Egress Process Research Articles

Rab27a GTPase and its effector Myosin Va are host factors required for efficient Oropouche virus cell egress.

Oropouche fever, a debilitating illness common in South America, is caused by Oropouche virus (OROV), an arbovirus. OROV belongs to the Peribunyaviridae family, a large group of RNA viruses. Little is known about the biology of Peribunyaviridae in host cells, especially assembly and egress processes. Our research reveals that the small GTPase Rab27a mediates intracellular transport of OROV induced compartments and viral release from infected cells. We show that Rab27a interacts with OROV glycoproteins and colocalizes with OROV during late phases of the infection cycle. Moreover, Rab27a activity is required for OROV trafficking to the cell periphery and efficient release of infectious particles. Consistently, depleting Rab27a's downstream effector, Myosin Va, or inhibiting actin polymerization also hinders OROV compartments targeting to the cell periphery and infectious viral particle egress. These data indicate that OROV hijacks Rab27a activity for intracellular transport and cell externalization. Understanding these crucial mechanisms of OROV's replication cycle may offer potential targets for therapeutic interventions and aid in controlling the spread of Oropouche fever.

Current Insights into the Maturation of Epstein-Barr Virus Particles.

The three subfamilies of herpesviruses (alphaherpesviruses, betaherpesviruses, and gammaherpesviruses) appear to share a unique mechanism for the maturation and egress of virions, mediated by several budding and fusion processes of various organelle membranes during replication, which prevents cellular membrane disruption. Newly synthesized viral DNA is packaged into capsids within the nucleus, which are subsequently released into the cytoplasm via sequential fusion (primary envelopment) and budding through the inner and outer nuclear membranes. Maturation concludes with tegumentation and the secondary envelopment of nucleocapsids, which are mediated by budding into various cell organelles. Intracellular compartments containing mature virions are transported to the plasma membrane via host vesicular trafficking machinery, where they fuse with the plasma membrane to extracellularly release mature virions. The entire process of viral maturation is orchestrated by sequential interactions between viral proteins and intracellular membranes. Compared with other herpesvirus subfamilies, the mechanisms of gammaherpesvirus maturation and egress remain poorly understood. This review summarizes the major findings, including recently updated information of the molecular mechanism underlying the maturation and egress process of the Epstein-Barr virus, a ubiquitous human gammaherpesvirus subfamily member that infects most of the population worldwide and is associated with a number of human malignancies.

Evaluating building egress safety for the elderly under different urgent-level circumstances in high-density cities: Based on ABEM-enabled simulation

In high-density cities, people usually live in mid-rise or high-rise residential buildings where indoor environments are quite complicated. Safety risks exist not only within homes, but also on the way to egress from residential buildings. As the aging issue becomes severer, it is essential to provide safe egress plans particularly for the elderly who are more fragile to risks and obstacles. Kinds of emergencies might occur in reality, some are hard to predict and extremely dangerous, while some can be warned in advance and would not cause damages immediately. However, previous research seldom considered the different urgent levels of circumstances during the egress process of the elderly. Consequently, this study contributes to proposing an agent-based egress model (ABEM)-based evaluation of building egress safety for the elderly under different urgent-level circumstances by introducing absolute and relative indicators. Absolute indicators evaluate building egress safety for the elderly, relative indicators reveal whether buildings hinder safe egress of the elderly compared with ordinary residents, and indicator weights are decided by AHP method to compute building egress safety index (BESI) as the evaluation results. Case study shows BESI scores of 8.696, 8.941 and 6.290 under three circumstances. BESI scores are valuable references for old home buyers to make decisions. Furthermore, this evaluation approach offers an effective tool to help designers to improve design schemes for safer egress, to assist property managers in updating egress plans and facilities based on new occupancy status in the operation stage, eventually to facilitate the development of healthy residential buildings.

