Biology subjects

Pokhrel, A.

Publications and source records attributed to Pokhrel, A..

12 recordsLinked to original sources

bioRxiv2026

Comparative genomics reveals shared accessory regions between members of two Fusarium species complexes virulent on garden pea

The contribution of accessory or conditionally dispensable chromosomes to host-specific virulence was first demonstrated in members of the Fusarium solani species complex (FSSC) that are pathogens of garden pea, Pisum sativum L. The phenomenon has since been shown to exist in many fungal plant pathogens, including the closely related F. oxysporum species complex (FOSC). Genome analysis of members of the FSSC and FOSC pathogenic on pea revealed a diverse size range of the accessory genome of these fungi. Despite the [~]65 million years of diverging time, regions on a chromosome known to carry host-specific virulence factors for pea, including the cytochrome P450 pisatin demethylase (PDA) and other pea pathogenicity (PEP) genes, were present in all genomes of these pea pathogens. Genes directly involved in virulence on pea - PEP2, PDA, and PEP5- were the most frequently clustered together. Transcriptome analysis of fungal mycelia treated with the pea phytoalexin pisatin, identified 1,155 differentially expressed genes where many were involved in cellular stress responses. As wilt pathogens that invade host xylem, members of the FOSC encode more putative effectors, when compared to those in the FSSC, and several FOSC effectors were identified to confer race specificity. The conservation of part of the accessory genomes across two evolutionarily diverged species complexes suggests a common origin. Horizontal transfer of accessory chromosomes containing genetic loci involved in pathogenesis for garden pea offers a parsimonious explanation of the polyphyletic origin of host specificity.

genomics↗

bioRxiv2025

Invasion and intracellular proliferation dynamics of non-pathogenic E. coli in model infection systems

Uropathogens are typically distinguished from commensal or laboratory E. coli strains by defined pathogenic behaviours. In this study, we compared the behaviour of non-pathogenic Escherichia coli to that of uropathogenic E. coli in diverse infection models at a single-cell level using live-cell fluorescence microscopy. Using in vitro microfluidics and ex vivo mouse urinary tract infection model systems, we demonstrated that the standard laboratory E. coli strain MG1655 can behave like a pathogen, invading and proliferating inside human and mouse urothelial cells. Upon exposure to stress by the addition of urine, we observed that MG1655 undergoes infection-related filamentation, and then subsequent reversal back to rod shape when the stress is removed. This observation supports that infection-related filamentation should be regarded as a stress response rather than a virulence factor. Taken together, our results show that under infection-favourable circumstances, even a well-studied non-pathogenic laboratory E. coli strain can exhibit what are considered pathogenic traits.

microbiology↗

bioRxiv2025

Cell-substrate friction controls biofilm development

Bacteria often live in biofilms, surface-attached communities that can form on nearly any surface, from coarse sands to smooth glass. It is often hypothesized that cell-substrate friction can impact biofilm growth and development on these disparate surfaces. However, the experimental difficulty in measuring the friction between a biofilm and its surface has limited our understanding of how friction forces impact emergent colony-level behaviors and morphologies. Here, we demonstrate that increasing cell-substrate friction increases the biofilm contact angle, which, in turn, decreases the horizontal biofilm range expansion rate. We first used individual-based simulations to isolate the impact of friction. In this simple model, we found that increasing friction increases biofilm contact angle, and, in turn, that the contact angle emerges from a simple dynamic force balance between cell-substrate friction, cell-cell steric forces, and cell-cell adhesion, reminiscent of Youngs equation for sessile liquid drops. We developed an approach to directly measure the friction between bacterial colonies and agar surfaces, and found that the friction between the substrate and the biofilm is higher on substrates made with higher agar weight percentage. Additionally, we observed that biofilm contact angles increase with friction, following a Young-equation-like balance. Finally, we found that contact angle increases with agar percentage for a wide range of bacteria, suggesting that the biophysical impact of friction may play a role for a wide array of microbes.

biophysics↗

bioRxiv2025

NCR13 peptide protects soybean against Cercospora sojina by multiple modes of action and additive interaction with chemical fungicides

