Curr Protoc Microbiol [JOURNAL]
Sun
200 papers
RSS
Smith MR, Schirtzinger EE, Wilson WC
… +1 more
, Davis AS
Curr Protoc Microbiol
· 2019 Dec · PMID 31763765
·
Publisher ↗
Rift Valley fever virus (RVFV) is an arthropod-borne, zoonotic disease endemic to sub-Saharan Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever have had up to 100% mortality rates in fetal and neonatal she...
Rift Valley fever virus (RVFV) is an arthropod-borne, zoonotic disease endemic to sub-Saharan Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever have had up to 100% mortality rates in fetal and neonatal sheep. Upon infection of ruminant and human hosts alike, RVFV infection causes an at times severe hepatitis and pathology in many other organs. The enveloped virion contains a tripartite, predominantly negative-sense, single-stranded RNA genome, which codes for the proteins the virus needs to replicate both in mammalian hosts and insect vectors. Endemic countries often use attenuated RVFV strains for vaccination of livestock but there are no commercially licensed vaccines for humans or livestock in non-endemic areas. In the laboratory, RVFV can be readily propagated and manipulated in vitro using cell culture systems. Presented in this article are techniques routinely used in RVFV research that have proven successful in our laboratories. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Propagation of Rift Valley fever virus in mammalian cells Basic Protocol 2: Quantification of Rift Valley fever virus by plaque assay Basic Protocol 3: Quantification of Rift Valley fever virus by 50% tissue culture infectious dose (TCID ) assay Basic Protocol 4: Quantification of Rift Valley fever virus by focus-forming assay Basic Protocol 5: Storage and disinfection Alternate Protocol 1: Propagation of Rift Valley fever virus in MRC-5 cells Alternate Protocol 2: Propagation of RVFV in mosquito-derived cells Alternate Protocol 3: TCID detection using fluorescence visualization Support Protocol 1: Calculation of the amount of virus needed to infect a flask at a chosen multiplicity of infection Support Protocol 2: Calculation of the virus titer by plaque assay or focus-forming assay Support Protocol 3: Calculation of the TCID titer by the method of Reed and Muench Support Protocol 4: Calculation of the antibody volume for the focus-forming assay.
Ashton GD, Dyer PS
Curr Protoc Microbiol
· 2019 Sep · PMID 31518066
·
Full text
Aspergillus fumigatus is an opportunistic human fungal pathogen, capable of causing invasive aspergillosis in patients with compromised immune systems. The fungus was long considered a purely asexual organism. However, a...
Aspergillus fumigatus is an opportunistic human fungal pathogen, capable of causing invasive aspergillosis in patients with compromised immune systems. The fungus was long considered a purely asexual organism. However, a sexual cycle was reported in 2009, with methods described to induce mating under laboratory conditions. The presence of a sexual cycle now offers a valuable tool for classical genetic analysis of the fungus, such as allowing determination of whether traits of interest are mono- or poly-genic in nature. For example, the sexual cycle is currently being exploited to determine the genetic basis of traits of medical importance such as resistance to azole antifungals and virulence, and to characterize the genes involved. The sexual cycle can also be used to assess the possibility of gene flow between isolates. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. This unit describes protocols for culturing of A. fumigatus and for inducing sexual reproduction between compatible MAT1-1 and MAT1-2 isolates of the species. The unit also provides working methods for harvesting sexual structures, isolating single-spore progeny and confirming whether sexual recombination has occurred. © The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Gallagher JR, Kim AJ, Gulati NM
… +1 more
, Harris AK
Curr Protoc Microbiol
· 2019 Sep · PMID 31518065
·
Full text
Negative-stain transmission electron microscopy (EM) is a technique that has provided nanometer resolution images of macromolecules for about 60 years. Developments in cryo-EM image processing have maximized the informat...
