Group B Streptococcus Drives Major Transcriptomic Changes in the Colonic Epithelium.

Group B Streptococcus (GBS) is a leading cause of infant sepsis worldwide. Colonization of the gastrointestinal tract is a critical precursor to late-onset disease in exposed newborns. Neonatal susceptibility to GBS intestinal translocation stems from intestinal immaturity; however, the mechanisms by which GBS exploits the immature host remain unclear. ß-hemolysin/cytolysin (ßH/C) is a highly conserved toxin produced by GBS capable of disrupting epithelial barriers. However, its role in the pathogenesis of late-onset GBS disease is unknown. Our aim was to determine the contribution of ßH/C to intestinal colonization and translocation to extraintestinal tissues. Using our established mouse model of late-onset GBS disease, we exposed animals to GBS COH-1 (WT), a ßH/C-deficient mutant (KO), or vehicle control (phosphate-buffered saline [PBS]) via gavage. Blood, spleen, brain, and intestines were harvested 4 days post-exposure for determination of bacterial burden and isolation of intestinal epithelial cells. RNA sequencing was used to examine the transcriptomes of host cells followed by gene ontology enrichment and KEGG pathway analysis. A separate cohort of animals was followed longitudinally to compare colonization kinetics and mortality between WT and KO groups. We demonstrate that dissemination to extraintestinal tissues occurred only in the WT exposed animals. We observed major transcriptomic changes in the colons of colonized animals, but not in the small intestines. We noted differential expression of genes that indicated the role of ßH/C in altering epithelial barrier structure and immune response signaling. Overall, our results demonstrate an important role of ßH/C in the pathogenesis of late-onset GBS disease.

Lineage-Specific Expansion of Plasmodium falciparum Parasites With pfhrp2 Deletion in the Greater Mekong Subregion.

Deletion of the pfhrp2 gene in Plasmodium falciparum can lead to false-negative rapid diagnostic test (RDT) results, constituting a major challenge for evidence-based malaria treatment. Here we analyzed the whole genome sequences of 138 P. falciparum clinical samples collected from the China-Myanmar boarder for pfhrp2 and pfhrp3 gene deletions. We found pfhrp2 and pfhrp3 deletions in 9.4% and 3.6% of samples, respectively, with no samples harboring deletions of both genes. The pfhrp2 deletions showed 2 distinct breakpoints, representing 2 different chromosomal deletion events. A phylogenetic analysis performed using genome-wide single-nucleotide polymorphisms revealed that the 2 pfhrp2 breakpoint groups as well as all the pfhrp3-negative parasites formed separate clades, suggesting they might have resulted from clonal expansion of pfhrp2- and pfhrp3-negative parasites. These findings highlight the need for urgent surveys to determine the prevalence of pfhrp2-negative parasites causing false-negative RDT results and a plan for switching of RDTs pending the survey results.

Light pollution increases West Nile virus competence of a ubiquitous passerine reservoir species.

Among the many anthropogenic changes that impact humans and wildlife, one of the most pervasive but least understood is light pollution. Although detrimental physiological and behavioural effects resulting from exposure to light at night are widely appreciated, the impacts of light pollution on infectious disease risk have not been studied. Here, we demonstrate that artificial light at night (ALAN) extends the infectious-to-vector period of the house sparrow (Passer domesticus), an urban-dwelling avian reservoir host of West Nile virus (WNV). Sparrows exposed to ALAN maintained transmissible viral titres for 2 days longer than controls but did not experience greater WNV-induced mortality during this window. Transcriptionally, ALAN altered the expression of gene regulatory networks including key hubs (OASL, PLBD1 and TRAP1) and effector genes known to affect WNV dissemination (SOCS). Despite mounting anti-viral immune responses earlier, transcriptomic signatures indicated that ALAN-exposed individuals probably experienced pathogen-induced damage and immunopathology, potentially due to evasion of immune effectors. A simple mathematical modelling exercise indicated that ALAN-induced increases of host infectious-to-vector period could increase WNV outbreak potential by approximately 41%. ALAN probably affects other host and vector traits relevant to transmission, and additional research is needed to advise the management of zoonotic diseases in light-polluted areas.

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

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.

Punctuated chromatin states regulate Plasmodium falciparum antigenic variation at the intron and 2 kb upstream regions.

