scholarly journals Single cell transcriptomic analysis of bloodstream form Trypanosoma brucei reconstructs cell cycle progression and differentiation via quorum sensing

2020 ◽  
Author(s):  
Emma Marie Briggs ◽  
Richard McCulloch ◽  
Keith Roland Matthews ◽  
Thomas Dan Otto
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Trypanosoma Brucei ◽  
Expression Patterns ◽  
Life Cycles ◽  
Bloodstream Form ◽  
Cycle Phase ◽  
Mammalian Host ◽  
Marker Genes ◽  
African Trypanosomes

The life cycles of African trypanosomes are dependent on several differentiation steps, where parasites transition between replicative and non-replicative forms specialised for infectivity and survival in mammal and tsetse fly hosts. Here, we use single cell transcriptomics (scRNA-seq) to dissect the asynchronous differentiation of replicative slender to transmissible stumpy bloodstream form Trypanosoma brucei. Using oligopeptide-induced differentiation, we accurately modelled stumpy development in vitro and captured the transcriptomes of 9,344 slender and stumpy stage parasites, as well as parasites transitioning between these extremes. Using this framework, we detail the relative order of biological events during development, profile dynamic gene expression patterns and identify putative novel regulators. Using marker genes to deduce the cell cycle phase of each parasite, we additionally map the cell cycle of proliferating parasites and position stumpy cell cycle exit at early G1, with subsequent progression to a distinct G0 state. We also explored the role of one gene, ZC3H20, with transient elevated expression at the key slender to stumpy transition point. By scRNA-seq analysis of ZC3H20 null parasites exposed to oligopeptides and mapping the resulting transcriptome to our atlas of differentiation, we identified the point of action for this key regulator. Using a developmental transition relevant for both virulence in the mammalian host and disease transmission, our data provide a paradigm for the temporal mapping of differentiation events and regulators in the trypanosome life cycle.

scholarly journals Active VSG Expression Sites in Trypanosoma brucei Are Depleted of Nucleosomes

2009 ◽  
Vol 9 (1) ◽  
pp. 136-147 ◽  
Author(s):  
Tara M. Stanne ◽  
Gloria Rudenko
Keyword(s):  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Histone H3 ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Micrococcal Nuclease ◽  
Pcr Analysis ◽  
Marker Genes ◽  
African Trypanosomes ◽  
Pol I

ABSTRACT African trypanosomes regulate transcription differently from other eukaryotes. Most of the trypanosome genome is constitutively transcribed by RNA polymerase II (Pol II) as large polycistronic transcription units while the genes encoding the major surface proteins are transcribed by RNA polymerase I (Pol I). In bloodstream form Trypanosoma brucei, the gene encoding the variant surface glycoprotein (VSG) coat is expressed in a monoallelic fashion from one of about 15 VSG bloodstream form expression sites (BESs). Little is known about the chromatin structure of the trypanosome genome, and the chromatin state of active versus silent VSG BESs remains controversial. Here, we determined histone H3 occupancy within the genome of T. brucei, focusing on active versus silent VSG BESs in the bloodstream form. We found that histone H3 was most enriched in the nontranscribed 50-bp and 177-bp repeats and relatively depleted in Pol I, II, and III transcription units, with particular depletion over promoter regions. Using two isogenic T. brucei lines containing marker genes in different VSG BESs, we determined that histone H3 is 11- to 40-fold depleted from active VSG BESs compared with silent VSG BESs. Quantitative PCR analysis of fractionated micrococcal nuclease-digested chromatin revealed that the active VSG BES is depleted of nucleosomes. Therefore, in contrast to earlier views, nucleosome positioning appears to be involved in the monoalleleic control of VSG BESs in T. brucei. This may provide a level of epigenetic regulation enabling bloodstream form trypanosomes to efficiently pass on the transcriptional state of active and silent BESs to daughter cells.

scholarly journals Single-cell transcriptomic analysis of bloodstream Trypanosoma brucei reconstructs cell cycle progression and developmental quorum sensing

