Plasmodium P-Type Cyclin CYC3 Modulates Endomitotic Growth during Oocyst Development in Mosquitoes

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei

Plasmodium P-type cyclin CYC3 modulates endomitotic growth during oocyst development in mosquitoes

Cell-cycle progression and cell division in eukaryotes are governed in part by the cyclin family and their regulation of cyclin-dependent kinases (CDKs). Cyclins are very well characterised in model systems such as yeast and human cells, but surprisingly little is known about their number and role in Plasmodium, the unicellular protozoan parasite that causes malaria. Malaria parasite cell division and proliferation differs from that of many eukaryotes. During its life cycle it undergoes two types of mitosis: endomitosis in asexual stages and an extremely rapid mitotic process during male gametogenesis. Both schizogony (producing merozoites) in host liver and red blood cells, and sporogony (producing sporozoites) in the mosquito vector, are endomitotic with repeated nuclear replication, without chromosome condensation, before cell division. The role of specific cyclins during Plasmodium cell proliferation was unknown. We show here that the Plasmodium genome contains only three cyclin genes, representing an unusual repertoire of cyclin classes. Expression and reverse genetic analyses of the single Plant (P)-type cyclin, CYC3, in the rodent malaria parasite, Plasmodium berghei, revealed a cytoplasmic and nuclear location of the GFP-tagged protein throughout the lifecycle. Deletion of cyc3 resulted in defects in size, number and growth of oocysts, with abnormalities in budding and sporozoite formation. Furthermore, global transcript analysis of the cyc3-deleted and wild type parasites at gametocyte and ookinete stages identified differentially expressed genes required for signalling, invasion and oocyst development. Collectively these data suggest that cyc3 modulates oocyst endomitotic development in Plasmodium berghei

Public health surveillance in the UK revolutionises our understanding of the invasive Salmonella Typhimurium epidemic in Africa

Background The ST313 sequence type of Salmonella Typhimurium causes invasive non-typhoidal salmonellosis and was thought to be confined to sub-Saharan Africa. Two distinct phylogenetic lineages of African ST313 have been identified. Methods We analysed the whole genome sequences of S. Typhimurium isolates from UK patients that were generated following the introduction of routine whole-genome sequencing (WGS) of Salmonella enterica by Public Health England in 2014. Results We found that 2.7% (84/3147) of S. Typhimurium from patients in England and Wales were ST313 and were associated with gastrointestinal infection. Phylogenetic analysis revealed novel diversity of ST313 that distinguished UK-linked gastrointestinal isolates from African-associated extra-intestinal isolates. The majority of genome degradation of African ST313 lineage 2 was conserved in the UK-ST313, but the African lineages carried a characteristic prophage and antibiotic resistance gene repertoire. These findings suggest that a strong selection pressure exists for certain horizontally acquired genetic elements in the African setting. One UK-isolated lineage 2 strain that probably originated in Kenya carried a chromosomally located blaCTX-M-15, demonstrating the continual evolution of this sequence type in Africa in response to widespread antibiotic usage. Conclusions The discovery of ST313 isolates responsible for gastroenteritis in the UK reveals new diversity in this important sequence type. This study highlights the power of routine WGS by public health agencies to make epidemiologically significant deductions that would be missed by conventional microbiological methods. We speculate that the niche specialisation of sub-Saharan African ST313 lineages is driven in part by the acquisition of accessory genome elements.</p

Two cases of monkeypox imported to the United Kingdom, September 2018

In early September 2018, two cases of monkeypox were reported in the United Kingdom (UK), diagnosed on 7 September in Cornwall (South West England) and 11 September in Blackpool (North West England). The cases were epidemiologically unconnected and had recently travelled to the UK from Nigeria, where monkeypox is currently circulating. We describe the epidemiology and the public health response for the first diagnosed cases outside the African continent since 2003

CYC3-GFP protein expression throughout most stages of the life cycle.

