Biology subjects

Ghai, R.

Publications and source records attributed to Ghai, R..

18 recordsLinked to original sources

bioRxiv2026

A remarkable case of conserved domain swapping in the COMMD family of proteins

The eukaryotic COMMD family of proteins are core subunits of the Commander protein complex, with a central role in endosomal membrane trafficking and signalling. Previous crystal and cryoEM structures show that COMMD and COMMD-like proteins form homo-oligomeric and hetero-decameric assemblies with a central ring structure formed by their small C-terminal COMM domains. Their -helical N-terminal (HN) domains decorate each side of these rings, and in the eukaryotic Commander complex engage the coiled-coil proteins CCDC22 and CCDC93. Here, we have determined new crystal structures of the isolated HN domains of human Commd4, Commd9 and Commd10, and find that all three proteins form domain swapped structures with a remarkably consistent topology. This occurs via a conformational change in a hinge-loop between helices 2 and 3, leading to exchange of helices 3 to 6 between protomers. The hinge-loops of Commd9 and Commd10 possess several serine and threonine residues that can be phosphorylated, and we find that specific phospho-mimicking mutations in Commd10 can promote or inhibit domain swapping. Whether the unique COMMD HN domains play any roles beyond assembly with CCDC proteins is unclear, but this work suggests a common conformational switch exists with a potential to regulate their function.

biochemistry↗

bioRxiv2023

Structural basis for inhibition of βFXIIa by garadacimab

FXIIa is the principal initiator of the plasma contact system and can activate both procoagulant and proinflammatory pathways. Its activity is important in the pathophysiology of Hereditary Angioedema (HAE). Here, we describe a high-resolution cryo-EM structure of the beta-chain from FXIIa ({beta}FXIIa) complexed with the Fab fragment of garadacimab. Garadacimab binds to {beta}FXIIa through an unusually long CDR-H3 that inserts into the S1 pocket in a non-canonical way. This atypical structural mechanism is likely the primary contributor in the inhibition of activated FXII proteolytic activity in HAE. Garadacimab Fab-{beta}FXIIa structure also reveals critical determinants of high-affinity binding of garadacimab to activated FXII. Structural analysis with other bonafide FXIIa inhibitors, such as benzamidine and C1-INH, reveals a conserved mechanism of {beta}FXIIa inhibition, a novel finding of this study. In summary, garadacimab Fab-{beta}FXIIa structure provides crucial insights into its mechanism of action and delineates primary and auxiliary paratopes/epitopes. This work reaffirms the benefits of cryo-EM as a primary tool to study antigen-antibody complexes in near native state, particularly where other methods have fallen short.

biochemistry↗

bioRxiv2023

Phenology and ecological role of Aerobic Anoxygenic Phototrophs in fresh waters

Aerobic anoxygenic phototrophic (AAP) bacteria are heterotrophic bacteria that supply their metabolism with light energy harvested by bacteriochlorophyll-a-containing reaction centres. Despite their substantial contribution to bacterial biomass, microbial food webs and carbon cycle, their phenology in freshwater lakes remains unknown. Hence, we investigated seasonal variations of AAP abundance and community composition biweekly across three years in a temperate, meso-oligotrophic freshwater lake. AAP bacteria displayed a clear seasonal trend with a spring maximum following the bloom of phytoplankton and a secondary maximum in autumn. As the AAP bacteria represent a highly diverse assemblage of species, we followed their seasonal succession using the amplicon sequencing of the pufM marker gene. To enhance the accuracy of the taxonomic assignment, we developed new pufM primers that generate longer amplicons and compiled the currently largest database of pufM gene, comprising 3633 reference sequences spanning all phyla known to contain AAP species. With this novel resource we demonstrated recurrent and dynamic seasonal succession of the AAP community. The majority of the species appeared during specific phases of the seasonal cycle, with less than 2% of AAP species detected during the whole year. Our results document the indigenous freshwater nature of the AAP community, characterized by high resilience and heterogenic adaptations to varying conditions of the freshwater environment. By integrating this information with the indicator of primary production (Chlorophyll-a) and existing ecological models, we show that AAP bacteria play a pivotal role in the recycling of dissolved organic matter released during spring phytoplankton bloom, contributing significantly to the ecological dynamics of lakes.

