Microbiology Virtual Week 2020

Asthma is an increasing health concern affecting more than 25 million individuals in the United States and more than 300 million individuals worldwide. In some cases, sensitization or exposure to specific microbes can exacerbate asthma. In 2006, a new asthma phenotype termed “severe asthma with fungal sensitization” (SAFS) was described for individuals whose asthma was poorly-controlled and who were sensitized to fungi. We have previously reported that asthmatics sensitized to fungi demonstrate worse lung function, higher serum IgE, blood eosinophil and exhaled nitric oxide levels. A growing area of interest is the identification of factors that contribute to immunopathogenesis in allergic asthma, including those sensitized to fungi, and determining whether these could be viable therapeutic targets. Indeed, biologics targeting the type 2 cytokines IL-4, IL-5 and IL-13 have been successful in treating individuals with severe asthma. To this end, we have reported that human asthmatics sensitized to fungi have increased levels of multiple cytokines, chemokines and growth factors in bronchoalveolar lavage fluid and sputum, some of which correlate with lung function or atopic measurements. This presentation will provide an overview of these findings and how some of these mediators mechanistically contribute to disease severity using an experimental animal model of fungal-associated allergic airway inflammation.

Learning Objectives:

1. To understand the clinical significance of allergic asthma as it relates to fungal sensitization

2. To understand experimental animal models as it relates to determining immunoprotective vs. immunopathogenic responses during fungal asthma

Epidemics are occurring at an increasing pace and scale. Our laboratory group has developed platform technologies for discovery of broad and potent neutralizing antibodies for many emerging infectious diseases. Studying diverse pathogens, we have discovered general principles about the genetic, molecular, and structural basis for virus neutralization by human antibodies. I will discuss mechanisms of neutralization and review specific case studies of antibiotic discovery programs for emerging infections leading to therapeutic drug candidates now in the clinic.

Learning Objectives:

1. Explain the concepts of sites of vulnerability for antibody-mediated neutralization

2. Define 3 common mechanisms of virus neutralization

3. Explain the rationale for rational selection of antibody combinations

HIV currently infects almost 40 million people worldwide. The virus is responsible for ~2 million new infections per year and ~1 million deaths. Like all retroviruses, HIV integrates a viral DNA copy of its RNA genome into host chromatin, thereby establishing a permanent and irreversible infection in the target cell. Integration depends on the obligate formation of a large oligomeric nucleoprotein complex containing the viral integrase enzyme assembled on viral DNA ends, commonly referred to as an intasome. Intasomes are also targeted by the latest generation antiretrovirals, integrase strand transfer inhibitors (INSTIs). Cryo-EM methods are beginning to shed light on the structures of HIV intasomes and how INSTIs bind, revealing how small changes in the integrase active site can have significant implications for drug binding. This work has implications for expanding effective treatments available for HIV-infected individuals. In this webcast, Dr. Dmitry Lyumkis will describe how advances in cryo-EM are facilitating structure-based drug design targeting HIV integration.

Learning Objectives:

1. The basics of single-particle cryo-EM, including the latest technical advances that are facilitating high-resolution structural studies

2. The biology of HIV integration, and why this step of the viral lifecycle is vulnerable to inhibition

3. How single-particle cryo-EM is becoming applicable to drug design and helping to combat the global HIV epidemic

My group is addressing fundamental questions in evolutionary biology, using both genome- and phenotype-first approaches. A few years ago, we discovered that Arabidopsis thaliana is a great model for the study of hybrid necrosis. This widespread syndrome of hybrid failure in plants is caused by plant paranoia – regardless of the presence of enemies, plants “think” they are being attacked by pathogens. Over the past decade, we have studied in detail the underlying genetics, finding that often one or two loci encoding NLR immune receptors are causal. NLRs make up the most variable gene family in plants, and it is not surprising that they are often involved in genome-genome conflicts. Hybrid necrosis results when NLR genes meet that have not been co-adapted. Our goal for the next decade is to understand the genomic and geographic patterns for immune system diversity. In 2018, we initiated a project, Pathodopsis, in which we aim to describe genetic diversity in the host A. thaliana and two of its important pathogens, the generalist Pseudomonas sp. and the specialist Hyaloperonospora arabidopsidis. The long-term vision is to produce maps of resistance alleles in the host, and of effector alleles in the pathogens, in order to learn when the pathogens win in a wild plant pathosystem – and when the hosts prevail. Additional information about our work can be found on our websites, http://weigelworld.org and http://pathodopsis.org.