<p>(A) Transcription of <i>cyc1</i>, <i>cyc3</i> and <i>cyc4</i> as analysed by qRT-PCR, normalised against two endogenous control genes, <i>arginine-tRNA synthetase</i> and <i>hsp70</i>. Each bar is the mean of three biological replicates ± SEM. All asexual blood stages: AS; schizonts: Sch; non-activated gametocytes: NAG; activated gametocytes: AG; ookinete: Ook; 14 dpi oocysts/sporozoites: Spor. (B) Expression of CYC3-GFP in trophozoites, schizonts, gametocytes, zygotes, ookinetes, oocysts and sporozoites. 13.1, a cy3-conjugated antibody which recognises P28 on the surface of activated females, zygotes, and ookinetes was used with the sexual stages. Scale bar = 5 μm. (C) Deconvolved 2D projections of live trophozoite, gametocyte, and ookinete expressing CYC3-GFP (green), co-stained with Hoechst 33342 (blue) and cy3-conjugated anti-P28 antibody, 13.1 as a marker for the ookinete surface (red). Scale bar = 5 μm. Line profiles (red) in the black and white images indicate pixel intensity for that channel.</p

Ultrastructure analysis of oocyst development in <i>∆cyc3 mutant</i>.

<p>(A) Low power ultrastructural images of oocyst development at 7–10 days (i, iv, vii) and 14–21 days post-infection (dpi) showing normal sporulation of WT (ii, iii) and certain <i>Δcyc3</i> (v, vi) parasites while other <i>Δcyc3</i> mutants show evidence of cytoplasmic vacuolation (V) and degeneration (vii, viii, ix). N–nucleus. Bars represent 10 μm. (B) Details showing progressive stages in sporozoite formation in wild type parasites (i-iii) and various abnormal developmental stages of the <i>Δcyc3</i> parasite (iv-vi). Bars represent 1 μm. i. Initiation of sporozoite formation with formation of the inner membrane complex (I) beneath the plasmalemma and above a peripherally located nucleus (N) with nuclear pole (NP). ii. Early sporozoite (S) with rhoptry anlagen (R) budding from the surface of the sporoblast. N–nucleus. iii. Late stage in sporozoite formation showing the elongated sporozoites (S). R–rhoptry. iv. Detail of the sporoblast cytoplasm showing nuclei (N) enclosed by abnormal membrane whorls v. Part of a nucleus with extensive nuclear spindle microtubule (Mt) not seen in WT parasite. vi. Detail of a late stage in parasite degeneration showing apoptotic-like nuclei (N) and dilated endoplasmic reticulum (ER).</p

CYC3 is dispensable in asexual and sexual stages but important for oocyst development.

<p>(A) Average number of nuclei per schizont measured using Giemsa stained slides at 100x magnification. Bar is the mean ± SEM. n = 4 independent experiments. (B) Microgametogenesis of <i>∆cyc3</i> compared with WT measured as the number of exflagellation centres per field. Means ± SEM are shown. n = 4 independent experiments. (C) Ookinete conversion as a percentage in <i>∆cyc3</i> and WT lines. Ookinetes were identified using the marker 13.1 and defined as those cells that successfully differentiated into elongated ‘banana shaped’ ookinetes. Bar is the mean ± SEM. n = 5 independent experiments. (D) Total number of GFP-positive oocysts per infected mosquito, including normal and small oocysts, at 5, 7, 10, 14, 21 dpi for <i>∆cyc3</i> and WT lines. Bar is the mean ± SEM. n = 3 independent experiments (20 mosquitoes for each). Example of relative oocyst size and numbers at (E) 10x and (F) 63x magnification in <i>∆cyc3</i> and WT lines. Images show DIC and GFP at 5, 7, 10, 14 and 21 dpi. Scale bar = 100 μm for 10x and 20 μm for 63x. (G) Individual <i>∆cyc3</i> and WT oocyst diameters in μm at 5, 7, 10, 14 and 21 dpi. Horizontal line indicates the mean from 3 independent experiments (20 mosquitoes for each) of <i>∆cyc3</i> and WT. <i>p</i> <0.001 for all time points. (H) Total number of sporozoites per mosquito from 14 and 21 dpi midguts for <i>∆cyc3</i> and WT lines. Three independent experiments are described, n = 20 mosquitoes for each replicate. ** <i>p</i> ≤ 0.01, *** <i>p</i> ≤ 0.001. (I) Genetic complementation of <i>∆cyc3</i>. Mosquitoes were fed with a combination of WT, <i>∆cyc3</i> or <i>∆cyc3</i> with either male (∆<i>p48/45 and ∆hap2</i>) or female (<i>∆dozi and ∆nek4</i>) mutants. Shown is a representation of one experiment (20 mosquitoes per line).</p

