Current Protocols in Microbiology最新文献

Integration of the human papillomavirus (HPV) genome into host cell chromosomes has been observed in a majority of HPV-positive cervical cancers and a subset of oral HPV-associated cancers. HPV integration also occurs in long-term cell culture. Screening for HPV integration can be labor intensive and yield results that are difficult to interpret. Here we describe an assay based on exonuclease V (ExoV/RecBCD) and quantitative polymerase chain reaction (qPCR) to determine if samples from cell lines and tissues contain episomal or integrated HPV. This assay can be applied to screen other small DNA viruses with episomal/linear genome configurations in their viral lifecycle and has the potential to be used in clinical settings to define viral genomic conformations associated with disease. © 2020 Wiley Periodicals LLC.

Basic Protocol: Exonuclease V genomic DNA digestion and qPCR for detection of HPV16 genome configuration in cells

Support Protocol: Exonuclease V analysis of HPV16 genome configuration in tissues

Alternate Protocol: Determining HPV integration type or integrity of HPV episome

Freshwater planarians are a powerful model organism for the study of animal regeneration, stem cell maintenance and differentiation, and the development and functions of several highly conserved complex tissues. At the same time, planarians are easy to maintain, inexpensive to propagate, and reasonably macroscopic (1 mm to 1 cm in length), making them excellent organisms to use in both complex academic research and hands-on teaching laboratories. Here, we provide a detailed description of how to maintain and propagate these incredibly versatile animals in any basic laboratory setting. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Salt solution preparation

Basic Protocol 2: Cleaning planarian housing

Basic Protocol 3: Food preparation

Basic Protocol 4: Feeding planarians

Basic Protocol 5: Expansion and amplification of colony

A technique to assess the ability of distinct Candida strains to efflux substrates, as well as to compare the effectiveness of efflux inhibitors, is important for analysis of antifungal drug resistance mechanisms and the mode of action of antifungals. We describe a method that measures the ability of Candida species to extrude the fluorescent dye Nile red as an output for efflux activity. This involves exposing cells to Nile red and using flow cytometry to quantify cellular fluorescence, enabling numerous samples to be processed in a limited time frame. This protocol provides a simple, yet effective method for quantifying efflux in drug-resistant Candida species. © 2020 Wiley Periodicals LLC

Basic Protocol 1: Growth and sample preparation of stained Candida

Basic Protocol 2: Quantitative measurement of fluorescence by flow cytometry

Alternate Protocol: Qualitative determination of fluorescence using microscopy

SARS-CoV-2, the causative agent of COVID-19, has been responsible for a million deaths worldwide as of September 2020. At the time of this writing, there are no available US FDA−approved therapeutics for the treatment of SARS-CoV-2 infection. Here, we describe a detailed protocol to generate recombinant (r)SARS-CoV-2 using reverse-genetics approaches based on the use of a bacterial artificial chromosome (BAC). This method will allow the production of mutant rSARS-CoV-2—which is necessary for understanding the function of viral proteins, viral pathogenesis and/or transmission, and interactions at the virus-host interface—and attenuated SARS-CoV-2 to facilitate the discovery of effective countermeasures to control the ongoing SARS-CoV-2 pandemic. © 2020 Wiley Periodicals LLC.

Basic Protocol: Generation of recombinant SARS-CoV-2 using a bacterial artificial chromosome

Support Protocol: Validation and characterization of rSARS-CoV-2

Germination is an important developmental process that supports resumption of growth in dormant spores. The study of the mechanisms underlying germination requires a pure spore population devoid of other cell types. This article describes the sporulation of wild Saccharomyces cerevisiae and Saccharomyces paradoxus strains, and the isolation and purification of ascospores. We also describe a method to synchronously induce germination in a spore population as well as to measure spore activation. This procedure can be applied, for example, to the study of environmental conditions that trigger germination. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Sporulation

Basic Protocol 2: Spore purification

Basic Protocol 3: Germination induction

Support Protocol 1: Flow cytometry analysis

Support Protocol 2: Heat-shock resistance measurement

Giardia is an enteric protozoan parasite that causes gastroenteritis in all classes of vertebrates. It is ranked among the leading causes of death in children under 5 years of age. Giardiasis affects approximately 280 million people worldwide annually, a situation exacerbated by the low availability of effective treatments and the lack of a vaccine. In addition, the parasite is difficult to manipulate in in vitro environments, which hampers the development of effective disease management strategies. This article highlights the development of a method for the purification of viable Giardia cysts from fecal samples, verified by a trypan blue dye exclusion test. This protocol produces a 10-fold increase in yield over current methods. By combining sucrose flotation with gated filtration, the protocol significantly reduces the amount of debris in the purified cysts suspension. Cyst viability is verified by a trypan blue dye exclusion test. The ability to purify large quantities of Giardia from fecal samples could advance the development of effective treatments to target this worldwide prevalent parasite. © 2020 Wiley Periodicals LLC.

