Biology subjects

Pujhari, S.

Publications and source records attributed to Pujhari, S..

11 recordsLinked to original sources

bioRxiv2025

In situ architecture of the endosymbiont Wolbachia pipientis

Hidden within host cells, the endosymbiont Wolbachia pipientis is the most prevalent bacterial infection in the animal kingdom. Scientific breakthroughs over the past century yielded fundamental mechanisms by which Wolbachia controls arthropod reproduction to shape dynamic ecological and evolutionary trajectories. However, the structure and spatial organization of symbiont machineries that underpin intracellular colonization and orchestrate maternal inheritance remain unknown. Here, we used cryo-electron tomography to directly image the nanoscale architecture of bacterial tools deployed for host manipulation and germline transmission. We discovered that Wolbachia assembles multiple structures at the host-endosymbiont interface including a filamentous ladder-like framework hypothesized to serve as a specialized motility mechanism that enables bacterial translocation to specific host cell compartments during embryogenesis and somatic tissue dissemination. In addition, we present the first in situ structure of the Rickettsiales vir homolog type IV secretion system (rvh T4SS). We provide evidence that the rvh T4SS nanomachine exhibits architectural similarities to the pED208-encoded T4SS apparatus including the biogenesis of rigid conjugative pili extending hundreds of nanometers beyond the bacterial cell surface. Coupled with integrative structural modeling, we demonstrate that in contrast to canonical T4SS architectures, the -proteobacterial T4SS outer membrane complex assembles a periplasmic baseplate structure predicted to comprise VirB9 oligomers complexed with cognate VirB10 subunits that form extended antennae projections surrounding the translocation channel pore. Collectively, these studies provide an unprecedented view into Wolbachia structural cell biology and unveil the molecular blueprints for architectural paradigms that reinforce ancient host-microbe symbioses.

microbiology↗

bioRxiv2023

Wolbachia-induced inhibition of O'nyong nyong virus in Anopheles mosquitoes is mediated by Toll signaling and modulated by cholesterol

Enhanced host immunity and competition for metabolic resources are two main competing hypotheses for the mechanism of Wolbachia-mediated pathogen inhibition in arthropods. Using an Anopheles mosquito - somatic Wolbachia infection - Onyong nyong virus (ONNV) model, we demonstrate that the mechanism underpinning Wolbachia-mediated virus inhibition is up-regulation of the Toll innate immune pathway. However, the viral inhibitory properties of Wolbachia were abolished by cholesterol supplementation. This result was due to Wolbachia-dependent cholesterol-mediated suppression of Toll signaling rather than competition for cholesterol between Wolbachia and virus. The inhibitory effect of cholesterol was specific to Wolbachia-infected Anopheles mosquitoes and cells. These data indicate that both Wolbachia and cholesterol influence Toll immune signaling in Anopheles mosquitoes in a complex manner and provide a functional link between the host immunity and metabolic competition hypotheses for explaining Wolbachia-mediated pathogen interference in mosquitoes. In addition, these results provide a mechanistic understanding of the mode of action of Wolbachia-induced pathogen blocking in Anophelines, which is critical to evaluate the long-term efficacy of control strategies for malaria and Anopheles-transmitted arboviruses. HIGHLIGHTSO_LIWolbachia inhibits Onyong nyong virus (ONNV) in Anopheles mosquitoes. C_LIO_LIEnhanced Toll signaling is responsible for Wolbachia-induced interference of ONNV. C_LIO_LICholesterol suppresses Toll signaling to modulate Wolbachia-induced ONNV interference. C_LI

microbiology↗

bioRxiv2023

Sindbis virus is suppressed in the yellow fever mosquito Aedes aegypti by ATG-6/Beclin-1 mediated activation of autophagy

