Publications by authors named "Utpal Pal"

92 Publications

Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection.

Nat Commun 2021 06 17;12(1):3696. Epub 2021 Jun 17.

Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA.

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases.
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http://dx.doi.org/10.1038/s41467-021-23900-8DOI Listing
June 2021

Tick host immunity: vector immunomodulation and acquired tick resistance.

Trends Immunol 2021 May 29. Epub 2021 May 29.

Department of Veterinary Medicine, University of Maryland, College Park, MD, USA; Virginia-Maryland College of Veterinary Medicine, College Park, MD, USA. Electronic address:

Ticks have an unparalleled ability to parasitize diverse land vertebrates. Their natural persistence and vector competence are supported by the evolution of sophisticated hematophagy and remarkable host immune-evasion activities. We analyze the immunomodulatory roles of tick saliva which facilitates their acquisition of a blood meal from natural hosts and allows pathogen transmission. We also discuss the contrasting immunological events of tick-host associations in non-reservoir or incidental hosts, in which the development of acquired tick resistance can deter tick attachment. A critical appraisal of the intricate immunobiology of tick-host associations can plant new seeds of innovative research and contribute to the development of novel preventive strategies against ticks and tick-transmitted infections.
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http://dx.doi.org/10.1016/j.it.2021.05.005DOI Listing
May 2021

Grappling with the tick microbiome.

Trends Parasitol 2021 May 4. Epub 2021 May 4.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06420, USA.

Ixodes scapularis and Ixodes pacificus are the predominant vectors of multiple human pathogens, including Borrelia burgdorferi, one of the causative agents of Lyme disease in North America. Differences in the habitats and host preferences of these closely related tick species present an opportunity to examine key aspects of the tick microbiome. While advances in sequencing technologies have accelerated a descriptive understanding of the tick microbiome, molecular and mechanistic insights into the tick microbiome are only beginning to emerge. Progress is stymied by technical difficulties in manipulating the microbiome and by biological variables related to the life cycle of Ixodid ticks. This review highlights these challenges and examines avenues to understand the significance of the tick microbiome in tick biology.
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http://dx.doi.org/10.1016/j.pt.2021.04.004DOI Listing
May 2021

Antibodies against EGF-like domains in Ixodes scapularis BM86 orthologs impact tick feeding and survival of Borrelia burgdorferi.

Sci Rep 2021 Mar 17;11(1):6095. Epub 2021 Mar 17.

Department of Veterinary Medicine, University of Maryland, College Park, MD, 20742, USA.

Ixodes scapularis ticks transmit multiple pathogens, including Borrelia burgdorferi sensu stricto, and encode many proteins harboring epidermal growth factor (EGF)-like domains. We show that I. scapularis produces multiple orthologs for Bm86, a widely studied tick gut protein considered as a target of an anti-tick vaccine, herein termed as Is86. We show that Is86 antigens feature at least three identifiable regions harboring EGF-like domains (termed as EGF-1, EGF-2, and EGF-3) and are differentially upregulated during B. burgdorferi infection. Although the RNA interference-mediated knockdown of Is86 genes did not show any influences on tick engorgement or B. burgdorferi sensu stricto persistence, the immunization of murine hosts with specific recombinant EGF antigens marginally reduced spirochete loads in the skin, in addition to affecting tick blood meal engorgement and molting. However, given the borderline impact of EGF immunization on tick engorgement and pathogen survival in the vector, it is unlikely that these antigens, at least in their current forms, could be developed as potential vaccines. Further investigations of the biological significance of Is86 (and other tick antigens) would enrich our knowledge of the intricate biology of ticks, including their interactions with resident pathogens, and contribute to the development of anti-tick measures to combat tick-borne illnesses.
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http://dx.doi.org/10.1038/s41598-021-85624-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971074PMC
March 2021

Interactions Between Ticks and Lyme Disease Spirochetes.

