Protocols in Current Issue
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1 Q&A 247 Views Jan 5, 2023

Sclerotinia sclerotiorum causes white mold, leading to substantial losses on a wide variety of hosts around the world. Many genes encoding effector proteins play important roles in the pathogenesis of S. sclerotiorum. Therefore, establishment of a transformation system for the exploration of gene function is necessarily significant. Here, we introduce a modified protocol to acquire protoplasts for transformation and generate knockout strains, which completements the transformation system of S. sclerotiorum.

0 Q&A 189 Views Dec 20, 2022

Group A streptococcus (GAS) is a Gram-positive human pathogen that causes invasive infections with mild to life-threatening severity, like toxic shock syndrome, rheumatic heart disease, and necrotizing fasciitis (NF). NF is characterized by a clinical presentation of widespread tissue destruction due to the rapid spread of GAS infection into fascial planes. Despite quick medical interventions, mortality from NF is high. The early onset of the disease is difficult to diagnose because of non-specific clinical symptoms. Moreover, the unavailability of an effective vaccine against GAS warrants a genuine need for alternative treatments against GAS NF. One endoplasmic reticulum stress signaling pathway (PERK pathway) gets triggered in the host upon GAS infection. Bacteria utilize asparagine release as an output of this pathway for its pathogenesis. We reported that the combination of sub-cutaneous (SC) and intraperitoneal (IP) administration of PERK pathway inhibitors (GSK2656157 and ISRIB) cures local as well as systemic GAS infection in a NF murine model, by reducing asparagine release at the infection site. This protocol's methodology is detailed below.

0 Q&A 189 Views Dec 20, 2022

Periodontal disease is a chronic multifactorial disease triggered by a complex of bacterial species. These interact with host tissues to cause the release of a broad array of pro-inflammatory cytokines, chemokines, and tissue remodelers, such as matrix metalloproteinases (MMPs), which lead to the destruction of periodontal tissues. Patients with severe forms of periodontitis are left with a persistent pro-inflammatory transcriptional profile throughout the periodontium, even after clinical intervention, leading to the destruction of teeth-supporting tissues. The oral spirochete, Treponema denticola , is consistently found at significantly elevated levels at sites with advanced periodontal disease. Of all T. denticola virulence factors that have been described, its chymotrypsin-like protease complex, also called dentilisin, has demonstrated a multitude of cytopathic effects consistent with periodontal disease pathogenesis, including alterations in cellular adhesion activity, degradation of various endogenous extracellular matrix–substrates, degradation of host chemokines and cytokines, and ectopic activation of host MMPs. Thus, the following model of T. denticola –human periodontal ligament cell interactions may provide new knowledge about the mechanisms that drive the chronicity of periodontal disease at the protein, transcriptional, and epigenetic levels, which could afford new putative therapeutic targets.

0 Q&A 287 Views Dec 5, 2022

Pathogen invasion of the central nervous system (CNS) is an important cause of infection-related mortality worldwide and can lead to severe neurological sequelae. To gain access to the CNS cells, pathogens have to overcome the blood–brain barrier (BBB), a protective fence from blood-borne factors. To study host–pathogen interactions, a number of cell culture and animal models were developed. However, in vitro models do not recapitulate the 3D architecture of the BBB and CNS tissue, and in vivo mammalian models present cellular and technical complexities as well as ethical issues, rendering systematic and genetic approaches difficult. Here, we present a two-pronged methodology allowing and validating the use of Drosophila larvae as a model system to decipher the mechanisms of infection in a developing CNS. First, an ex vivo protocol based on whole CNS explants serves as a fast and versatile screening platform, permitting the investigation of molecular and cellular mechanisms contributing to the crossing of the BBB and consequences of infection on the CNS. Then, an in vivo CNS infection protocol through direct pathogen microinjection into the fly circulatory system evaluates the impact of systemic parameters, including the contribution of circulating immune cells to CNS infection, and assesses infection pathogenicity at the whole host level. These combined complementary approaches identify mechanisms of BBB crossing and responses of a diversity of CNS cells contributing to infection, as well as novel virulence factors of the pathogen.

Graphical abstract

Procedures flowchart.
Mammalian neurotropic pathogens could be tested in two Drosophila central nervous system (CNS) infection setups (ex vivo and in vivo) for their ability to: (1) invade the CNS (pathogen quantifications), (2) disturb blood–brain barrier permeability, (3) affect CNS host cell behaviour (gene expression), and (4) alter host viability.