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Emma M. Briggs ◽  
Federico Rojas ◽  
Richard McCulloch ◽  
Keith R. Matthews ◽  
Thomas D. Otto
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Trypanosoma Brucei ◽  
Cell Cycle Progression ◽  
Expression Patterns ◽  
Tsetse Fly ◽  
Cycle Progression ◽  
Progressive Development ◽  
Asynchronous Development

AbstractDevelopmental steps in the trypanosome life-cycle involve transition between replicative and non-replicative forms specialised for survival in, and transmission between, mammalian and tsetse fly hosts. Here, using oligopeptide-induced differentiation in vitro, we model the progressive development of replicative ‘slender’ to transmissible ‘stumpy’ bloodstream form Trypanosoma brucei and capture the transcriptomes of 8,599 parasites using single cell transcriptomics (scRNA-seq). Using this framework, we detail the relative order of biological events during asynchronous development, profile dynamic gene expression patterns and identify putative regulators. We additionally map the cell cycle of proliferating parasites and position stumpy cell-cycle exit at early G1 before progression to a distinct G0 state. A null mutant for one transiently elevated developmental regulator, ZC3H20 is further analysed by scRNA-seq, identifying its point of failure in the developmental atlas. This approach provides a paradigm for the dissection of differentiation events in parasites, relevant to diverse transitions in pathogen biology.

scholarly journals Developmental competence and antigen switch frequency can be uncoupled in Trypanosoma brucei

2019 ◽  
Vol 116 (45) ◽  
pp. 22774-22782 ◽  
Author(s):  
Kirsty R. McWilliam ◽  
Alasdair Ivens ◽  
Liam J. Morrison ◽  
Monica R. Mugnier ◽  
Keith R. Matthews
Keyword(s):  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Experimental Studies ◽  
Developmental Competence ◽  
Parasite Development ◽  
Mammalian Host ◽  
Mechanical Transmission ◽  
African Trypanosomes ◽  
Infection Dynamic ◽  
Developmental Capacity

African trypanosomes use an extreme form of antigenic variation to evade host immunity, involving the switching of expressed variant surface glycoproteins by a stochastic and parasite-intrinsic process. Parasite development in the mammalian host is another feature of the infection dynamic, with trypanosomes undergoing quorum sensing (QS)-dependent differentiation between proliferative slender forms and arrested, transmissible, stumpy forms. Longstanding experimental studies have suggested that the frequency of antigenic variation and transmissibility may be linked, antigen switching being higher in developmentally competent, fly-transmissible, parasites (“pleomorphs”) than in serially passaged “monomorphic” lines that cannot transmit through flies. Here, we have directly tested this tenet of the infection dynamic by using 2 experimental systems to reduce pleomorphism. Firstly, lines were generated that inducibly lose developmental capacity through RNAi-mediated silencing of the QS signaling machinery (“inducible monomorphs”). Secondly, de novo lines were derived that have lost the capacity for stumpy formation by serial passage (“selected monomorphs”) and analyzed for their antigenic variation in comparison to isogenic preselected populations. Analysis of both inducible and selected monomorphs has established that antigen switch frequency and developmental capacity are independently selected traits. This generates the potential for diverse infection dynamics in different parasite populations where the rate of antigenic switching and transmission competence are uncoupled. Further, this may support the evolution, maintenance, and spread of important trypanosome variants such as Trypanosoma brucei evansi that exploit mechanical transmission.

scholarly journals Identification of cell-type-specific marker genes from co-expression patterns in tissue samples

2021 ◽  
Author(s):  
Yixuan Qiu ◽  
Jiebiao Wang ◽  
Jing Lei ◽  
Kathryn Roeder
Keyword(s):  
Single Cell ◽  
Expression Patterns ◽  
R Package ◽  
Supplementary Information ◽  
Marker Genes ◽  
Specific Marker ◽  
Cell Type ◽  
Correlation Pattern ◽  
Tissue Samples ◽  
Bulk Data