Curr Issues Mol Biol 2021 8;42:113-144. Epub 2020 Dec 8.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.

sensu lato causes Lyme borreliosis in a variety of animals and humans. These atypical bacterial pathogens are maintained in a complex enzootic life cycle that primarily involves a vertebrate host and spp. ticks. In the Northeastern United States, is the main vector, while wild rodents serve as the mammalian reservoir host. As is transmitted only by and closely related ticks, the spirochete-tick interactions are thought to be highly specific. Various borrelial and arthropod proteins that directly or indirectly contribute to the natural cycle of infection have been identified. Discrete molecular interactions between spirochetes and tick components also have been discovered, which often play critical roles in pathogen persistence and transmission by the arthropod vector. This review will focus on the past discoveries and future challenges that are relevant to our understanding of the molecular interactions between and ticks. This information will not only impact scientific advancements in the research of tick- transmitted infections but will also contribute to the development of novel preventive measures that interfere with the life cycle.
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http://dx.doi.org/10.21775/cimb.042.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045411PMC
December 2020

Acquired tick resistance: The trail is hot.

Parasite Immunol 2021 May 15;43(5):e12808. Epub 2020 Dec 15.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA.

Acquired tick resistance is a phenomenon wherein the host elicits an immune response against tick salivary components upon repeated tick infestations. The immune responses, potentially directed against critical salivary components, thwart tick feeding, and the animal becomes resistant to subsequent tick infestations. The development of tick resistance is frequently observed when ticks feed on non-natural hosts, but not on natural hosts. The molecular mechanisms that lead to the development of tick resistance are not fully understood, and both host and tick factors are invoked in this phenomenon. Advances in molecular tools to address the host and the tick are beginning to reveal new insights into this phenomenon and to uncover a deeper understanding of the fundamental biology of tick-host interactions. This review will focus on the expanding understanding of acquired tick resistance and highlight the impact of this understanding on anti-tick vaccine development efforts.
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http://dx.doi.org/10.1111/pim.12808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058238PMC
May 2021

Epigenetic Regulation of Tick Biology and Vectorial Capacity.

Trends Genet 2021 01 2;37(1):8-11. Epub 2020 Oct 2.

Department of Veterinary Medicine, University of Maryland, College Park, MD, USA; Virginia-Maryland College of Veterinary Medicine, College Park, MD, USA. Electronic address:

Ticks exist across diverse environments and transmit numerous pathogens. Due to their long and unique life cycles, these arthropods likely evolved robust epigenetic mechanisms that provide sustainable responses and buffers against extreme environmental conditions. Herein, we highlight how the study of the epigenetic basis of tick biology and vectorial capacity will enrich our knowledge of tick-borne infections.
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http://dx.doi.org/10.1016/j.tig.2020.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008791PMC
January 2021

A novel tick protein supports integrity of gut peritrophic matrix impacting existence of gut microbiome and Lyme disease pathogens.

Cell Microbiol 2021 02 21;23(2):e13275. Epub 2020 Oct 21.

Department of Veterinary Medicine, University of Maryland, College Park and Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland, USA.

The peritrophic matrix (PM) is an acellular membrane that covers the gut epithelium in arthropods and physically separates it from the lumen. The structure is thought to play an important role in tick biology. The PM is also known to impact the persistence of tick-borne pathogens like Borrelia burgdorferi, although limited information is available about its molecular constituents or their biological significance. Herein, we characterise a novel PM-associated gut protein in Ixodes scapularis ticks, annotated as Peritrophic Membrane Chitin Binding Protein (PM_CBP), for its role in the integrity and function of the matrix. The PM_CBP displays homology to the chitin deacetylase metalloenzyme, shows upregulation during tick feeding, and is localized at the luminal surface of the gut epithelium. The structural integrity of the PM was impaired both by the knock down of PM_CBP expression via RNA interference and by treatment with anti-PM_CBP antibodies, as revealed by its electron microscopic appearance. Additionally, the duration of tick engorgement on mice and the passage of experimentally-inoculated fluorescent dextran molecules across the PM are affected by the knock down of PM_CBP expression. The transfer of anti-PM_CBP antibodies into the tick gut impacted the overall composition of the resident microbiome, and also influenced B. burgdorferi acquisition in ticks and its transmission to mice. Taken together, these data highlight the biological significance of the Ixodes PM and suggest that the targeting of its molecular constituents may contribute to the development of novel interventions against tick-borne infections.
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http://dx.doi.org/10.1111/cmi.13275DOI Listing
February 2021

An Ixodes scapularis Protein Disulfide Isomerase Contributes to Borrelia burgdorferi Colonization of the Vector.

Infect Immun 2020 11 16;88(12). Epub 2020 Nov 16.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.

causes Lyme disease, the most common tick-transmitted illness in North America. When feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, rotein isulfide somerase (IsPDIA3), enhances colonization of the tick gut. ticks in which has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on -infected mice. Moreover, administration of IsPDIA3 antiserum to -infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.
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http://dx.doi.org/10.1128/IAI.00426-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671890PMC
November 2020

Interactions between Borrelia burgdorferi and ticks.