Learning Objectives:

1. Understand drivers of plant immune receptor diversity

2. Understand limits of plant immune receptor diversity

Human chromosome 19q13.4 contains genes encoding killer-cell immunoglobulin-like receptors (KIR). The region has certain properties such as single nucleotide variation, structural variation, homology, and repetitive elements that make it hard to align accurately beyond single gene alleles. The 68 reference haplotypes in the human genome reference range in length from 67 to 269 kilobases and contain 4 to 18 genes. We leveraged these references and tools from our long-read KIR haplotype assembly algorithm to define and align KIR haplotypes at <5 kb resolution on average. We then used a standard alignment algorithm to refine that alignment down to single base resolution. This processing demonstrated that the high-level alignment recapitulates human-curated annotation of the human haplotypes as well as a chimpanzee haplotype. Further, assignments and alignments of gene alleles were consistent with their human curation in haplotype and allele databases. These results define KIR haplotypes as 14 loci containing 9 genes. The multiple sequence alignments have been applied in two computational workflows as in silico gene markers and in vitro capture probes. The first workflow is an efficient computational approach for in silico KIR probe interpretation (KPI) to accurately interpret individual’s KIR genes and haplotype-pairs from KIR sequencing reads. The second workflow efficiently captures, sequences, and assembles diploid human KIR haplotypes from PacBio HiFi reads.

Learning Objectives:

1. Identify the types of variation in the KIR region

2. Explain how this variation affects sequence interpretation

3. Define the difference in the output between the two WGS interpretation algorithms

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen–host interactions.

Learning Objectives:

1. What are the state of the art methods for mapping bacterial DNA methylation?

2. How can we exploit bacterial DNA methylation to better understand bacteria and microbiome?

Traditionally, virology has been focused in studying the pathogenic effect of viruses. In the recent years, however, this perception is changing and viruses are being studied as mutualistic components of ecosystems. Even pathogenic viruses can be beneficial for its host under certain circumstances: e.g., virus-infected plants showed a higher drought tolerance than the non-infected ones. We are studying the evolutionary dynamics of a pathosystem under drought stress conditions. To do so, we evolved turnip mosaic potyvirus (TuMV) in the model plant Arabidopsis thaliana. Viruses have been subjected to five evolutionary passages in four ecotypes of A. thaliana that were previously shown to have different transcriptomic responses to potyviral infection. In each accession, three independent viral lineages were evolved in normal conditions (SD) and another three were evolved in water-deficient conditions (DT). After evolution, DT-evolved viruses showed a higher infectivity than SD-evolved viruses. All infections increased the drought tolerance of the host, although DT-evolved viruses induced a significantly higher tolerance to water deficit than SD-evolved viruses. DT-evolved viruses provided its host with higher survival rates when facing water withdrawal. As TuMV is a sterilizing virus, plants infected with SD-evolved viruses barely produced seeds. However, plants infected with DT-evolved viruses produced larger amount of seeds than plants infected by SD-evolved viruses, a direct measure of increased plant fitness. Interestingly, the magnitude of this beneficial effects is ecotype-specific. The genetic and physiological causes of these differential host responses to both viruses are being characterized with targeted metabolomic and transcriptomic approaches. Results are consistent with a virus-induced activation of salicylic acid signaling pathway, which is also involved in drought tolerance. This research shows how environmental conditions shape the evolutionary dynamics in a plant-virus pathosystem, modifying the relationships between the virus and the host. Effects are variable among natural ecotypes but in all cases DT-evolved viruses were more virulent but less pathogenic for the host, providing the host an increased tolerance to drought.

