微生物学

We recently developed an approach for cell type–specific CRISPR/Cas9 editing and transgene expression using a single viral vector. Here, we present a protocol describing how to design and generate plasmids and adeno-associated viruses (AAVs) compatible with this single-vector gene editing approach. This protocol has four components: (1) guide RNA (gRNA) design to target specific genes of interest, (2) ligation and cloning of CRISPR-competent AAV vectors, (3) production of vector-containing AAVs, and (4) viral titer quantification. The resultant vectors are compatible for use with mouse lines expressing the Cas9 protein from Streptococcus pyogenes (SpCas9) and Cre recombinase to enable selective co-expression of standard neuroscience tools in edited cells. This protocol can produce AAVs of any serotype, and the resulting AAVs can be used in the central and peripheral nervous systems. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection.

Trypanosoma cruzi, the causative agent of Chagas disease, faces significant metabolic challenges due to fluctuating nutrient availability and oxidative stress within its insect vector. Metabolomic techniques, such as gas chromatography–mass spectrometry (GC–MS), have been widely used to study the adaptive mechanisms of the parasite. This article describes a standardized method for the untargeted metabolomics analysis of T. cruzi epimastigote, covering parasite cultivation, sample deproteinization with methanol, metabolite extraction, derivatization with BSTFA, and GC–MS analysis. To ensure robustness and reproducibility, statistical analysis uses univariate tests, as well as multivariate approaches such as principal component analysis (PCA) and partial least squares (PLS) regression. The protocol offers a reliable and sensitive method to study metabolic responses in T. cruzi under environmental stress, with low biological variability and high reproducibility.

Malaria remains a major public health threat, especially in tropical and subtropical regions. Accurate and rapid diagnosis is essential for effective disease management and control, yet conventional malaria diagnostics, including blood smear microscopy using Giemsa staining, PCR, and rapid diagnostic tests (RDTs), are limited by the need for trained personnel, reliance on laboratory infrastructure, and reduced sensitivity at low parasite densities, respectively. This protocol details an innovative, rapid, and economical diagnostic platform combining a simplified Chelex-100 resin-based nucleic acid extraction method with a multiplex loop-mediated isothermal amplification microscanner (LAMP-MS) assay. The malaria diagnostic platform enables simultaneous detection of Plasmodium falciparum (Pf), Plasmodium vivax (Pv), pan-malaria (Pan), and an internal control (IC) within 40 min, from DNA extraction to result interpretation. It demonstrates sensitivity and specificity comparable to traditional PCR-based diagnostics, making it a practical and scalable solution for use in resource-constrained environments.

Since the creation of the Global Polio Eradication Initiative (GPEI) in 1988, significant progress has been made toward attaining a poliovirus-free world. This has resulted in the eradication of wild poliovirus (WPV) serotypes two (WPV2) and three (WPV3) and limited transmission of serotype one (WPV1) in Pakistan and Afghanistan. However, the increased emergence of circulating vaccine-derived poliovirus (cVDPV) and the continued circulation of WPV1, although limited to two countries, pose a continuous threat of international spread of poliovirus. These challenges highlight the need to further strengthen surveillance and outbreak responses, particularly in the African Region (AFRO). Phylogeographic visualization tools may provide insights into changes in poliovirus epidemiology, which can in turn guide the implementation of more strategic and effective supplementary immunization activities and improved outbreak response and surveillance. We created a comprehensive protocol for the phylogeographic analysis of polioviruses using Nextstrain, a powerful open-source tool for real-time interactive visualization of virus sequencing data. It is expected that this protocol will support poliovirus elimination strategies in AFRO and contribute significantly to global eradication strategies. These tools have been utilized for other pathogens of public health importance, for example, SARS-CoV-2, human influenza, Ebola, and Mpox, among others, through real-time tracking of pathogen evolution (https://nextstrain.org), harnessing the scientific and public health potential of pathogen genome data.

The DNA double-strand breaks (DSBs) generated by exogenous and endogenous factors are repaired by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). These two pathways compete for DSB repair, and the choice of pathway depends on the context of the DNA lesion, the stage of the cell cycle, and the ploidy in the yeast Saccharomyces cerevisiae. However, the mechanistic details of the DSB repair pathway choice and its consequences for S. cerevisiae genome stability remain unclear. Here, we present PCR-based and cell-based assays as well as data analysis methods to quantitatively measure the efficiency of HR and NHEJ at DSBs in S. cerevisiae. An intermolecular recombination assay between plasmid and chromosomal DNA involving G-quadruplex DNA and a “suicide-deletion” assay have been utilized to evaluate the efficiency of HR and NHEJ, respectively. These streamlined protocols and optimized growth conditions can be used to identify the NHEJ- and HR-deficient S. cerevisiae mutant strains.

The study of carbohydrate–protein interactions is crucial for clarifying biological processes and identifying potential drug candidates. However, due to the complex structure of carbohydrates, such as high molecular weight, dynamic flexibility, and high solution viscosity, it is challenging to study their interactions with diverse proteins. Conventional analytical techniques like isothermal titration calorimetry (ITC), X-ray crystallography, molecular dynamics (MD) simulations, and nuclear magnetic resonance (NMR) spectroscopy have limitations in revealing these molecular interactions. Surface plasmon resonance (SPR), an advanced optical biosensor technique, overcomes these limitations. It enables real-time, label-free monitoring of the interaction dynamics between carbohydrates and proteins through a continuous flow over a chip surface. In this study, we utilized SPR-based techniques to explore the interaction of capsular polysaccharides (CPS) of Klebsiella pneumoniae and the enzyme KpACE (K. pneumoniae acetylated capsule esterase). Our SPR-based analytical platform has several advantages, including shorter experimental time, a simulated physiological state, and minimal sample requirements for investigating carbohydrate–protein interactions. This approach expands the applicability scope of SPR technology and provides a valuable tool for a wide range of research. By using SPR, we successfully verified that KpACE acts on the acetyl groups of CPS, demonstrating its enzymatic activity, which is crucial for understanding the pathogenic mechanism of K. pneumoniae and developing potential antibacterial drugs.

