Biology subjects

Alam, M.

Publications and source records attributed to Alam, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2020

Temporal Shifts in Antibiotic Resistance Elements Govern Virus-Pathogen Conflicts

Bacteriophage predation selects for diverse anti-phage systems that frequently cluster on mobilizable defense islands in bacterial genomes. However, there remains a lack of molecular insight into the reciprocal dynamics of phage-bacterial adaptations in nature, particularly in clinical contexts where there is need to inform phage therapy efforts and understand how phages drive pathogen evolution. Here, using time-shift experiments we show that fluctuations in SXT integrative and conjugative elements (ICEs), which notoriously confer antibiotic resistance, govern Vibrio choleraes susceptibility to phages in clinical samples. We find that SXT ICEs, which are widespread in Gammaproteobacteria, invariably encode phage defense and function to protect other genera from phage attack following conjugation. We discover phage counter-adaptation to SXT-mediated restriction in clinical samples, and show that heterogeneity in SXT ICEs allows for re-emergence of phage resistance. Further, phage infection stimulates high frequency SXT ICE conjugation, leading to the concurrent dissemination of phage and antibiotic resistance.

microbiology↗

bioRxiv2020

Megasphaera in the stool microbiota is negatively associated with diarrheal cryptosporidiosis

BackgroundThe protozoan parasites in the Cryptosporidium genus cause both acute diarrheal disease and subclinical (i.e. non-diarrheal) disease. It is unclear if the microbiota can influence the manifestation of diarrhea during a Cryptosporidium infection. MethodsTo characterize the role of the gut microbiota in diarrheal cryptosporidiosis, the microbiome composition of both diarrheal and surveillance Cryptosporidium-positive fecal samples was evaluated using 16S rRNA gene sequencing. Additionally, the microbiome composition prior to infection was examined to test whether a preexisting microbiome profile could influence the Cryptosporidium infection phenotype. ResultsFecal microbiome composition was associated with diarrheal symptoms at two timepoints. Megasphaera was significantly less abundant in diarrheal samples when compared to subclinical samples at the time of Cryptosporidium detection (log2(fold change) = -4.3, p=10-10) and prior to infection (log2(fold change) = -2.0, p=10-4). Random forest classification also identified Megasphaera abundance in the pre- and post-exposure microbiota.as predictive of a subclinical infection. ConclusionsMicrobiome composition broadly, and specifically low Megasphaera abundance, was associated with diarrheal symptoms prior to and at the time of Cryptosporidium detection. This observation suggests that the gut microenvironment may play a role in determining the severity of a Cryptosporidium infection. SummaryMegasphaera abundance in the stool of Bangladeshi infants is associated with the development of diarrhea upon infection with the Cryptosporidium parasite.

microbiology↗

bioRxiv2020

Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

Missense mutations in p53 are severely deleterious and occur in over 50% of all human cancers. The vast majority of these mutations are located in the inherently unstable DNA-binding domain (DBD), many of which destabilize the domain further and expose its aggregation-prone hydrophobic core, prompting self-assembly of mutant p53 into inactive cytosolic amyloid-like aggregates. Screening an oligopyridylamide library, previously shown to inhibit amyloid formation associated with Alzheimers disease and type II diabetes, identified a tripyridylamide, ADH-6, that potently abrogates self-assembly of the aggregation-nucleating subdomain of mutant p53 DBD. Moreover, ADH-6 effectively targets and dissociates mutant p53 aggregates in human cancer cells, which restores p53s transcriptional activity, leading to cell cycle arrest and apoptosis. Notably, ADH-6 treatment substantially shrinks xenografts harboring mutant p53 and prolongs survival, while exhibiting no toxicity to healthy tissue. This study demonstrates the first successful application of a bona fide small-molecule amyloid inhibitor as an anticancer agent.

cancer biology↗

bioRxiv2020

Cold sensitivity of the SARS-CoV-2 spike ectodomain

The SARS-CoV-2 spike (S) protein, a primary target for COVID-19 vaccine development, presents its Receptor Binding Domain in two conformations: receptor-accessible "up" or receptor-inaccessible "down" conformations. Here, we report that the commonly used stabilized S ectodomain construct "2P" is sensitive to cold temperature, and that this cold sensitivity is resolved in a "down" state stabilized spike. Our results will impact structural, functional and vaccine studies that use the SARS-CoV-2 S ectodomain.