Frogeye Leaf Spot (FLS) disease, caused by a fungal pathogen Cercospora sojina, is a serious threat to soybean production globally. The control of FLS is facing a major challenge due to the rapid emergence of pathogen resistance to Quinone outside Inhibitor (QoI) fungicides. Effective long-term management of FLS in soybean calls for the discovery of antifungal compounds with new modes of action (MoA), durability and safety. Here, we showed that chickpea nodule-specific cysteine-rich peptide, NCR13_Peptide Folding Variant1 (NCR13_PFV1), exhibited antifungal activity against QoI-sensitive and -resistant field isolates of C. sojina at nanomolar concentrations representing the first antifungal NCR peptide reported effective against C. sojina. Spray-application of this peptide showed no phytotoxicity and effectively protected soybean against FLS. When combined with the QoI fungicide azoxystrobin, NCR13_PFV1 provided additive control of FLS. NCR13_PFV1 induced plasma membrane disruption and production of reactive oxygen species (ROS) in C. sojina. NCR13_PFV1 was rapidly internalized into fungal cells where it accumulated in the cytoplasm, localized inside nucleus, bound to fungal ribosomal RNA and inhibited protein translation in vivo. RNA-seq studies revealed the upregulation of several genes encoding heme binding proteins in peptide-challenged C. sojina. Notably, iron supplementation in the growth medium reduced the peptide-induced ROS and antifungal activity, revealing the importance of iron homeostasis in protection or recovery of C. sojina from oxidative stress. Overall, NCR13_PFV1 with multiple MoA holds potential as a bio-fungicide for FLS control complementing conventional QoI fungicides and overcoming fungicide resistance in C. sojina.

plant biology↗

bioRxiv2024

Modes of action and bio-fungicide potential of peptides derived from the bi-domain plant defensin MtDef5

Medicago truncatula bi-domain defensin MtDef5 exhibits antifungal activity at sub-micromolar concentrations against some fungal pathogens. It comprises two single-domain defensins, MtDef5A and MtDef5B, connected with a linker APKKVEP. MtDef5B is a more potent antifungal defensin than MtDef5A. We identified amino acid residues important for antifungal activity of MtDef5B and elucidated its modes of action (MoA). MtDef5B inhibited spore germination of Botrytis cinerea (Bc) at low micromolar concentrations. However, it did not inhibit spore germination of Colletotrichum gloeosporioides (Cg). MtDef5B permeabilized the plasma membrane, induced reactive oxygen species and traveled to the nucleoli in germlings of Bc. Furthermore, a carboxy-terminal MtDef5A-derived GMA5AC peptide was selected for mutagenesis because of its lower cationicity than the corresponding MtDef5B-derived peptide. GMA5AC inhibited spore germination of Bc, but not of Cg. However, GMA5AC_V2, a variant of GMA5AC, inhibited spore germination of Cg and exhibited multi-faceted MoA. Spray-application of GMA5AC_V2 on the leaves of pepper plants demonstrated preventive and curative control of the gray mold disease. Furthermore, when applied topically on tomato fruits pre-inoculated with the pathogen Cg, this peptide reduced anthracnose disease symptoms. This study highlights the potential of short chain defensin-derived peptides for management of fungal diseases. HighlightsThe bi-domain MtDef5-derived antifungal peptides exhibit multiple modes of action and confer resistance against the gray mold and anthracnose diseases in pepper plants and tomato fruits, respectively.

pathology↗

bioRxiv2024

Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action

Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted-repeat lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form 2 or 3 intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +9 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea, causal agent of gray mold disease in fruits, vegetables and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides structural framework for design of novel, potent antifungal peptides for agricultural use. Author SummaryFungal pathogens cause significant pre-harvest and post-harvest losses of crop yield, making them a serious biological threat to global food security. Chemical fungicides are effective in controlling fungal diseases across various crops. However, rapid evolution of fungal pathogen resistance to single-site chemical fungicides in agriculture has created an urgent need for development of safe, sustainable, and cost-effective multi-site fungicides. Nodule-specific cysteine-rich (NCR) peptides expressed in the inverted-repeat lacking clade legume plants exhibit potent antifungal activity; however, their modes of action (MoA) are poorly understood. Particularly, the specific contribution of disulfide pairing to the potency and spectrum of antifungal activity against fungal plant pathogens and MoA of these peptides remains to be identified. Chickpea NCR13 expressed in P. pastoris generates two peptide variants that differ in their disulfide cross-linking pattern. These variants exhibit striking differences in their three-dimensional structures and potency of antifungal activity against multiple fungal pathogens and MoA. They also differ in their ability to confer resistance to gray mold in pepper and tomato plants. Our study highlights the major impact a specific pattern of disulfide pairing can have on the in vitro and in planta antifungal activity of an NCR peptide.

plant biology↗

bioRxiv2024

E. coli division machinery drives cocci development inside host cells

Escherichia coli is arguably one of the most studied bacterial model systems in modern biology. Under normal laboratory conditions E. coli adopts its characteristic rod-shape. However, during stress conditions E. coli has been shown to undergo conditional morphology changes to inhibit division and grow into highly elongated forms. Here, on the other end of the morphology spectra, using an in-vitro infection model system combined with advanced imaging we show uropathogenic E. coli rods dividing to form and proliferate as cocci inside human bladder epithelial cells. In these intracellular bacterial communities, the frequency of cell division outpaced the rate of cell growth, resulting in smaller cocci cells. This mechanism was guided by an active FtsZ-governed division machinery, directed to midcell by division-site placement systems. These results show how a previously uncharacterised level of morphological plasticity occurs in bacteria with traditionally well-defined rod shape.