Ni Ingress and Egress in SrTiO3 Single Crystals of Different Facets

Metal-ion surface interactions and/or doping in host perovskite-oxides are techniques that are widely employed for electronic structure tuning purposes and in developing novel heterogeneous catalysts; however, an in-depth understanding of the different elementary steps and factors involved in these processes is lacking. Herein, we use Atomic Force Microscopy (AFM), Scanning Transmission Electron Microscopy (STEM), and ab initio thermodynamics through density functional theory (DFT) to specifically investigate Ni surface adsorption, ingress, migration, segregation, and egress processes across different SrTiO3 (STO) single-crystal facets and terminations, specifically the (001), (110), and the (111). Under oxidizing and reducing conditions at different temperatures, Ni egress is observed on (110) STO samples, but not the (001). DFT results demonstrate Ni to have a higher thermodynamic egress propensity, specifically through an oxygen-terminated (110) facet in comparison to other (001) terminations, whereas for the (111)-Ti terminated facet, Ni is likely to remain in the bulk post ingress. We suggest that the observed uniqueness of the (110) surface facet toward Ni egress is possibly a consequence of a surface phase transition. These results can help guide design interests with regard to Ni surface stabilization, ingress/egress suppression, or facilitation in STO by elucidating the nuances involved across different facets.

Mesoscale simulations illuminate the functional interplay and the vulnerabilities of influenza virus glycoproteins.

Influenza A virus (IAV) has menacingly resurfaced during the last flu season, posing an additional threat to public health as it started overlapping with the ongoing COVID-19 pandemic. This concerning scenario foregrounds the urgency of a universal influenza vaccine that is not affected by the antigenic drift and would put an end to the flu. The main targets are the two principal membrane glycoproteins, hemagglutinin (HA) and neuraminidase (NA), whose functional balance is at the basis of IAV virulence and transmissibility. Here, we have used the ‘computational microscope’ to provide unseen views of the glycoprotein interplay and dynamics. By integrating experimental structural data with computational approaches, we have built two massive models of the whole H1N1 IAV accounting for different strains and glycosylation profiles. Mesoscale, all-atom molecular dynamics simulations of these systems tallying ∼160 million atoms have handed over unique insights into the glycoprotein conformational dynamics that would not have been accessible through individual protein simulations. On top of deciphering the glycoprotein interplay and cooperativity, we have characterized the functional motions of HA and NA in a crowded, realistic environment, shedding light on the recondite viral egress process. Interestingly, these motions are found to transiently expose cryptic epitopes on the NA and HA surface, offering new opportunities for vaccine development.

‘Erythritol’, a safe natural sweetener exhibits multi-stage anti-malarial activity by permeating into Plasmodium falciparum through aquaglyceroporin channel

The increased resistance of human malaria parasite Plasmodium falciparum (Pf) to currently used drugs necessities the development of novel anti-malarials. Here, we examine the potential of erythritol, a sugar substitute for therapeutic intervention. Erythritol is a permeant of Plasmodium falciparum aquaglyceroporin (PfAQP) which is a multifunctional channel responsible for maintaining hydro-homeostasis. We show that erythritol effectively inhibited growth and progression of asexual blood stage malaria parasite, and effect invasion and egress processes. It also inhibited the liver stage (sporozoites) and transmission stage parasite (gametocytes) development. Interestingly, erythritol inhibited in vivo growth of malaria parasite in mouse experimental model. It was more effective in inhibiting parasite growth both in vivo and in vitro when tested together with a known anti-malarial ‘artesunate’. Additionally, erythritol showed cytokine-modulating effect which suggests its direct effect on the host immune system. Ammonia detection assay demonstrated that erythritol uptake effects the amount of ammonia release across the parasite. Our functional complementation assays suggest that PfAQP expression in yeast mutant restores its growth in hyperosmotic conditions but showed reduced growth in the presence of erythritol. Osmotic lysis assay suggests that erythritol creates osmotic stress for killing the parasite. Overall, our data bestow erythritol as a promising lead compound with an attractive antimalarial profile and could possibly be combined with known drugs without losing its efficacy. We propose the use of erythritol based sweet candies for protection against malaria specially in children living in the endemic area.