Nat Rev Microbiol 2020 10 10;18(10):587-600. Epub 2020 Jul 10.

Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT, USA.

Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted to vertebrate hosts by Ixodes spp. ticks. The spirochaete relies heavily on its arthropod host for basic metabolic functions and has developed complex interactions with ticks to successfully colonize, persist and, at the optimal time, exit the tick. For example, proteins shield spirochaetes from immune factors in the bloodmeal and facilitate the transition between vertebrate and arthropod environments. On infection, B. burgdorferi induces selected tick proteins that modulate the vector gut microbiota towards an environment that favours colonization by the spirochaete. Additionally, the recent sequencing of the Ixodes scapularis genome and characterization of tick immune defence pathways, such as the JAK-STAT, immune deficiency and cross-species interferon-γ pathways, have advanced our understanding of factors that are important for B. burgdorferi persistence in the tick. In this Review, we summarize interactions between B. burgdorferi and I. scapularis during infection, as well as interactions with tick gut and salivary gland proteins important for establishing infection and transmission to the vertebrate host.
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http://dx.doi.org/10.1038/s41579-020-0400-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351536PMC
October 2020

The Factor H-Binding Site of CspZ as a Protective Target against Multistrain, Tick-Transmitted Lyme Disease.

Infect Immun 2020 04 20;88(5). Epub 2020 Apr 20.

Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, New York, USA

The spirochete is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.
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http://dx.doi.org/10.1128/IAI.00956-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171238PMC
April 2020

A Novel Laminin-Binding Protein Mediates Microbial-Endothelial Cell Interactions and Facilitates Dissemination of Lyme Disease Pathogens.

J Infect Dis 2020 04;221(9):1438-1447

Department of Veterinary Medicine, University of Maryland, College Park, Maryland, USA.

Borrelia burgdorferi conserved gene products BB0406 and BB0405, members of a common B. burgdorferi paralogous gene family, share 59% similarity. Although both gene products can function as potential porins, only BB0405 is essential for infection. Here we show that, despite sequence homology and coexpression from the same operon, both proteins differ in their membrane localization attributes, antibody accessibility, and immunogenicity in mice. BB0406 is required for spirochete survival in mammalian hosts, particularly for the disseminated infection in distant organs. We identified that BB0406 interacts with laminin, one of the major constituents of the vascular basement membrane, and facilitates spirochete transmigration across host endothelial cell barriers. A better understanding of how B. burgdorferi transmigrates through dermal and tissue vascular barriers and establishes disseminated infections will contribute to the development of novel therapeutics to combat early infection.
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http://dx.doi.org/10.1093/infdis/jiz626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137894PMC
April 2020

Protective Immunity and New Vaccines for Lyme Disease.

Clin Infect Dis 2020 04;70(8):1768-1773

Department of Medicine, Rutgers New Jersey Medical School, Newark, New Jersey, USA.

Lyme disease, caused by some Borrelia burgdorferi sensu lato, is the most common tick-borne illness in the Northern Hemisphere and the number of cases, and geographic spread, continue to grow. Previously identified B. burgdorferi proteins, lipid immunogens, and live mutants lead the design of canonical vaccines aimed at disrupting infection in the host. Discovery of the mechanism of action of the first vaccine catalyzed the development of new strategies to control Lyme disease that bypassed direct vaccination of the human host. Thus, novel prevention concepts center on proteins produced by B. burgdorferi during tick transit and on tick proteins that mediate feeding and pathogen transmission. A burgeoning area of research is tick immunity as it can unlock mechanistic pathways that could be targeted for disruption. Studies that shed light on the mammalian immune pathways engaged during tick-transmitted B. burgdorferi infection would further development of vaccination strategies against Lyme disease.
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http://dx.doi.org/10.1093/cid/ciz872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155782PMC
April 2020

Characterization of tick organic anion transporting polypeptides (OATPs) upon bacterial and viral infections.

Parasit Vectors 2018 Nov 14;11(1):593. Epub 2018 Nov 14.

Department of Biological Sciences, Old Dominion University, Norfolk, VA, USA.