microbiology↗

bioRxiv2023

Cross-sector collaboration reduces SARS-CoV-2 risk in deer

One Health helps achieve optimal health outcomes for people, animals, plants, and their shared environments. We describe a multidisciplinary effort to better understand and mitigate SARS-CoV-2 spread in white-tailed deer across One Health sectors. We first framed the risk problem with three governance sectors that manage captive and wild deer and human public health. The framing included the objectives for each sector, interactions that facilitate human-to-deer and deer-to-deer transmission, and alternatives intended to reduce risk. We then developed a dynamic compartmental model that linked wild and captive deer herds and humans and simulated SARS-CoV-2 dynamics. For baseline conditions, we estimated that median SARS-CoV-2 prevalence in wild and captive herds varied between 0.03 - 0.07, incidence between 0.68 - 1.46, and probability of persistence between 0.64 - 0.97 across 120-day simulations. We then tested single-sector alternatives alone and in combination with other sector actions. We found that single sector alternatives varied in their ability to reduce transmission and that the best performing alternative required collaborative actions among wildlife management, agricultural management, and public health agencies.

ecology↗

bioRxiv2023

Rickettsiales' deep evolutionary history sheds light on the emergence of intracellular lifestyles

Ancient bacteria largely lived and flourished as free-living cells till the rise of eukaryotes triggered their adaptation to a new habitat: the intracellular milieu. Rickettsiales bacteria are the most prevalent intracellular microorganisms discovered and the culprits behind some of mankinds worst pestilential diseases. Here, we show that intracellularity is not a defining feature of the group and describe the eco-evolutionary processes that transformed harmless free-living bacteria into obligate intracellular symbionts and parasites. We found that the evolution of free-living lineages towards enhanced cross-feeding interactions with microbial eukaryotes trapped them in a nutritional bind to their trophic partners. We discovered that the oldest Rickettsiales lineages are the closest relatives to modern eukaryotes and are enriched in proteins predicted to have been present in the mitochondrial ancestor. This study not only opens avenues for the detection and surveillance of emerging diseases but also expands our understanding of the origins of complex life. One-Sentence SummaryCross-feeding interactions shaped Rickettsiales genomic architectures along the parasite/free-living spectrum

microbiology↗

bioRxiv2023

Structure of the Commander endosomal trafficking complex linked to X-linked intellectual disability/Ritscher-Schinzel syndrome

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy and in silico predictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

biochemistry↗

bioRxiv2021

Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins

The sorting nexin SNX17 controls endosome-to-cell surface recycling of diverse transmembrane cargo proteins including integrins, the amyloid precursor protein and lipoprotein receptors. This requires association with the multi-subunit Commander trafficking complex, which depends on the C-terminus of SNX17 through unknown mechanisms. Using affinity enrichment proteomics, we find that a C-terminal peptide of SNX17 is not only sufficient for Commander interaction but also associates with members of the actin-associated PDZ and LIM domain (PDLIM) family. We show that SNX17 contains a type III PSD95/Dlg/Zo1 (PDZ) binding motif (PDZbm) that binds specifically to the PDZ domains of PDLIM family proteins but not to other PDZ domains tested. The structure of the PDLIM7 PDZ domain bound to the SNX17 C-terminus was determined by NMR spectroscopy and reveals an unconventional perpendicular peptide interaction. Mutagenesis confirms the interaction is mediated by specific electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

biochemistry↗

bioRxiv2021

Quorum sensing signals from epibiont mediate the induction of bioactive peptides in mat-forming cyanobacteria Nostoc

The regulation of oligopeptides production is essential in understanding their ecological role in complex microbial communities including harmful cyanobacterial blooms. The role of chemical communication between the cyanobacterium and the microbial community harboured as epibionts within its phycosphere is at an initial stage of research and little is understood about its specificity. Herein, we present insight into the role of a bacterial epibiont in regulating production of cyanobacterial oligopeptides microviridins, well-known elastase inhibitors with presumed anti-grazing effects, in an ecologically important cyanobacterial genus Nostoc. Heterologous expression and identification of specific signal molecules from the epibiont suggest the role of a quorum sensing-based interaction. Further, physiological experiments show an increase in microviridin production without affecting cyanobacterial growth and photosynthetic activity. Simultaneously, oligopeptides presenting a selective inhibition pattern provide support for their specific function in response to the presence of cohabitant epibionts. Thus, the chemical interaction revealed in our study provides an example of an interspecies signalling pathway monitoring the bacterial flora around the cyanobacterial filaments and induction of intrinsic species-specific metabolic responses. IMPORTANCEThe regulation of cyanopeptide production beyond microcystin is essential to understand their ecological role in complex microbial communities, e.g. harmful cyanobacterial blooms. The role of chemical communication between the cyanobacterium and the epibionts within its phycosphere is at an initial stage of research and little is understood about its specificity. The frequency of cyanopeptide occurrence also demonstrates the need to understand the contribution of cyanobacterial peptides to the overall biological impact of cyanopeptides on aquatic organisms and vertebrates including humans. Our results shed light on the epibiont control of cyanopeptide production via quorum sensing mechanisms and we posit that such mechanisms may be widespread in natural cyanobacterial bloom community regulation.