Uba1: A Potential Ubiquitin-like Activator Protein of Urm1 in Toxoplasma gondii.

We had shown in our previous study that TgUrm1 (ubiquitin-related Modifier 1) was involved in the regulation of anti-oxidant stress in Toxoplasma gondii by conjugating with TgAhp1. It is generally believed that Urm1 binds to target proteins through a mechanism involving Uba (ubiquitin-like activator protein). Here, we identified the TgUrm1-exclusive ubiquitin-like activator-TgUba1, which was located in the cytoplasm of Toxoplasma. TgUba1 contained three domains, including the atrophin-1 domain (ANT1), the E1-like domain (AD), and the rhodanese homology domain (RHD). We explored the interaction of TgUba1 with TgUrm1, and the AD domain was essential for the interaction of the two proteins. The TgUba1 knockout and complementary mutants were obtained based on CRISPR/Cas9 gene editing technology. The knockout of TgUba1 attenuated parasite proliferation and virulence in mice, but not invasion and egress processes, revealing the pivotal role played by TgUba1 in T. gondii survival. Meanwhile, the conjugate band of TgUrm1 was significantly reduced under oxidative stress stimulation without TgUba1, indicating that TgUba1 enhanced the targeted conjugation ability of TgUrm1 in response to oxidative stress, especially under diamide (Dia) stimulation. Furthermore, eleven TgUba1-interacting proteins were identified by proximity-based protein labeling techniques, relating them to ubiquitin-like modifications, anti-oxidative stress and metabolic regulation processes. In conclusion, TgUba1 was essential for T. gondii survival and might be a potential ubiquitin-like activator protein for TgUrm1.

Nanoconfined vanadium nitride in 3D porous reduced graphene oxide microspheres as high-capacity cathode for aqueous zinc-ion batteries

Aqueous zinc-ion batteries (ZIBs) are receiving considerable research highlights owing to their high safety and environment-friendliness. To implement this promising technology for grid-scale energy storage, effective cathode materials with high capacity, cycle stability, and electrochemical kinetics should be developed. Herein, the synthesis of uniquely structured porous VN-reduced graphene oxide composite (VN-rGO) microspheres through a facile spray pyrolysis process and their application as cathodes for ZIBs are introduced. The electrochemical reaction mechanism of VN-rGO microspheres with zinc ions is investigated through various in situ and ex situ analyses. During the initial charge process, VN phase transforms into the Zn3(OH)2V2O7·2H2O (ZVOH) phase. From the second cycle and on, the ZVOH phase undergoes zinc-ion ingress and egress processes. VN-rGO microspheres exhibit an unprecedented high capacity (809 mA h g−1 at 0.1 A g−1), high energy density (613 W h kg−1), and good rate capability (467 mA h g−1 at 2.0 A g−1). The cathode delivers a reversible capacity of 445 mA h g−1 after 400 cycles at 1.0 A g−1, which ascertains the robustness of the structure. The 3D porous rGO matrix to which VN nanocrystals are homogenously anchored accelerates the zinc-ion storage kinetics and endows the cathode with structural robustness.

A Simultaneous Analysis of the User Safety and Resilience of a Twin-Tube Road Tunnel

This study complements our previous work with a simultaneous analysis of user safety and road tunnel resilience. We developed a computational fluid dynamics (CFD) model and simulated the corresponding egress process to evaluate the risk level of users exposed to different types of fire, such those which might occur on the undisrupted lane of a partially closed tube tunnel due to a traffic accident, or in the adjacent tube when used for two-way traffic in the case of the complete closure of the tube involved in the accident. The CFD results showed that: (i) better environmental conditions were found with the partial closure of the tube rather than the complete one; (ii) additional benefits can be achieved by activating variable message signs (VMSs) that suggest an alternative itinerary for heavy good vehicles (HGVs) only; (iii) safety issues for human health may arise only in the case of a 100 MW fire, occurring during the complete closure of the tube and the use of the parallel one for two-way traffic. The findings of the CFD simulations were subsequently used to perform a quantitative risk analysis (QRA) based on a probabilistic approach. The findings of the QRA were found to be consistent with those obtained by the tunnel resilience analysis. In particular, the lowest risk level for user safety was found with the partial closure of the tube instead of the complete one, and by activating the VMSs to redirect HGVs only towards an alternative itinerary. This finding was found to correspond to a higher resilience index of the tunnel (i.e., a lower resilience loss due to a traffic accident occurring in a tube). This study increases our knowledge on certain relevant aspects of the operating conditions of tunnels and can serve as a possible reference for tunnel management agencies (TMAs) in their choice of the most appropriate arrangement to recover the functionality of a tunnel taking into account both user safety and resilience at the same time.

