Department of Microbiology and Immunology Faculty

Research Summary

Bordetella pertussis, Bordetella bronchiseptica and Bordetella parapertussis are mammalian respiratory pathogens that are highly genetically-related Gram-negative members of the family Alcaligenaceae. B. pertussis, the agent of whooping cough (pertussis), is an obligate human pathogen. B. parapertussis causes respiratory infections in humans and sheep, and B. bronchiseptica infects a wide range of nonhuman mammals. These bacteria adhere to the cilia of the host respiratory tract, obtain nutrients, replicate and elaborate virulence factors that enable them to persist and become transmitted to new hosts.To determine how these organisms successfully live in the host, we must understand their biology. Nearly all organisms require nutritive iron and its assimilation is essential for invading pathogenic bacteria to establish infection in the iron-limiting environment of the host. Bordetella species obtain iron supplied by their native iron-chelating siderophore, alcaligin, as well as siderophores produced by other microbial species, and from host iron containing compounds including heme. These iron retrieval systems are expressed when the bacteria are starved for iron and expression is further activated by cognate transcriptional regulators that respond to the presence of the specific iron compound. Recent studies have determined that these bacteria can utilize host neuroendocrine hormones, such as norepinephrine, for iron retrieval. Our studies are aimed at understanding the processes governing the expression of different Bordetella iron acquisition systems in the host during the course of infection.

Publications

• Hanawa, T., H. Yonezawa, H. Kawakami, S. Kamiya and S.K. Armstrong. 2013. Role of Bordetella pertussis RseA in the cell envelope stress response and adenylate cyclase toxin release. Pathog. Dis. In press.
• Brickman, T.J. and S.K. Armstrong. 2012. Iron and pH-responsive FtrABCD ferrous iron utilization system of Bordetella species. Mol. Microbiol. 86:580-593.
• Armstrong, S.K., T.J. Brickman and R.J. Suhadolc. 2012. Involvement of multiple distinct Bordetella receptor proteins in the utilization of iron liberated from transferrin by host catecholamine stress hormones. Mol. Microbiol. 84:446-462.
• Brickman, T.J., C. A. Cummings, S-Y. Liew , D. A. Relman and S.K. Armstrong. 2011. Transcriptional profiling of the iron starvation response in Bordetella pertussis provides new insights into siderophore utilization and virulence gene expression. J. Bacteriol. 193:4798-4812.
• Brickman, T.J. and S.K. Armstrong. 2009. Temporal signaling and differential expression of Bordetella iron transport systems: the role of ferrimones and positive regulators. BioMetals. 22:33-41.
• Brickman, T.J., Hanawa T., Anderson M.T., Suhadolc, R.J. and S.K. Armstrong. 2008. Differential expression of Bordetella pertussis iron transport system genes during infection. Mol. Microbiol. 70:3-14.
• Anderson, M.T. and S.K. Armstrong. 2008. Norepinephrine mediates acquisition of transferrin-iron in Bordetella bronchiseptica. J. Bacteriol. 190:3940-3947.

Research Summary

Metabolic requirements for bacterial persistenceThe ability of Mycobacterium tuberculosis (Mtb) to persist in the presence of anti-mycobacterial drugs and immune effector functions is the greatest obstacle in our attempt to eradicate tuberculosis (TB). My laboratory is investigating the hypothesis that persistence is enabled by entrance into and maintenance of a physiological state that is metabolically distinct from that of actively replicating bacilli. The aim of this work is to define this state (or series of states) and characterize pathways that are essential for its establishment and maintenance. My laboratory uses a variety of classical and novel approaches in mycobacterial genetics, biochemistry, physiology, and metabolomics to characterize i) novel aspects of intermediary metabolism in Mtb, ii) metabolic requirements for phenotypic drug tolerance, and iii) drug targets and drug-like compounds effective against persistent bacilli. While these studies address basic science questions about unique aspects of Mtb, they will also provide important contributions to the collaborative and global effort to eradicate TB.

