Genomic diversity of bacteriophages infecting Microbacterium spp

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics

Instructional Models for Course-Based Research Experience (CRE) Teaching

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis

Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated gene editing to Blastomyces, including to simultaneously target multiple genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium gene transfer. We succeeded in disrupting several fungal genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces gene disruption. Simultaneous dual-gene targeting proceeded with efficiencies similar to those of single-gene-targeting frequencies for the respective targets. CRISPR/Cas9 disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined disruption of both genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient gene disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivo

Novel Identity and Functional Markers for Human Corneal Endothelial Cells

PURPOSE: Human corneal endothelial cell (HCEC) density decreases with age, surgical complications, or disease, leading to vision impairment. Such endothelial dysfunction is an indication for corneal transplantation, although there is a worldwide shortage of transplant-grade tissue. To overcome the current poor donor availability, here we isolate, expand, and characterize HCECs in vitro as a step toward cell therapy. METHODS: Human corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro. Cell identity was evaluated based on morphology and immunocytochemistry, and gene expression analysis and flow cytometry were used to identify novel HCEC-specific markers. The functional ability of HCEC to form barriers was assessed by transendothelial electrical resistance (TEER) assays. RESULTS: Cultured HCECs demonstrated canonical morphology for up to four passages and later underwent endothelial-to-mesenchymal transition (EnMT). Quality of donor tissue influenced cell measures in culture including proliferation rate. Cultured HCECs expressed identity markers, and microarray analysis revealed novel endothelial-specific markers that were validated by flow cytometry. Finally, canonical HCECs expressed higher levels of CD56, which correlated with higher TEER than fibroblastic HCECs. CONCLUSIONS: In vitro expansion of HCECs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers. Markers described correlated with function in culture, suggesting a basis for cell therapy for corneal endothelial dysfunction

The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major.

For many arthropod vectors, the diverse bacteria and fungi that inhabit the gut can negatively impact pathogen colonization. Our attempts to exploit antibiotic treatment of colonized Phlebotomus duboscqi sand flies in order to improve their vector competency for Leishmania major resulted instead in flies that were refractory to the development of transmissible infections due to the inability of the parasite to survive and to colonize the anterior midgut with infective, metacyclic stage promastigotes. The parasite survival and development defect could be overcome by feeding the flies on different symbiont bacteria but not by feeding them on bacterial supernatants or replete medium. The inhibitory effect of the dysbiosis was moderated by lowering the concentration of sucrose (<30% w/v) used in the sugar feeds to maintain the colony. Exposure of promastigotes to 30% sucrose was lethal to the parasite in vitro. Confocal imaging revealed that the killing in vivo was confined to promastigotes that had migrated to the anterior plug region, corresponding to the highest concentrations of sucrose. The data suggest that sucrose utilization by the microbiota is essential to promote the appropriate osmotic conditions required for the survival of infective stage promastigotes in vivo

Investigation of Genetic Susceptibility to Blastomycosis Reveals Interleukin-6 as a Potential Susceptibility Locus

Blastomycosis is a potentially life-threatening infection caused by the fungus Blastomyces dermatitidis. As with related fungal diseases, blastomycosis is noted to affect some populations more than others. These patterns of illness are often not related to predisposing conditions or exposure risks; thus, genetic differences are thought to underlie these health disparities. People of Hmong ancestry in Wisconsin are at elevated risk of blastomycosis compared to the general population. We studied the genetic codes of Hmong blastomycosis patients and identified candidate sites in their genomes that may explain their susceptibility to this infection. We further studied one particular region of the genome that is involved with the immune processes that fight B. dermatitidis. Our work revealed population differences in the response to fungi. A better understanding of the genetic underpinnings of susceptibility to infectious diseases has broader implications for community health, especially in the paradigm of personalized medicine.Genetic differences are hypothesized to underlie ethnic disparities in incidence rates of the endemic systemic mycoses, including blastomycosis. Individuals of Hmong ancestry display elevated risk for this serious fungal infection. Here, we interrogated the genomes of Wisconsin (WI) Hmong blastomycosis patients using homozygosity mapping to uncover regions of the genome that are likely shared among the greater Hmong population and filtered for variants with high potential to affect disease susceptibility. This approach uncovered 113 candidate susceptibility variants, and among the most promising are those in genes involved in the interleukin-17 (IL-17) response. In particular, we identified 25 linked variants near the gene encoding IL-6 (IL6). We validated differences in cytokine production between Hmong and European volunteers and formally demonstrated a critical role for IL-6 in the development of adaptive immunity to Blastomyces dermatitidis. Our findings suggest that the dysregulation of IL-17 responses underlies a recently reported and poorly understood ethnic health disparity

