Ulrike G. Munderloh - US grants

The human granulocytic ehrlichiosis (HGE) agent is a newly identified zoonotic pathogen capable of causing a potentially fatal illness. In the USA, it is transmitted by Ixodes ticks in areas that essentially overlap the distribution of Lyme disease owing to the fact that both agents share the same vector. Serologic surveys indicate that the disease is widespread also in Europe where it has recently been identified in a human patient. Last year, two research groups isolated the etiologic agent from human patients (Goodman et al. 1996) and a horse (Munderloh et al. 1996), utilizing cell culture systems that represent the spectrum of the life cycle of the agent in nature, a human promyelocytic leukemia cell line and an Ixodes scapularis tick cell line, respectively. These achievements have opened the way to investigate the cellular and molecular biology of the agent in vitro in human and vector cell systems. The development of the agent in tick cell culture differs strikingly from its growth in human cells, hinting at specific adaptations to these divergent hosts. In collaboration with Dr. Goodman, I have identified hamsters as a small laboratory animal that is susceptible to the agent from human and tick cell culture, and I. scapularis ticks have experimentally transmitted it to hamsters. Hamsters developed infections of bone-marrow cells and developed hematologic signs similar to those seen in humans. This proposal aims 1) to elucidate in detail the interaction of the HGE agent with its vector in vivo and in vitro, focussing on environmental cues that stimulate development and expression of surface proteins which could serve as ligands during invasion. These changes will be related to infectivity and pathogenicity for cultured cells and mammalian hosts; 2) to determine the role of transstadial vs transovarial maintenance in the natural history of the HGE agent; 3) to characterize the antigenic profile of the agent in tick cell culture and in ticks which are presumably representative of the make-up of immunogens first encountered by the mammal, and which may have implications for diagnosis and vaccine development.

DESCRIPTION: (provided by the applicant): Tick-borne diseases are increasingly diagnosed in humans and animals. Some are due to the resurgence of previously known illnesses, like Rocky Mountain spotted fever (Walker 1995). but others are due to new, emerging pathogens. Among the spotted fever group (SFG) alone, 8 new human pathogens have been described in the last 15 years (Stenos et al. 1997; Nilsson et al. 1999; Fournier et al. 2000), but they also include viruses, ehilichias and Babesia (Dawson et al. 1991; Thomford et al. 1994; Bakken et al. 1994; Telford et al 1991). Novel, efficient, specific and environmentally acceptable methods that interfere with disease transmission by ticks are urgently needed. Using paratransgenic ticks that carry symbiotic prokaryotes expressing an antimicrobial substance, as has been achieved with the symbiote of the Chagas disease vector, Rhodnius prolixus (Durvasula et aL 1997), could offer a safe and effective way to reduce disease transmission by ticks. A major obstacle to accomplishing this goal has been the lack of culture systems for tick symbiotes. Our laboratory has the largest collection of tick cell lines. We have successfully used these to isolate tick-associated rickettsiae (Munderloh et al. 1998; Weller et al. 1998; Palmer et al. 1999; Simser et al. 2001a,b) from the Lone Star tick (MOAa), the Rocky Mountain wood tick (R. peacockli DAE100R), and the Castor Bean tick (Rmoreli T2). We have characterized these microbes by light and electron microscopy, by using specific antibodies, as well as by PCR and nucleotide sequence analysis of 165 rDNA and other key genes. We are now in the process of defining the cultures to facilitate genetic manipulation of the symbiotes. Ourlong-termaim is the stabletransformation of Rickettsiapeacockii with cecropinA. an insect ponn gene (Hultmark et al. 1983). Infection of ticks with the transformed rickettsia, and interference with pathogen transmission. We plan to target the non-functional rompA gene of R. peacockli as a site for homologous transformation, avoiding deleterious effects associated with disruption of a vital gene, e.g. the rpoB gene (troyer et al. 1999). We will take the recent advances in successful transformation of insect-borne rickettsiae as a guide (Rachek et al. 1998; Troyer et al. 1999). and also apply transposome technology (Epicentre). Specifically, we will 1. optimize culture conditions for production of R. peacockii in tick cell culture, exanine its behavior in tick and mammalian cell culture by light and electron microscopy. 2. We will analyze cultured R. peacockii in ticks in terms of tissue tropism and transstadial/transovanal passage, and sensitivity to Cecropin A. Finally, we will work towards 3. stable transformation of R. peacockii with cecropinA. We will then test the transfonnants for antimicrobial activity in vitro and in ticks, and characterize them by sequence analysis.

