Cryptococcus neoformans Transcriptome Analysis to Identify Differentially Expressed Genes Using the STAR Pipeline in CyVerse Discovery Environment.

RNA sequencing is a next-generation sequencing approach that may be used to investigate many aspects of gene expression changes between cells. Analysis of the data is typically a multistep process using several bioinformatics tools. The following protocol utilizes a reliable pipeline for identifying differentially expressed genes among samples of Cryptococcus neoformans that is approachable for the adventurous beginner.

Regulation of macrophage IFNγ-stimulated gene expression by the transcriptional coregulator CITED1.

Macrophages serve as a first line of defense against microbial pathogens. Exposure to interferon-γ (IFNγ) increases interferon-stimulated gene (ISG) expression in these cells, resulting in enhanced antimicrobial and proinflammatory activity. Although this response must be sufficiently vigorous to ensure the successful clearance of pathogens, it must also be carefully regulated to prevent tissue damage. This is controlled in part by CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2), a transcriptional coregulator that limits ISG expression by inhibiting STAT1 and IRF1. Here, we show that the closely related Cited1 is an ISG, which is expressed in a STAT1-dependent manner, and that IFNγ stimulates the nuclear accumulation of CITED1 protein. In contrast to CITED2, ectopic CITED1 enhanced the expression of a subset of ISGs, including Ccl2, Ifit3b, Isg15 and Oas2. This effect was reversed in a Cited1-null cell line produced by CRISPR-based genomic editing. Collectively, these data show that CITED1 maintains proinflammatory gene expression during periods of prolonged IFNγ exposure and suggest that there is an antagonistic relationship between CITED proteins in the regulation of macrophage inflammatory function. This article has an associated First Person interview with the first author of the paper.

Teaching in the Time of COVID-19: Creation of a Digital Internship to Develop Scientific Thinking Skills and Create Science Literacy Exercises for Use in Remote Classrooms.

The extreme academic and social disruption caused by COVID-19 in the spring and summer of 2020 led to the loss of many student internships. We report here our creation of a novel internship for students majoring in the biological sciences. Student interns worked together to systematically categorize multiple episodes of This Week in Microbiology (TWiM). They annotated episodes by labeling relevant ASM fundamental curricular guidelines and the microbiology techniques described in several podcast episodes. Interns worked together, which advanced their written and oral communication skills while improving their scientific thinking skills. Faculty then enhanced each annotation by adding short figure-reading exercises that can be used in a variety of educational settings to teach science literacy. When surveyed, students reported greater confidence in analyzing and interpreting results from a variety of microbiological methods, improved communication of fundamental microbiology concepts in written and oral form, and enhanced ability to collaborate with others. Combined, this digital internship provided a unique opportunity for students to develop critical technical and scientific thinking skills and generated useful open education resources for teaching general microbiology in the form of annotated podcasts.

Serial Passage of Cryptococcus neoformans in Galleria mellonella Results in Increased Capsule and Intracellular Replication in Hemocytes, but Not Increased Resistance to Hydrogen Peroxide.

To gain insight into how pathogens adapt to new hosts, Cryptococcus neoformans (H99W) was serially passaged in Galleria mellonella. The phenotypic characteristics of the passaged strain (P15) and H99W were evaluated. P15 grew faster in hemolymph than H99W, in vitro and in vivo, suggesting that adaptation had occurred. However, P15 was more susceptible to hydrogen peroxide in vitro, killed fewer mouse macrophages, and had less fungal burden in human ex vivo macrophages than H99W. Analysis of gene expression changes during Galleria infection showed only a few different genes involved in the reactive oxygen species response. As P15 sheds more GXM than H99W, P15 may have adapted by downregulating hemocyte hydrogen peroxide production, possibly through increased capsular glucuronoxylomannan (GXM) shedding. Hemocytes infected with P15 produced less hydrogen peroxide, and hydrogen peroxide production in response to GXM-shedding mutants was correlated with shed GXM. Histopathological examination of infected larvae showed increased numbers and sizes of immune nodules for P15 compared to H99W, suggesting an enhanced, but functionally defective, response to P15. These results could explain why this infection model does not always correlate with murine models. Overall, C. neoformans' serial passage in G. mellonella resulted in a better understanding of how this yeast evolves under selection.