Background: Ixodes scapularis organic anion transporting polypeptides (OATPs) play important roles in tick-rickettsial pathogen interactions. In this report, we characterized the role of these conserved molecules in ticks infected with either Lyme disease agent Borrelia burgdorferi or tick-borne Langat virus (LGTV), a pathogen closely related to tick-borne encephalitis virus (TBEV).

Results: Quantitative real-time polymerase chain reaction analysis revealed no significant changes in oatps gene expression upon infection with B. burgdorferi in unfed ticks. Synchronous infection of unfed nymphal ticks with LGTV in vitro revealed no significant changes in oatps gene expression. However, expression of specific oatps was significantly downregulated upon LGTV infection of tick cells in vitro. Treatment of tick cells with OATP inhibitor significantly reduced LGTV loads, kynurenine amino transferase (kat), a gene involved in the production of tryptophan metabolite xanthurenic acid (XA), levels and expression of several oatps in tick cells. Furthermore, bioinformatics characterization of OATPs from some of the medically important vectors including ticks, mosquitoes and lice revealed the presence of several glycosylation, phosphorylation and myristoylation sites.

Conclusions: This study provides additional evidence on the role of arthropod OATPs in vector-intracellular pathogen interactions.
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http://dx.doi.org/10.1186/s13071-018-3160-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6236954PMC
November 2018

Vector Immunity and Evolutionary Ecology: The Harmonious Dissonance.

Trends Immunol 2018 11 6;39(11):862-873. Epub 2018 Oct 6.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA. Electronic address:

Recent scientific breakthroughs have significantly expanded our understanding of arthropod vector immunity. Insights in the laboratory have demonstrated how the immune system provides resistance to infection, and in what manner innate defenses protect against a microbial assault. Less understood, however, is the effect of biotic and abiotic factors on microbial-vector interactions and the impact of the immune system on arthropod populations in nature. Furthermore, the influence of genetic plasticity on the immune response against vector-borne pathogens remains mostly elusive. Herein, we discuss evolutionary forces that shape arthropod vector immunity. We focus on resistance, pathogenicity and tolerance to infection. We posit that novel scientific paradigms should emerge when molecular immunologists and evolutionary ecologists work together.
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http://dx.doi.org/10.1016/j.it.2018.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218297PMC
November 2018

Borrelia burgdorferi protein interactions critical for microbial persistence in mammals.

Cell Microbiol 2019 02 8;21(2):e12885. Epub 2018 Jul 8.

Department of Veterinary Medicine, University of Maryland, College Park, Maryland.

Borrelia burgdorferi is the causative agent of Lyme disease that persists in a complex enzootic life cycle, involving Ixodes ticks and vertebrate hosts. The microbe invades ticks and vertebrate hosts in spite of active immune surveillance and potent microbicidal responses, and establishes long-term infection utilising mechanisms that are yet to be unravelled. The pathogen can cause multi-system disorders when transmitted to susceptible mammalian hosts, including in humans. In the past decades, several studies identified a limited number of B. burgdorferi gene-products critical for pathogen persistence, transmission between the vectors and the host, and host-pathogen interactions. This review will focus on the interactions between B. burgdorferi proteins, as well as between microbial proteins and host components, protein and non-protein components, highlighting their roles in pathogen persistence in the mammalian host. A better understanding of the contributions of protein interactions in the microbial virulence and persistence of B. burgdorferi would support development of novel therapeutics against the infection.
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http://dx.doi.org/10.1111/cmi.12885DOI Listing
February 2019

Immune Responses Against Lyme Disease Pathogens.

Front Cell Infect Microbiol 2018 29;8:176. Epub 2018 May 29.

Department of Veterinary Medicine and Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD, United States.

Although and other related tick species are considered prolific vectors for a number of important human diseases, many aspects of their biology, microbial interactions, and immunity are largely unknown; in particular, how these ancient vectors recognize invading pathogens like and influence their persistence. The analysis of the genome and a limited set of transcriptomic data have established that ticks encode many components of classical immune pathways; yet at the same time, they lack many key orthologs of these recognition networks. Therefore, whether a given immune pathway is active in ticks and how precisely they exert its microbicidal functions are only incompletely delineated. A few recent studies have suggested that classical pathways like the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) as well as immunodeficiency (IMD) pathways are fully functional in , and upon challenge with microbes, generate potent microbicidal responses against diverse tick-borne pathogens including . These studies also highlight novel concepts of vector immunity that include both a direct and an indirect mode of recognition of pathogens, as well as the influence of the gut microbiome, which ultimately dictates the outcome of a robust microbicidal response. Further understanding of how ticks recognize and suppress invading microbes like will enrich our fundamental knowledge of vector immunobiology, thereby contributing to the development of future interventions to better control the tick-borne pathogen.
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http://dx.doi.org/10.3389/fcimb.2018.00176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986905PMC
June 2019

Borrelia burgdorferi surface-located Lmp1 protein processed into region-specific polypeptides that are critical for microbial persistence.