Specific sub fractions from Terminalia mantaly (H. Perrier) extracts potently inhibit Plasmodium falciparum rings, merozoite egress and invasion

Ethnopharmacological relevanceTerminalia mantaly (H. Perrier) and Terminalia superba (Engl. & Diels) are sources of treatment for various diseases, including malaria and/or related symptoms in parts of Southwestern Cameroon. However, there is limited information on the extent of the antiplasmodial potential of their extracts. Aim of the studyThe present study was designed to investigate the antiplasmodial potential of chromatographic sub fractions (SFs) from promising fractions of Terminalia mantaly (Tm) [TmsbwChl, the chloroform fraction from water extract of Tm, IC50 (μg/mL) PfINDO: 0.56, Pf3D7: 1.12; SI > 357 (HEK/PfINDO) & 178 (HEK/Pf3D7)] and Terminalia superba (Ts) [TsrmEA, the ethyl acetate fraction from methanolic extract of Ts, IC50 (μg/mL) PfINDO: 1.82, Pf3D7: 1.65; SI > 109 (HEK/PfINDO) & 121 (HEK/Pf3D7)] obtained from previous studies. The SFs were tested against Plasmodium falciparum 3D7 (Pf3D7-chloroquine sensitive) and INDO (PfINDO-chloroquine resistant) strains in culture. Also, the phytochemical profile of potent SFs was determined and finally, the inhibition of the asexual blood stages of Plasmodium falciparum by the SFs with the highest promise was assessed. Material and methodsSelected SFs were submitted to a second bio-guided fractionation using silica gel column chromatography. The partial phytochemical composition of potent antiplasmodial SFs was determined using gas chromatography coupled to mass spectrometry (GC–MS). The SYBR Green I-based fluorescence microtiter plate assay was used to monitor the growth of Plasmodium falciparum parasites in culture in the presence or absence of extracts. Microscopy and flow cytometry counting was used to assess the Plasmodium falciparum stage–specific inhibition and post–drug exposure growth suppression by highly potent extracts. ResultsTwenty-one of the 39 SFs afforded from TmsbwChl showed activity (IC50: 0.29–4.74 μg/mL) against both Pf3D7 and PfINDO strains. Of note, eight SFs namely, Tm25, Tm28-30, Tm34-36 and Tm38, exerted highly potent antiplasmodial activity (IC50 < 1 μg/mL) with IC50PfINDO: 0.41–0.84 μg/mL and IC50Pf3D7: 0.29–0.68 μg/mL. They also displayed very high selectivity (50 < SIPfINDO, SIPf3D7 > 344) on the two Plasmodial strains. On the other hand, 7 SFs (SFs Ts03, Ts04, Ts06, Ts09, Ts10, Ts12 and Ts13) from TsrmEA showed promising inhibitory potential against both parasite strains (IC50: 2.01–5.14 μg/mL). Sub fraction Tm36 (IC50PfINDO: 0.41 μg/mL, SIPfINDO > 243; IC50Pf3D7: 0.29 μg/mL, SIPf3D7 > 344) showed the highest promise. The GC–MS analysis of the 8 selected SFs led to the identification of 99 phytometabolites, with D-limonene (2), benzaldehyde (12), carvone (13), caryophyllene (35), hexadecanoic acid, methyl ester (74) and 9-octadecenoic acid, methyl ester (82) being the main constituents. Sub fractions Tm28, Tm29, Tm30, Tm36 and Tm38 inhibited all the three intraerythrocytic stages of P. falciparum, with strong potency against ring stage development, merozoite egress and invasion processes. ConclusionsThis study has identified highly potent antiplasmodial SFs from Terminalia mantaly with significant activity on the intraerythrocytic development of Plasmodium falciparum. These SFs qualify as promising sources of novel antiplasmodial lead compounds. Further purification and characterization studies are expected to unravel molecular targets in rings and merozoites.