Negative-stain transmission electron microscopy (EM) is a technique that has provided nanometer resolution images of macromolecules for about 60 years. Developments in cryo-EM image processing have maximized the information gained from averaging large numbers of particles. These developments can now be applied back to negative-stain image analysis to ascertain domain level molecular structure (10 to 20 Å) more quickly and efficiently than possible by atomic resolution cryo-EM. Using uranyl acetate stained molecular complexes of influenza hemagglutinin bound to Fab 441D6, we describe a simple and efficient means to collect several hundred micrographs with SerialEM. Using RELION, we illustrate how tens of thousands of complexes can be auto-picked and classified to accurately describe the domain level topology of this unconventional hemagglutinin head-domain epitope. By comparing to the cryo-EM density map of the same complex, we show that questions about epitope mapping and conformational heterogeneity can readily be answered by this negative-stain method. © 2019 The Authors.
Zhao C, Fraczek MG, Dineen L
… +7 more
, Lebedinec R, Macheleidt J, Heinekamp T, Delneri D, Bowyer P, Brakhage AA, Bromley M
Curr Protoc Microbiol
· 2019 Sep · PMID 31518064
·
Full text
Aspergillus fumigatus is a human pathogen and the principal etiologic agent of invasive and chronic aspergillosis leading to several hundreds of thousands of deaths every year. Very few antifungals are available to treat...
Aspergillus fumigatus is a human pathogen and the principal etiologic agent of invasive and chronic aspergillosis leading to several hundreds of thousands of deaths every year. Very few antifungals are available to treat infections caused by A. fumigatus, and resistance is developing to those we have. Our understanding of the molecular mechanisms that drive pathogenicity and drug resistance have been hampered by the lack of large mutant collections, which limits our ability to perform functional genomics analysis. Here we present a high-throughput gene knockout method that combines a highly reproducible fusion PCR method to enable generation of gene replacement cassettes with a multiwell format transformation procedure. This process can be used to generate 96 null mutants within 5 days by a single person at a cost of less than £18 ($24) per mutant and is being employed in our laboratory to generate a barcoded genome-wide knockout library in A. fumigatus. © 2019 The Authors.
Thesseling FA, Bircham PW, Mertens S
… +2 more
, Voordeckers K, Verstrepen KJ
Curr Protoc Microbiol
· 2019 Sep · PMID 31518063
·
Full text
Beer would not exist without microbes. During fermentation, yeast cells convert cereal-derived sugars into ethanol and CO . Yeast also produces a wide array of aroma compounds that influence beer taste and aroma. The com...
Beer would not exist without microbes. During fermentation, yeast cells convert cereal-derived sugars into ethanol and CO . Yeast also produces a wide array of aroma compounds that influence beer taste and aroma. The complex interaction between all these aroma compounds results in each beer having its own distinctive palette. This article contains all protocols needed to brew beer in a standard lab environment and focuses on the use of yeast in beer brewing. More specifically, it provides protocols for yeast propagation, brewing calculations and, of course, beer brewing. At the end, we have also included protocols for analyses that can be performed on the resulting brew, with a focus on yeast-derived aroma compounds. © 2019 The Authors.
Fraczek MG, Zhao C, Dineen L
… +4 more
, Lebedinec R, Bowyer P, Bromley M, Delneri D
Curr Protoc Microbiol
· 2019 Sep · PMID 31518062
·
Full text
Aspergillus fumigatus is an opportunistic human pathogenic mold. DNA extraction from this fungus is usually performed by mechanical perturbation of cells, as it possesses a rigid and complex cell wall. While this is not...
Aspergillus fumigatus is an opportunistic human pathogenic mold. DNA extraction from this fungus is usually performed by mechanical perturbation of cells, as it possesses a rigid and complex cell wall. While this is not problematic for single isolates, it can be time consuming for large numbers of strains if using traditional DNA extraction procedures. Therefore, in this article we describe a fast and efficient thermal-shock method to release DNA from spores of A. fumigatus and other filamentous fungi without the need for complex extraction methods. This is especially important for high-throughput PCR analyses of mutants in 96- or 384-well formats in a very short period of time without any concern about sample cross-contamination. This method is currently being used to validate the protein-coding gene and non-coding RNA knockout libraries in A. fumigatus generated in our laboratory, and could be used in the future for diagnostics purposes. © 2019 The Authors.