Cell Microbiol 2018 09 25;20(9):e12855. Epub 2018 May 25.

Department of Veterinary Medicine, University of Maryland, College Park, MD, USA.

One of the Borrelia burgdorferi virulence determinants, annotated as Lmp1, is a surface-exposed, conserved, and potential multi-domain protein involved in various functions in spirochete infectivity. Lmp1 contributes to host-pathogen interactions and evasion of host adaptive immunity by spirochetes. Here, we show that in diverse B. burgdorferi species, Lmp1 exists as distinct, region-specific, and lower molecular mass polypeptides encompassing 1 or more domains, including independent N-terminal and middle regions and a combined middle and C-terminal region. These polypeptides originate from complex posttranslational maturation events, partly supported by a periplasmic serine protease termed as BbHtrA. Although spirochete persistence in mice is independently supported by domain-specific Lmp1 polypeptides, transmission of B. burgdorferi from ticks to mammals requires essential contributions from both N-terminal and middle regions. Interference with the functions of Lmp1 domains or their complex posttranslational maturation events may aid in development of novel therapeutic strategies to combat infection and transmission of pathogens.
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http://dx.doi.org/10.1111/cmi.12855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113067PMC
September 2018

Identification of Lyme borreliae proteins promoting vertebrate host blood-specific spirochete survival in Ixodes scapularis nymphs using artificial feeding chambers.

Ticks Tick Borne Dis 2018 07 4;9(5):1057-1063. Epub 2018 Apr 4.

Department of Biomedical Sciences, State University of New York at Albany, NY, USA; Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, NY, USA. Electronic address:

Lyme borreliosis, the most common vector-borne illness in Europe and the United States, is caused by spirochetes of the Borrelia burgdorferi sensu lato complex and transmitted by Ixodes ticks. In humans, the spirochetes disseminate from the tick bite site to multiple tissues, leading to serious clinical manifestations. The ability of spirochetes to survive in ticks during blood feeding is thought to be essential for Lyme borreliae to be transmitted to different vertebrate hosts. This ability is partly attributed to several B. burgdorferi proteins, including BBA52 and Lp6.6, which promote spirochete survival in nymphal ticks feeding on mice. One of the strategies to identify such proteins without using live animals is to feed B. burgdorferi-infected ticks on blood via artificial feeding chambers. In previous studies, ticks were only fed on bovine blood in the feeding chambers. In this study, we used this chamber model and showed that I. scapularis ticks will not only acquire bovine blood but human and quail blood as well. The latter two are the incidental host and an avian host of Lyme borreliae, respectively. We also investigated the roles that BBA52 and Lp6.6 play in promoting spirochete survival in nymphal ticks fed on human or quail blood. After feeding on human blood, spirochete burdens in ticks infected with an lp6.6-deficient B. burgdorferi were significantly reduced, while bba52-deficient spirochete burdens in ticks remained unchanged, similar to the wild-type strain. No strain showed a change in spirochete burdens in ticks fed on quail blood. These results indicate that Lp6.6 plays a role for B. burgdorferi in nymphs fed on human but not quail blood. Such information also demonstrates that the artificial feeding chamber is a powerful tool to identify B. burgdorferi proteins that promote vertebrate host blood-specific spirochete survival in I. scapularis ticks.
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http://dx.doi.org/10.1016/j.ttbdis.2018.03.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035877PMC
July 2018

Plasticity in early immune evasion strategies of a bacterial pathogen.

Proc Natl Acad Sci U S A 2018 04 2;115(16):E3788-E3797. Epub 2018 Apr 2.