The Complex Regulatory Role of Cytomegalovirus Nuclear Egress Protein pUL50 in the Production of Infectious Virus.

The regulation of the nucleocytoplasmic release of herpesviral capsids is defined by the process of nuclear egress. Due to their large size, nuclear capsids are unable to traverse via nuclear pores, so that herpesviruses evolved to develop a vesicular transport pathway mediating their transition through both leaflets of the nuclear membrane. This process involves regulatory proteins, which support the local distortion of the nuclear envelope. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that initiates multicomponent assembly with NEC-associated proteins and capsids. Hereby, pUL50 serves as a multi-interacting determinant that recruits several viral and cellular factors by direct and indirect contacts. Recently, we generated an ORF-UL50-deleted recombinant HCMV in pUL50-complementing cells and obtained first indications of putative additional functions of pUL50. In this study, we produced purified ΔUL50 particles under both complementing (ΔUL50C) and non-complementing (ΔUL50N) conditions and performed a phenotypical characterization. Findings were as follows: (i) ΔUL50N particle preparations exhibited a clear replicative defect in qPCR-based infection kinetics compared to ΔUL50C particles; (ii) immuno-EM analysis of ΔUL50C did not reveal major changes in nuclear distribution of pUL53 and lamin A/C; (iii) mass spectrometry-based quantitative proteomics showed a large concordance of protein contents in the NIEP fractions of ΔUL50C and ΔUL50N particles, but virion fraction was close to the detection limit for ΔUL50N; (iv) confocal imaging of viral marker proteins of immediate early (IE) and later phases of ΔUL50N infection indicated a very low number of cells showing an onset of viral lytic protein expression; and, finally (v) quantitative measurements of encapsidated genomes provided evidence for a substantial reduction in the DNA contents in ΔUL50N compared to ΔUL50C particles. In summary, the results point to a complex and important regulatory role of the HCMV nuclear egress protein pUL50 in the maturation of infectious virus.

Bunyavirus SFTSV exploits autophagic flux for viral assembly and egress

ABSTRACT Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging negatively stranded enveloped RNA bunyavirus that causes SFTS with a high case fatality rate of up to 30%. Macroautophagy/autophagy is an evolutionarily conserved process involved in the maintenance of host homeostasis, which exhibits anti-viral or pro-viral responses in reaction to different viral challenges. However, the interaction between the bunyavirus SFTSV and the autophagic process is still largely unclear. By establishing various autophagy-deficient cell lines, we found that SFTSV triggered RB1CC1/FIP200-BECN1-ATG5-dependent classical autophagy flux. SFTSV nucleoprotein induced BECN1-dependent autophagy by disrupting the BECN1-BCL2 association. Importantly, SFTSV utilized autophagy for the viral life cycle, which not only assembled in autophagosomes derived from the ERGIC and Golgi complex, but also utilized autophagic vesicles for exocytosis. Taken together, our results suggest a novel virus–autophagy interaction model in which bunyavirus SFTSV induces classical autophagy flux for viral assembly and egress processes, suggesting that autophagy inhibition may be a novel therapy for treating or releasing SFTS.

TRP channels, the missing link for Ca2+ tuning by a unicellular eukaryotic parasite?

