Current Protocols in Microbiology最新文献

Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene-knockdown tool that uses an RNA-protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile-CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile-CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile-CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile-CRISPRi vectors, Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria, and ICEBs1 transfer of Mobile-CRISPRi to Bacillales. © 2020 The Authors.

Basic Protocol 1: sgRNA design

Basic Protocol 2: Cloning of new sgRNA spacers into Mobile-CRISPRi vectors

Basic Protocol 3: Tn7 transfer of Mobile-CRISPRi to Gram-negative bacteria

Basic Protocol 4: ICEBs1 transfer of Mobile-CRISPRi to Bacillales

Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters

Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates

Support Protocol 3: Transformation of E. coli by electroporation

Support Protocol 4: Transformation of CaCl₂-competent E. coli

Dimorphic fungi in the genera Blastomyces, Histoplasma, Coccidioides, and Paracoccidioides are important human pathogens that affect human health in many countries throughout the world. Understanding the biology of these fungi is important for the development of effective treatments and vaccines. Gene editing is a critically important tool for research into these organisms. In recent years, gene targeting approaches employing RNA-guided DNA nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), have exploded in popularity. Here, we provide a detailed description of the steps involved in applying CRISPR/Cas9 technology to dimorphic fungi, with Blastomyces dermatitidis in particular as our model fungal pathogen. We discuss the design and construction of single guide RNA and Cas9-expressing targeting vectors (including multiplexed vectors) as well as introduction of these plasmids into Blastomyces using Agrobacterium-mediated transformation. Finally, we cover the outcomes that may be expected in terms of gene-editing efficiency and types of gene alterations produced. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Construction of CRISPR/Cas9 targeting vectors

Support Protocol 1: Choosing protospacers in the target gene

Basic Protocol 2: Agrobacterium-mediated transformation of Blastomyces

Support Protocol 2: Preparation of electrocompetent Agrobacterium

Support Protocol 3: Preparation and recovery of Blastomyces frozen stocks

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium and opportunistic pathogen of humans and shrimp. Investigating the mechanisms of V. parahaemolyticus infection and the multifarious virulence factors it employs requires procedures for bacterial culture, genetic manipulation, and analysis of virulence phenotypes. Detailed protocols for growth assessment, generation of mutants, and phenotype assessment are included in this article. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Assessment of growth of V. parahaemolyticus

Alternate Protocol 1: Assessment of growth of V. parahaemolyticus using a plate reader

Basic Protocol 2: Swimming/swarming motility assay

Basic Protocol 3: Genetic manipulation

Alternate Protocol 2: Natural transformation

Basic Protocol 4: Secretion assay and sample preparation for mass spectrometry analysis

Basic Protocol 5: Invasion assay (gentamicin protection assay)

Basic Protocol 6: Immunofluorescence detection of intracellular V. parahaemolyticus

Basic Protocol 7: Cytotoxicity assay for T3SS2

Human papillomavirus (HPV) infection occurs in differentiating epithelial tissues. Cancers caused by high-risk types (e.g., HPV16 and HPV18) typically occur at oropharyngeal and anogenital anatomical sites. The HPV life cycle is differentiation-dependent, requiring tissue culture methodology that is able to recapitulate the three-dimensional (3D) stratified epithelium. Here we report two distinct and complementary methods for growing differentiating epithelial tissues that mimic many critical morphological and biochemical aspects of in vivo tissue. The first approach involves growing primary human epithelial cells on top of a dermal equivalent consisting of collagen fibers and living fibroblast cells. When these cells are grown at the liquid-air interface, differentiation occurs and allows for epithelial stratification. The second approach uses a rotating wall vessel bioreactor. The low-fluid-shear microgravity environment inside the bioreactor allows the cells to use collagen-coated microbeads as a growth scaffold and self-assemble into 3D cellular aggregates. These approaches are applied to epithelial cells derived from HPV-positive and HPV-negative oral and cervical tissues. The second part of the article introduces potential downstream applications for these 3D tissue models. We describe methods that will allow readers to start successfully culturing 3D tissues from oral and cervical cells. These tissues have been used for microscopic visualization, scanning electron microscopy, and large omics-based studies to gain insights into epithelial biology, the HPV life cycle, and host-pathogen interactions. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Establishing human primary cell–derived 3D organotypic raft cultures

Support Protocol 1: Isolation of epithelial cells from patient-derived tissues

Support Protocol 2: Growth and maintenance of primary human epithelial cells in monolayer culture

Support Protocol 3: PCR-based HPV screening of primary cell cultures

Basic Protocol 2: Establishing human 3D cervical tissues using the rotating wall vessel bioreactor

Support Protocol 4: Growth and maintenance of human A2EN cells in monolayer culture

Support Protocol 5: Preparation of the slow-turning lateral vessel bioreactor

Support Protocol 6: Preparation of Cytodex-3 microcarrier beads

Basic Protocol 3: Histological assessment of 3D organotypic raft tissues

Basic Protocol 4: Spatial analysis of protein expression in 3D organotypic raft cultures

Basic Protocol 5: Immunofluorescence imaging of RWV-derived 3D tissues

Basic Protocol 6: Ultrastructural visualization and imaging of RWV-derived 3D tissues

Basic Protocol 7: Characterization of gene expression by RT-qPCR

The fungus Zymoseptoria tritici is one of the most devastating pathogens of wheat. Aside from its importance as a disease-causing agent, this species has emerged as a powerful model system for evolutionary genetic studies of crop-infecting fungal pathogens. Z. tritici exhibits exceptionally high levels of genetic and phenotypic diversity as well as morphological plasticity, which can make experimental studies and comparability of results obtained in different laboratories, e.g., from infection assays, challenging. Therefore, standardized experimental methods are crucial for research on Z. tritici biology and the interaction of this fungus with its wheat host. Here, we describe a suite of well-tested and optimized protocols ranging from isolation of Z. tritici field specimens to analyses of virulence assays under controlled conditions. Several biological and technical aspects of working with Z. tritici under laboratory conditions are considered and carefully described in each protocol. © 2020 The Authors.