Autophagy is a critical modulator of pathogen invasion response in vertebrates and invertebrates. However, how it affects mosquito-borne viral pathogens that significantly burden public health remains underexplored. To address this gap, we use a genetic approach to activate macroautophagy/autophagy in the yellow fever mosquito (Aedes aegypti), infected with a recombinant Sindbis virus (SINV) expressing an autophagy activator. We first demonstrate a 17- amino acid peptide derived from the Ae. aegypti autophagy-related protein 6 (ATG-6/beclin-1-like protein) is sufficient to induce autophagy in C6/36 mosquito cells, as marked by lipidation of ATG- 8 and puncta formation. Next, we engineered a recombinant SINV expressing this bioactive beclin- 1-like peptide and used it to infect and induce autophagy in adult mosquitoes. We find that modulation of autophagy using this recombinant SINV negatively regulated production of infectious viruses. The results from this study improve our understanding of the role of autophagy in arboviruses in invertebrate hosts and also highlight the potential for the autophagy pathway to be exploited for arboviral control.

microbiology↗

bioRxiv2023

Variable effects of Wolbachia on alphavirus infection in Aedes aegypti

2.Wolbachia pipientis (=Wolbachia) has promise as a tool to suppress virus transmission by Aedes aegypti mosquitoes. However, Wolbachia can have variable effects on mosquito-borne viruses. This variation remains poorly characterized, yet the multimodal effects of Wolbachia on diverse pathogens could have important implications for public health. Here, we examine the effects of somatic infection with two strains of Wolbachia (wAlbB and wMel) on the alphaviruses Sindbis virus (SINV), Onyong-nyong virus (ONNV), and Mayaro virus (MAYV) in Ae. aegypti. We found variable effects of Wolbachia including enhancement and suppression of viral infections, with some effects depending on Wolbachia strain. Both wAlbB- and wMel-infected mosquitoes showed enhancement of SINV infection rates one week post-infection, with wAlbB-infected mosquitoes also having higher viral titers than controls. Infection rates with ONNV were low across all treatments and no significant effects of Wolbachia were observed. The effects of Wolbachia on MAYV infections were strikingly strain-specific; wMel strongly blocked MAYV infections and suppressed viral titers, while wAlbB did not influence MAYV infection. The variable effects of Wolbachia on vector competence underscore the importance of further research into how this bacterium impacts the virome of wild mosquitoes including the emergent human pathogens they transmit. Impact statementIn recent years, wild populations of Aedes aegypti mosquitoes have been deliberately infected with Wolbachia--a bacterium that helps to curb the spread of some pathogens including dengue virus. But how does Wolbachia affect the ability of mosquitoes to become infected with and spread the many different viruses they encounter in nature? Here, we characterize the effects of Wolbachia on three alphaviruses that cause illness in humans-- Sindbis virus, Onyong-nyong virus, and Mayaro virus. We find Wolbachia has variable effects on these pathogens, including significant enhancement of Sindbis virus infections. Our research has important implications for the design of vector control strategies, and suggests further research is needed to understand how Wolbachia shapes the replication and transmission of diverse viruses. 3. Data SummaryAll data in the study are available in the Figures and supplementary material.

microbiology↗

bioRxiv2021

Identification of microRNAs in the West Nile virus vector Culex tarsalis

BackgroundmicroRNAs (miRNAs) represent a group of small non-coding RNAs that are crucial gene regulators of important biological functions including development and pathogen defense in most living organisms. Presently, there is a lack of availability of information regarding the miRNAs in the mosquito Culex tarsalis, which is one of the most important vectors of West Nile virus (WNV) in the United States. We used small RNA sequencing data and in vitro and in vivo experiments to identify and validate the presence of a repertoire of miRNAs in Cx. tarsalis mosquitoes. ResultsUsing bioinformatic approaches we analyzed small RNA sequencing data from the Cx. tarsalis CT embryonic cell line to discover 86 miRNAs. Consistent with other mosquitoes such as Aedes albopictus and Cx. quinquefasciatus, mi-184 was found to be the most abundant miRNA in Cx tarsalis. We also identified an additional 20 novel miRNAs from the recently sequenced Cx. tarsalis genome, for a total of 106 miRNAs identified in this study. The presence of selected miRNAs was biologically validated in both cell line and adult Cx. tarsalis mosquitoes using RT-qPCR and sequencing. ConclusionsCx. tarsalis is an important vector of many medically important pathogens including WNV and Western Equine encephalitis. Here we report a detailed insight into the miRNA population in Cx. tarsalis mosquitoes. These results will open new avenues of research deciphering the role of miRNAs in different Cx. tarsalis biological events such as development, metabolism, immunity and pathogen infection.

