Predicting Functional and Regulatory Divergence of a Drug Resistance Transporter Gene in the Human Malaria Parasite

Background: The paradigm of resistance evolution to chemotherapeutic agents is that a key coding mutation in a specific gene drives resistance to a particular drug. In the case of resistance to the anti-malarial drug chloroquine (CQ), a specific mutation in the transporter pfcrt is associated with resistance. Here, we apply a series of analytical steps to gene expression data from our lab and leverage 3 independent datasets to identify pfcrt-interacting genes. Resulting networks provide insights into pfcrt’s biological functions and regulation, as well as the divergent phenotypic effects of its allelic variants in different genetic backgrounds. Results: To identify pfcrt-interacting genes, we analyze pfcrt co-expression networks in 2 phenotypic states - CQ-resistant (CQR) and CQ-sensitive (CQS) recombinant progeny clones - using a computational approach that prioritizes gene interactions into functional and regulatory relationships. For both phenotypic states, pfcrt co-expressed gene sets are associated with hemoglobin metabolism, consistent with CQ’s expected mode of action. To predict the drivers of co-expression divergence, we integrate topological relationships in the co-expression networks with available high confidence protein-protein interaction data. This analysis identifies 3 transcriptional regulators from the ApiAP2 family and histone acetylation as potential mediators of these divergences. We validate the predicted divergences in DNA mismatch repair and histone acetylation by measuring the effects of small molecule inhibitors in recombinant progeny clones combined with quantitative trait locus (QTL) mapping. Conclusions: This work demonstrates the utility of differential co-expression viewed in a network framework to uncover functional and regulatory divergence in phenotypically distinct parasites. pfcrt-associated co-expression in the CQ resistant progeny highlights CQR-specific gene relationships and possible targeted intervention strategies. The approaches outlined here can be readily generalized to other parasite populations and drug resistances

MMWR. Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports / Centers for Disease Control

This revision of the General Recommendations on Immunization updates the 1989 statement. Changes in the immunization schedule for infants and children include recommendations that the third dose of oral polio vaccine be administered routinely at 6 months of age rather than at age 15 months and that measles-mumps-rubella vaccine be administered routinely to all children at 12-15 months of age. Other updated or new sections include a) a listing of vaccines and other immunobiologics available in the United States by type and recommended routes, advice on the proper storage and handling of immunobiologics, a section on the recommended routes for administration of vaccines, and discussion of the use of jet injectors; b) revisions in the guidelines for spacing administration of immune globulin preparations and live virus vaccines, a discussion of vaccine interactions and recommendations for the simultaneous administration of multiple vaccines, a section on the interchangeability of vaccines from different manufacturers, and a discussion of hypersensitivity to vaccine components; c) a discussion of vaccination during pregnancy, a section on breast-feeding and vaccination, recommendations for the vaccination of premature infants, and updated schedules for immunizing infants and children (including recommendations for the use of Haemophilus influenzae type b conjugate vaccines); d) sections on the immunization of hemophiliacs and immunocompromised persons; e) discussion of the Standards for Pediatric Immunization Practices (including a new table of contraindications and precautions to vaccination), information on the National Vaccine Injury Compensation Program, the Vaccine Adverse Events Reporting System, and Vaccine Information Pamphlets; and f) guidelines for vaccinating persons without documentation of immunization, a section on vaccinations received outside the United States, and a section on reporting of vaccine-preventable diseases. These recommendations are based on information available before publishing and are not comprehensive for each vaccine. The most recent Advisory Committee on Immunization Practices (ACIP) recommendations for each specific vaccine should be consulted for more details.INFECTIOUS DISEASEPrevention and controlSUPERSEDEDACI

Altered expression of K13 disrupts DNA replication and repair in Plasmodium falciparum

Abstract Background Plasmodium falciparum exhibits resistance to the artemisinin component of the frontline antimalarial treatment Artemisinin-based Combination Therapy in South East Asia. Millions of lives will be at risk if artemisinin resistance (ART-R) spreads to Africa. Single non-synonymous mutations in the propeller region of PF3D7_1343700,“K13” are implicated in resistance. In this work, we use transcriptional profiling to characterize a laboratory-generated k13 insertional mutant previously demonstrated to have increased sensitivity to artemisinins to explore the functional role of k13. Results A set of RNA-seq and microarray experiments confirmed that the expression profile of k13 is specifically altered during the early ring and early trophozoite stages of the mutant intraerythrocytic development cycle. The down-regulation of k13 transcripts in this mutant during the early ring stage is associated with a transcriptome advance towards a more trophozoite-like state. To discover the specific downstream effect of k13 dysregulation, we developed a new computational method to search for differential gene expression while accounting for the temporal sequence of transcription. We found that the strongest biological signature of the transcriptome shift is an up-regulation of DNA replication and repair genes during the early ring developmental stage and a down-regulation of DNA replication and repair genes during the early trophozoite stage; by contrast, the expressions of housekeeping genes are unchanged. This effect, due to k13 dysregulation, is antagonistic, such that k13 levels are negatively correlated with DNA replication and repair gene expression. Conclusion Our results support a role for k13 as a stress response regulator consistent with the hypothesis that artemisinins mode of action is oxidative stress and k13 as a functional homolog of Keap1 which in humans regulates DNA replication and repair genes in response to oxidative stress