Learning Objectives:

1. The triangle of pathology: disease is the outcome of an interplay between host and virus genotypes and environment

2. Host-virus interactions can evolve from parasitic to mutualistic as a consequence of environmental stress

Over the last several decades, antibodies (Abs) have become a valuable weapon in the fight against viral infections, with several studies demonstrating the importance of both neutralizing and non-neutralizing Ab responses in preventing or controlling viral infections in-vivo. Fc receptor mediated non-neutralizing effector functions such as, Ab-dependent Cellular Cytotoxicity (ADCC), phagocytosis and cytokine expression, have all been shown to contribute to effective immune responses in-vivo. However, in the case of some viral infections, such as dengue, Ab/Fc receptor mediated interactions enhance the severity of dengue hemorrhagic fever via Antibody Dependent Enhancement (ADE), which is hypothesized to primarily occur via Fc gamma receptor (FcγR) interactions. Importantly, in the context of the current SARS CoV-2 pandemic, researchers have observed that disease severity correlates with anti-SARS CoV-2 spike protein levels. Further, previous studies on a SARS1 vaccine suggest that ADE may contribute to mortality in animal models. These findings have important implications for anti-spike based SARS CoV-2 therapeutics and vaccine approaches. Therefore, our laboratory has begun to profile the levels of FcγR activation in a cohort of SARS CoV-2 convalescent patients versus severe hospitalized patients, to begin to understand whether ADE is a primary driver of severity and mortality during COVID-19 disease.

Learning objectives:

1. To learn how non-neutralizing Ab responses contribute to immune regulation during viral infections

2. To learn how in the majority of viral infections, non-neutralizing antibodies help to potently control infections in-vivo

3. To learn that in the context of certain viral infections, such as dengue, non-neutralizing Ab functions can be detrimental to the host and enhance severity of disease

The statement by Dimitri Ivanovsky in 1882 that "the sap of leaves infected with tobacco mosaic disease retains its infectious properties even after filtration through Chamberland filter candles" gave birth to a new field of science; Virology. Since that time, and well-beyond the essential goal of crop protection, virology studies conducted in plants have made seminal contributions to science with such landmark demonstrations as the infectious nature of nucleic acids, RNA silencing, and identification of host factors with roles in viral replication and recombination.  This engaging presentation considers how plant virology can inform the most pressing epidemiological concern of our time under the hypothesis that; if plant virology can inform zoonotic virus emergence, then the two should share in common: “outbreak” phenomena (sudden appearance), “spillover” phenomena (transfer from wild to domesticated areas), common genetics (similar types of viruses), reliance on ”vectors” for viral spread, and maintenance of viruses in “reservoir” species.

Learning Objectives:

1. Identify three plant viruses that are genetically similar to viruses that infect humans

2. Identify common vectors and reservoir species for plant viruses

3. Explain how ecology and population biology studies of plant viruses parallel those for zoonotic viruses for informing risk of virus emergence

As a response to various inflammatory stimuli, neutrophils and macrophages expel a mixture of their nuclear and granular elements in the form of extracellular traps (ETs). These web-like substances represent an evolutionarily conserved defense mechanism across humans, animals and even plants. ETs are composed of DNA, histones, and granular enzymes, and are capable of the entrapment of invading pathogens. Besides their protective function, ETs have been detected in a variety of pathological scenarios (from infections and autoimmune conditions to thrombosis) with detrimental effects, therefore they have been often referred to as ‘double-edged swords of immunity’. Most recently, neutrophil ETs have been identified as major contributors to the widespread immune-thrombosis in COVID-19 induced acute respiratory distress syndrome.