Gulati NM, Torian U, Gallagher JR
… +1 more
, Harris AK
Curr Protoc Microbiol
· 2019 Jun · PMID 31219685
·
Full text
Immunoelectron microscopy is a powerful technique for identifying viral antigens and determining their structural localization and organization within vaccines and viruses. While traditional negative staining transmissio...
Immunoelectron microscopy is a powerful technique for identifying viral antigens and determining their structural localization and organization within vaccines and viruses. While traditional negative staining transmission electron microscopy provides structural information, identity of components within a sample may be confounding. Immunoelectron microscopy allows for identification and visualization of antigens and their relative positions within a particulate sample. This allows for simple qualitative analysis of samples including whole virus, viral components, and viral-like particles. This article describes methods for immunogold labeling of viral antigens in a liquid suspension, with examples of immunogold-labeled influenza virus glycoproteins, and also discusses the important considerations for sample preparation and determination of morphologies. Together, these methods allow for understanding the antigenic makeup of viral particulate samples, which have important implications for molecular virology and vaccine development. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Pastor-Fernández I, Pegg E, Macdonald SE
… +3 more
, Tomley FM, Blake DP, Marugán-Hernández V
Curr Protoc Microbiol
· 2019 Jun · PMID 30811108
·
Publisher ↗
Eimeria is a genus of apicomplexan parasites that contains a large number of species, most of which are absolutely host-specific. Seven species have been recognized to infect chickens. Infection of susceptible chickens r...
Eimeria is a genus of apicomplexan parasites that contains a large number of species, most of which are absolutely host-specific. Seven species have been recognized to infect chickens. Infection of susceptible chickens results in an intestinal disease called coccidiosis, characterized by mucoid or hemorrhagic enteritis, which is associated with impaired feed conversion or mortality in severe cases. Intensive farming practices have increased the significance of coccidiosis since parasite transmission is favored by high-density housing of large numbers of susceptible chickens. Routine chemoprophylaxis and/or vaccination with live parasite vaccines provides effective control of Eimeria, although the emergence of drug resistance and the relative cost and production capacity of current vaccine lines can prove limiting. As pressure to reduce drug use in livestock production intensifies, novel vaccination strategies are needed. Development of effective protocols supporting genetic complementation of Eimeria species has until recently been hampered by their inability to replicate efficiently in vitro. Now, the availability of such protocols has raised the prospect of generating transgenic parasite lines that function as vaccine vectors to express and deliver heterologous antigens. For example, this technology has the potential to streamline the production of live anticoccidial vaccines through the generation of parasite lines that co-express immunoprotective antigens derived from multiple Eimeria species. In this paper we describe detailed protocols for genetic manipulation, laboratory growth, and in vivo propagation of Eimeria tenella parasites, which will encourage future work from other researchers to expand biological understanding of Eimeria through reverse genetics. © 2019 by John Wiley & Sons, Inc.
Chang AL, Hole CR, Doering TL
Curr Protoc Microbiol
· 2019 Jun · PMID 30802005
·
Full text
Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis, which kills 200,000 individuals worldwide each year. It is ubiquitous in the environment and is first inhaled into the lungs of...
Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis, which kills 200,000 individuals worldwide each year. It is ubiquitous in the environment and is first inhaled into the lungs of the host, where it is taken up by phagocytes. The interaction of these fungal cells with host phagocytes, therefore, is a critical step in the pathogenesis of this disease. One characteristic of this initial step in host-pathogen interactions is the avidity with which fungal cells are taken up by phagocytes, described by the phagocytic index. In this chapter, we detail a high-throughput method of directly assessing the phagocytic index of fungal cells using an imaging-based paradigm. By automating image collection and processing, this method permits rapid assessment of this critical host interaction. © 2019 by John Wiley & Sons, Inc.