Abstract Motivation Marker genes, defined as genes that are expressed primarily in a single cell type, can be identified from the single cell transcriptome; however, such data are not always available for the many uses of marker genes, such as deconvolution of bulk tissue. Marker genes for a cell type, however, are highly correlated in bulk data, because their expression levels depend primarily on the proportion of that cell type in the samples. Therefore, when many tissue samples are analyzed, it is possible to identify these marker genes from the correlation pattern. Results To capitalize on this pattern, we develop a new algorithm to detect marker genes by combining published information about likely marker genes with bulk transcriptome data in the form of a semi-supervised algorithm. The algorithm then exploits the correlation structure of the bulk data to refine the published marker genes by adding or removing genes from the list. Availability and implementation We implement this method as an R package markerpen, hosted on CRAN (https://CRAN.R-project.org/package=markerpen). Supplementary information Supplementary data are available at Bioinformatics online.

Differentiation of African trypanosomes is controlled by a density sensing mechanism which signals cell cycle arrest via the cAMP pathway

1997 ◽  
Vol 110 (21) ◽  
pp. 2661-2671 ◽  
Author(s):  
E. Vassella ◽  
B. Reuner ◽  
B. Yutzy ◽  
M. Boshart
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Mammalian Host ◽  
Intracellular Camp ◽  
Quiescent State ◽  
Camp Content ◽  
African Trypanosomes ◽  
Sensing Mechanism ◽  
Camp Signalling ◽  
Cycle Arrest

Differentiation of African trypanosomes from replicating slender bloodstream forms to nondividing stumpy forms limits the parasite population size, allowing survival of the mammalian host and establishment of a stable host-parasite relationship. Using a novel in vitro culture system we have shown that slender to stumpy differentiation is induced by parasite density alone and thus is independent of host cues. Here we investigate the density sensing mechanism and show that trypanosomes release a soluble activity of low relative molecular mass, termed stumpy induction factor (SIF), which accumulates in conditioned medium. SIF activity triggers cell cycle arrest in G1/G0 phase and induces differentiation with high efficiency and rapid kinetics. Membrane-permeable derivates of cAMP or the phosphodiesterase inhibitor etazolate perfectly mimic SIF activity. Furthermore, SIF activity elicits an immediate two- to threefold elevation of intracellular cAMP content upon addition to slender forms. We conclude that SIF and hence density sensing operate through the cAMP signalling pathway. Temporal correlation of markers indicates that cell cycle arrest invariably precedes differentiation. Thus, our results indicate that the cell cycle regulation of bloodstream forms is under dominant control of cAMP signalling. Irreversible commitment to the quiescent state is elicited by a cAMP agonist within a period shorter than one complete cell cycle.

scholarly journals Revealing immune responses in the Mycobacterium avium subsp. paratuberculosis-infected THP-1 cells using single cell RNA-sequencing

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254194
Author(s):  
Hong-Tae Park ◽  
Woo Bin Park ◽  
Suji Kim ◽  
Jong-Sung Lim ◽  
Gyoungju Nah ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Single Cell ◽  
Mycobacterium Avium ◽  
Expression Patterns ◽  
Cell Types ◽  
Marker Genes ◽  
Specific Marker ◽  
Cell Type ◽  
Cytokines And Chemokines

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease, which is a chronic and debilitating disease in ruminants. MAP is also considered to be a possible cause of Crohn’s disease in humans. However, few studies have focused on the interactions between MAP and human macrophages to elucidate the pathogenesis of Crohn’s disease. We sought to determine the initial responses of human THP-1 cells against MAP infection using single-cell RNA-seq analysis. Clustering analysis showed that THP-1 cells were divided into seven different clusters in response to phorbol-12-myristate-13-acetate (PMA) treatment. The characteristics of each cluster were investigated by identifying cluster-specific marker genes. From the results, we found that classically differentiated cells express CD14, CD36, and TLR2, and that this cell type showed the most active responses against MAP infection. The responses included the expression of proinflammatory cytokines and chemokines such as CCL4, CCL3, IL1B, IL8, and CCL20. In addition, the Mreg cell type, a novel cell type differentiated from THP-1 cells, was discovered. Thus, it is suggested that different cell types arise even when the same cell line is treated under the same conditions. Overall, analyzing gene expression patterns via scRNA-seq classification allows a more detailed observation of the response to infection by each cell type.