DESCRIPTION (Adapted from the applicant's abstract): Human granulocytic ehrlichiosis (HGE) is an acute febrile illness described 2 years ago in Minnesota and Wisconsin and now rapidly emerging in areas infested by its likely vector, Ixodes ticks. HGE appears in blood granulocytes, causes depression of leukocyte and platelet counts, and can result in serious complications. Diagnosis has been difficult and serologic testing, using leukocytes from horses infected with the related Ehrlichia equi, is insensitive at presentation. The applicant recently isolated the etiologic agent of HGE from patients by cultivation in HL60 cells (Goodman, et al, 1996). In addition, the applicant has grown E. equi (Munderloh et al, 1996), and now HGE, in I. scapularis cell lines, where it develops different forms than in human cells. These findings provided the basis for studies aimed at better understanding the biology of the agent. In his laboratory, HGE has now also been grown in its presumed natural target cells, human bone marrow progenitors, with infection of both granulocytic and monocytic cells and precursors. The applicant's studies also strongly suggest that sialyl Lewis x (CD15s), which is present on both cell types, is a major cell surface receptor for HGE. The agent manifests unique iron/siderophore interactions and appears capable of survival within endosomes. I. scapularis was infected with cultured HGE and used to tick bite infect hamsters (resulting in blood findings similar to those in humans). The agent was then reisolated from infected hamsters, completing its life-cycle. HGE-based IFA and immunoblot assays have been developed to begin to define major immunogens, their temporal evolution during infection, and possible differences in antigenic expression in tick vs. human cells. Working in an endemic area, the applicant has formed a multidisciplinary team to build rapidly upon these advances. Dr. Goodman's aims are: 1) to characterize, at the molecular level, the interaction of the HGE agent with both its human target cells and receptor(s), 2) to characterize the biologically critical interaction of HGE with its tick vector and cultured tick cells and compare these interactions with those defined in human cells and 3) to identify and characterize the key antigenic proteins of HGE and the immune response to these antigens.

Anaplasma phagocytophilum (Ap) is an intracellular pathogen causing human granulocytic anaplasmosis (HGA, formerly HGE), an emerging tickborne infection that can be fatal. Ap invades neutrophil granulocytes (PMN), the first cells to infiltrate the tick bite site, via PSGL-1 which normally initiates rolling on inflamed endothelium, leading to extravasation, phagocytosis and death of PMN. But because infected PMN remain in the blood stream and are too short-lived to maintain Ap over extended periods, we hypothesize that other cells may be involved. We found that Ap multiplies in human primary microvascular endothelial cells and lines, causing upregulation of adhesion molecules, adherence of PMN, and increased expression of TNFalpha and CD83 by HL-60 cells. Infected human endothelial cells alter the ratio of MHC-I and II expression, and upregulate galectin-1, which has been found to inhibit PMN transmigration. We hypothesize that Ap infects endothelium, disrupting adhesion-rolling and transmigration to infect PMN, and induces production or display of immunomodulatory substances by infected endothelium. We will examine gene expression with human gene arrays and RNA from endothelial cells, HL-60 and PMN exposed to Ap for various times and conditions, focussing on genes linked to immuneregulation, leukocyte function, and inflammation. We will use confocal microscopy with FISH, antibodies to Ap and cell surface markers to follow Ap from tick to mammal, identify sites of infection, detail endothelial cell invasion and passage to PMN. Our DsRed-expressing tick and endothelial cells will facilitate imaging. Ap will be electroporated with fluorescent protein genes, as already achieved by us with Rickettsiae. Until available Ap are labeled with live cell dyes. Endothelial and PMN receptors will be identified in adhesion assays with antibodies to cell surface markers and lectins. Surface association of Ap and relationship to cellular markers will be studied by FESEM. To identify Ap adhesion proteins, we will screen a cDNA library with blocking antisera, but focus on p100, p130 and MSP2 variants.