Santiago-Tirado FH, Klein RS, Doering TL
Curr Protoc Microbiol
· 2019 Jun · PMID 30776307
·
Full text
Cryptococcus neoformans is an environmental yeast found worldwide that causes lethal brain infections, particularly in immunocompromised hosts. In 2016, there were 280,000 cases of cryptococcal meningitis in the HIV+ pop...
Cryptococcus neoformans is an environmental yeast found worldwide that causes lethal brain infections, particularly in immunocompromised hosts. In 2016, there were 280,000 cases of cryptococcal meningitis in the HIV+ population, two-thirds of them fatal; other immunocompromised patients are also affected. The burden of cryptococcal disease and the limits of current chemotherapy create a pressing need for improved treatment. One hindrance to the development of new therapies is lack of understanding of how this pathogen breaches the barriers protecting the brain. Here we describe a tool for investigating this process. This simple in vitro blood-brain-barrier (BBB) model, based on a human brain endothelial cell line grown on a permeable membrane, may be used to assay the BBB transmigration of C. neoformans or other neurotropic pathogens. © 2019 by John Wiley & Sons, Inc.
Frazer C, Hernday AD, Bennett RJ
Curr Protoc Microbiol
· 2019 Jun · PMID 30747494
·
Full text
Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches includi...
Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white-opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.
Jossé L, Bones AJ, Purton T
… +2 more
, Michaelis M, Tsaousis AD
Curr Protoc Microbiol
· 2019 Jun · PMID 30735306
·
Publisher ↗
Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa. Cryptosporidium species are the second largest cause of childhood diarrhea and are associated with increased morbidity....
Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa. Cryptosporidium species are the second largest cause of childhood diarrhea and are associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods. Recently, our lab has successfully cultivated C. parvum in the esophageal cancer-derived cell line COLO-680N, and has been able to maintain infection for several weeks. The success of this cell line was assessed with a combination of various techniques including fluorescent microscopy and qPCR. In addition, to tackle the issue of long-term oocyst production in vitro, a simple, low-cost bioreactor system using the COLO-680N cell line was established, which produced infectious oocysts for 4 months. This chapter provides details on the methodologies used to culture, maintain, and assess Cryptosporidium infection and propagation in COLO-680N. © 2019 by John Wiley & Sons, Inc.
Kay C, Peacock L, Gibson W
Curr Protoc Microbiol
· 2019 Jun · PMID 30707507
·
Publisher ↗
Trypanosoma congolense, together with T. vivax and T. brucei, causes African animal trypanosomiasis (AAT), or nagana, a livestock disease carried by bloodsucking tsetse flies in sub-Saharan Africa. These parasitic protis...
Trypanosoma congolense, together with T. vivax and T. brucei, causes African animal trypanosomiasis (AAT), or nagana, a livestock disease carried by bloodsucking tsetse flies in sub-Saharan Africa. These parasitic protists cycle between two hosts: mammal and tsetse fly. The environment offered by each host to the trypanosome is markedly different, and hence the metabolism of stages found in the mammal differs from that of insect stages. For research on new diagnostics and therapeutics, it is appropriate to use the mammalian life cycle stage, bloodstream forms. Insect stages such as procyclics are useful for studying differentiation and also serve as a convenient source of easily cultured, non-infective organisms. Here, we present protocols in current use in our laboratory for the in vitro culture of different life cycle stages of T. congolense-procyclics, epimastigotes, and bloodstream forms-together with methods for transfection enabling the organism to be genetically modified. © 2019 by John Wiley & Sons, Inc.