BACKGROUND: Understanding the regulation mechanism of var gene expression is crucial for explaining antigenic variation in Plasmodium falciparum. Recent work observed that while all var genes produce transcripts, only a few var genes exhibit high expression levels. However, the global regulation of var expression and the relationship between epigenetic and genetic control remains to be established. RESULT: We have systematically reanalyzed the existing genomic data including chromatin configurations and gene expressions; and for the first time used robust statistical methods to show that the intron and 2 kb upstream regions of each endogenous var gene always maintain high chromatin accessibility, with high potential to bind transcription factors (TFs). The levels of transcripts for different var gene family members are associated with this chromatin accessibility. Any given var gene thus shows punctuated chromatin states throughout the asexual life cycle. This is demonstrated by three independent transcript datasets. Chromatin accessibility in the var intron and 2 kb upstream regions are also positively correlated with their GC content, suggesting the level of var genes silencing might be encoded in their intron sequences. Interestingly, both var intron and 2 kb upstream regions exhibit higher chromatin accessibility when the genes have relatively lower transcription levels, suggesting a punctuated repressive function for these regulatory elements. CONCLUSION: By integrating and analyzing epigenomic, genomic and transcriptomic data, our work reveals a novel distal element in var control. We found dynamic modulations of specific epigenetic marks around the var intron and distal upstream regions are involved in the general var gene expression patterns in malarial antigenic variation.

Heat-shock responses: systemic and essential ways of malaria parasite survival.

Fever is a part of the human innate immune response that contributes to limiting microbial growth and development in many infectious diseases. For the parasite Plasmodium falciparum, survival of febrile temperatures is crucial for its successful propagation in human populations as well as a fundamental aspect of malaria pathogenesis. This review discusses recent insights into the biological complexity of the malaria parasite's heat-shock response, which involves many cellular compartments and essential metabolic processes to alleviate oxidative stress and accumulation of damaged and unfolded proteins. We highlight the overlap between heat-shock and artemisinin resistance responses, while also explaining how the malaria parasite adapts its fever response to fight artemisinin treatment. Additionally, we discuss how this systemic and essential fight for survival can also contribute to parasite transmission to mosquitoes.

Protein KIC5 is a novel regulator of artemisinin stress response in the malaria parasite Plasmodium falciparum.

Artemisinin combination therapies (ACTs) have led to a significant decrease in Plasmodium falciparum malaria mortality. This progress is now threatened by emerging artemisinin resistance (ART-R) linked originally in SE Asia to polymorphisms in the Kelch propeller protein (K13) and more recently to several other seemingly unrelated genetic mutations. To better understand the parasite response to ART, we are characterizing a P. falciparum mutant with altered sensitivity to ART that was created via piggyBac transposon mutagenesis. The transposon inserted near the putative transcription start site of a gene defined as a "Plasmodium-conserved gene of unknown function," now functionally linked to K13 as the Kelch13 Interacting Candidate 5 protein (KIC5). Phenotype analysis of the KIC5 mutant during intraerythrocytic asexual development identified transcriptional changes associated with DNA stress response and altered mitochondrial metabolism, linking dysregulation of the KIC5 gene to the parasite's ability to respond to ART exposure. Through characterization of the KIC5 transcriptome, we hypothesize that this gene may be essential under ART exposure to manage gene expression of the wild-type stress response at early ring stage, thereby providing a better understanding of the parasite's processes that can alter ART sensitivity.

MalariaSED: a deep learning framework to decipher the regulatory contributions of noncoding variants in malaria parasites.

Malaria remains one of the deadliest infectious diseases. Transcriptional regulation effects of noncoding variants in this unusual genome of malaria parasites remain elusive. We developed a sequence-based, ab initio deep learning framework, MalariaSED, for predicting chromatin profiles in malaria parasites. The MalariaSED performance was validated by published ChIP-qPCR and TF motifs results. Applying MalariaSED to ~ 1.3 million variants shows that geographically differentiated noncoding variants are associated with parasite invasion and drug resistance. Further analysis reveals chromatin accessibility changes at Plasmodium falciparum rings are partly associated with artemisinin resistance. MalariaSED illuminates the potential functional roles of noncoding variants in malaria parasites.

A novel Modulator of Ring Stage Translation (MRST) gene alters artemisinin sensitivity in Plasmodium falciparum.