genomics↗

bioRxiv2021

In vitro and in vivo co-infection and super-infection dynamics of Mayaro and Zika viruses in mosquito and vertebrate backgrounds

Factors related to increasing globalization and climate change have contributed to the simultaneous increase and spread of arboviral diseases. Co-circulation of multiple arboviruses in the same geographic regions provides impetus to study the impacts of multiple arbovirus infections in a single vector. In the present study we describe co-infection and super-infection with Mayaro virus (Family Togaviridae, genus Alphavirus) and Zika virus (family Flaviviridae, genus Flavivirus) in vertebrate cells, mosquito cells, and Aedes aegypti mosquitoes to understand the interaction dynamics of these pathogens and effects on viral infection, dissemination and transmission. In Aedes aegypti mosquitoes, co-infection has a negative impact on infection and dissemination rates for Zika virus, but not Mayaro virus, when compared to single infection scenarios, and super-infection of Mayaro virus with a previous Zika virus infection resulted in increased Mayaro virus infection rates. We found that co-infection and super-infection negatively affected Zika viral replication in vertebrate cells (Vero and Huh), resulting in the complete blockage of Zika virus replication in some scenarios. At the cellular level, we demonstrate that single vertebrate and insect cells can be simultaneously infected with Zika and Mayaro viruses. This study highlights the dynamics of arboviral co- and super-infections and emphasizes the importance of considering these dynamics during risk assessment in epidemic areas.

microbiology↗

bioRxiv2020

Everything you wanted to know about Mayaro virus but were afraid to ask: Characterization and lifecycle of Mayaro virus in vertebrate and invertebrate cellular backgrounds

Mayaro virus (MAYV) is an emerging new world alphavirus (genus Alphavirus, family Togaviridae) that causes acute multiphasic febrile illness, skin rash, polyarthritis, and occasional severe clinical phenotypes. The virus lifecycle alternates between invertebrate and vertebrate hosts. Here we characterize the replication features, cell entry, life cycle, and virus-related cell pathology of MAYV using vertebrate and invertebrate in vitro models. Electron dense clathrin-coated pits in infected cells, and reduced viral production in the presence of dynasore, ammonium chloride, and bafilomycin, indicates that viral entry occurs through pH-dependent endocytosis. Increase in FITC-dextran uptake (an indicator of macropinocytosis) in MAYV-infected cells, and dose-dependent infection inhibition by 5-(N-ethyl-N-isopropyl) amiloride (a macropinocytosis inhibitor), indicated that macropinocytosis is an additional entry mechanism of MAYV in vertebrate cells. Acutely infected vertebrate and invertebrate cells formed cytoplasmic or membrane-associated extracytoplasmic replication complexes. Mosquito cells showed modified hybrid cytoplasmic vesicles that supported virus replication, nucleocapsid production, and maturation. Mature virus particles were released from cells by both exocytosis and budding from the cell membrane. MAYV replication was cytopathic and associated with induction of apoptosis by the intrinsic pathway, and later by the extrinsic pathway in infected vertebrate cells. Given that MAYV is expanding its geographical existence as a potential public health problem, this study lays the foundation of biological understanding valuable for therapeutic and preventive interventions.

microbiology↗

bioRxiv2020

Vectorchip: Microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

Mosquito bites transmit a number of human pathogens resulting in potentially fatal diseases including malaria, dengue, chikungunya, West Nile encephalitis, and Zika. Although female mosquitoes transmit pathogens via salivary droplets deposited during blood feeding on a host, very little is known about the genomic content of these nanoliter scale droplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform for molecular interrogation of individual mosquito bites in a high-throughput fashion. An ultra-thin PDMS membrane coupled to a microfluidic chip acts as a biting interface, through which freely-behaving mosquitoes deposit saliva droplets by biting into isolated arrayed micro-wells enabling molecular interrogation of individual bites. By modulating membrane thickness, the device enables on-chip comparison of biting capacity and provides a mechanical filter allowing selection of a specific mosquito species. Utilizing Vectorchip, we show on-chip simultaneous detection of mosquito DNA as well as viral RNA from Zika infected Aedes aegypti mosquitoes - demonstrating multiplexed high-throughput screening of vectors and pathogens. Focus-forming assays performed on-chip quantify number of infectious viral particles transmitted during mosquito bites, enabling assessment of active virus transmission. The platform presents a promising approach for single-bite-resolution laboratory and field characterization of vector pathogen communities, to reveal the intricate dynamics of pathogen transmission, and could serve as powerful early warning artificial "sentinel" for mosquito-borne diseases.