biochemistry↗

bioRxiv2020

Fab-dimerized glycan-reactive antibodies neutralize HIV and are prevalent in humans and rhesus macaques

SummaryThe HIV-1 envelope (Env) is comprised by mass of over 50% glycans. A goal of HIV-1 vaccine development is the induction of Env glycan-reactive broadly neutralizing antibodies (bnAbs). The 2G12 bnAb recognizes an Env glycan cluster using a unique variable heavy (VH) domain-swapped conformation that results in fragment antigen-binding (Fab) dimerization. Here we describe Fab-dimerized glycan (FDG)-reactive antibodies without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques that neutralized heterologous HIV-1 isolates. FDG precursors were boosted by vaccination in macaques, and were present in HIV-1-naïve humans with an average estimated frequency of one per 340,000 B cells. These data demonstrate frequent HIV-1 Env glycan-reactive bnAb B cell precursors in macaques and humans and reveal a novel strategy for their induction by vaccination.HighlightsDiscovery of Fab-dimerized HIV-1 glycan-reactive antibodies with a non-domain-swapped architectureFab-dimerized antibodies neutralize heterologous HIV-1 isolates.Antibodies with this architecture can be elicited by vaccination in macaques.Fab-dimerized antibodies are found in HIV-1 naïve humans.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

bioRxiv2020

Bioinformatic analysis based genome-wide identification, characterization, diversification and regulatory transcription components of RNA silencing machinery genes in wheat (Triticum aestivum L.)

Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) gene families are known as RNA silencing machinery genes or RNAi genes. They have important activities at post-transcriptional and chromatin modification levels. They regulate gene expression relating to different stresses, growth, and development in eukaryotes. A complete cycle of gene silencing is occurred by the collaboration of these three families. However, these gene families are not yet rigorously studied in the economically important wheat genome. Our bioinformatic analysis based genome-wide identification, characterization, diversification and regulatory components of these gene families identified 7 TaDCL, 39 TaAGO and 16 TaRDR genes from wheat genome against RNAi genes of Arabidopsis thaliana. Phylogenetic analysis of wheat genome with Arabidopsis and rice RNAi genes showed that TaDCL, TaAGO and TaRDR proteins are clustered into four, eight and four subgroups respectively. Domain, motif and exon-intron structure analyses showed that the TaDCL, TaAGO and TaRDR proteins conserve identical characteristics within groups while retain diverse differences between groups. GO annotations implied that a number of biological and molecular pathways are linked to RNAi mechanism in wheat. Gene networking between transcription factors and RNAi proteins indicates that ERF is the leading family linked to maximum RNAi genes followed by MIKC-MADS, C2H2, BBR-BPC, MYB, and Dof. Cis-regulatory elements associated to RNAi genes are predicted to act as regulatory components against various environmental conditions. Expressed sequence tag analysis showed that larger numbers of RNAi genes are expressed in different tissues and organs predicted to play roles for healthy plants and grains. Expression analysis of 7 TaDCL genes using qRT-PCR showed that only TaDCL3a and TaDCL3b had root specific significant expression (p-value<0.05) with no expression in leaf validated EST results. Besides, TaDCL3b and TaDCL4 significantly prompted in drought condition indicating their potential role in drought stress tolerance. Overall results would however help researchers for in-depth biological investigation of these RNAi genes in wheat crop improvement.

genomics↗

bioRxiv2020

Single-cell transcriptomic profile reveals macrophage heterogeneity in medulloblastoma and their treatment-dependent recruitment

The role of macrophages in medulloblastoma, the most common malignant pediatric brain tumor, is unclear. Using single-cell RNA sequencing in a mouse model of sonic hedgehog medulloblastoma and analysis of bulk RNA sequencing of human medulloblastoma, we investigated macrophage heterogeneity. Our findings reveal differential recruitment of macrophages with molecular-targeted versus radiation therapy and identify an immunosuppressive monocyte-derived macrophages following radiation treatment of mouse medulloblastoma, uncovering potential strategies for immunomodulation as adjunctive therapy.

cancer biology↗

bioRxiv2020

A Vibrio Cholerae Core Genome Multilocus Sequence Typing Scheme to Facilitate the Epidemiological Study of Cholera