Research Summary

Molecular mechanisms of human cytomegalovirus replication and pathogenesis My laboratory is focused on understanding the mechanisms of human cytomegalovirus (HCMV) replication and pathogenesis. The long term goal of my laboratory is to provide a molecular understanding of HCMV replication and pathogenesis that will lead to the development of novel approaches or therapeutics to help fight HCMV infection. HCMV is a ubiquitous herpesvirus that infects greater than 70% of the world's population. Although HCMV infection is usually sub-clinical in healthy individuals, it is a major health problem for neonates, infants, and immunocompromised individuals. HCMV is the most common cause of viral induced birth defects in the US, with over 40,000 infants infected at birth each year. HCMV pathology in adults is most frequently encountered in immunocompromised individuals such as transplant recipients and AIDS patients. HCMV is the single most important infectious agent affecting organ transplant recipients; with at least two-thirds of these patients developing an HCMV-infection 1-4 months after transplantation. The increasing use of therapeutic immunosuppression, organ transplantation and the incidence of AIDS have focused our attention upon the HCMV life cycle, with a view towards achieving a thorough understanding of virus/cell interactions and viral replication processes, and thereby identifying approaches to attenuate primary and/or reactivated HCMV infection. Our research focuses on three areas of HCMV biology that include: 1) the identification and functional characterization of viral tegument proteins, 2) identifying the mechanisms by which HCMV subverts the host innate immune system, and 3) using genomic approaches to study HCMV gene regulation.One major interest of our laboratory is to identify the mechanisms by which HCMV subverts the host's innate antiviral defenses. Results from our laboratory indicate that HCMV rapidly and effectively blocks the induction of IFN-b and chemokines following infection of the host cell. The inhibition of IFN-b presumably inhibits the cells ability to induce interferon responsive genes and mount an effective antiviral response, thereby providing HCMV a mechanism to evade the host immune system and persist within the host. Using numerous biochemical and genetic approaches we have determined that the HCMV immediate early-2 gene product IE86 effectively blocks the induction of IFN-b and chemokines following HCMV infection. We are currently working to identify the molecular mechanism by which IE86 blocks the induction of these proteins.A second interest in the laboratory involves determining what role HCMV tegument proteins play during HCMV replication. Tegument proteins are packaged within the virus particle and delivered to the host cell immediately upon infection. These tegument proteins are key regulators of viral and cellular gene expression, modulators of the host anti-viral response, and are critical for "kick starting" the viral infection. Our laboratory has taken biochemical and genetic approaches to decipher the functions of individual HCMV tegument proteins and examine what role they play in viral replication and pathogenesis

Research Summary

C. albicans genetics and pathogenesisMy primary research focus of my laboratory is to understand how the fungal pathogenC. albicans responds to changes in the extracellular environment and how these changes are related to virulence. We have identified two distinct signal transduction pathways that govern adaptation to extracellular pH in vitro and are critical for virulencein vivo. To further this research we are addressing the following questions: What are mechanistic details of these pathways and how do they relate to each other to govern pH responses? What are the targets of these pathways and how do these targets affect the phenotypes seen in pathway mutants? How do these pathway promote virulence? To answer these questions, we have developed both forward and reverse genetic techniques which provide powerful approaches to address questions 1 and 2. Further, we are using an ex vivo models, including C. albicans stimulated host cell damage models, to address question 3 .

Bio

Ashley T. Haase is a Regents' Professor and Head of the Department Microbiology and Immunology at the University of Minnesota, Minneapolis. Dr. Haase has devoted the past 25 years of his career to investigating human (HIV-1/AIDS) and non-human primate (SIV) lentivirus infections, and his laboratory is currently investigating the globally predominant sexual route of HIV transmission in the SIV rhesus macaque model with the goal of developing effective vaccines and microbicides. Dr. Haase is an NIH NINDS Javits Awardee and two-time recipient of an NIH MERIT Award for his work on HIV, and a member of the Institute of Medicine of the National Academy of Sciences.