Sun S, Priest SJ, Heitman J
Curr Protoc Microbiol
· 2019 Jun · PMID 30661293
·
Full text
The Cryptococcus pathogenic species complex is a group of opportunistic human fungal pathogens that cause cryptococcal meningoencephalitis, an infection associated with unacceptably high mortality rates. The public healt...
The Cryptococcus pathogenic species complex is a group of opportunistic human fungal pathogens that cause cryptococcal meningoencephalitis, an infection associated with unacceptably high mortality rates. The public health relevance of these pathogens has galvanized extensive research over the past several decades and led to characterization of their sexual cycles. This research has allowed several Cryptococcus species to develop into model fungal organisms for both pathogenesis and basic science studies. Many of these studies require observation of the meiotic process and its associated mating structures as well as generation of meiotic progeny with novel phenotypes and genotypes. Herein, we describe how to set up genetic crosses between Cryptococcus strains and observe their mating phenotypes as well as how to recover progeny from these crosses for further analysis. © 2019 by John Wiley & Sons, Inc.
Fernandez N, Waters CM
Curr Protoc Microbiol
· 2019 Feb · PMID 30489040
·
Full text
Bacterial biofilms are notorious for their deleterious effects on human health and industrial biofouling. Key processes in biofilm formation are regulated by the second messenger signal cyclic dimeric guanosine monophosp...
Bacterial biofilms are notorious for their deleterious effects on human health and industrial biofouling. Key processes in biofilm formation are regulated by the second messenger signal cyclic dimeric guanosine monophosphate (c-di-GMP); accumulation of c-di-GMP promotes biofilm formation, while lowering c-di-GMP promotes motility. Complex networks of modular enzymes are involved in regulating c-di-GMP homeostasis. Understanding how these enzymes function in bacterial cells can help enlighten how bacteria use environmental cues to modulate c-di-GMP and cell physiology. In this article, we describe a workflow that utilizes Escherichia coli as a heterologous host to allow the researcher to identify genes encoding potential c-di-GMP-metabolizing proteins, to express the gene of interest from an inducible plasmid, and to directly detect changes in intracellular c-di-GMP using ultra-performance liquid chromatography-tandem mass spectrometry. © 2018 by John Wiley & Sons, Inc.
Han B, Moretto M, M Weiss L
Curr Protoc Microbiol
· 2019 Feb · PMID 30444582
·
Full text
Microsporidia are eukaryotic unicellular parasites that have been studied for more than 150 years. They are found throughout the world and are capable of infecting various invertebrate and vertebrate hosts. They can caus...
Microsporidia are eukaryotic unicellular parasites that have been studied for more than 150 years. They are found throughout the world and are capable of infecting various invertebrate and vertebrate hosts. They can cause disease in both immune-compromised and immune-competent humans. In immune-compromised individuals, infections can be severe and often fatal. Microsporidia possess a unique, highly specialized invasion mechanism that involves a structure known as the polar tube as well as the spore wall. During spore germination, the polar tube rapidly discharges from the spore and deliver the sporoplasm into the host cell. Spores are the only stage of microsporidia that can survive outside of host cells. Since the first attempt to culture microsporidia in vitro in 1930s, their cultivation has served a critical role in the study and diagnosis of these parasites. In this chapter, we include methods on the cultivation, isolation, and cryopreservation of Encephalitozoon cuniculi, which can infect humans and provides a useful model for other microsporidia. These methods can also be utilized for the culture of Encephalitozoon hellem or Encephalitozoon intestinalis. © 2018 by John Wiley & Sons, Inc.
Janowski AB, Wang D
Curr Protoc Microbiol
· 2019 Feb · PMID 30444308
·
Full text
Astrovirus VA1/HMO-C (VA1) is the representative genotype of mamastrovirus 9, a species of the single-stranded, positive-sense RNA viral family, Astroviridae. Astroviruses have been traditionally considered pathogens of...
