Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female <named-content content-type="genus-species">Plasmodium falciparum</named-content> Gametocytes

ABSTRACT Sexual reproduction is an obligate step in the Plasmodium falciparum life cycle, with mature gametocytes being the only form of the parasite capable of human-to-mosquito transmission. Development of male and female gametocytes takes 9 to 12 days, and although more than 300 genes are thought...

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Autores principales:	Katelyn A. Walzer, Danielle M. Kubicki, Xiaohu Tang, Jen-Tsan Ashley Chi
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	single cell RNA-FISH Plasmodium falciparum sexual development gene expression Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/f8a40361e0b34bc1be42838b9624b503
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spelling	oai:doaj.org-article:f8a40361e0b34bc1be42838b9624b5032021-11-15T15:22:14ZSingle-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female <named-content content-type="genus-species">Plasmodium falciparum</named-content> Gametocytes10.1128/mSphere.00130-182379-5042https://doaj.org/article/f8a40361e0b34bc1be42838b9624b5032018-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00130-18https://doaj.org/toc/2379-5042ABSTRACT Sexual reproduction is an obligate step in the Plasmodium falciparum life cycle, with mature gametocytes being the only form of the parasite capable of human-to-mosquito transmission. Development of male and female gametocytes takes 9 to 12 days, and although more than 300 genes are thought to be specific to gametocytes, only a few have been postulated to be male or female specific. Because these genes are often expressed during late gametocyte stages and for some, male- or female-specific transcript expression is debated, the separation of male and female populations is technically challenging. To overcome these challenges, we have developed an unbiased single-cell approach to determine which transcripts are expressed in male versus female gametocytes. Using microfluidic technology, we isolated single mid- to late-stage gametocytes to compare the expression of 91 genes, including 87 gametocyte-specific genes, in 90 cells. Such analysis identified distinct gene clusters whose expression was associated with male, female, or all gametocytes. In addition, a small number of male gametocytes clustered separately from female gametocytes based on sex-specific expression independent of stage. Many female-enriched genes also exhibited stage-specific expression. RNA fluorescent in situ hybridization of male and female markers validated the mutually exclusive expression pattern of male and female transcripts in gametocytes. These analyses uncovered novel male and female markers that are expressed as early as stage III gametocytogenesis, providing further insight into Plasmodium sex-specific differentiation previously masked in population analyses. Our single-cell approach reveals the most robust markers for sex-specific differentiation in Plasmodium gametocytes. Such single-cell expression assays can be generalized to all eukaryotic pathogens. IMPORTANCE Most human deaths that result from malaria are caused by the eukaryotic parasite Plasmodium falciparum. The only form of this parasite that is transmitted to the mosquito is the sexual form, called the gametocyte. The production of mature gametocytes can take up to 2 weeks and results in phenotypically distinct males and females, although what causes this gender-specific differentiation remains largely unknown. Here, we demonstrate the first use of microfluidic technology to capture single gametocytes and determine their temporal sex-specific gene expression in an unbiased manner. We were able to determine male or female identity of single cells based on the upregulation of gender-specific genes as early as mid-stage gametocytes. This analysis has revealed strong markers for male and female gametocyte differentiation that were previously concealed in population analyses. Similar single-cell analyses in eukaryotic pathogens using this method may uncover rare cell types and heterogeneity previously masked in population studies.Katelyn A. WalzerDanielle M. KubickiXiaohu TangJen-Tsan Ashley ChiAmerican Society for Microbiologyarticlesingle cellRNA-FISHPlasmodium falciparumsexual developmentgene expressionMicrobiologyQR1-502ENmSphere, Vol 3, Iss 2 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	single cell RNA-FISH Plasmodium falciparum sexual development gene expression Microbiology QR1-502
spellingShingle	single cell RNA-FISH Plasmodium falciparum sexual development gene expression Microbiology QR1-502 Katelyn A. Walzer Danielle M. Kubicki Xiaohu Tang Jen-Tsan Ashley Chi Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female <named-content content-type="genus-species">Plasmodium falciparum</named-content> Gametocytes
description	ABSTRACT Sexual reproduction is an obligate step in the Plasmodium falciparum life cycle, with mature gametocytes being the only form of the parasite capable of human-to-mosquito transmission. Development of male and female gametocytes takes 9 to 12 days, and although more than 300 genes are thought to be specific to gametocytes, only a few have been postulated to be male or female specific. Because these genes are often expressed during late gametocyte stages and for some, male- or female-specific transcript expression is debated, the separation of male and female populations is technically challenging. To overcome these challenges, we have developed an unbiased single-cell approach to determine which transcripts are expressed in male versus female gametocytes. Using microfluidic technology, we isolated single mid- to late-stage gametocytes to compare the expression of 91 genes, including 87 gametocyte-specific genes, in 90 cells. Such analysis identified distinct gene clusters whose expression was associated with male, female, or all gametocytes. In addition, a small number of male gametocytes clustered separately from female gametocytes based on sex-specific expression independent of stage. Many female-enriched genes also exhibited stage-specific expression. RNA fluorescent in situ hybridization of male and female markers validated the mutually exclusive expression pattern of male and female transcripts in gametocytes. These analyses uncovered novel male and female markers that are expressed as early as stage III gametocytogenesis, providing further insight into Plasmodium sex-specific differentiation previously masked in population analyses. Our single-cell approach reveals the most robust markers for sex-specific differentiation in Plasmodium gametocytes. Such single-cell expression assays can be generalized to all eukaryotic pathogens. IMPORTANCE Most human deaths that result from malaria are caused by the eukaryotic parasite Plasmodium falciparum. The only form of this parasite that is transmitted to the mosquito is the sexual form, called the gametocyte. The production of mature gametocytes can take up to 2 weeks and results in phenotypically distinct males and females, although what causes this gender-specific differentiation remains largely unknown. Here, we demonstrate the first use of microfluidic technology to capture single gametocytes and determine their temporal sex-specific gene expression in an unbiased manner. We were able to determine male or female identity of single cells based on the upregulation of gender-specific genes as early as mid-stage gametocytes. This analysis has revealed strong markers for male and female gametocyte differentiation that were previously concealed in population analyses. Similar single-cell analyses in eukaryotic pathogens using this method may uncover rare cell types and heterogeneity previously masked in population studies.
format	article
author	Katelyn A. Walzer Danielle M. Kubicki Xiaohu Tang Jen-Tsan Ashley Chi
author_facet	Katelyn A. Walzer Danielle M. Kubicki Xiaohu Tang Jen-Tsan Ashley Chi
author_sort	Katelyn A. Walzer
title	Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female <named-content content-type="genus-species">Plasmodium falciparum</named-content> Gametocytes
title_short	Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female <named-content content-type="genus-species">Plasmodium falciparum</named-content> Gametocytes
title_full	Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female <named-content content-type="genus-species">Plasmodium falciparum</named-content> Gametocytes
title_fullStr	Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female <named-content content-type="genus-species">Plasmodium falciparum</named-content> Gametocytes
title_full_unstemmed	Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female <named-content content-type="genus-species">Plasmodium falciparum</named-content> Gametocytes
title_sort	single-cell analysis reveals distinct gene expression and heterogeneity in male and female <named-content content-type="genus-species">plasmodium falciparum</named-content> gametocytes
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/f8a40361e0b34bc1be42838b9624b503
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Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female <named-content content-type="genus-species">Plasmodium falciparum</named-content> Gametocytes

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