Research Summary

Viral pathogenesis, HIV My laboratory investigates the pathogenesis, treatment and prevention of lentiviral immunodeficiency infections caused by HIV-1 and its simian relative, SIV, using such technologies as in situ hybridization, in situ tetramer staining and quantitative image analysis to visualize infection and the hosts' cellular immune response in tissues. Much of our recent work has focused on sexual mucosal transmission and the acute stage of SIV infection, the roles of "resting" and activated CD4 T cells in establishing infection, and the mechanisms of the massive depletion of CD4 T cells in the gut. Going forward, these studies provide a foundation for studies of the correlates of protection for attenuated vaccines, and the development of vaccines and microbicides to prevent transmission. My laboratory has also undertaken a comprehensive microarray analysis of HIV-1 and SIV infections with the objectives of understanding pathogenesis and identifying novel targets for treatment and prevention. Current efforts focus on broadening the microarray analysis to encompass the early through late stages of HIV-1 infection, and mapping genes identified in the analysis to gain insight into their function in HIV-1 infected lymphatic tissues, the principal sites of virus production, persistence and pathology.

Research Summary

Host-microbe interactions, microbial community dynamicsResearch in the Hunter lab is focused on host-microbe interactions and seeks to characterize the effect of in vivo environmental chemistry on the dynamics of polymicrobial infections. While recent culture-independent studies have provided a comprehensive survey of the host microbiome and its association with health and disease, there is a major gap in our understanding of the environmental conditions under which pathogens live, how and why they adapt to specific niches within the host, and how microbe-environment relationships co-evolve throughout the course of infection. Our research combines molecular genetics, environmental chemistry, high-resolution microscopy (in situ hybridization and cryo-electron microscopy) and culture-independent approaches to address these ecological questions. The long-term goal is to gain a better understanding of disease etiology, predictors of clinical outcomes, and novel avenues for antimicrobial therapy. We are primarily interested in the opportunistic pathogen Pseudomonas aeruginosa and its co-habitants in microbial communities of chronic respiratory infections (chronic obstructive pulmonary disease, sinusitis, and cystic fibrosis).

Research Summary

Immunity to influenza virus infectionsInfluenza A virus (IAV) represents a major global health burden. Despite yearly vaccinations the virus is able to escape seasonal immunity requiring yearly vaccination and the threat of novel pandemics loom. Therefore continued understanding of the host-pathogen interactions and protective immune responses are critical for broadly protective vaccine development. Our overall research goal is to address fundamental questions in virology and viral immunology that have been difficult to dissect using conventional approaches. We utilized host-derived microRNAs, small non-coding RNA capable of mediating silencing of mRNA, to restrict the natural tropism of IAV. This allows for previously unavailable insights into immune responses to the virus. Additionally, we generate novel reporter viruses to further define the relationship between cellular tropism and immunity as well as to determine infected cell fate. A more comprehensive understanding of virus infection requirements that dictate immunity will be critical for the design of next generation vaccines and therapeutics

Research Summary

T cell migration, differentiation, and memory development in response to infectionsMy laboratory studies CD8 and CD4 T cell responses to a variety of viral and bacterial infections to help understand the development of immunological protection from re-infection. We observe and manipulate pathogen specific T cell responses over time by using MHC tetramers, adoptive transfer of transgenic T cells, fluorescence flow cytometry and sorting, and gene microarry analysis. Upon activation in lymphoid tissue, rare pathogen-specific naïve T cells proliferate, acquire effector functions, disseminate throughout the organism, and contribute to the eradication of pathogens. In situations where antigen is successfully eliminated, most effector T cells die by apoptosis. However, a fraction of effector T cells escape death and differentiate into long-lived memory T cells that contribute to protective immunity. We are currently dedicated to elucidating the developmental cues that govern T cell migration to different anatomical locations, commitment to the memory lineage, and the contribution of memory T cell differentiation state and location to protection from re-infection. Memory T cells that reside at common portals of pathogen entry or infection, especially the intestinal mucosa, are of particular interest. By understanding these issues, we hope to contribute to the development of better vaccination strategies, and are currently focused on informing development of a protective HIV vaccine.