Astrovirus VA1/HMO-C (VA1) is the representative genotype of mamastrovirus 9, a species of the single-stranded, positive-sense RNA viral family, Astroviridae. Astroviruses have been traditionally considered pathogens of the gastrointestinal tract but they have been recently associated with neurological diseases in humans, cattle, mink, sheep, and pigs. VA1 is the astrovirus genotype most commonly identified from human cases of meningoencephalitis and has been recently propagated in cell culture. VA1 can now be used as a model system to study pathogenesis of the neurological diseases associated with astrovirus infection. In this article, we describe two fundamental assays to quantify replication and propagation of VA1, a quantitative reverse transcription-PCR (qRT-PCR) to measure viral RNA and a 50% tissue culture infectious dose (TCID ) assay to measure infectious viral particles. © 2018 by John Wiley & Sons, Inc.
Videau P, Cozy LM
Curr Protoc Microbiol
· 2019 Feb · PMID 30398694
·
Publisher ↗
Anabaena sp. strain PCC 7120 is a multicellular, filamentous, freshwater cyanobacterium that is capable of differentiating specialized heterocyst cells for nitrogen fixation. This unit includes protocols for the growth a...
Anabaena sp. strain PCC 7120 is a multicellular, filamentous, freshwater cyanobacterium that is capable of differentiating specialized heterocyst cells for nitrogen fixation. This unit includes protocols for the growth and maintenance of Anabaena appropriate for a research or teaching laboratory. Controlled induction and assessment of heterocyst development is also covered. © 2018 by John Wiley & Sons, Inc.
Kelley BR, Ellis JC, Hyatt D
… +2 more
, Jacobson D, Johnson J
Curr Protoc Microbiol
· 2018 Nov · PMID 30369079
·
Publisher ↗
As a leading cause of bacterial-derived gastroenteritis worldwide, Campylobacter has a significant impact on human health. In the developed world, most campylobacteriosis cases are attributed to the consumption of underc...
As a leading cause of bacterial-derived gastroenteritis worldwide, Campylobacter has a significant impact on human health. In the developed world, most campylobacteriosis cases are attributed to the consumption of undercooked, contaminated poultry; however, it has been shown that Campylobacter can be transmitted to humans through contaminated water and other types of food, including beef and milk. As such, high-resolution microbial source-tracking is essential for health department officials to determine the source(s) of Campylobacter outbreaks. For these reasons, this protocol provides the techniques needed for isolation of Campylobacter from agricultural and environmental sources, as well as human clinical specimens. Additionally, we describe a simple method for preparing high-quality genomic DNA that can be used for whole-genome sequencing and downstream bioinformatics analyses of Campylobacter genotypes. © 2018 by John Wiley & Sons, Inc.
Chaudhri G, Kaladimou G, Pandey P
… +1 more
, Karupiah G
Curr Protoc Microbiol
· 2018 Nov · PMID 30281950
·
Publisher ↗
Ectromelia virus (ECTV) is an orthopoxvirus that causes mousepox in mice. Members of the genus orthopoxvirus are closely related and include variola (the causative agent of smallpox in humans), monkeypox, and vaccinia. C...
Ectromelia virus (ECTV) is an orthopoxvirus that causes mousepox in mice. Members of the genus orthopoxvirus are closely related and include variola (the causative agent of smallpox in humans), monkeypox, and vaccinia. Common features of variola virus and ECTV further include a restricted host range and similar disease progression in their respective hosts. Mousepox makes an excellent small animal model for smallpox to investigate pathogenesis, vaccine and antiviral agent testing, host-virus interactions, and immune and inflammatory responses. The availability of a wide variety of inbred, congenic, and gene-knockout mice allows detailed analyses of the host response. ECTV mutant viruses lacking one or more genes encoding immunomodulatory proteins are being used in numerous studies in conjunction with wild-type or gene-knockout mice to study the functions of these genes in host-virus interactions. The methods used for propagation of ECTV in cell culture, purification, and quantification of infectious particles through viral plaque assay are described. © 2018 by John Wiley & Sons, Inc.