The implementation of artemisinin (ART) combination therapies (ACTs) has greatly decreased deaths caused by Plasmodium falciparum malaria, but increasing ACT resistance in Southeast Asia and Africa could reverse this progress. Parasite population genetic studies have identified numerous genes, single-nucleotide polymorphisms (SNPs), and transcriptional signatures associated with altered artemisinin activity with SNPs in the Kelch13 (K13) gene being the most well-characterized artemisinin resistance marker. However, there is an increasing evidence that resistance to artemisinin in P. falciparum is not related only to K13 SNPs, prompting the need to characterize other novel genes that can alter ART responses in P. falciparum. In our previous analyses of P. falciparum piggyBac mutants, several genes of unknown function exhibited increased sensitivity to artemisinin that was similar to a mutant of K13. Further analysis of these genes and their gene co-expression networks indicated that the ART sensitivity cluster was functionally linked to DNA replication and repair, stress responses, and maintenance of homeostatic nuclear activity. In this study, we have characterized PF3D7_1136600, another member of the ART sensitivity cluster. Previously annotated as a conserved Plasmodium gene of unknown function, we now provide putative annotation of this gene as a Modulator of Ring Stage Translation (MRST). Our findings reveal that the mutagenesis of MRST affects gene expression of multiple translation-associated pathways during the early ring stage of asexual development via putative ribosome assembly and maturation activity, suggesting an essential role of MRST in protein biosynthesis and another novel mechanism of altering the parasite's ART drug response.IMPORTANCEPlasmodium falciparum malaria killed more than 600,000 people in 2021, though ACTs have been critical in reducing malaria mortality as a first-line treatment for infection. However, ACT resistance in Southeast Asia and emerging resistance in Africa are detrimental to this progress. Mutations to Kelch13 (K13) have been identified to confer increased artemisinin tolerance in field isolates, however, genes other than K13 are implicated in altering how the parasite responds to artemisinin prompts additional analysis. Therefore, in this study we have characterized a P. falciparum mutant clone with altered sensitivity to artemisinin and identified a novel gene (PF3D7_1136600) that is associated with alterations to parasite translational metabolism during critical timepoints for artemisinin drug response. Many genes of the P. falciparum genome remain unannotated, posing a challenge for drug-gene characterizations in the parasite. Therefore, through this study, we have putatively annotated PF3D7_1136600 as a novel MRST gene and have identified a potential link between MRST and parasite stress response mechanisms.

Transcriptomic Analysis of the Acute Skeletal Muscle Effects after Intramuscular DNA Electroporation Reveals Inflammatory Signaling.

Skeletal muscle is a promising tissue for therapeutic gene delivery because it is highly vascularized, accessible, and capable of synthesizing protein for therapies or vaccines. The application of electric pulses (electroporation) enhances plasmid DNA delivery and expression by increasing membrane permeability. Four hours after plasmid electroporation, we evaluated acute gene and protein expression changes in mouse skeletal muscle to identify regulated genes and genetic pathways. RNA sequencing followed by functional annotation was used to evaluate differentially expressed mRNAs. Our data highlighted immune signaling pathways that may influence the effectiveness of DNA electroporation. Cytokine and chemokine protein levels in muscle lysates revealed the upregulation of a subset of inflammatory proteins and confirmed the RNA sequencing analysis. Several regulated DNA-specific pattern recognition receptor mRNAs were also detected. Identifying unique molecular changes in the muscle will facilitate a better understanding of the underlying molecular mechanisms and the development of safety biomarkers and novel strategies to improve skeletal muscle targeted gene therapy.

IKACh is constitutively active via PKC epsilon in aging mediated atrial fibrillation.

Atrial fibrillation (AF), the most common abnormal heart rhythm, is a major cause for stroke. Aging is a significant risk factor for AF; however, specific ionic pathways that can elucidate how aging leads to AF remain elusive. We used young and old wild-type and PKC epsilon- (PKCϵ) knockout mice, whole animal, and cellular electrophysiology, as well as whole heart, and cellular imaging to investigate how aging leads to the aberrant functioning of a potassium current, and consequently to AF facilitation. Our experiments showed that knocking out PKCϵ abrogates the effects of aging on AF by preventing the development of a constitutively active acetylcholine sensitive inward rectifier potassium current (IKACh). Moreover, blocking this abnormal current in the old heart reduces AF inducibility. Our studies demonstrate that in the aging heart, IKACh is constitutively active in a PKCϵ-dependent manner, contributing to the perpetuation of AF.

A Pilot Study To Establish an In Vitro Model To Study Premature Intestinal Epithelium and Gut Microbiota Interactions.