Ligation of Dectin-2 with a novel microbial ligand promotes adjuvant activity for vaccination

<div><p>The development of vaccines against fungi and other intracellular microbes is impeded in part by a lack of suitable adjuvants. While most current vaccines against infectious diseases preferentially induce production of antibodies, cellular immunity is essential for the resolution of fungal infections. Microbes such as fungi and <i>Mycobacterium tuberculosis</i> require Th17 and Th1 cells for resistance, and engage the C-type lectin receptors including Dectin-2. Herein, we discovered a novel Dectin-2 ligand, the glycoprotein <i>Blastomyces</i> Eng2 (Bl-Eng2). Bl-Eng2 triggers robust signaling in Dectin-2 reporter cells and induces IL-6 in human PBMC and BMDC from wild type but not Dectin-2^-/- and Card9^-/- mice. The addition of Bl-Eng2 to a pan-fungal subunit vaccine primed large numbers of Ag-specific Th17 and Th1 cells, augmented activation and killing of fungi by myeloid effector cells, and protected mice from lethal fungal challenge, revealing Bl-Eng2’s potency as a vaccine adjuvant. Thus, ligation of Dectin-2 by Bl-Eng-2 could be harnessed as a novel adjuvant strategy to protect against infectious diseases requiring cellular immunity.</p></div

Bl-Eng2 is a bona-fide, superior Dectin-2 ligand.

<p><b>(A)</b><i>Pichia</i>-expressed proteins were plate-bound and tested for ligand activity using CLR expressing B3Z reporter cells expressing FcRγ chain, Dectin-2 + FcRγ, MCL + FcRγ, and Mincle + FcRγ, and BWZ cells and a subline expressing Dectin-1-CD3ζ (Dectin-1). <b>(B)</b> Supernatants from murine BMDCs (2 × 10⁵ per well) co-cultured with plate-bound Bl-Eng2 or PDIA1 were analyzed for IL-6 by ELISA. <i>Blastomyces</i> vaccine yeast (4 × 10⁵ per well) was used as positive control. <b>(C)</b> Supernatants from BMDCs (10⁵ per well) co-cultured with 1, 10, or 100 ng and 0.01, 0.1 or 1 pmol plate-bound Bl-Eng2, Man-LAM, Furfurman or MP98 were analyzed for IL-6 by ELISA. <i>Blastomyces</i> vaccine yeast (10⁴, 10⁵ or 10⁶ per well) was used as positive control. Data in A-C represent the mean ± SEM of one representative experiment of 3 independent experiments. <b>(D)</b> Bl-Eng2 induces IL-6 and IL-1β by human PBMCs. Human PBMCs were stimulated with plate-bound Bl-Eng2 for 24h and cytokines in cell culture supernatants were measured by ELISA. Data represent the mean ± SEM of 5 healthy individuals. *, p < 0.05 vs. no Bl-Eng2.</p

Myeloid effector mechanisms by Bl-Eng-2.

<p>Mice received 1807 cells prior to vaccination and were vaccinated and boosted with indicated adjuvants and formulated calnexin. Two weeks after the boost, mice were challenged i.t. with 10⁵ DsRed yeast and lungs were harvested 3 days later. The percentage of dead (DsRed^-Uvitex⁺)(blue) yeast among total neutrophil-associated yeast (all Uvitex⁺ events)(blue and red together) (see gating strategy in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006568#ppat.1006568.s006" target="_blank">S6A Fig</a>) were analyzed and calculated (dot plots are concatenates from 5 mice/group) to depict the amount of killing by neutrophils <b>(A+B)</b>. The percentage of killing by alveolar macrophages is shown in (<b>C</b>). The number of live yeast was depicted by showing the total number of DsRed⁺ events <b>(D)</b> or plating lung CFU <b>(E)</b>. The numbers indicate the n-fold reduction in live yeast (DsRed⁺ or CFU) vs. the calnexin control groups. *p<0.05 control groups without Bl-Eng2. Cnx denotes calnexin.</p