Department of Veterinary Medicine, University of Maryland, College Park, MD 20742;

is one of the few extracellular pathogens capable of establishing persistent infection in mammals. The mechanisms that sustain long-term survival of this bacterium are largely unknown. Here we report a unique innate immune evasion strategy of , orchestrated by a surface protein annotated as BBA57, through its modulation of multiple spirochete virulent determinants. BBA57 function is critical for early infection but largely redundant for later stages of spirochetal persistence, either in mammals or in ticks. The protein influences host IFN responses as well as suppresses multiple host microbicidal activities involving serum complement, neutrophils, and antimicrobial peptides. We also discovered a remarkable plasticity in BBA57-mediated spirochete immune evasion strategy because its loss, although resulting in near clearance of pathogens at the inoculum site, triggers nonheritable adaptive changes that exclude detectable nucleotide alterations in the genome but incorporate transcriptional reprograming events. Understanding the malleability in spirochetal immune evasion mechanisms that ensures their host persistence is critical for the development of novel therapeutic and preventive approaches to combat long-term infections like Lyme borreliosis.
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http://dx.doi.org/10.1073/pnas.1718595115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910839PMC
April 2018

Eliminating Factor H-Binding Activity of CspZ Combined with Virus-Like Particle Conjugation Enhances Its Efficacy as a Lyme Disease Vaccine.

Front Immunol 2018 8;9:181. Epub 2018 Feb 8.

Latvian Biomedical Research and Study Centre, Riga, Latvia.

The spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the US and Europe. No potent human vaccine is currently available. The innate immune complement system is vital to host defense against pathogens, as complement activation on the surface of spirochetes results in bacterial killing. Complement system is inhibited by the complement regulator factor H (FH). To escape killing, produces an outer surface protein CspZ that binds FH to inhibit complement activation on the cell surface. Immunization with CspZ alone does not protect mice from infection, which we speculate is because FH-binding cloaks potentially protective epitopes. We modified CspZ by conjugating to virus-like particles (VLP-CspZ) and eliminating FH binding (modified VLP-CspZ) to increase immunogenicity. We observed greater bactericidal antibody titers in mice vaccinated with modified VLP-CspZ: A serum dilution of 1:395 (modified VLP-CspZ) vs 1:143 (VLP-CspZ) yielded 50% borreliacidal activity. Immunizing mice with modified VLP-CspZ cleared spirochete infection, as did passive transfer of elicited antibodies. This work developed a novel Lyme disease vaccine candidate by conjugating CspZ to VLP and eliminating FH-binding ability. Such a strategy of conjugating an antigen to a VLP and eliminating binding to the target ligand can serve as a general model for developing vaccines against other bacterial infectious agents.
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http://dx.doi.org/10.3389/fimmu.2018.00181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809437PMC
April 2019

Borrelia burgdorferi surface protein Lmp1 facilitates pathogen dissemination through ticks as studied by an artificial membrane feeding system.

Sci Rep 2018 01 30;8(1):1910. Epub 2018 Jan 30.

Department of Veterinary Medicine and Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD, 20742, USA.

In its natural infection cycle, the pathogen of Lyme borreliosis transits between a tick vector and a mammalian host. As relatively a minor fraction of spirochetes transits between the host and the vector precluding their reliable detection at early infection, artificial membrane feeders emerged as useful tools to study roles of spirochete proteins in pathogen entry, persistence, and exit through ticks. Here we report the development of a modified membrane feeder to study the role of a Borrelia burgdorferi surface protein called Lmp1 in spirochete transitions between the murine host and ticks. We show that our membrane feeder supports the blood meal engorgement process where ticks can acquire spirochetes from the feeder containing extremely low levels of pathogens (10 cells/ml of blood). Our data revealed that in comparison to wild-type spirochetes, lmp1 deletion mutants are significantly impaired for acquisition in naïve ticks as well as transmission from infected ticks. Taking together, our data suggest that Lmp1 plays an essential role in spirochete transitions between hosts and the vector. These studies also underscore the usefulness of artificial membrane feeding system as a valuable tool to study the role of B. burgdorferi gene-products in pathogen persistence in and passage through vector ticks.
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http://dx.doi.org/10.1038/s41598-018-20208-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790009PMC
January 2018

Analysis of Borrelia burgdorferi Proteome and Protein-Protein Interactions.

Methods Mol Biol 2018 ;1690:259-277

Department of Veterinary Medicine, University of Maryland, College Park, MD, USA.