Core genome multilocus sequence typing (cgMLST) has gained popularity in recent years in epidemiological research and subspecies level classification. cgMLST retains the intuitive nature of traditional MLST but offers much greater resolution by utilizing significantly larger portions of the genome. Here, we introduce a cgMLST scheme for Vibrio cholerae, a bacterium abundant in marine and freshwater environments and the etiologic agent of cholera. A set of 2,443 core genes ubiquitous in V. cholerae were used to analyze a comprehensive dataset of 1,262 clinical and environmental strains collected from 52 countries, including 65 newly sequenced genomes in this study. We established a sublineage threshold based on 133 allelic differences that creates clusters nearly identical to traditional MLST types, providing backwards compatibility to new cgMLST classifications. We also defined an outbreak threshold based on seven allelic differences that is capable of identifying strains from the same outbreak and closely related isolates which could give clues on outbreak origin. Using cgMLST, we confirmed the South Asian origin of modern epidemics and identified clustering affinity among sublineages of environmental isolates from the same geographic origin. Advantages of this method are highlighted by direct comparison with existing classification methods, such as MLST and single nucleotide polymorphism-based methods. cgMLST outperforms all existing methods in terms of resolution, standardization, and ease-of-use. We anticipate this scheme will serve as a basis for a universally applicable and standardized classification system for V. cholerae research and epidemiological surveillance in the future. This cgMLST scheme is publicly available on PubMLST (https://pubmlst.org/vcholerae/). IMPORTANCEToxigenic Vibrio cholerae of the O1 and O139 serogroups are the causative agent of cholera, an acute diarrheal disease that plagued the world for centuries, if not millennia. Here, we introduce a core genome multilocus sequence typing (cgMLST) scheme for V. cholerae. Using cgMLST, we established an outbreak threshold that can efficiently identify outbreak related strains and potential sources of introduction. We also defined a sublineage threshold that is similar to traditional MLST sequence type which will provide context to this new typing method by relating it to previous MLST results. cgMLST outperforms all existing methods in terms of resolution, standardization, and ease-of-use, making this scheme the most suitable method for V. cholerae typing and surveillance worldwide.

microbiology↗

bioRxiv2019

Simultaneous quantification of Vibrio metoecus and Vibrio cholerae with its O1 serogroup and toxigenic subpopulations in environmental reservoirs

Vibrio metoecus is a recently described and little studied causative agent of opportunistic infections in humans, often coexisting with V. cholerae in aquatic environments. However, the relative abundance of V. metoecus with V. cholerae and their population dynamics in aquatic reservoirs is still unknown. We developed a multiplex qPCR assay with a limit of detection of three copies per reaction to simultaneously quantify total V. metoecus and V. cholerae abundance, as well as the toxigenic and O1 serogroup subpopulations of V. cholerae from environmental samples. Four different genes were targeted as specific markers for individual Vibrio species or subpopulations; viuB, a gene encoding a vibriobactin utilization protein, was used to quantify the total V. cholerae population. The cholera toxin gene ctxA provided an estimation of toxigenic V. cholerae abundance, while the rfbO1 gene specifically detected and quantified V. cholerae belonging to the O1 serogroup, which includes almost all lineages of the species responsible for the majority of past and ongoing cholera pandemics. To measure V. metoecus abundance, the gene mcp, encoding methyl accepting chemotaxis protein, was used. Marker specificity was confirmed by testing several isolates of V. cholerae and V. metoecus alongside negative controls of isolates within and outside of the Vibrio genus. Analysis of environmental water samples collected from four different geographic locations including cholera-endemic (Dhaka, Kuakata and Mathbaria in Bangladesh) and non-endemic (Oyster Pond in Falmouth, Massachusetts, USA) regions showed that V. metoecus was only present in the USA site, recurring seasonally. Within the coastal USA site, the non-toxigenic O1 serogroup represented up to [~]18% of the total V. cholerae population. V. cholerae toxigenic O1 serogroup was absent or present in low abundance in coastal Bangladesh (Kuakata and Mathbaria) but constituted a relatively high proportion of the total V. cholerae population sustained throughout the year in inland Bangladesh (Dhaka). A preference for host/particle attachment was observed, as the majority of cells from both Vibrio species (>90%) were identified in the largest water size fraction sampled, composed of particles or organisms >63 m and their attached bacteria. This is the first study to apply a culture-independent method to quantify V. cholerae or V. metoecus directly in environmental reservoirs of areas endemic and non-endemic for cholera on significant temporal and spatial scales.\n\nSIGNIFICANCECholera is a life-threatening disease that requires immediate intervention; it is of prime importance to have fast, accurate and sensitive means to detect V. cholerae. Consistent environmental monitoring of the abundance of V. cholerae along with its toxigenic and O1 serogroup subpopulations could facilitate the determination of the actual distribution of this organism in aquatic reservoirs and thus help to predict an outbreak before it strikes. The lack of substantial temporal and spatial environmental sampling, along with specific quantitative measures, has made this goal elusive so far. The same is true for V. metoecus, a close relative of V. cholerae which has been associated with several clinical infections and could likely pose an emerging threat, readily exchanging genetic material with its more famous relative.