[unreadable] DESCRIPTION (provided by applicant): The biology of Anaplasma phagocytophilum (Ap) and its host are intertwined. Specific co-adaptations which have co-evolved over millennia govern the interactions between disease agents and humans. This proposal will address the symbiosis of parasite and host at the molecular level using human cells and a human pathogen. To understand how Ap exploits its host cells requires an understanding of what the host provides for the microbes. Our central hypothesis is that Ap redirects the cellular metabolic machinery for its own use. We hypothesize that Ap engages in molecular parasitism to complement its streamlined genome, that is deficient in many metabolic pathways. Therefore, we propose to determine the role of human host cells in supporting Ap parasitism through systematic knockdown of 47,400 human genes using a pseudoviral library of short interfering RNA (siRNA), followed by transfeetion of cells with specific siRNA constructs. Silencing expression of cellular genes critical for Ap infection will render cells unable to support Ap. We will conduct a genomic screen using an siRNA library to identify host factors required for adhesion, invasion, and growth of Ap in human cells. Host cells with gene knockdown(s) that interfere with Ap infection can be isolated, and the siRNA species responsible identified using human genomic DNA arrays. To evaluate the resultant list of genes, we will map them to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database to link the silenced genes to metabolic pathways. Selected genes identified on microarrays will be targeted using the pMaleficent Sleeping Beauty transposon based RNAi delivery system to produce heritable integration and expression of specific siRNA in human host cell lines. The ability of Ap to enter and multiply in host cells will be assessed by light and fluorescent microscopy, time-lapse microscopy, FACS analysis and by using a fluorescent plate reader. We expect to generate human cell lines with defined and stable gene knockdown phenotypes. Remapping affected genes on the KEGG database will reveal and confirm specific dependencies of Ap on human metabolites that may lead to new treatments and vaccines against these and other intracellular pathogens. Although the human genome has been sequenced, a significant proportion of ORFs are of unknown function. We anticipate that the interaction of the human host cells with these highly specialized intracellular pathogens will shed light,on the nature of some unidentified human genes. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The genus Rickettsia contains important pathogens and consists of small gram-negative bacteria that replicate within eukaryotic host cells. Many Rickettsia species are associated with blood-feeding arthropods (ticks, lice, fleas and mites) that serve as vectors for their transmission to vertebrate hosts. Various Rickettsia species are the etiologic agents of typhus and spotted fevers in humans but others are apparently confined to arthropod hosts as endosymbionts. Members of the Rickettsia are the closest known microbial relatives of mitochondria with which they share the characteristics of highly reduced genomes and intimate metabolic interactions with their host cells. Historically, the slow and obligate intracellular growth of rickettsiae and their resistance to genetic manipulation presented substantial technical challenges to their study. Our laboratory has been at the forefront of both the recently developed capability to genetically manipulate rickettsiae and the discovery of rickettsial plasmids, which are extra-chromosomal autonomously replicating genetic elements. Plasmids in pathogenic bacteria are often associated with virulence functions involving particular metabolic capabilities or antibiotic resistance and are an important source of genetic diversity because they may be mobile, or genetically transmissible, via conjugation between bacteria of the same or different species. We hypothesize that plasmids are involved in rickettsial host adaptation/virulence and genetic plasticity. We propose a comprehensive analysis of the distribution, phylogeny and role of plasmids in the biology of rickettsiae associated with blood-feeding arthropods. We will use pulsed field gel electrophoresis and Southern blot techniques to define the distribution of plasmids within the genus. We will physically isolate and clone plasmids from Rickettsia species that range from non-pathogenic arthropod endosymbionts to human pathogens. The cloned plasmids will be sequenced with high throughput pyro-sequencing technology (454 Life Science Corp.) and subjected to phylogenetic analysis to determine the probable origin(s) and evolutionary history of plasmids within the genus, and obtain clues to their potential host adaptive functions. We will test our hypotheses that plasmids confer host adaptive functions and genetic diversity within rickettsiae through plasmid curing and phenotype analyses and plasmid conjugation experiments. The proposed research will delineate the distribution of plasmids and test their role in host adaptation/virulence in a group of medically important obligate intracellular bacteria that, until recently, were not believed to harbor plasmids. PUBLIC HEALTH RELEVANCE: Obligate intracellular bacteria of the genus Rickettsia are transmitted by blood-feeding insects and ticks. Members of the Rickettsia cause disease in humans that includes typhus and spotted fevers. The proposed research will characterize newly discovered genetic components (plasmids) of the Rickettsia and how they may contribute to the ability of these bacteria to infect insects, ticks and humans. The results of this research may provide new tools to control Rickettsia infections of humans.