0 Q&A 438 Views Nov 20, 2022

The study of haloarchaea provides an opportunity to expand understanding of the mechanisms used by extremophiles to thrive in and respond to harsh environments, including hypersaline and oxidative stress conditions. A common strategy used to investigate molecular mechanisms of stress response involves the deletion and/or site-directed mutagenesis of genes identified through omics studies followed by a comparison of the mutant and wild-type strains for phenotypic differences. The experimental methods used to monitor these differences must be controlled and reproducible. Current methods to examine recovery of halophilic archaea from extreme stress are complicated by extended incubation times, nutrients not typically encountered in the environment, and other related limitations. Here we describe a method for assessing the function of genes during hypochlorite stress in the halophilic archaeon Haloferax volcanii that overcomes these types of limitations. The method was found reproducible and informative in identifying genes needed for H. volcanii to recover from hypochlorite stress.

0 Q&A 731 Views Nov 20, 2022

Genome-wide screens using yeast or phage displays are powerful tools for identifying protein–ligand interactions, including drug or vaccine targets, ligand receptors, or protein–protein interactions. However, assembling libraries for genome-wide screens can be challenging and often requires unbiased cloning of 105–107 DNA fragments for a complete representation of a eukaryote genome. A sub-optimal genomic library can miss key genomic sequences and thus result in biased screens. Here, we describe an efficient method to generate genome-wide libraries for yeast surface display using Gibson assembly. The protocol entails genome fragmentation, ligation of adapters, library cloning using Gibson assembly, library transformation, library DNA recovery, and a streamlined Oxford nanopore library sequencing procedure that covers the length of the cloned DNA fragments. We also describe a computational pipeline to analyze the library coverage of the genome and predict the proportion of expressed proteins. The method allows seamless library transfer among multiple vectors and can be easily adapted to any expression system.

0 Q&A 972 Views Nov 20, 2022

Membrane transporters and soluble binding proteins recognize particular nutrients, metabolites, vitamins, or ligands. By modifying genetically engineered single cysteine residues near the active sites of such proteins with extrinsic maleimide fluorophores, the approaches that we report create sensitive fluorescent sensors that detect, quantify, and monitor molecules that are relevant to the biochemistry, physiology, microbiology, and clinical properties of pro- and eukaryotic organisms.

Graphical abstract:

0 Q&A 383 Views Nov 20, 2022

Babesiosis is a tick-borne disease caused by pathogens belonging to the genus Babesia. In humans, the disease presents as a malaria-like illness and can be fatal in immunocompromised and elderly people. In the past few years, human babesiosis has been a rising concern worldwide. The disease is transmitted through tick bite, blood transfusion, and transplacentally in rare cases, with several species of Babesia causing human infection. Babesia microti, Babesia duncani, and Babesia divergens are of particular interest because of their important health impact and amenability to research inquiries. B. microti, the most commonly reported Babesia pathogen infecting humans, can be propagated in immunocompetent and immunocompromised mice but so far has not been successfully continuously propagated in vitro in human red blood cells (hRBCs). Conversely, B. divergens can be propagated in vitro in hRBCs but lacks a mouse model to study its virulence. Recent studies have highlighted the uniqueness of B. duncani as an ideal model organism to study intraerythrocytic parasitism in vitro and in vivo. An optimized B. duncani in culture and in mouse (ICIM) model has recently been described, combining long-term continuous in vitro culture of the parasite in human red blood cells with an animal model of parasitemia (P) and lethal infection in C3H/HeJ mice. Here, we provide a detailed protocol for the use of the B. duncani ICIM model in research. This model provides a unique and sound foundation to gain further insights into the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites, and has been validated as an efficient system to evaluate novel strategies for the treatment of human babesiosis and possibly other parasitic diseases.

Graphical abstract:

ICIM model [Adapted and modified from Pal et al. (2022)]

0 Q&A 592 Views Nov 5, 2022

Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a ubiquitin-conjugating E2, and an E3 ubiquitin ligase. Cooperation between E2s and E3s is required for substrate ubiquitination, but some ubiquitin-conjugating E2s are also able to catalyze by themselves the formation of free di-ubiquitin, independently or in cooperation with a ubiquitin E2 variant. Here, we describe a method for assessing (i) di-ubiquitin formation by an E1 together with an E2 and an E2 variant, and (ii) the cooperation of an E3 with an E1 and E2 (with or without the E2 variant). Reaction products are assessed using western blotting with one of two antibodies: the first detects all ubiquitin conjugates, while the second specifically recognizes K63-linked ubiquitin. This allows unambiguous identification of ubiquitinated species and assessment of whether K63 linkages are present. We have developed these methods for studying ubiquitination proteins of Leishmania mexicana, specifically the activities of the E2, UBC2, and the ubiquitin E2 variant UEV1, but we anticipate the assays to be applicable to other ubiquitination systems with UBC2/UEV1 orthologues.

0 Q&A 690 Views Oct 20, 2022

The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM.

Graphical abstract:

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