Commitment to differentiation and cell cycle re-entry are coincident but separable events in the transformation of African trypanosomes from their bloodstream to their insect form

1997 ◽  
Vol 110 (20) ◽  
pp. 2609-2618 ◽  
Author(s):  
K.R. Matthews ◽  
K. Gull
Keyword(s):  
Cell Cycle ◽  
Surface Antigen ◽  
Temperature Drop ◽  
Antigen Expression ◽  
Tsetse Fly ◽  
Bloodstream Form ◽  
Cellular Morphology ◽  
African Trypanosomes ◽  
Surface Antigen Expression ◽  
Stumpy Form

African trypanosomes undergo extensive changes in cellular morphology, biochemistry and surface antigen expression as they differentiate from their bloodstream form to those forms that colonise the midgut of their tsetse fly vector. If initiated with stumpy-form cells, a non-dividing sub-type of the bloodstream parasite, differentiation and cell cycle re-entry occur synchronously in the population and provide a means to dissect the respective controls of proliferation and transformation. We have exploited this synchrony to determine the respective importance and hierarchy of the known triggers for differentiation (cis aconitate, temperature drop) for individual components of both differentiation and the cell cycle. This has revealed the pre-eminence of cis aconitate as a primary trigger for parasite differentiation, and has allowed us to determine that the cellular commitment to both differentiation and cell-cycle re-entry are precisely co-incident processes.

scholarly journals Characterizing and inferring quantitative cell cycle phase in single-cell RNA-seq data analysis

2020 ◽  
Vol 30 (4) ◽  
pp. 611-621 ◽  
Author(s):  
Chiaowen Joyce Hsiao ◽  
PoYuan Tung ◽  
John D. Blischak ◽  
Jonathan E. Burnett ◽  
Kenneth A. Barr ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Data Analysis ◽  
Single Cell ◽  
Cell Cycle Phase ◽  
Cycle Phase ◽  
Rna Seq

Escape mechanisms of African trypanosomes: why trypanosomosis is keeping us awake

Parasitology ◽  
2014 ◽  
Vol 142 (3) ◽  
pp. 417-427 ◽  
Author(s):  
JENNIFER CNOPS ◽  
STEFAN MAGEZ ◽  
CARL De TREZ
Keyword(s):  
Immune Response ◽  
Trypanosoma Brucei ◽  
Parasitic Infection ◽  
Mammalian Host ◽  
Tsetse Flies ◽  
African Trypanosomes ◽  
Transmission Potential ◽  
Highly Sensitive ◽  
Commercially Important ◽  
And Control

SUMMARYAfrican trypanosomes have been around for more than 100 million years, and have adapted to survival in a very wide host range. While various indigenous African mammalian host species display a tolerant phenotype towards this parasitic infection, and hence serve as perpetual reservoirs, many commercially important livestock species are highly disease susceptible. When considering humans, they too display a highly sensitive disease progression phenotype for infections withTrypanosoma brucei rhodesienseorTrypanosoma brucei gambiense, while being intrinsically resistant to infections with other trypanosome species. As extracellular trypanosomes proliferate and live freely in the bloodstream and lymphatics, they are constantly exposed to the immune system. Due to co-evolution, this environment however no longer poses a hostile threat, but has become the niche environment where trypanosomes thrive and obligatory await transmission through the bites of tsetse flies or other haematophagic vectors, ideally without causing severe side infection-associated pathology to their host. Hence, African trypanosomes have acquired various mechanisms to manipulate and control the host immune response, evading effective elimination. Despite the extensive research into trypanosomosis over the past 40 years, many aspects of the anti-parasite immune response remain to be solved and no vaccine is currently available. Here we review the recent work on the different escape mechanisms employed by African Trypanosomes to ensure infection chronicity and transmission potential.

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