microbiology↗

bioRxiv2023

The 3'UTR of vigR is required for virulence in Staphylococcus aureus and has expanded through STAR sequence repeat insertions

Staphylococcus aureus is an adaptable human pathogen causing life-threatening endocarditis and bacteraemia. Methicillin-resistant S. aureus (MRSA) is alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteraemia and vancomycin treatment failure is often associated with vancomycin-intermediate S. aureus strains termed VISA. The regulatory 3 UTR of vigR mRNA contributes to vancomycin tolerance in the clinical VISA isolate JKD6008 and upregulates the lytic transglycosylase IsaA. Using MS2-affinity purification coupled with RNA sequencing (MAPS), we find that the vigR 3 UTR also interacts with mRNAs involved in carbon metabolism, amino acid biogenesis, cell wall biogenesis, and virulence. The vigR 3 UTR was found to repress dapE, a succinyl-diaminopimelate desuccinylase required for lysine and cell wall peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the vigR 3 UTR increased VISA virulence in a wax moth larvae model, and we find that an isaA mutant is completely attenuated in the larvae model. Sequence and structural analysis of the vigR 3 UTR indicates that the UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions (STAR repeats) that partly contribute sequence for the isaA interaction seed and may functionalise the 3 UTR. Our findings reveal an extended regulatory network for vigR, uncovering a novel mechanism of regulation of cell wall metabolism and virulence in a clinical S. aureus isolate.

microbiology↗

bioRxiv2023

Application of nanotags and nanobodies for live cell single-molecule imaging of the Z-ring in Escherichia coli

Understanding where proteins are localized in a bacterial cell is essential for understanding their function and regulation. This is particularly important for proteins that are involved in cell division, which localize at the division septum and assemble into highly regulated complexes. Current knowledge of these complexes has been greatly facilitated by super-resolution imaging using fluorescent protein fusions. Herein we demonstrate with FtsZ that single-molecule PALM images can be obtained in-vivo using a genetically fused nanotag (ALFA) and a corresponding nanobody fused to mEos3.2. The methodology presented is applicable to other bacterial proteins.

microbiology↗

bioRxiv2023

Direct visualization of bacterial multispecies infection and symbiotic interactions within human bladder epithelial cells

Urinary tract infections (UTI) are one of the most common bacterial infections worldwide. While the overall infection course is known on a macroscale, bacterial behavior is not fully understood at the cellular level and bacterial pathophysiology during multispecies infection is not well characterized. Here we establish co-infection models combined with high resolution imaging to compare single- and multi-species bladder cell invasion events in three common uropathogens: uropathogenic Escherichia coli (UPEC), Klebsiella pneumoniae and Enterococcus faecalis. While all three species invaded the bladder cells, under flow conditions the Gram-positive E. faecalis was significantly less invasive compared to the Gram-negative UPEC and K. pneumoniae. When introduced simultaneously during an infection experiment, all three bacterial species sometimes invaded the same bladder cell, at differing frequencies suggesting complex interactions between bacterial species and bladder cells. Inside host cells, we observed encasement of E. faecalis colonies specifically by UPEC. During subsequent dispersal from the host cells, only the Gram-negative bacteria underwent infection-related filamentation (IRF). Taken together, our data suggest that bacterial multispecies invasions of single bladder cells are frequent and support earlier studies showing intraspecies cooperation on a biochemical level during UTI.

microbiology↗

bioRxiv2021

Engineering Bacillus subtilis for the formation of a durable living biocomposite material

Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis was engineered to display SpyTags on polar flagella for cell attachment and cross-linking of SpyCatcher modified secreted scaffolds. Through deletion of the autolysis LytC, endospore limited B. subtilis cells become a structural component of the material with spores for long-term storage of genetic programming. Known silica biomineralization peptides were screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of silica material and that new functions can be readily incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs as functional, self-healing materials for use as responsive coatings and plasters.

synthetic biology↗

bioRxiv2021

Unique features of magic spot metabolism in Clostridioides difficile

The magic spot alarmones (pp)pGpp, previously implicated in Clostridioides difficile antibiotic survival, are synthesized by CdRSH and CdRelQ. These enzymes are transcriptionally activated by diverse environmental stresses, but both exclusively synthesize pGpp rather than ppGpp as has previously been reported. While direct synthesis of pGpp from a GMP substrate and (p)ppGpp hydrolysis into pGpp by NUDIX hydrolases have previously been reported, there is no precedent for a bacterium synthesizing pGpp exclusively. Hydrolysis of the 5 phosphate or pyrophosphate from GDP or GTP substrates is necessary for activity by the clostridial enzymes, neither of which can utilize GMP as a substrate. Both enzymes are remarkably insensitive to the size of their metal ion cofactor, tolerating a broad array of metals that do not allow activity in (pp)pGpp synthetases from other organisms. It is clear that while C. difficile utilizes magic spot signaling, its mechanisms of alarmone synthesis are not directly homologous to those in more completely characterized organisms.

microbiology↗