Sensing and responding to changes in the cellular environments are essential for the diverse family of Apicomplexan parasites, which undergo complex life cycles comprised of both extracellular and obligate intracellular stages. Despite evidence of paramount roles for Ca2+, the molecular players behind how parasites sense Ca2+ and initiate Ca2+ signaling cascades have remained enigmatic. In a recent publication, Marquez-Nogueras et al., identify a transient receptor potential (TRP)-like channel in Toxoplasma gondii and show its implication in the crucial processes of parasite invasion and egress from host cells.

Detecting aggressive agents in egress process by using conflict data in cellular automaton model

Aggressive behaviors at exits cause conflicts and increase egress times. To deter agents from aggressive behaviors, we first need to know who the aggressive ones are. Therefore, we developed a method for detecting aggressive agents. We focused on only egress simulations with a cellular-automata model in this article since we would like to deeply investigate theoretical characteristics of our method. There are two types of agents, which are normal agents and aggressive agents in the simulations. Aggressive agents tend to push out others in conflicts and try to move to their target cell aggressively. We considered all the possible combinations of agent types, labeled them, and computed the joint probabilities of the labels from the conflict data obtained from the egress simulations. The label which achieved the maximum joint probability was regarded as the predicted label. Our detecting method succeeded in detecting the aggressive agents perfectly with the reasonable number of observations. Moreover, there were no false accusations. We have also investigated how the restriction of the usage of the conflict data affect the results. By only using the conflict data of successes in solving conflicts, the accuracy failed to achieve 1.0 when there are many aggressive agents. However, if there are a few very aggressive agents, the progress rate of the accuracy increases by the restriction of the usage of the conflict data. We elucidated this counterintuitive phenomenon theoretically by exploiting a simple probabilistic calculation.

P300-mediated NEDD4 acetylation drives ebolavirus VP40 egress by enhancing NEDD4 ligase activity.

The final stage of Ebola virus (EBOV) replication is budding from host cells, where the matrix protein VP40 is essential for driving this process. Many post-translational modifications such as ubiquitination are involved in VP40 egress, but acetylation has not been studied yet. Here, we characterize NEDD4 is acetylated at a conserved Lys667 mediated by the acetyltransferase P300 which drives VP40 egress process. Importantly, P300-mediated NEDD4 acetylation promotes NEDD4-VP40 interaction which enhances NEDD4 E3 ligase activity and is essential for the activation of VP40 ubiquitination and subsequent egress. Finally, we find that Zaire ebolavirus production is dramatically reduced in P300 knockout cell lines, suggesting that P300-mediated NEDD4 acetylation may have a physiological effect on Ebola virus life cycle. Thus, our study identifies an acetylation-dependent regulatory mechanism that governs VP40 ubiquitination and provides insights into how acetylation controls EBOV VP40 egress.

CAMP-Dependent Signaling Pathways as Potential Targets for Inhibition of Plasmodium falciparum Blood Stages.

We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites.

Enhancing Construction Truck Safety at Work Zones: A Microscopic Traffic Simulation Study

Freeway work zones are generally considered dangerous, causing hundreds of fatalities every year. One significant task of work zone safety issue is how to manage construction trucks egress from work zones. Previous studies have been conducted to improve truck egress safety at work zones, yet few proactive safety engineering countermeasures have been implemented which might have significant effects. In this study, a microsimulation network of freeway work zone interval is built and calibrated using field data in Florida. The safety impact of work zone truck egress speed is investigated based on the simulation model, and the results indicate that it is significant to provide sufficient acceleration distance for trucks when egressing. Afterwards, the truck egress system (TES) that aims at providing a sufficient gap for trucks to merging into when egress from the work zone is developed. Then, the truck egress process is simulated with different parameter settings of TES under various traffic volumes. The results show that the introduction of TES improved safety, and the optimal settings for different traffic volume scenarios are recommended afterwards considering both safety and efficiency. The findings of this study could help practitioners understand the effect of the TES before implementing it in the real field.