Characterisation of the cyclin repertoire of the <i>Apicomplexa</i>.

<p>(A) Maximum likelihood phylogeny based on an alignment of cyclins from <i>Plasmodium falciparum</i>, <i>Toxoplasma gondii</i>, <i>Cryptosporidium parvum</i> and <i>Homo sapiens</i>. CYCP1 and CYCH1 from <i>Arabidopsis thaliana</i> have been included for clarity. Topology support from bootstrapping is shown at nodes. (B) Distribution of cyclin families across <i>Apicomplexa</i>. Presence (filled dot) or absence (empty circle) of specific families of cyclin in each predicted proteome is shown. Dot area is proportional to number of putative proteins. The Group I cyclins in <i>Cryptosporidium</i> cannot be placed reliably into any specific families within the group (“Orphaned”). *The <i>Plasmodium berghei</i> predicted proteome (release 9.3; plasmodb.org/) contains no apparent orthologue of CYC1, as the likely gene encoding the protein on Chromosome 13 (downstream of PBANKA_132730, syntenic with <i>cyc1</i> in other <i>Plasmodium</i> species) is interrupted by a sequence gap.</p

Expression of CYC3-GFP during sporogony in mosquitoes.

<p>Fluorescence microscopy of CYC3-GFP at different time points: 5, 7, 10, 14 and 21 dpi during development in the mosquito. Scale bar = 20 μm. Representative percentage of oocysts that either: do not express GFP (black number), have a low expression of GFP (red number) or have a high expression of GFP (green number).</p

Transcript analysis of genes involved in parasite development.

<p>(A) Ratio-Intensity scatter plots for ∆<i>cyc3</i> activated gametocytes and ookinetes. The y-axis shows the log₂ fold change between wild-type and mutant and the x-axis shows the average of normalised FPKM (See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005273#ppat.1005273.s008" target="_blank">S2 Table</a>). Significantly up-regulated genes are highlighted in green while down-regulated genes are highlighted in red. (B) Heatmaps for invasion, kinase and phosphatase gene clusters based on their log₂ fold change in <i>Δcyc3</i> activated gametocytes (inner track) and <i>Δcyc3</i> ookinetes (outer track) relative to WT. Functional groups were inferred from annotations available in GeneDB (<a href="http://www.genedb.org/" target="_blank">http://www.genedb.org/</a>). Genes that were found significantly misregulated are shown in bold and those validated by qRT-PCR are shown in red. Full gene list and functional clusters are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005273#ppat.1005273.s009" target="_blank">S3 Table</a>. (C) Log₂ fold transcript change in Δ<i>cyc3</i> at different life-cycle stages of cell cycle, signalling and transcription genes, and invasion and oocyst development genes, studied in ∆<i>cyc3</i> (compared against WT) using qRT-PCR. Data were normalised against an endogenous control gene, <i>hsp70</i> (PBANKA_081890). Each bar is the mean of relative expression in comparison to WT from three biological replicates ± SEM. Sch: schizonts; AG: activated gametocytes; Ook: ookinetes; Spor: 14 dpi oocysts/sporozoites. * <i>p</i> ≤0.05; ** <i>p</i> ≤0.01, *** <i>p</i> ≤0.001. (D) Comparison of qRT-PCR and RNA-seq data (Cuffdiff2 analysis) using Log₂ values for activated gametocyte and ookinete samples.</p