Basic Protocol 1: Purification of Z. tritici field isolates from leaf material

Basic Protocol 2: Molecular identification of Z. tritici isolates

Support Protocol 1: Rapid extraction of Z. tritici genomic DNA

Support Protocol 2: Extraction of high-quality Z. tritici genomic DNA

Basic Protocol 3: In vitro culture and long-term storage of Z. tritici isolates

Basic Protocol 4: Analysis of Z. tritici virulence in wheat

Support Protocol 3: Preparation of Z. tritici inoculum

Bordetella bronchiseptica is a gram-negative bacterium that causes respiratory tract infections. It is a natural pathogen of a wide variety of mammals, including some used as laboratory models. This makes B. bronchiseptica an ideal organism to study pathogen–host interactions in order to unveil molecular mechanisms behind pathogenic processes. Even though genetic engineering is an essential tool in this area, there are just a few reports about genome manipulation techniques in this organism. In this article we describe an allelic exchange protocol based on double crossover recombination facilitated by the Bacillus subtilis sacB gene that can be applied for partial or complete gene knockouts, single-nucleotide mutations, or even introduction of coding sequences for transcriptional fusions. In contrast to previously employed techniques, this protocol renders genetically manipulated chromosomes without foreign DNA and enables the construction of successive genome manipulation using the same vector backbone. The entire procedure has been developed for fast and reliable manipulations with a total duration of 2 weeks. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Setting up strains

Basic Protocol 2: Homologous recombination (first crossing-over)

Alternate Protocol: B. bronchiseptica electroporation

Basic Protocol 3: Screening for sucrose-sensitive clones

Basic Protocol 4: Homologous recombination (second crossing-over)

Basic Protocol 5: PCR screening of putative marker-exchange mutants

Support Protocol: Electrocompetent cell preparation

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira species that are maintained in sylvatic and domestic environments by transmission among rodents and other carriers. Humans become infected after contact of breached skin or mucosa with contaminated water or soil. Understanding persistent or sublethal infection in a host is critical for controlling human risk of exposure to pathogenic Leptospira. Animal models that recapitulate disease progression after infection via natural transmission routes are more appropriate for validation of vaccines and therapeutics. Furthermore, the ability to measure shedding of live Leptospira in urine of reservoir and carrier hosts can be used to develop new diagnostic assays and sensors to evaluate human risk of exposure. We developed inbred mouse models of Leptospirosis, that bypass survival as a criterion, in which we can analyze both pathogen and host factors affecting sublethal infection (<1 month), including shedding of Leptospira in urine. Mice are infected with pathogenic Leptospira using a physiologic route, and the clinical, histological, and molecular scores of disease are measured. Furthermore, the host immune response to Leptospira is evaluated. This mouse model also provides a tool in which to test fundamental hypotheses related to host-pathogen interactions and the immune mechanisms engaged in protective and pathogenic immune responses. © 2020 Wiley Periodicals LLC

Basic Protocol 1: Culture and maintenance of virulent Leptospira

Basic Protocol 2: Infection of mice through a physiologic route and collection of clinical scores and biological samples

Basic Protocol 3: Analysis of pathogenesis after Leptospira infection

Candida albicans is a common mucosal colonizer, as well as a cause of lethal invasive fungal infections. The major predisposing factor for invasive fungal disease is a compromised immune system. One component of the host immune response to fungal infection is the activation of the inflammasome, a multimeric protein complex that is critical for regulating host pro-inflammatory responses. Here, we describe methods for investigating the interactions between C. albicans and host macrophages, with a focus on the inflammasome. C. albicans isolates differ in the degree to which they activate the inflammasome due to differences in internalization, morphogenic switching, and inflammasome priming. Therefore, we include protocols for identifying these factors. This simple in vitro model can be used to elucidate the contributions of specific C. albicans strains or mutants to different aspects of interactions with macrophages. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Measuring inflammasome priming in response to Candida albicans

Basic Protocol 2: Measuring inflammasome activation in response to Candida albicans

Support Protocol: Controlling for phagocytosis

Yersinia pseudotuberculosis has been studied for many decades, and research on this microbe has taught us a great deal about host-pathogen interactions, bacterial manipulation of host cells, virulence factors, and the evolution of pathogens. This microbe should not be cultivated at 37°C because this is a trigger that the bacterium uses to sense its presence within a mammalian host and results in expression of genes necessary to colonize a mammalian host. Prolonged growth at this temperature can result in accumulation of mutations that reduce the virulence of the strain, so all protocols need to be modified for growth at room temperature, or 26°C. This article describes protocols for cultivating this microbe and for its long-term storage and its genetic manipulation by transformation and conjugation. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Growth of Y. pseudotuberculosis from a stock

Basic Protocol 2: Growth of Y. pseudotuberculosis in liquid medium from a single colony

Basic Protocol 3: Freezing Y. pseudotuberculosis in glycerol for long-term storage

Basic Protocol 4: Transformation of Y. pseudotuberculosis by electroporation

Basic Protocol 5: Tri-parental mating/conjugation

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