Intestinal microbiota has emerged as an important player in the health and disease of preterm infants. The interactions between intestinal flora and epithelium can lead to local injury and systemic diseases. A suitable in vitro cell model is needed to enhance our understanding of these interactions. In this study, we exposed fetal epithelial cell cultures (FHs-74 int cells, human, ATCC CCL 241) to sterile fecal filtrates derived from stool collected from preterm infants at <2 and at 3 to 4 weeks of age. We measured the cytokine levels from the culture media after 4, 24, and 48 h of exposure to the fecal filtrates. We analyzed the 16S rRNA V4 gene data of the fecal samples and transcriptome sequencing (RNA-seq) data from the fetal epithelial cells after 48 h of exposure to the same fecal filtrates. The results showed correlations between inflammatory responses (both cytokine levels and gene expression) and the Proteobacteria-to-Firmicutes ratio and between fecal bacterial genera and epithelial apoptosis-related genes. Our in vitro cell model can be further developed and applied to study how the epithelium responds to different microbial flora from preterm infants. Combining immature epithelial cells and preterm infant stool samples into one model allows us to investigate disease processes in preterm infants in a way that had not been previously reported. IMPORTANCE The gut bacterial flora influences the development of the immune system and long-term health outcomes in preterm infants. Studies of the mechanistic interactions between the gut bacteria and mucosal barrier are limited to clinical observations, animal models, and in vitro cell culture models for this vulnerable population. Most in vitro cell culture models of microbe-host interactions use single organisms or adult origin cell lines. Our study is innovative and significant in that we expose immature epithelial cells derived from fetal tissues to fecal filtrates from eight stool samples from four preterm infants to study the role of intestinal epithelial cells. In addition, we analyzed epithelial gene expression to examine multiple cellular processes simultaneously. This model can be developed into patient-derived two- or three-dimensional cell cultures exposed to their own fecal material to allow better prediction of patient physiological responses to support the growing field of precision medicine.

The human malaria parasite genome is configured into thousands of coexpressed linear regulatory units.

The human malaria parasite Plasmodium falciparum thrives in radically different host environments in mosquitoes and humans, with only a limited set of transcription factors. The nature of regulatory elements or their target genes in the P. falciparum genome remains elusive. Here, we found that this eukaryotic parasite uses an efficient way to maximally use genetic and epigenetic regulation to form regulatory units (RUs) during blood infections. Genes located in the same RU tend to have the same pattern of expression over time and are associated with open chromatin along regulatory elements. To precisely define and quantify these RUs, a novel hidden Markov model was developed to capture the regulatory structure in a genome-wide fashion by integrating expression and epigenetic evidence. We successfully identified thousands of RUs and cross-validated with previous findings. We found more genes involved in red blood cell (RBC) invasion located in the same RU as the PfAP2-I (AP2-I) transcription factor, demonstrating that AP2-I is responsible for regulating RBC invasion. Our study has provided a regulatory mechanism for a compact eukaryotic genome and offers new insights into the in vivo transcriptional regulation of the P. falciparum intraerythrocytic stage.

Iron Hack - A symposium/hackathon focused on porphyrias, Friedreich's ataxia, and other rare iron-related diseases.

Background: Basic and clinical scientific research at the University of South Florida (USF) have intersected to support a multi-faceted approach around a common focus on rare iron-related diseases. We proposed a modified version of the National Center for Biotechnology Information's (NCBI) Hackathon-model to take full advantage of local expertise in building "Iron Hack", a rare disease-focused hackathon. As the collaborative, problem-solving nature of hackathons tends to attract participants of highly-diverse backgrounds, organizers facilitated a symposium on rare iron-related diseases, specifically porphyrias and Friedreich's ataxia, pitched at general audiences. Methods: The hackathon was structured to begin each day with presentations by expert clinicians, genetic counselors, researchers focused on molecular and cellular biology, public health/global health, genetics/genomics, computational biology, bioinformatics, biomolecular science, bioengineering, and computer science, as well as guest speakers from the American Porphyria Foundation (APF) and Friedreich's Ataxia Research Alliance (FARA) to inform participants as to the human impact of these diseases. Results: As a result of this hackathon, we developed resources that are relevant not only to these specific disease-models, but also to other rare diseases and general bioinformatics problems. Within two and a half days, "Iron Hack" participants successfully built collaborative projects to visualize data, build databases, improve rare disease diagnosis, and study rare-disease inheritance. Conclusions: The purpose of this manuscript is to demonstrate the utility of a hackathon model to generate prototypes of generalizable tools for a given disease and train clinicians and data scientists to interact more effectively.