bioengineering↗

bioRxiv2019

Cas9-mediated gene-editing in the malaria mosquito Anopheles stephensi by ReMOT Control

Innovative tool development is essential for continued advancement in malaria control and depends on a deeper understanding of the molecular mechanisms that govern transmission of malaria parasites by Anopheles mosquitoes. Targeted disruption of genes in mosquito vectors is a powerful method to uncover the underlying biology of vector-pathogen interactions, and genome manipulation technologies can themselves form the basis of mosquito and pathogen control strategies. However, the embryo injection methods used to genetically manipulate mosquitoes, and in particular Anopheles species, are difficult and inefficient, particularly for non-specialist laboratories. We have adapted a strategy called ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) to deliver the Cas9 ribonucleoprotein complex to adult mosquito ovaries and generate targeted and heritable mutations in the malaria vector Anopheles stephensi. We found that gene editing by ReMOT Control in Anopheles mosquitoes was comparable to the technique in Ae. aegypti and as efficient in editing as standard embryo injections. The adaptation of this technology to Anopheles mosquitoes opens up the power of reverse genetics to malaria vector labs that do not have the equipment or technical expertise to perform embryo injections and establishes the flexibility of ReMOT Control for gene-editing in non-Aedes species.

genetics↗

bioRxiv2018

Anopheles Mosquitoes May Drive Invasion and Transmission of Mayaro Virus across Geographically Diverse Regions

The Togavirus (Alphavirus) Mayaro virus (MAYV) was initially described in 1954 from Mayaro County (Trinidad) and has been responsible for outbreaks in South America and the Caribbean. Imported MAYV cases are on the rise, leading to invasion concerns similar to Chikungunya and Zika viruses. Little is known about the range of mosquito species that are competent MAYV vectors. We tested vector competence of 2 MAYV genotypes for six mosquito species (Aedes aegypti, Anopheles gambiae, An. stephensi, An. quadrimaculatus, An. freeborni, Culex quinquefasciatus). Ae. aegypti and Cx. quinquefasciatus were poor MAYV vectors, and either were poorly infected or poorly transmitted. In contrast, all Anopheles species were able to transmit MAYV, and 3 of the 4 species transmitted both genotypes. The Anopheles species tested are divergent and native to widely separated geographic regions, suggesting that Anopheles may be important in the invasion and spread of MAYV across diverse regions of the world.

epidemiology↗

bioRxiv2017

Heat Shock Protein 70 (Hsp70) Is Involved In The Zika Virus Cellular Infection Process

Zika virus (ZIKV) is a historically neglected flavivirus that has recently caused epidemics in the western hemisphere. ZIKV has been associated with severe symptoms including infant microcephaly and Guillain Barre syndrome, stimulating interest in understanding factors governing ZIKV infection. Heat shock protein 70 (Hsp70) has been shown to be an infection factor for multiple viruses. We investigated the role of Hsp70 in the ZIKV infection process. We localized Hsp70 protein to the Vero cell membrane surface by confocal microscopy and demonstrated that, inside the cell, there is significant co-localization between Hsp70 and ZIKV E protein. Inducing and suppressing Hsp70 expression increased and decreased ZIKV production, respectively. Antibody blocking cell surface-localized Hsp70 decreased ZIKV cell infection rates and production of infectious virus particles, as did competition with recombinant Hsp70 protein. Our data suggest that Hsp70 is an important factor in the ZIKV infection process. Understanding the interactions between Hsp70 and ZIKV may lead to novel therapeutics for ZIKV infection, particularly for pregnant women and fetuses.

microbiology↗