microbiology↗

bioRxiv2021

Heliorhodopsin evolution is driven by photosensory promiscuity in monoderms

The ability to harness Suns electromagnetic radiation by channeling it into high-energy phosphate bonds empowered microorganisms to tap into a cheap and inexhaustible source of energy. Lifes billion-years history of metabolic innovations led to the emergence of only two biological complexes capable of harvesting light: one based on rhodopsins and the other on (bacterio)chlorophyll. Rhodopsins encompass the most diverse and abundant photoactive proteins on Earth and were until recently canonically split between type-1 (microbial rhodopsins) and type-2 (animal rhodopsins) families. Unexpectedly, the long-lived type-1/type-2 dichotomy was recently amended through the discovery of heliorhodopsins (HeRs) (Pushkarev et al. 2018), a novel and exotic family of rhodopsins (i.e. type-3) that evaded recognition in our current homology-driven scrutiny of lifes genomic milieu. Here, we bring to resolution the debated monoderm/diderm occurrence patterns by conclusively showing that HeR distribution is restricted to monoderms. Furthermore, through investigating protein domain fusions, contextual genomic information, and gene co-expression data we show that HeRs likely function as generalised light-dependent switches involved in the mitigation of light-induced oxidative stress and metabolic circuitry regulation. We reason that HeRs ability to function as sensory rhodopsins is corroborated by their photocycle dynamics (Pushkarev et al. 2018) and that their presence and function in monoderms is likely connected to the increased sensitivity to light-induced damage of these organisms (Maclean et al. 2009).

microbiology↗

bioRxiv2020

De novo macrocyclic peptides for inhibiting, stabilising and probing the function of the Retromer endosomal trafficking complex

The Retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signalling. Mutations in Retromer cause late-onset Parkinsons disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function we have created a novel series of macrocyclic peptides that bind Retromer with high affinity and specificity. Crystal structures show the majority of cyclic peptides bind to Vps29 via a Pro-Leu-containing sequence, structurally mimicking known interactors such as TBC1D5, and blocking their interaction with Retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds Retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting Retromer. Finally, tagged peptides can be used to probe the cellular localisation of Retromer and its functional interactions in cells, providing novel tools for studying Retromer function.

biochemistry↗

bioRxiv2020

A freshwater radiation of diplonemids

Diplonemids are considered marine protists and have been reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Lake Biwa, Japan. However, their distribution and abundances in freshwater ecosystems remain unknown. We assessed abundance and diversity of diplonemids from several geographically distant deep freshwater lakes of the world by amplicon-sequencing, shotgun metagenomics and CARD-FISH. We found diplonemids in all the studied lakes, albeit with low abundances and diversity. We assembled long 18S rRNA sequences from freshwater diplonemids and showed that they form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a sister-group to marine isolates from coastal and bay areas, suggesting a recent habitat transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved from single cell genomes of marine diplonemids shows they encode multiple rRNA copies that may be very divergent from each other, suggesting that marine diplonemid abundance and diversity both have been overestimated. These results have wider implications on assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone.

microbiology↗

bioRxiv2019

Phage-centric ecological interactions in aquatic ecosystems revealed through ultra-deep metagenomics