DESCRIPTION (provided by applicant): The international community of researchers who are members of the American Society for Rickettsiology (ASR) is represented in countries from Australia to Zimbabwe, including, of course, the USA and Europe. This wide geographic representation is fed by the global emergence of vector-borne rickettsial pathogens. The annual meetings of the ASR foster scientific exchange of discoveries and resources even before publication. This has resulted in an interwoven fabric of cooperating members that is highly conducive to the advancement of the field itself and facilitates connections between young investigators with established scientists. At every meeting, the organizers sponsor a junior member (primarily students, postdoctoral researchers and assistant professors) from each attending laboratory, specifically with the aim to strengthen the flow of new information, and to encourage young rickettsiologists to remain in the field. At the end of last year's meeting the organizers invited a discussion among all attendees to identify research areas viewed to be of highest priority for driving advances in rickettsiology. As a result, we propose to organize a 1 and 1/2-day workshop within the framework of the annual ASR meeting that addresses the central theme of Regulation of Gene Expression in Rickettsiae. This will broadly include molecular determinants of successful colonization of the arthropod vector (Abdu Azad UMD, Kevin Macaluso LSU) and the mammalian host (Jere McBride UTMB, Matt Welch UCB, Yasuko Rikihisa OSU), defensive responses of arthropods (Joao Pedra UCR, Viveka Vadyvaloo WSU) and mammals (Guy Palmer WSU, Nahed Ismail UPMC), and genetic manipulation of pathogens to identify the regulatory mechanisms that coordinate this interplay (Ulrike Munderloh UMN, Dan Rockey OR, Daniel Voth AR). The workshop will comprise oral presentations by the key speakers, and additional speakers who will be selected by each key speaker from junior faculty, postdoctoral scientists and students who have submitted abstracts. An in-depth discussion session will follow oral presentations. Oral presentation sessions are complemented by poster sessions that fall within the central theme. Members from underrepresented groups will be actively recruited as speakers and poster presenters. Key speakers will be asked to organize posters into groups of about five - six each, and will lead their discussion among poster presenters and participating meeting attendees. With the help of session chairs, and in collaboration with presenters, the major impacts of oral and poster presentations will be summarized, including the discussions, and published following the meeting.

? DESCRIPTION (provided by applicant): Human anaplasmosis caused by Anaplasma phagocytophilum (Ap) is now the second most prevalent tick-borne illness in North America, with increasing incidence also in Europe and Asia. The acute, pro-inflammatory syndrome is characterized by high fever, pancytopenia and elevated serum transaminases, requiring hospitalization in >36% of patients, with an overall mortality rate of ~1%. A major impediment to understanding the mechanisms whereby these obligate intracellular bacteria cause disease has been the lack of reproducible systems for mutagenesis to study gene function. This has been partially overcome with the establishment of a library containing ~1,200 intra- and intergenic insertions using human cells to select mutants. The broad, long-term goals of this renewal application are to use the principles of functional genomics to understand how Ap bacteria use their genome and specific genes to thrive in two biologically vastly different hosts, mammals and ticks, and cause human disease. Our central hypothesis is that many mutations that are tolerated in an in vitro human cell culture system will nevertheless produce a deficient phenotype in human and tick cells in vitro, and in mice and ticks. Therefore, we 1) plan to catalogue and develop the currently uncharacterized library into a resource for the scientific community. We will test the hypothesis that mutants selected in a human promyelocyte cell line will allow detailed analyses of the molecular basis of gene function in cell cultures from humans and ticks. We propose a high throughput screen of candidate mutants for defective infectivity in mice and ticks, and analyses for changes in phenotype in vitro using human and tick cell culture systems. 2) We will use advanced imaging technology, proteomics and 2-hybrid bacterial screens to reveal intracellular trafficking of secreted effectors and their interactions with host targets, with emphasis on T4SS structures and effectors, other secreted proteins, outer membrane proteins and hypothetical proteins that were selected using bioinformatics-based predictions. 3) We hypothesize that mutants selected in vector tick cell culture will additionally identify genes that are essential for growth in human cells, and which are not represented in the current library. To do this, we will refine methods for selection of transposon mutants in tick cel culture using constructs designed for facile complementation of mutants with wild-type gene sequences to restore function, and develop site-directed mutagenesis using CRISPR/Cas9 mediated allelic exchange. Investigators from three collaborating laboratories will join in this effort to maximize advancement of research plans. The knowledge gained will broadly impact the field of rickettsiology and identify the molecular basis underlying pathogenic mechanisms and survival strategies of arthropod-borne intracellular bacteria. These efforts will enable a gian step forward in functional genomics and enable rational design of vaccines and drugs for the Rickettsiales, a group that includes severe pathogens, e.g., Ap, Ehrlichia chaffeensis, Rickettsia rickettsii and R. prowazekii for which such tools are either lacking or in great need of refinement