microbiology↗

bioRxiv2019

Mammary epithelial cells have lineage-restricted metabolic identities

Cancer metabolism adapts the metabolic network of its tissue-of-origin. However, breast cancer is not a disease of a singular origin. Multiple epithelial populations serve as the culprit cell-of-origin for specific breast cancer subtypes, yet knowledge surrounding the metabolic network of normal mammary epithelial cells is limited. Here, we show that mammary populations have cell type-specific metabolic programs. Primary human breast cell proteomes of basal, luminal progenitor, and mature luminal populations revealed their unique enrichment of metabolic proteins. Luminal progenitors had higher abundance of electron transport chain subunits and capacity for oxidative phosphorylation, whereas basal cells were more glycolytic. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed distinct metabolic vulnerabilities of the mammary lineages. Computational analysis indicated that breast cancer subtypes retain metabolic features of their putative cell-of-origin. Lineage-restricted metabolic identities of normal mammary cells partly explain breast cancer metabolic heterogeneity and rationalize targeting subtype-specific metabolic vulnerabilities to advance breast cancer therapy.

cell biology↗

bioRxiv2019

Electrospun cellulose acetate/gelatin nanofibrous wound dressing containing berberine for diabetic foot ulcer healing: in vitro and in vivo studies

Functional dressing with tailored physicochemical and biological properties is vital for diabetic foot ulcer (DFU) treatment. Our main objective in the current study was to fabricate Cellulose Acetate/Gelatin (CA/Gel) electrospun nanofibrous mat loaded with berberine (Beri) as the DFU dressing. The results demonstrated that the diameter of the nanofibers was around 502 nm, the tensile strength, contact angle, porosity, water vapor permeability, and water uptake ratio of CA/Gel nanofibers were around 2.83 MPa, 58.07, 78.17 %, 11.23 mg/cm2 hr, and 12.78 respectively, while these values for CA/Gel/Beri nanofibers were 2.69 {+/-} 0.05 MPa, 56.93 {+/-} 1, 76.17 {+/-} 0.76 %, 10.17 {+/-} 0.21 mg/cm2 hr, 14.37 {+/-} 0.42 respectively. The bacterial evaluations demonstrated that the dressings are an excellent barrier against bacterial penetration with potent antibacterial activity. The animal studies depicted that the collagen density and angiogenesis score in the CA/Gel/Beri treated group were 88.8{+/-}6.7 % and 19.8{+/-}3.8, respectively. These findings implied that the incorporation of berberine did not compromise the physical properties of dressing, while improving the biological activates. In conclusion, our findings implied that the prepared mat is a proper wound dressing for DFU management and treatment.

bioengineering↗

bioRxiv2019

Neuromodulation of pelvic nerve during early phase of spinal cord injury in rats using implantable prototype devices - a preliminary study.

ObjectivesThe bladder becomes retentive during the early phase of spinal cord injury, and requires proper bladder management to prevent damage to the lower urinary tract and kidney. We investigated the effects of on-demand pelvic nerve stimulation on the areflexive bladder during the earliest phase of complete spinal cord injury in rats and the use of pelvic nerve signals as a proxy to estimate intravesical pressure for closed-loop applications.\n\nMaterials and MethodsIn order to stimulate the pelvic nerves in female Sprague-Dawley rats with complete spinal cord transection (T7 level), a flexible electrode was implanted unilaterally on pelvic nerve, and electrical stimulation was provided by a custom-built nerve stimulator. Stimulation-evoked voiding was monitored in the awake state while size, capacity and spontaneous contractions of the bladder were analysed under anaesthesia. Separately, recordings of the pelvic nerve signals, external urethral sphincter activity and intravesical pressure were performed in animals with intact and transected spinal cord under anaesthesia.\n\nResultsSuccessful pelvic nerve stimulation enabled more frequent voiding, reduced overdistension of bladder, and preserved non-voiding spontaneous bladder contractions. Typical bladder management protocol for SCI rats (manual expression every 8 - 12 hours) resulted in more severe bladder overdistention. Signal processing of the recorded extraneural pelvic nerve signals successfully reconstructed changes in intravesical pressure, demonstrating their use in estimating the fullness and contractions of the bladder.\n\nConclusionsThe preliminary results suggest that pelvic nerve stimulators can serve as an alternative method for frequent emptying of the areflexive bladder. Simultaneous recording of the same pelvic nerve will be useful for development of a closed-loop neuroprosthesis.