Research Summary

Research in my laboratory is focused on the virulence factors and pathogenic mechanisms of the gram-negative bacterium Burkholderia cepacia. B. cepacia has recently emerged as an important opportunistic pathogen of the lower respiratory tract, affecting immunocompromised individuals and particularly cystic fibrosis (CF) patients.

Projects in My Laboratory:

Type III Secretion Pathogenesis in a growing number of gram-negative bacteria is dependent on the secretion of virulence proteins via a conserved secretory system, termed type III. We have identified and characterized a genetic locus encoding multiple type III secretion genes in B. cepacia. Using allelic exchange mutagenesis, we generated defined null mutations in central components of the type III secretion system, and demonstrated that a type III mutant is severely attenuated in virulence in a murine model of infection. We are currently pursuing proteomic and genomic-based approaches to identify the secreted substrates of the B. cepacia type III translocon, as well as further characterize the role of type III secretion in modulating the host response to B. cepacia infection. Cable Pilus Biogenesis Cable pili are unique peritrichous adherence organelles expressed by certain strains of B. cepacia. Cable pili have been shown to mediate binding to both cellular and acellular receptors, and likely promote B. cepacia colonization of the respiratory tract of compromised hosts. We have undertaken a genetic analysis of cable pili and have identified and characterized a locus ( cbl) encoding structural, accessory, and regulatory components of the cable pilus biosynthetic pathway. We have also initiated studies to define the regulatory control of cable pilus gene expression, and have evidence that control is manifested at both the transcriptional and posttranscriptional levels. We have recently identified within the cbl locus three additional genes, designated cblS, cblT and cblR, predicted to encode new members of the superfamily of environmentally responsive two-component signal transduction systems. We are currently utilizing a combined genetic and biochemical approach to characterize the function of these genes and to define the signal transduction events controlling cable pilus gene expressionElectron micrographs of macrophages infected with Burkholderia cepaciaElectron micrographs of wild-type Burkholderia cepacia expressing cable pili (A-D) and a cblA mutant strain (E,F) blocked in cable pilus expression

Research Summary

Central Nervous System Penetration by Cryptococcus neoformans Fungal infections affect billions of people every year, often causing lethal disease in immunocompromised individuals. Of particular concern are invasive fungal infections, estimated to kill one and a half million people annually. Cryptococcus neoformans infections cause almost half of all deaths due to fungal infection. Even with access to the best available antifungals, Cn mortality rates of 20-40% remain unacceptably high. Although natural immunity to fungal infection is quite efficient in immune replete humans, disease in immune deficient individuals is often a complex interaction between failure to control fungal replication and an inappropriate immune response. My research program focuses on understanding how Cryptococcus causes disease, with the goal of developing better treatment strategies that improve patient survival. Our research primarily focuses on studies to understand the novel "titan cell" morphology during Cryptococcus infection. Upon inhalation, Cryptococcus establishes an initial pulmonary infection that eventually disseminates to the central nervous system (CNS) to cause meningitis. In response to the host pulmonary environment, a subset of Cryptococcus cells become titan cells that are 5-10x larger than typical-sized cells. These titan cells are critical for virulence, impact dissemination to the CNS, and their production during the infection dramatically alters the host response by reducing phagocytosis and stimulating a detrimental Th2-mediated response. Our current research is aimed at understanding the molecular mechanisms and host-pathogen interactions underlying the activity of titan cells during infection.

Research Summary

Herpes simplex virus gene expression The infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in dramatic alterations to the host cell nucleus, so that viral genes are expressed at high levels, while cellular genes are nearly completely suppressed. This genetic subversion is accomplished by a small set of HSV-1 regulatory proteins, which are amenable to biochemical and genetic analysis. Currently, our laboratory is studying two of these proteins, ICP27 and ICP22. ICP27 is conserved in all known herpesviruses and is absolutely essential for HSV-1 late gene expression. Although its mechanism of action is unknown, a variety of evidence suggests that it is an unusual type of gene regulator which affects pre-mRNA processing and transport. Consistent with its suspected post-transcriptional role, we have shown that ICP27 is an RNA-binding protein that continuously shuttles between the nucleus and cytoplasm. We are also studying ICP22, an HSV-1 protein which is required for viral growth in some cell lines. We have found that HSV-1 infection alters the phosphorylation of the large subunit of RNA polymerase II, and that this effect requires ICP22. Our current goal is to delineate the molecular pathway by which ICP22 alters RNA polymerase II and to understand the functional significance of this change.