Intracellular Cryptococcus neoformans disrupts the transcriptome profile of M1- and M2-polarized host macrophages.

Macrophages serve as a first line of defense against infection with the facultative intracellular pathogen, Cryptococcus neoformans (Cn). However, the ability of these innate phagocytic cells to destroy ingested Cn is strongly influenced by polarization state with classically (M1) activated macrophages better able to control cryptococcal infections than alternatively (M2) activated cells. While earlier studies have demonstrated that intracellular Cn minimally affects the expression of M1 and M2 markers, the impact on the broader transcriptome associated with these states remains unclear. To investigate this, an in vitro cell culture model of intracellular infection together with RNA sequencing-based transcriptome profiling was used to measure the impact of Cn infection on gene expression in both polarization states. The gene expression profile of both M1 and M2 cells was extensively altered to become more like naive (M0) macrophages. Gene ontology analysis suggested that this involved changes in the activity of the Janus kinase-signal transducers and activators of transcription (JAK-STAT), p53, and nuclear factor-κB (NF-κB) pathways. Analyses of the principle polarization markers at the protein-level also revealed discrepancies between the RNA- and protein-level responses. In contrast to earlier studies, intracellular Cn was found to increase protein levels of the M1 marker iNos. In addition, common gene expression changes were identified that occurred post-Cn infection, independent of polarization state. This included upregulation of the transcriptional co-regulator Cited1, which was also apparent at the protein level in M1-polarized macrophages. These changes constitute a transcriptional signature of macrophage Cn infection and provide new insights into how Cn impacts gene expression and the phenotype of host phagocytes.

Development of a Lac Operon Concept Inventory (LOCI).

Concept inventories (CIs) are valuable tools for educators that assess student achievement and identify misconceptions held by students. Results of student responses can be used to adjust or develop new instructional methods for a given topic. The regulation of gene expression in both prokaryotes and eukaryotes is an important concept in genetics and one that is particularly challenging for undergraduate students. As part of a larger study examining instructional methods related to gene regulation, the authors developed a 12-item CI assessing student knowledge of the lac operon. Using an established protocol, the authors wrote open-ended questions and conducted in-class testing with undergraduate microbiology and genetics students to discover common errors made by students about the lac operon and to determine aspects of item validity. Using these results, we constructed a 12-item multiple-choice lac operon CI called the Lac Operon Concept Inventory (LOCI), The LOCI was reviewed by two experts in the field for content validity. The LOCI underwent item analysis and was assessed for reliability with a sample of undergraduate genetics students (n = 115). The data obtained were found to be valid and reliable (coefficient alpha = 0.994) with adequate discriminatory power and item difficulty.

Evolutionary analysis of the mammalian M1 aminopeptidases reveals conserved exon structure and gene death.

The members of the M1 aminopeptidase family share conserved domains, yet show functional divergence within the family as a whole. In order to better understand this family, this study analyzed the mammalian members in depth at exon, gene, and protein levels. The twelve human members, eleven rat members, and eleven mouse members were first analyzed in multiple alignments to visualize both reported and unreported conserved domains. Phylogenetic trees were then generated for humans, rats, mice, and all mammals to determine how closely related the homologs were and to gain insight to the divergence in the family members. This produced three groups with similarity within the family. Next, a synteny study was completed to determine the present locations of the genes and changes that had occurred. It became apparent that gene death likely resulted in the lack of one member in mouse and rat. Finally, an in-depth analysis of the exon structure revealed that nine members of the human family and eight in mouse, are highly conserved within the exon structure. Taken together, these results indicate that the M1 aminopeptidase family is a divergent family with three subgroups and that genetic evidence mirrors categorization of the family by enzymatic function.