The proteome of Borrelia burgdorferi undergoes dynamic alterations as the microbe cycles through and persists in diverse host or vector environments. Therefore, studies of B. burgdorferi proteome and protein-protein interactions, which play central roles in biological processes in diverse organisms, are critical in understanding biology and infectivity of spirochetes. Here, we describe the proteomic analysis of B. burgdorferi by two-dimensional (2-D) gel electrophoresis followed by protein identification via liquid chromatography-mass spectrometry and database searching. We also describe assays for studying the interaction between borrelial proteins: a novel high-throughput luciferase assay, yeast two-hybrid assay, and a far-Western assay that are routinely used in our laboratories.
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http://dx.doi.org/10.1007/978-1-4939-7383-5_19DOI Listing
June 2018

Artificial Infection of Ticks with Borrelia burgdorferi Using a Microinjection Method and Their Detection In Vivo Using Quantitative PCR Targeting flaB RNA.

Methods Mol Biol 2018 ;1690:105-114

Department of Veterinary Medicine, University of Maryland, College Park, MD, USA.

Borrelia burgdorferi is maintained in nature by a tick-rodent infection cycle where it traverses and colonizes a variety of host and vector tissues. A tick-borne murine model has been developed to study Lyme disease in the laboratory, which has a substantial impact in advancing our knowledge of spirochete infectivity and pathogenesis. Here, we detail a microinjection-based method for rapid and efficient infection of ticks with B. burgdorferi. While laboratory generation of B. burgdorferi-infected nymphs via natural larval engorgement on infected hosts and subsequent molting could take several weeks to months, the microinjection-based infection procedure requires only a few hours to generate infected ticks and allows introduction of defined quantities of spirochetes, including mutant isolates that are attenuated for infection in mice and thus cannot be naturally acquired by ticks. We also describe a quantitative PCR-based protocol for the measurement of B. burgdorferi in tick and murine hosts targeting spirochete RNA that is highly efficient, reproducible, and a better surrogate of active infection.
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http://dx.doi.org/10.1007/978-1-4939-7383-5_9DOI Listing
June 2018

Citrate Anticoagulant Improves the Sensitivity of Borreliella (Borrelia) burgdorferi Plasma Culture.

J Clin Microbiol 2017 11 6;55(11):3297-3299. Epub 2017 Sep 6.

Department of Veterinary Medicine, University of Maryland, College Park, and Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland, USA.

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http://dx.doi.org/10.1128/JCM.01046-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5654915PMC
November 2017

A protein-protein interaction dictates Borrelial infectivity.

Sci Rep 2017 06 7;7(1):2932. Epub 2017 Jun 7.

Department of Veterinary Medicine, University of Maryland, College Park and Virginia-Maryland Regional College of Veterinary Medicine, College Park, MD, USA.

Two Borrelia burgdorferi interacting proteins, BB0238 and BB0323, play distinct roles in pathogen biology and infectivity although a significance of their interaction remained enigmatic. Here we identified the polypeptide segment essential for BB0238-BB0323 interaction and examined how it supports spirochete infectivity. We show that the interaction region in BB0323 requires amino acid residues 22-200, suggesting that the binding encompasses discontinuous protein segments. In contrast, the interaction region in BB0238 spans only 11 amino acids, residues 120-130. A deletion of these 11 amino acids neither alters the overall secondary structure of the protein, nor affects its stability or oligomerization property, however, it reduces the post-translational stability of the binding partner, BB0323. Mutant B. burgdorferi isolates producing BB0238 lacking the 11-amino acid interaction region were able to persist in ticks but failed to transmit to mice or to establish infection. These results suggest that BB0238-BB0323 interaction is critical for post-translational stability of BB0323, and that this interaction is important for mammalian infectivity and transmission of B. burgdorferi. We show that saturation or inhibition of BB0238-BB0323 interaction could be studied in a luciferase assay, which could be amenable for future identification of small molecule inhibitors to combat B. burgdorferi infection.
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http://dx.doi.org/10.1038/s41598-017-03279-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462797PMC
June 2017

Borrelia burgdorferi BBI39 Paralogs, Targets of Protective Immunity, Reduce Pathogen Persistence Either in Hosts or in the Vector.

J Infect Dis 2017 03;215(6):1000-1009

Department of Veterinary Medicine, University of Maryland and Virginia-Maryland, Regional College of Veterinary Medicine, College Park, Maryland, USA.