Basic Protocol: Purification of Giardia cysts from fecal samples

Support Protocol: Cyst viability test

Tick-borne viruses cause thousands of cases of disease worldwide every year. Specific countermeasures to many tick-borne viruses are not commercially available. Very little is known regarding tick-virus interactions and increasing this knowledge can lead to potential targets for countermeasure development. Virus infection of ex vivo organ cultures from ticks can provide an approach to identify susceptible cell types of tissue to infection. Additionally, these organ cultures can be used for functional genomic studies to pinpoint tick-specific genes involved in the virus lifecycle. Provided here are step-by-step procedures to set up basic tick organ cultures in combination with virus infection and/or functional genomic studies. These procedures can be adapted for future use to characterize other tick-borne pathogen infections as well as tick-specific biological processes. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Loading 96-well plates with gelfoam substrate

Basic Protocol 2: Step-by-step aseptic dissection of unfed female/male Ixodes scapularis ticks for multiple organs

Basic Protocol 3: Step-by-step aseptic dissection of fed female Ixodes scapularis ticks to remove salivary glands

Basic Protocol 4: Metabolic viability analyses of tick organ cultures

Basic Protocol 5: Virus infection of tick organ cultures

Basic Protocol 6: Functional RNA interference analyses using tick organ cultures

Cryptococcus neoformans, an opportunistic yeast-like fungal pathogen, has demonstrated resistance to all major classes of antifungals used to treat cryptococcal meningitis. However, combatting this fungal disease is an ongoing challenge among clinicians due to the evolution of antifungal-resistant strains. The limited availability of clinically approved antifungals has heightened the urgency to investigate the molecular mechanisms underscoring resistance. Studying how a fungal pathogen evolves to an antifungal drug in vitro using experimental evolution provides a simple, yet powerful approach to study the mechanisms of antifungal resistance. Experimental evolution involves the serial passaging of microbial populations under laboratory conditions, such that adaptive mutations can occur and be monitored in real time. This technique plays a key role in investigating the mechanisms of antifungal resistance in C. neoformans, and this can help in developing novel strategies to combat the emergence of resistance. Here, we outline how to make overnight cultures of C. neoformans and how to perform experimental evolution, and we present a spectrophotometric analysis to evaluate the evolution of antifungal resistance. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Growth and sample preparation of Cryptococcus neoformans

Basic Protocol 2: Experimental evolution of antifungal resistance

Basic Protocol 3: Analyzing the evolution of antifungal resistance

Basic Protocol 4: Glycerol stock preparation

Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species. The bacterium has been used as a model organism to study symbiosis in the context of its association with its host, the Hawaiian bobtail squid Euprymna scolopes. The genetic tractability of this bacterium has facilitated the mapping of pathways that mediate interactions between these organisms. The protocols included here describe methods for genetic manipulation of V. fischeri. Following these protocols, the researcher will be able to introduce linear DNA via transformation to make chromosomal mutations, to introduce plasmid DNA via conjugation and subsequently eliminate unstable plasmids, to eliminate antibiotic resistance cassettes from the chromosome, and to randomly or specifically mutagenize V. fischeri with transposons. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Transformation of V. fischeri with linear DNA

Basic Protocol 2: Plasmid transfer into V. fischeri via conjugation

Support Protocol 1: Removing FRT-flanked antibiotic resistance cassettes from the V. fischeri genome

Support Protocol 2: Eliminating unstable plasmids from V. fischeri

Alternate Protocol 1: Introduction of exogenous DNA using a suicide plasmid

Alternate Protocol 2: Site-specific transposon insertion using a suicide plasmid

Alternate Protocol 3: Random transposon mutagenesis using a suicide plasmid

Candida albicans is an opportunistic fungal pathogen and a model organism to study fungal pathogenesis. It exists as a harmless commensal organism and member of the healthy human microbiome, but can cause life-threatening mucosal and systemic infections. A model host to study C. albicans infection and pathogenesis is the nematode Caenorhabditis elegans. C. elegans is frequently used as a model host to study microbial-host interactions because it can be infected by many human pathogens and there are also close morphological resemblances between the intestinal cells of C. elegans and mammals, where C. albicans infections can occur. This article outlines a detailed methodology for exploiting C. elegans as a host to study C. albicans infection, including a C. elegans egg preparation protocol and an agar-based C. elegans killing protocol to monitor fungal virulence. These protocols can additionally be used to study C. albicans genetic mutants in order to further our understanding of the genes involved in pathogenesis and virulence in C. albicans and the mechanisms of host-microbe interactions. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Preparation of Caenorhabditis elegans eggs

Support Protocol 1: Freezing and recovering Caenorhabditis elegans

Support Protocol 2: Making superfood agar and OP50 plates

Basic Protocol 2: Caenorhabditis elegans/Candida albicans agar killing assay

Support Protocol 3: Constructing a worm pick