single cell RNA sequencing was performed which showed different cell layers expressed the appropriate markers. Together, these analyses show that human skin equivalents have key features of native human skin.

2. Human skin equivalents could be infected by trypanosome-infected tsetse flies

To demonstrate whether skin equivalents could be infected by T. brucei, tsetse flies infected with metacyclic forms were allowed to bite and penetrate the skin with their proboscis. The bite produced a skin lesion, and analysis of this lesion showed that an average of 4000 parasites had been deposited per bite. Several days after infection, the parasites had doubled in population size, with dividing trypanosomes detected and very low numbers of dead trypanosomes found. These analyses demonstrate for the first time a ‘natural’ infection of human skin by trypanosomes in laboratory conditions.

3. Human skin infected trypanosomes are fully developmentally competent

Analysis of the cell cycle at different time points post-infection showed that within 24 hours the average parasite population exhibited bloodstream form size, morphology, motility and protein synthesis rates. However, it took at least 3 days for bloodstream VSG isoforms to be detected on the surface of the parasites, meaning the differentiation process was not quite complete until this time.

Single-cell RNA sequencing was performed at different time points post-infection, which found 24 genes to be upregulated in the early stages of infection and 92 genes upregulated later in infection. As gene regulation in trypanosomes is solely post-transcriptional, RNA- binding proteins are key in altering gene expression in and for adaptation to different environments. Interestingly, many of the genes differentially expressed were RNA-binding proteins, including some encoding for proteins of unknown function. These analyses give key insights into the regulators of trypanosome differentiation for the first time in a human model system.

As parasite density increases, quorum sensing results in the differentiation from ‘slender’ to ‘stumpy’ forms that are growth-arrested. In the infected skin equivalents, very few stumpy forms were detected (<0.1%). Despite this, the infected skin-equivalents successfully infected tsetse flies – showing that the skin-residing parasites are fully developmentally competent and can complete a full life cycle.

4. Identification of a novel quiescent skin tissue form

In later stages of infection, the parasites exhibited reduced protein synthesis and growth rates, both of which are hallmarks of quiescence. RNA sequencing of these cells revealed an upregulation in the expression of genes found to be expressed in quiescent cells of other organisms. These cells could be reactivated by culturing them in medium, meaning that the quiescence programme is reversible in human skin-infected trypanosomes.

This novel finding leads the authors to conclude that trypanosomes transmitted by the tsetse fly into skin enter a “persister-like state, the quiescent skin tissue form”. They suggest this form is different from other quiescent stages (metacyclic and stumpy) and may be specifically adapted for skin infections. As this quiescent skin tissue form can be bypassed by infection straight into the bloodstream, it is not a key developmental stage but an example of evolutionary adaptation that could be important for trypanosomes to sustain long infections.

What I like about this preprint

This study demonstrates an advanced skin tissue model that allows analysis of both host cells and parasites during different stages of vector-borne infection. It is a meticulous study that uses a combination of experimental approaches to precisely define the lifecycle stages of the parasites over the course of infection, which allowed the identification of novel regulators of gene expression and a novel quiescence programme in skin-infected trypanosomes. Most importantly, it provides possibility for future studies of different hosts, vectors and parasites using similar model systems. This study and future studies will inevitably further our understanding of infection and could lead to the development of novel treatments for diseases that cause significant public health and economic burdens.

Tags: human skin, quorum sensing, trypanosome, tsetse fly

Posted on: 30th June 2021 , updated on: 2nd July 2021

doi: https://doi.org/10.1242/prelights.29839

Read preprint (4 votes)




Author's response

Christian Reuter shared

It appears that trypanosomes do not actively leave the skin model into the surrounding system. Do you think this is an artefact or do you think the major way trypanosomes enter the bloodstream is by direct inoculation?

We observed that fibroblasts were quite capable of leaving the skin model. Therefore, in principle, the trypanosomes should also be able to leave the model. However, we found only very low numbers of trypanosomes in the surrounding culture medium. This suggests that the model may exert some tropism on the trypanosomes. In the mammalian host T. brucei invades several tissues, e.g. the skin, adipose tissue, gonads, brain, skeletal muscle, lung and heart. However, it is not clear what could be the basis for the tissue tropism. Regarding the skin, we hypothesize that the main route into the bloodstream is through the lymphatic system. Previous studies have shown that after an infection by a tsetse fly, trypanosomes were first detected in the lymph and then in the blood. However, transendothelial migration into the bloodstream is by no means excluded.

Do the quiescent skin tissue forms undergo quorum-sensing and reactivate their cell cycle in the skin equivalents?

It would be interesting to see if the skin tissue forms (STFs) change their behavior at a lower cell density in the skin (e.g. reactivate at low cell concentrations in the skin). However, we hypothesize that adaptation as STF to the skin is induced by the skin microenvironment. In axenic cultured and differentiated metacyclic forms, we did not detect any evidence of similar behavior. Whether the STFs do quorum sensing in the skin is an interesting point. If so, this should have resulted in a much larger proportion of stumpy parasites in the confined skin environment. This suggests that STFs are resistant to stumpy induction or do not secrete quorum sensing signals. However, the infected skin equivalents were cultured with a large medium reservoir of 7 ml and the medium was changed every 2 -3 days. The reported size of the quorum sensing signal is < 500 Da. The small size allows fast diffusion from the skin into the surrounding medium reservoir and the signal might not be able to accumulate to an effective concentration in the skin.

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