Learning Objectives:

1. Understand the basic types of ETs and the route of their formation

2. Name three pathological conditions where ETs play a role

3. Outline the suspected steps of ET contribution to (COVID-19 triggered) thrombosis

The regions of our genome responsible for encoding the genes that regulate our immune response are some of the most complex and polymorphic known. This complexity encompasses multiple types of genetic variation, from enrichment of single nucleotide variants through large structural differences. In several instances this variation has been shown to contribute to differential, individual susceptibility to disease. Investigating these regions is incredibly difficult using traditional short read approaches, as these reads often do not span entire genes or structural variants. Highly accurate long reads, such as those generated by single-molecule, real time (SMRT) sequencing, provide resolution of full-length immune genes without imputation and assembly of large, complex immune loci in a haplotype-specific manner. This presentation will use two examples, the human leukocyte antigen (HLA) locus, which is critically involved in effective T cell immunity, as well as the immunoglobulin heavy chain (IGH) locus, which encodes antibody responses, to demonstrate the power of SMRT sequencing for unparalleled resolution of these loci. In both cases, end-to-end genotyping pipelines have been developed and are available to leverage the power of long read sequencing to provide HLA and IGH genotyping data for most samples of interest.

Learning Objectives:

1. Identify mechanisms of genomic variation in immune loci that provide the extreme diversity of B and T cell responses

2. Understand the benefits of long read (vs. short read) sequencing approaches for resolving immune loci

3. Design an experiment utilizing HLA and/or IGH genotyping to understand the impact of immune loci variation in your specific disease model

Influenza severity is determined by the interplay between the virus and the host response. Previously, we identified a three-pronged lung gene expression signature that predicted severe influenza outcomes in mice. We hypothesized that this signature resulted from differential activation or infiltration of immune cells in the lungs during mild versus severe influenza infection. In order to build a comprehensive model of lung immune dynamics in response to influenza virus infections, we employed tissue deconvolution and linear regression to model the lung immune cell changes that occurred during mild or severe influenza virus infections. Modeling accurately predicted known macrophage dynamics that occur during influenza infection in vivo, and further predicted a novel population that transcriptionally resembled small serosal macrophages. This macrophage population was predicted to be present in the lungs of animals that recovered from influenza infection and absent from the lungs of those that succumbed. Overall, we provide a multidimensional analysis that delineates how immune cell responses relate to immunopathological consequences of influenza infection. We also demonstrate the utility of bulk tissue gene expression data in generating hypotheses about immune cell dynamics.

Learning Objectives:

1. Understand how different lung immune cell populations impact influenza severity

2. Understand how gene expression data can be used to generate hypotheses about immune cell dynamics

To establish productive infection, plant viruses need to be able to efficiently invade and spread within a plant. Most viruses are introduced into a plant via the epidermal or mesophyll cells where they undergo disassembly, replication, translation of their proteins, and assemble new virions. Viruses then move to the adjacent cells until they reach the phloem, which serves as the highway by which viruses spread throughout their hosts. It was shown that the phloem has evolved additional protection against these molecular intruders. Virus entry into, translocation through, and exit from the phloem involve additional viral factors and complex virus-host interactions. Although the majority of plant viruses are capable of infecting most types of cells within their hosts, there are several groups of plant viruses whose tissue tropism is limited to the phloem. These viruses are typically introduced directly into the phloem by vectoring phloem-feeding insects. Interestingly, the movement systems of such viruses are complex and involve multiple viral proteins. The factors and processes that control phloem restriction of these viruses are not well understood. We are working with Citrus tristeza virus (CTV), the largest non-segmented positive-sense RNA virus of plants, which belongs to the family Closteroviridae. In our recent study, we showed that the p33 protein of CTV is a viral effector, which affects viral pathogenicity by modulating a host immune response. Upon CTV infection, p33 triggers the accumulation of reactive oxygen species (ROS) and plant cell death. Deletion of the p33 gene from the CTV genome resulted in a significant decrease in the ROS production during virus invasion of the host plants and an increase in the virus accumulation and spread.  Remarkably, the p33 deletion mutant was able to spread beyond the phloem and enter the immature xylem cells where it perturbed the vascular tissue differentiation leading to the enhancement of the stem pitting syndrome. We hypothesize that the plant recognizes p33 and activates the host immune response to restrict CTV into the phloem tissue and minimize the disease.

Learning Objectives:

1. Learn how plant viruses move cell-to-cell and long-distance in their hosts

2. Understand how plant viruses modulate phloem to their advantage

3. Get familiar with mechanisms plants have developed to perceive and defeat viruses

4. Learn what factors mediate phloem restriction of certain plant viruses.