Simultaneous genome-wide gene expression and transcript isoform profiling in the human malaria parasite

<div><p>Gene expression DNA microarrays have been vital for characterizing whole-genome transcriptional profiles. Nevertheless, their effectiveness relies heavily on the accuracy of genome sequences, the annotation of gene structures, and the sequence-dependent performance of individual probes. Currently available gene expression arrays for the malaria parasite <i>Plasmodium falciparum</i> rely on an average of 2 probes per gene, usually positioned near the 3′ end of genes; consequently, existing designs are prone to measurement bias and cannot capture complexities such as the occurrence of transcript isoforms arising from alternative splicing or alternative start/ stop sites. Here, we describe two novel gene expression arrays with exon-focused probes designed with an average of 12 and 20 probes spanning each gene. This high probe density minimizes signal noise inherent in probe-to-probe sequence-dependent hybridization intensity. We demonstrate that these exon arrays accurately profile genome-wide expression, comparing favorably to currently available arrays and RNA-seq profiling, and can detect alternatively spliced transcript isoforms as well as non-coding RNAs (ncRNAs). Of the 964 candidate alternate splicing events from published RNA-seq studies, 162 are confirmed using the exon array. Furthermore, the exon array predicted 330 previously unidentified alternate splicing events. Gene expression microarrays continue to offer a cost-effective alternative to RNA-seq for the simultaneous monitoring of gene expression and alternative splicing events. Microarrays may even be preferred in some cases due to their affordability and the rapid turn-around of results when hundreds of samples are required for fine-scale systems biology investigations, including the monitoring of the networks of gene co-expression in the emergence of drug resistance.</p></div

Biological replicates show high reproducibility on the exon arrays.

<p>Spearman correlations of biological replicates show highly reproducible signal for 12 hpi samples hybridized to the (A) Nimblegen and (B) Agilent HD exon arrays. (C) Hierarchical clustering of correlation values across all samples hybridized to the high-density exon arrays show reproducibility of signal between chip platforms across the intra-erythrocytic lifecycle.</p

Differentially expressed genes are replicated with high concordance on the exon arrays.

<p>Log₂ ratios of 12 hpi vs. 36 hpi samples were calculated to determine differentially expressed genes. The correlations between log₂ ratios on the Nimblegen and Agilent HD exon (0.905, A and C) and Agilent 15K arrays (0.873, B and D) show that biologically relevant information is replicated across array platforms. The mean differential expression values are comparable between chip platforms, though both Agilent arrays have an overall larger dynamic range (C and D). The correlation between the Agilent 15K and Agilent HD exon array is 0.676 (E). Different biological samples were hybridized to these two arrays and the correlation value matches that of these two biological replicates when hybridized on the same platform (F, 0.692) suggesting that the variation seen is due to differences in the samples.</p

Gene set enrichment analysis (GSEA) of up-regulated genes between exon arrays and previously published 15K agilent array show high reproducibility of biological information.

<p>GSEA plots for chip to chip comparisons of unregulated genes for HB3 12 hpi vs 36 hpi samples. Black lines indicate the location of “hits” from the rank order list of the up or down-regulated genes from the query list. Correlation values between ranked ordered and query list is progressively calculated and indicated by the red bar (with stronger correlations indicated by darker hue). The “zero crosses at” dotted line indicates when the enrichment score decays back to zero and there are no more potential matches between the query and rank ordered lists. (A and C) Comparing the top up-regulated and down-regulated genes on the Agilent 15K array to the rank ordered list of all genes on the Nimblegen exon array. (B and D) Comparing the top up-regulated and down-regulated genes on the Agilent HD exon array to the rank ordered differential expression of all genes on the comparably designed Nimblegen exon array. These comparisons show that the top up-regulated genes between 12 hpi and 36 hpi are consistent across all 3 platforms.</p

Exon array designs.

<p>Exon array designs.</p

Exon arrays can detect exon skipping transcript isoforms.

<p>Comparison of genes with transcript isoforms (A) Differential exon splicing is detected on both Nimblegen and Agilent HD exon array platforms using splicing index with a correlation of 0.626. (B) Venn Diagram showing the overlap of genes with transcript isoforms reported by the Agilent HD Exon Array (blue) and RNA-seq studies (Otto (yellow), Sorber (orange), Broadbent (green), and Lopez-Barragen (purple) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187595#pone.0187595.ref024" target="_blank">24</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187595#pone.0187595.ref025" target="_blank">25</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187595#pone.0187595.ref036" target="_blank">36</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187595#pone.0187595.ref037" target="_blank">37</a>]), (C) Alternative start sites for PF3D7_0929200 produce time point dependent exon skipping of exons 1 and 2 during the schizont parasite stage at 36 and 48 hpi.</p