A Comprehensive Gender-related Secretome of Plasmodium berghei Sexual Stages

Plasmodium, the malaria parasite, undergoes a complex life cycle alternating between a vertebrate host and a mosquito vector of the genus Anopheles In red blood cells of the vertebrate host, Plasmodium multiplies asexually or differentiates into gamete precursors, the male and female gametocytes, responsible for parasite transmission. Sexual stage maturation occurs in the midgut of the mosquito vector, where male and female gametes egress from the host erythrocytes to fuse and form a zygote. Gamete egress entails the successive rupture of two membranes surrounding the parasite, the parasitophorous vacuole membrane and the erythrocyte plasma membrane. In this study, we used the rodent model parasite Plasmodium berghei to design a label-free quantitative proteomic approach aimed at identifying gender-related proteins differentially released/secreted by purified mature gametocytes when activated to form gametes. We compared the abundance of molecules secreted by wild type gametocytes of both genders with that of a transgenic line defective in male gamete maturation and egress. This enabled us to provide a comprehensive data set of egress-related molecules and their gender specificity. Using specific antibodies, we validated eleven candidate molecules, predicted as either gender-specific or common to both male and female gametocytes. All of them localize to punctuate, vesicle-like structures that relocate to cell periphery upon activation, but only three of them localize to the gametocyte-specific secretory vesicles named osmiophilic bodies. Our results confirm that the egress process involves a tightly coordinated secretory apparatus that includes different types of vesicles and may put the basis for functional studies aimed at designing novel transmission-blocking molecules.

In-silico analysis of Calcium Dependent Protein Kinase 6 of Cryptosporidium parvum through molecular modeling, docking, and dynamics simulation study

Calcium Dependent Protein Kinases are found in the Apicomplexan, algae, and plants; however, they are not reported in vertebrates and are regarded as excellent drug targets for pharmaceutical interventions. Calcium Dependent Protein Kinases of Cryptosporidium are probably involved in the regulation of invasion and egress process during the infection of the host cells. The previous study reported that after the Calcium Dependent Protein Kinase 1 gene, Calcium Dependent Protein Kinase 6 of Cryptosporidium parvum is expressed in all stages of the parasite (merozoites/schizonts as well as sexual stages) at a comparable level and makes it as a valid drug target. In this study, an attempt is made to address the similarity in sequences and phylogenetic study of Calcium Dependent Protein Kinase 6 (CDPK6) among Calcium Dependent Protein Kinases of Apicomplexans. Further, the three-dimensional structure determination of CDPK6 of C. parvum was performed through a molecular modeling approach followed by virtual screening of small-molecule inhibitors from different datasets. The best inhibitor from Tres Cantos Antimalarial Set with ID 11730 reported a binding affinity of −8.2 kcal/mol against CDPK6 of C. parvum. Furthermore, the reliability of the binding mode of the inhibitor is validated through a complex molecular dynamics simulation study for a time interval of 100 ns. The simulation study advocates that the inhibitor Tres Cantos Antimalarial Set_11730 formed a stable interaction with the predicted active site residues and can be considered for industrial pharmaceutical research in future. Communicated by Ramaswamy H. Sarma

Plasmodium Perforin-Like Protein Pores on the Host Cell Membrane Contribute in Its Multistage Growth and Erythrocyte Senescence

The pore forming Plasmodium Perforin Like Proteins (PPLP), expressed in all stages of the parasite life cycle are critical for completion of the parasite life cycle. The high sequence similarity in the central Membrane Attack Complex/ Perforin (MACPF) domain among PLPs and their distinct functional overlaps define them as lucrative target for developing multi-stage antimalarial therapeutics. Herein, we evaluated the mechanism of Pan-active MACPF Domain (PMD), a centrally located and highly conserved region of PPLPs, and deciphered the inhibitory potential of specifically designed PMD inhibitors. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to hemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence which can be hypothesized to account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing malaria anemia. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress processes and protected erythrocytes against rPMD induced senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage, transmission-blocking potential of these inhibitors. Concievably, our study has introduced a novel set of anti-PMD inhibitors with pan-inhibitory activity against all the PPLPs members which can be developed into potent cross-stage antimalarial therapeutics along with erythrocyte senescence protective potential to occlude PPLPs mediated anemia in severe malaria.