Bio

As Associate Dean for the Undergraduate Curriculum in the Office of Undergraduate Education, Leslie Schiff oversees activities of the Campus Curriculum Committee, the Council on Liberal Education, and the Campus Writing Board, and serves as a liaison to collegiate-level curriculum committees. In addition, she is a member of the OUE Executive Committee for Assessment and serves as an advisor on educational policy issues related to the curriculum.Schiff earned her Sc.B in Biology from Brown University in 1979 and her Ph.D. in Immunology from Tufts University in 1986. She joined the faculty at the University of Minnesota in 1990 after a post-doctoral fellowship at Harvard Medical School. A Professor in the Department of Microbiology, Leslie has served for many years as Director of Undergraduate Studies in Microbiology and as faculty liaison for the College of Biological Sciences Writing-Enriched Curriculum. She developed and teaches a two-semester support course for thesis writers in biology, as well as a writing-intensive course on the biology of viruses. Schiff has received University and national awards for her teaching, including the Carski Award from the American Society for Microbiology, and the Morse-Alumni Distinguished Teaching Award. Her disciplinary research focuses on the cell entry of mammalian reoviruses, which are being developed as therapeutic agents to treat human cancers.

Research Summary

Virus-host interactions, viral entry, translational control in virus-infected cells, viral immune avoidance mechanisms

Research Summary

Understanding the dynamics of how growth-promoting mutations arise and accumulate in a population of cells is a fundamental problem underlying our understanding of drug resistance, tumorigenesis, and the treatment of cancer. We use experimental evolution, mathematical modeling, and comparative genomics to understand the impact of mutations on the adaptation of a cell and its surrounding population.We employ diverse yeast model systems (Saccharomyces cerevisiae, Candida albicans, Candida auris, etc.) to understand how genome instability contributes to adaptation (eg. antifungal drug resistance) and determine the underlying mechanisms that promote genome instability. In the human fungal pathogen Candida albicans, genome rearrangements resulting in copy number variation (CNV) and loss of heterozygosity (LOH) confer increased virulence and antifungal drug resistance, yet the mechanisms driving these rearrangements are not completely understood. We recently identified an extensive array of long repeat sequences (65–6499 bp) that are associated with CNV, LOH, and chromosomal inversions and are a significant source of genome plasticity across diverse strain backgrounds - including clinical, environmental, and experimentally evolved C. albicans isolates. Many of these long repeat sequences were uncharacterized and encompass one or more coding sequences that are actively transcribed. Further, the repeats associated with genome rearrangements were predominantly inverted and separated by up to ~1.6 Mb, an extraordinary distance for homology-based DNA repair/recombination in yeast!We also utilize flow cytometry-based systems that enable us to detect the acquisition and spread of beneficial mutations within populations. We found that polyploid S. cerevisiae adapted more rapidly than isogenic haploid or diploid cells in poor carbon medium, and that polyploid cells acquired more mutations, including point mutations, large segmental aneuploidies, and whole chromosome aneuploidies (Selmecki et al., Nature 2015). Additionally, polyploid cells acquired a broader spectrum of beneficial mutations than lower ploidy cells (Scott et al., MBE 2017). We continue to use these ploidy lineages to study how changes in chromosome number (ploidy and aneuploidy), cell size, and environment affect genome stability and evolvability. Our previous research identified chromosome aneuploidy as a driver for the acquisition of antifungal drug resistance in the pathogenic yeast C. albicans (Selmecki et al., Science 2006). We found that aneuploid cells arose within a population very rapidly in the presence of antifungal drug (Selmecki et al., PLoS Genetics 2009), and that increased copy number of two specific genes found on the most common aneuploid chromosome provided the drug resistance phenotype (Selmecki et al., Molecular Microbiology 2008).