Borrelia burgdorferi genome harbors several paralogous gene families (pgf) that can encode immunogenic proteins of unknown function. Protein-protein interaction assays using a transmission-blocking vaccine candidate, BBA52, as bait identified an interacting partner in spirochetes-a member of pgf 54, annotated as BBI39. We show that BBI39 is a surface-exposed membrane antigen that is immunogenic during spirochete infection, despite the gene being primarily transcribed in the vector with a transient expression in the host only at tick-bite sites. Immunization of rodents with BBI39, or a diverse paralog, BBI36, or their combination impaired pathogen acquisition by the vector, transmission from ticks to hosts, or induction of disease. High-titer BBI39 immunoglobulin G antibodies, which have borreliacidal properties, could be generated through routine subcutaneous or oral immunization, further highlighting use of BBI39 proteins as novel Lyme disease vaccines that can target pathogens in the host or in ticks.
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http://dx.doi.org/10.1093/infdis/jix036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407057PMC
March 2017

Infection-derived lipids elicit an immune deficiency circuit in arthropods.

Nat Commun 2017 02 14;8:14401. Epub 2017 Feb 14.

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.
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http://dx.doi.org/10.1038/ncomms14401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316886PMC
February 2017

Lyme disease spirochaete Borrelia burgdorferi does not require thiamin.

Nat Microbiol 2016 Nov 21;2:16213. Epub 2016 Nov 21.

Department of Oral Biology, State University of New York at Buffalo, Buffalo, New York 14214, USA.

Thiamin pyrophosphate (ThDP), the active form of thiamin (vitamin B), is believed to be an essential cofactor for all living organisms. Here, we report the unprecedented result that thiamin is dispensable for the growth of the Lyme disease pathogen Borrelia burgdorferi (Bb). Bb lacks genes for thiamin biosynthesis and transport as well as known ThDP-dependent enzymes, and we were unable to detect thiamin or its derivatives in Bb cells. We showed that eliminating thiamin in vitro and in vivo using BcmE, an enzyme that degrades thiamin, has no impact on Bb growth and survival during its enzootic infectious cycle. Finally, high-performance liquid chromatography analysis reveals that the level of thiamin and its derivatives in Ixodes scapularis ticks, the enzootic vector of Bb, is extremely low. These results suggest that by dispensing with use of thiamin, Borrelia, and perhaps other tick-transmitted bacterial pathogens, are uniquely adapted to survive in tick vectors before transmitting to mammalian hosts. To our knowledge, such a mechanism has not been reported previously in any living organisms.
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http://dx.doi.org/10.1038/nmicrobiol.2016.213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5157048PMC
November 2016

Characterization of a Chikungunya virus strain isolated from banked patients' sera.

Virol J 2016 09 2;13(1):150. Epub 2016 Sep 2.

Department of Molecular Biotechnology and Bioinformatics, Faculty of Science, Prince of Songkla University, Hatyai, Songkhla, 90112, Thailand.

Background: Chikungunya virus (CHIKV) is a prevalent mosquito-borne pathogen that is emerging in many parts of the globe causing significant human morbidity. Here, we report the isolation and characterization of an infectious CHIKV from banked serum specimens of suspected patients from the 2009 epidemic in Thailand.

Methods: Standard plaque assay was used for CHIKV isolation from the banked serum specimens. Isolated CHIKV was identified base on E1 structural gene sequence. Growth kinetic, infectivity, cell viability and cytokine gene expression throughout CHIKV infection in a permissive cell line, 293T cells, was performed using several approaches, including standard plaque assay, immunofluorescence assay, classical MTT assay, and quantitative real-time PCR. Two tailed Student's t test was used for evaluation statistically significance between the mean values of the groups.

Results: Based on the E1 structural gene sequence and phylogenetic analysis, we identified the virus as the CHIK/SBY8/10 isolate from Indonesia. Assessment of the growth kinetics, cytopathic effects as well as its ability to induce cellular immune responses suggested that the currently isolated CHIK/SBY8/10 virus is relatively more virulent than a known CHIKV vaccine strain, which also induces more dramatic proinflammatory responses.

Conclusions: Our studies further add to the infectivity of a less-studied yet infectious CHIKV isolate as well as underscored the importance and utility of 293T cells as an excellent cell culture model for studying viral growth, CHIKV-induced inflammatory cellular responses and cell death. Together, these studies provide novel information on the CHIKV biology, infectivity and virus-cell interaction, which would help develop novel interventions against the infection.
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http://dx.doi.org/10.1186/s12985-016-0606-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009685PMC
September 2016