The persistent inertia in the ability to culture environmentally abundant microbes from aquatic ecosystems represents an obstacle in disentangling the complex web of ecological interactions spun by a diverse assortment of participants (pro- and eukaryotes and their viruses). In aquatic microbial communities, the numerically most abundant actors, the viruses, remain the most elusive, and especially in freshwaters their identities and ecology remain obscure. Here, using ultra-deep metagenomic sequencing from freshwater habitats we recovered complete genomes of >2000 phages, including small \"miniphages\" and large \"megaphages\" infecting iconic prokaryotic lineages. We also show that many phages encode genes that likely enhance survival of infected microbes during strong eukaryotic grazing pressure. For instance, we describe genes that afford protection to their host from reactive oxygen species (ROS) in the environment and from the oxidative burst in protist phagolysosomes (phage-mediated ROS defense) or those that directly effect targeted killing of the predators upon ingestion of a phage-infected microbe (Trojan horse). Spatiotemporal abundance analyses of phage genomes revealed evanescence as the primary dynamic in upper water layers, where they displayed short-lived existences. In contrast, persistence was characteristic for the deeper layers where many identical phage genomes were recovered repeatedly. Phage and host abundances corresponded closely, with distinct populations displaying preferential distributions in different seasons and depths, closely mimicking overall stratification and mixis.

microbiology↗

bioRxiv2019

Evolution in action: Habitat-transition leads to genome-streamlining in Methylophilaceae (Betaproteobacteriales)

The most abundant aquatic microbes are small in cell and genome size. Genome-streamlining theory predicts gene loss caused by evolutionary selection driven by environmental factors, favouring superior competitors for limiting resources. However, evolutionary histories of such abundant, genome-streamlined microbes remain largely unknown. Here we reconstruct the series of steps in the evolution of some of the most abundant genome-streamlined microbes in freshwaters ( Ca. Methylopumilus) and oceans (marine lineage OM43). A broad genomic spectrum is visible in the family Methylophilaceae (Betaproteobacteriales), from sediment microbes with medium-sized genomes (2-3 Mbp genome size), an occasionally blooming pelagic intermediate (1.7 Mbp), and the most reduced pelagic forms (1.3 Mbp). We show that a habitat transition from freshwater sediment to the relatively oligotrophic pelagial was accompanied by progressive gene loss and adaptive gains. Gene loss has mainly affected functions not necessarily required or advantageous in the pelagial or are encoded by redundant pathways. Likewise, we identified genes providing adaptations to oligotrophic conditions that have been transmitted horizontally from pelagic freshwater microbes. Remarkably, the secondary transition from the pelagial of lakes to the oceans required only slight modifications, i.e., adaptations to higher salinity, gained via horizontal gene transfer from indigenous microbes. Our study provides first genomic evidence of genome-reduction taking place during habitat transitions. In this regard, the family Methylophilaceae is an exceptional model for tracing the evolutionary history of genome-streamlining as such a collection of evolutionarily related microbes from different habitats is practically unknown for other similarly abundant microbes (e.g., Ca. Pelagibacterales, Ca. Nanopelagicales).

microbiology↗

bioRxiv2019

Visualization of Loki- and Heimdallarchaeia (Asgardarchaeota) by fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH)

Metagenome-assembled genomes (MAGs) of Asgardarchaeota are starting to be recovered from a variety of habitats, broadening their environmental distribution and providing access to the genetic makeup of this archaeal lineage. Despite their singular phylogenetic position at the base of the eukaryotic tree of life, the morphology of these bewildering organisms remains a mystery. In order to visualize this elusive group, we applied a combination of CARD-FISH and epifluorescence microscopy on coastal hypersaline sediment samples, using specifically designed CARD-FISH probes for Heimdallarchaeia and Lokiarchaeia lineages and provide the first visual evidence for both these groups. Here, we show that while Heimdallarchaeia are characterized by a uniform cellular morphology typified by central DNA localization, Lokiarchaeia display a plethora of shapes and sizes that likely reflect their broad phylogenetic diversity and ecological distribution.

microbiology↗

bioRxiv2018

A Swiftian Voyage from Brobdingnag to Lilliput: Freshwater Planctomycetes drifting towards the poles of the genome size spectrum

Freshwater environments teem with microbes. Currently, our apprehension of evolutionary ecology of freshwater bacteria is hampered by the difficulty to establish organism models for the most representative clades. To circumvent the bottlenecks inherent to the cultivation-based techniques, we applied ecogenomics approaches in order to unravel the evolutionary history and the processes that drive genome architecture in hallmark freshwater lineages from Planctomycetes phylum. The evolutionary history inferences showed that sediment/soil Planctomycetes transitioned to aquatic environments were, through processes mostly associated with reductive genome evolution, gave rise to new freshwater-specific clades. The most successful lineage was found to simultaneously have the most specialized lifestyle (increased regulatory genetic circuits; metabolism tuned for mineralization of proteinaceous sinking aggregates; psychrotrophic behavior) and to harbor the smallest genomes, highlighting a genomic architecture shaped by niche-directed evolution.