Tick-borne bacteria in the family Anaplasmataceae cause emerging zoonoses across the globe, such as human anaplasmosis and ehrlichioses, where competent vector ticks occur. Due to the non-specific signs and symptoms they cause, and absence of diagnostic antibodies at the time of illness onset, they may be confused with better recognized febrile illnesses such as rickettsioses or viral diseases. Severe human ehrlichiosis is characterized by a pro- inflammatory syndrome, but the bacterial gene products responsible for inducing adverse host reactions are unknown. Determination of gene function in the Anaplasmataceae is complicated by their obligately intracellular nature, yet, this would allow identification of targets for prevention and treatment of disease. Previous animal models of ehrlichiosis do not reproduce features of human disease, which makes it difficult to investigate virulence factors. Our group has isolated a novel human ehrlichiosis agent (Ehrlichia muris-like agent, EMLA) in vitro from both a human patient and a blacklegged tick. EMLA causes disease in laboratory mice that recapitulates human monocytic ehrlichiosis, making it now possible to investigate the factors that cause clinical ehrlichiosis in a mouse model. To elucidate the molecular basis of pathogenicity and tick transmission in ehrlichiae, we propose to generate a library of mutant EMLA using random mutagenesis and characterization by Illumina sequencing-based insertion site determination. We will use a transposon containing a cassette that can subsequently be replaced with a functional gene copy for complementation. We will implement our plan through completion of the following specific aims: AIM 1: Produce a library of Ehrlichia muris-like agent (EMLA) mutants with a replaceable selection cassette in tick and human cell cultures. AIM 2a: Map insertion sites of transposons by Illumina-based sequence analysis of mutant pools followed by assignment to individual mutant lines using PCR. 2b. Identification of mutants defective for invasion and successful colonization of human and tick cells using cell invasion/colonization screens, bioinformatics analysis, and genetic complementation. Ehrlichia species differentially express genes depending on the host cell in which they reside. We will raise mutants in human and tick cells to recover bacteria with disrupted genes that are essential for colonization of only one host cell type, as well as those that govern pathogenicity but are not essential. We realize that genes required for colonization of and transmission by ticks are important for ability of ehrlichiae to infect mammals. With the availability of the human, the EMLA, and the Ixodes scapularis genomes, the conditions are now right to identify ehrlichia genes that mediate interactions with its host and vector in vitro, setting the stage for more comprehensive studies in a subsequent project.

TICK RESOURCES CORE Abstract: The overarching goal of the Tick Resources Core within this P01 application is to provide support to all three research projects and share reagents, materials and new technologies with the scientific community. Requests from the community will be handled through the Biodefense and Emerging Infections Research Resources Repository (BEI) website (www.beiresources.org). BEI was established by the National Institute of Allergy and Infectious Diseases and has been managed under contract by the American Type Culture Collection for fifteen years. Dr. Ulrike Munderloh, a Professor at the University of Minnesota, will be the director of this technical core and is ideally suited to manage the facility. First, she has been a pioneer in the field of tick-pathogen interactions by developing and refining techniques for mutagenesis, plasmid-based complementation, and functional genomics for rickettsial agents in addition to inventing a membrane feeder system for ticks. Second, her laboratory has contributed to 22 out of 30 Rickettsiales species for sequencing in the ?Rickettsiales Genomes? project, and supplied 10 of the 14 isolates deposited in BEI. Third, Dr. Munderloh has an outstanding record of distributing tick cell lines and microbial isolates to the scientific community. Aim#1 of the Tick Resources Core will provide currently available tick-vector-based tools to support the research for all three research projects within this P01 application. Aim#2 of the Tick Resources Core will develop novel cell lines suitable for analyses of immune pathways that are activated or repressed in response to pathogens, symbionts and selected microbiota. This will be achieved through RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9), reporter tags and overexpression assays. Model organisms have a myriad of tools that facilitate the study of pathogen-arthropod interactions. They also provide critical insights to the field of entomology, microbiology and immunology. However, we present strong evidence in this P01 application that Ixodes scapularis ticks seem to be built on entirely different principles that govern microbial pathogenesis and immunity. Therefore, a strong Tick Resources Core is of utmost importance to support all three Projects within this P01 application. By providing existing I. scapularis-based tools and developing novel technologies to investigators, the Tick Resources Core will contribute to uncover novel paradigms in I. scapularis-microbial interactions. This scientific knowledge has potential implications for basic and translational science.