Research Summary

Human Retrovirus Transmission and Pathogenesis I am interested in the cellular and molecular mechanisms underlying virus transmission and the initiation of virus-induced diseases. After working for many years with experimental infections of mice with an RNA virus, lymphocytic choriomeningitis virus (LCMV), my research focus has shifted recently to the study of infections with the human retroviruses, human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus types I and II (HTLV-I and HTLV-II). These retroviruses can be transmitted either vertically or horizontally in human populations and we are beginning to define essential parameters, in both the donor and recipient of infectivity, that impact on the overall probability of transmission. Our experiments are based on primary human cell populations, human organ cultures, and body fluids from seropositive patients. Our goal is to develop manipulable experimental systems that duplicate the natural routes for virus transmission. More specifically, we are investigating the nature of HIV-1 infections in male reproductive tissue to understand variability in the shedding of HIV-1 infectivity in seminal fluid. In recipients, we are interested in defining the mechanisms whereby HIV-1 infectivity can breach epithelial barriers at mucosal surfaces and gain access to intraepithelial leukocytes for the initiation and expansion of primary infections. New insight into HIV transmission, with both cell-free and cell-associated HIV infectivity, will be invaluable in refining vaccine studies and may contribute significantly to the design of novel therapeutic agents to reduce the probability of HIV transmission.

Research Summary

Cancer immunology and immunothereapy; T cell engineering The Stromnes laboratory is focused on advancing the understanding of cancer immunology and immunotherapy, with a particular emphasis on properties of the tumor and the tumor microenvironment that influence antigen-specific T lymphocyte migration and function. The laboratory aims to uncover how tumors and their mutations coordinate a suppressive microenvironment and elicit a program of T cell dysfunction. The lab is also focused on understanding the cellular and acellular components of tumor microenvironment that influence immunotherapy response. The laboratory combines studies of human tumors, genetically engineered mouse models that faithfully recapitulate human cancer, and novel cell engineering approaches evaluated in both mouse and human T cells to develop next generation cellular therapies for intractable malignancies, with a major focus on carcinomas including pancreatic cancer. Preclinical studies performed by Dr. Stromnes and colleagues have led to a novel engineered T cell therapy for pancreatic and ovarian cancer patient treatment.

Research Summary

Mycobacterium tuberculosis Counter-immune MechanismsThe bacterial pathogen Mycobacterium tuberculosis (Mtb) is unusual in its ability to persist in the lungs of infected humans for many years, despite triggering a robust immune response. My laboratory seeks to identify and characterize factors that Mtb requires for evasion of host adaptive immunity and persistence. We use a combination of bacterial genetics and the mouse infection model to identify these "counter-immune" factors.The laboratory is currently focused on characterization of an Mtb signal transduction system, Pst/SenX3-RegX3, which regulates gene expression in response to phosphate availability. Mtb mutants that lack components of this system are specifically sensitive to host adaptive immunity and fail to persist in the lungs of mice. We aim to identify the genes controlled by this system that directly contribute to avoidance of host immune responses. Knowledge of these interactions between host and pathogen may contribute to vaccine design, as well as provide novel targets for therapeutic intervention.

Research Summary

T cell responses in autoimmunity and chronic infections My laboratory studies CD8 and CD4 T cell responses to proteins, which are persistently present in an organism to elucidate how chronic interaction with cognate antigen impacts T cell selection, differentiation and survival. We observe and manipulate chronic pathogen or self-specific T cell responses over time by using MHC tetramers, adoptive transfer of transgenic T cells and fluorescence flow cytometry. We are currently interested in understanding what maintains the population of memory T cells specific for persistent pathogens, such as polyoma virus. In addition, we are interested in elucidating how tolerance is induced and maintained to intestinal protein, as breakdown of this can lead to diseases such as ulcerative colitis and Crohn's disease. By understanding these processes, we can learn how to manipulate the immune system for eradication of persistent infections, as well as interfering with the development and progression of autoimmunity.