neuroscience↗

bioRxiv2019

Metabolically-active obligate aerobes in anoxic (sulfidic) marine sediments

Metabolically-active obligate aerobes are unheard-of in tightly-anoxic environments. Present culture-independent and culture-dependent investigations revealed aerobic microbial communities along two, ~3-meter-long sediment-cores underlying the eastern Arabian Sea oxygen minimum zone, where high H2S disallows O2 influx from the water-column. While genes for aerobic respiration by aa3-/cbb3-type cytochrome-c oxidases and cytochrome-bd ubiquinol oxidase, and aerobic oxidation of methane/ammonia/alcohols/thiosulfate/sulfite/organosulfur-compounds, were present across the cores, so were live aerobic, sulfur-chemolithoautotrophs and chemoorganoheterotrophs. The 8820-years-old, highly-sulfidic, methane-containing sediment-sample from 275 cmbsf of 530 mbsl yielded many such obligately-aerobic bacterial-isolates that died upon anaerobic incubation with alternative electron-acceptors/fermentative-substrates. Several metatranscriptomic reads from this sediment-sample matched aerobic-respiration-/oxidase-reaction-/transcription-/translation-/DNA-replication-/membrane-transport-/cell-division-related genes of the obligately-aerobic isolates, thereby corroborating their active aerobic metabolic-status in situ. Metagenomic and metatranscriptomic detection of perchlorate-/chlorate-reduction genes, plus anaerobic growth of an obligately-aerobic Halothiobacillus isolate in the presence of perchlorate and perchlorate-reducing-consortia, suggested that cryptic O2 produced by perchlorate-respirers could be sustaining obligately-aerobes in this environment.

microbiology↗

bioRxiv2019

Two pathways for thiosulfate oxidation in the alphaproteobacterial chemolithotroph Paracoccus thiocyanatus SST

Chemolithotrophic bacteria oxidize various sulfur species for energy and electrons, thereby operationalizing biogeochemical sulfur cycles in nature. The best-studied pathway of bacterial sulfur-chemolithotrophy, involving direct oxidation of thiosulfate to sulfate (without any free intermediate) by the SoxXAYZBCD multienzyme system, is apparently the exclusive mechanism of thiosulfate oxidation in facultatively chemolithotrophic alphaproteobacteria. Here we explore the molecular mechanisms of sulfur oxidation in the thiosulfate- and tetrathionate-oxidizing alphaproteobacterium Paracoccus thiocyanatus SST, and compare them with the prototypical Sox process characterized in Paracoccus pantotrophus. Our results revealed the unique case where, an alphaproteobacterium has Sox as its secondary pathway of thiosulfate oxidation, converting [~]10% of the thiosulfate supplied whilst 90% of the substrate is oxidized via a Tetrathionate-Intermediate pathway. Knock-out mutation, followed by the study of sulfur oxidation kinetics, showed that thiosulfate-to-tetrathionate conversion, in SST, is catalyzed by a thiosulfate dehydrogenase (TsdA) homolog that has far-higher substrate-affinity than the Sox system of this bacterium, which, remarkably, is also less efficient than the P. pantotrophus Sox. soxB-deletion in SST abolished sulfate-formation from thiosulfate/tetrathionate while thiosulfate-to-tetrathionate conversion remained unperturbed. Physiological studies revealed the involvement of glutathione in SST tetrathionate oxidation. However, zero impact of the knock-out of a thiol dehydrotransferase (thdT) homolog, together with no production of sulfite as an intermediate, indicated that tetrathionate oxidation in SST is mechanistically novel, and distinct from its betaproteobacterial counterpart mediated by glutathione, ThdT, SoxBCD and sulfite:acceptor oxidoreductase. All the present findings collectively highlight extensive functional diversification of sulfur-oxidizing enzymes across phylogenetically close, as well as distant, bacteria.