microbiology↗

bioRxiv2018

The sunlit microoxic niche of the archaeal eukaryotic ancestor comes to light

Recent advances in phylogenomic analyses and increased genomic sampling of uncultured prokaryotic lineages brought compelling evidence in support of the emergence of eukaryotes from within the Archaea domain of life. The discovery of Asgardaeota archaea and their recognition as the closest extant relative of eukaryotes fuelled the revival of a decades-old debate regarding the topology of the tree of life. While it is apparent that Asgardaeota encode a plethora of eukaryotic-specific proteins (the highest number identified to date in prokaryotes), the lack of genomic information and metabolic characterization has precluded inferences about their lifestyles and the metabolic landscape that may have favoured the emergence of the hallmark eukaryotic subcellular architecture. Here, we use advanced phylogenetic analyses to infer the deep ancestry of eukaryotes and genome-scale metabolic reconstructions to shed light on the metabolic milieu of the closest archaeal eukaryotic ancestors discovered till date. In doing so, we: i) generate the largest Asgardaeota genomic dataset available so far, ii) describe a new clade of rhodopsins encoded within the recovered genomes, iii) provide unprecedented evidence for mixotrophy within Asgardaeota, iv) present first-ever proofs that the closest extant archaeal relatives to all eukaryotes (Heimdallarchaeia) have microoxic lifestyles with aerobic metabolic pathways unique among Archaea (i.e. kynurenine pathway) and v) generate the first images of Asgardaeota.

microbiology↗

bioRxiv2018

Hidden in plain sight - highly abundant and diverse planktonic freshwater Chloroflexi

BackgroundRepresentatives of the phylum Chloroflexi, though reportedly highly abundant (up to 30% of total prokaryotes) in the extensive deep water habitats of both marine (SAR202) and freshwater (CL500-11), remain uncultivated and uncharacterized. There are few metagenomic studies on marine Chloroflexi representatives, while the pelagic freshwater Chloroflexi community is largely unknown except for a single metagenome-assembled genome of CL500-11.\n\nResultsHere we provide the first extensive examination of the community composition of this cosmopolitan phylum in a range of pelagic habitats (176 datasets) and highlight the impact of salinity and depth on their phylogenomic composition. Reconstructed genomes (53 in total) provide a perspective on the phylogeny, metabolism and distribution of three novel classes and two family-level taxa within the phylum Chloroflexi. We unraveled a remarkable genomic diversity of pelagic freshwater Chloroflexi representatives that thrive not only in the hypolimnion as previously suspected, but also in the epilimnion. Our results suggest that the lake hypolimnion provides a globally stable habitat reflected in lower species diversity among hypolimnion specific CL500-11 and TK10 clusters in distantly related lakes compared to a higher species diversity of the epilimnion specific SL56 cluster. Cell volume analyses show that the CL500-11 are amongst the largest prokaryotic cells in the water column of deep lakes and with a biomass:abundance ratio of two they significantly contribute to the deep lake carbon flow. Metabolic insights indicate participation of JG30-KF-CM66 representatives in the global cobalamin production via cobinamide to cobalamin salvage pathway.\n\nConclusionsExtending phylogenomic comparisons to brackish and marine habitats suggests salinity as the major influencer of the community composition of the deep-dwelling Chloroflexi in marine (SAR202) and freshwater (CL500-11) habitats as both counterparts thrive in intermediate brackish salinity however, freshwater habitats harbor the most phylogenetically diverse community of pelagic Chloroflexi representatives that reside both in epi- and hypolimnion.

microbiology↗

bioRxiv2018

Heliorhodopsins are absent in diderm (Gram-negative) bacteria: Some thoughts and possible implications for activity

Microbial heliorhodopsins are a new type of rhodopsins with an opposite membrane topology compared to type-1 and -2 rhodopsins, currently believed to engage in light sensing. We determined their presence/absence is monoderms and diderms representatives from the Tara Oceans and freshwater 25 lakes metagenomes. Heliorhodopsins were absent in diderms, confirming our previous observations in cultured Proteobacteria. Based on these observations, we speculate on the putative role of heliorhodopsins in light-driven transport of amphiphilic molecules.

microbiology↗