The target of rapamycin complex 1 (TORC1) is a highly conserved protein complex whose primary function is to link nutrient availability to cell growth in eukaryotes, particularly nitrogen sources. It was originally identified during the screening of Saccharomyces cerevisiae strains resistant to rapamycin treatment. For its part, S. cerevisiae is well known for being a key model organism in biological research and an essential microorganism for the fermentation of food and beverages. This yeast is widely distributed in nature, with domesticated and wild strains existing. However, little is known about what effects domestication has had on its different phenotypes; for example, how nitrogen sources are sensed for TORC1 activation and what impact domestication has had on TORC1 activation are questions that still have no complete answer. To study the genetic basis of TORC1 activation associated with domestication through approaches such as quantitative trait loci (QTL) mapping or genome-wide association studies (GWAS), and more generally for any study requiring TORC1 activity as a readout for a large number of individuals, it is necessary to have a high-throughput methodology that allows monitoring the activation of this pathway in numerous yeast strains. In this context, the present protocol was designed to assess phenotypical differences in TORC1 activation using a new reporter plasmid, the pTOMAN-G plasmid, specifically designed to monitor TORC1 activation. As a proof of concept, this methodology allowed phenotyping a large population of yeast strains derived from the 1002 Yeast Genomes Project, the most complete catalog of genetic variation in yeasts. This protocol proved to be an efficient alternative to assess TORC1 pathway activation compared to techniques based on immunoblot detection, which, although effective, are considerably more laborious. Briefly, the protocol involves the design and construction of the pTOMAN-G plasmid, which carries a construct containing the firefly luciferase gene (Luc) under the control of the TORC1-regulated RPL26A gene promoter (P_RPL26A). The protocol then details the process for selecting subgroups of yeasts based on their ability to grow under nutrient-limited conditions, using proline as the sole nitrogen source. These yeasts are then transformed with the TOMAN-G plasmid, using two alternative transformation methods. Finally, those yeasts that emit luminescence are selected, whose phenotype for TORC1 activation is measured by a nitrogen-upshift experiment in microculture. This approach, using the pTOMAN-G plasmid, offers a rapid and consistent method for assessing TORC1 signaling pathway activation in a large number of yeast strains, highlighting its usefulness to study the activation of the TORC1 pathway and the domestication process associated with it. In the future, a redesign of the plasmid could extend its use as a reporter tool to monitor the activation of the TORC1 pathway, or other pathways, in other yeast species.

Human intestinal barrier function is crucial for health. Beneficial microbes, such as commensal gut bacteria and probiotics, are known to contribute to the regulation of this barrier function. Interactions between bacteria and human intestinal cells can be analyzed by co-culturing bacteria with mammalian cells in vitro. Here, we describe a method to assess the effect of individual bacterial strains on intestinal barrier function using automated transepithelial electrical resistance (TEER) measurements. Caco-2 cells are used as a model of the intestinal epithelium, as these cells spontaneously differentiate into small intestinal epithelial-like cells characterized by tight junctions between adjacent cells. These cells are seeded on polyester filter inserts and cultured for 17 days to form a differentiated monolayer prior to the co-culture experiment. Bacteria are grown on agar, and a single colony is used to prepare a liquid culture in bacterial broth appropriate for the bacteria of interest. On the day of the co-culture experiment, the bacterial culture is resuspended in cell culture medium at the desired concentration. Inserts are transferred to cellZscope cell modules to enable automated TEER measurements, and the medium in the insert is replaced with cell culture medium containing the bacteria of interest. This method allows for intestinal tight junction barrier function to be assessed non-invasively and in real-time in response to probiotics. The use of the automated cellZscope system eliminates the need for labor-intensive manual TEER measurements, which reduces the variability in data that results from human handling and temperature changes that occur when cells are removed from the incubator.

It has been discovered that many phytopathogenic fungi can absorb exogenous double-stranded RNAs (dsRNAs) to silence target genes, inhibiting fungal growth and pathogenicity for plant protection. In our recent report, the beneficial arbuscular mycorrhizal (AM) fungi are capable of acquiring external naked dsRNAs; however, whether the dsRNAs can be delivered into AM fungi through nanocarriers remains to be investigated. Here, we introduce a simple and advanced method for in vitro synthesizing chitosan (CS)/dsRNA polyplex nanoparticles (PNs) to silence the target gene in the AM fungus Rhizophagus irregularis. This method is straightforward, requiring minimal modifications, and is both efficient and eco-friendly, offering potential for rapid application in elucidating gene functions in AM fungi.

Since the establishment of the iSLK-BAC16 cell culture system, iSLK-BAC16 cells and their derivatives have been widely used for Kaposi’s sarcoma-associated herpesvirus (KSHV) studies. However, iSLK-BAC16 cells can be difficult to work with, in part due to the lack of standardized protocols and conflicting troubleshooting suggestions. Here, we describe the protocol for general iSLK-BAC16 cell culture and reactivation, which induces lytic KSHV replication and virion production. This protocol achieves robust levels of KSHV reactivation in our hands and can be readily used for studies of KSHV lytic infection mechanisms.