microbiology↗

bioRxiv2019

Inorganic salts and compatible solutes help mesophilic bacteria inhabit the high temperature waters of a Trans-Himalayan sulfur-borax spring

While geographically-/geologically-distinct hot springs harbor different levels of microbial diversity, some of them encompass several such taxa which have no strain reported for laboratory growth at >45{degrees}C. We, therefore, hypothesized that native geomicrobial factors could be potent determinants of the microbial habitability of hot spring environments. To test this hypothesis, aquatic microbial communities were revealed metataxonomically, and considered in the context of spring-water chemistry, along the 85-14{degrees}C hydrothermal gradient of a sulfur-boron spring named Lotus Pond located at 4,436 m, within the Puga geothermal area of the Indian Trans-Himalayan region of Ladakh. Water samples were studied from four distinct sites along Lotus Ponds spring-water transit from the vent to an adjacent river called Rulang. Insinuations obtained from geomicrobiological data were tested via pure-culture growth experiments in habitat-inspired media. Microbial diversities were found to be high at all the sample-sites; majority of the genera identified at the 70-85{degrees}C sites were found to have no report of laboratory growth at >45{degrees}C; concurrently, these sample-sites had high concentrations of the kosmotropic solutes boron, lithium, sodium, sulfide, thiosulfate and sulfate, which are known to biophysically stabilize macromolecules. Based on the universal thermodynamic status of these solutes, we conjectured that they may be instrumental in helping mesophiles withstand high in situ temperatures. Corroboratively, growth experiments with a mesophilic, 80{degrees}C-isolate, Paracoccus SMMA_5 showed that at 50{degrees}C and 70{degrees}C, depending on the incubation-time, lithium/boron/sulfate/sodium/glycine-betaine either increases the number of colony-forming units present in the culture or arrests decline of the same. Incubations at 70{degrees}C, followed by fluorescein diacetate staining and flow cytometry, showed that these solutes keep more cells under viable condition than in ready-to-divide state. We concluded that kosmotropes and compatible solutes help mesophiles overcome the chaotropic effects of heat by augmenting such indigenous, entropy-minimizing biophysical mechanisms that apparently trade-off cell division for cell viability.

microbiology↗

bioRxiv2019

Viral satellites exploit phage proteins to escape degradation of the bacterial host chromosome

Phage defense systems are often found on mobile genetic elements (MGEs), where they constitutively defend against invaders or are induced to respond to new assaults. Some MGEs, the phage satellites, exploit phages for their own transmission after induction, reducing phage production and protecting their hosts in the process. One such satellite in Vibrio cholerae, PLE, is triggered by the lytic phage ICP1 to excise from the chromosome, replicate, and transduce to neighboring cells, completely sabotaging phage production. Here, we found that ICP1 has evolved to possess one of two syntenic loci encoding an SF1B-type helicase, either of which PLE can exploit to directly drive PLE replication. Further, loss of PLE mobilization limits anti-phage activity due to phage-mediated degradation of the bacterial genome. Our work provides insight into the unique challenges imposed on the parasites of lytic phages and underscores the adaptions of these satellites to their ever-evolving target phage.

microbiology↗

bioRxiv2018

Validation of the protein kinase PfCLK3 as a multi-stage cross species malarial drug target

The requirement for next generation anti-malarials to be both curative and transmission blockers necessitate the identification of molecular pathways essential for viability of both asexual and sexual parasite life stages. Here we identify a selective inhibitor to the Plasmodium falciparum protein kinase PfCLK3 which we use in combination with chemogenetics, whole genome sequencing and transcriptomics to validate PfCLK3 as a druggable target acting at multiple parasite life stages. Consistent with the proposed role of PfCLK3 as a regulator of RNA splicing, inhibition results in the down-regulation of >400 genes essential for parasite survival. Through this mechanism, blocking PfCLK3 activity not only results in rapid killing of asexual blood stage parasites but is also effective on sporozoites and gametocytes as well as showing parasiticidal activity in all Plasmodium species tested. Hence, our data establishes PfCLK3 as a target with the potential to deliver both symptomatic treatment and transmission blocking in malaria.

microbiology↗

bioRxiv2018

Competition between mobile genetic elements drives optimization of a phage-encoded CRISPR-Cas system: Insights from a natural arms-race

CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defense against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not equally interfere with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses.

microbiology↗