Browsing by Author "Chaconas, George"

Now showing 1 - 17 of 17
  • Thumbnail Image
    ItemOpen Access
    Analysis of recombinational switching at the antigenic variation locus of the Lyme spirochete using a novel PacBio sequencing pipeline
    (Wiley, 2018-01) Verhey, Theodore B; Castellanos, Mildred; Chaconas, George
    The Lyme disease spirochete evades the host immune system by combinatorial variation of VlsE, a surface antigen. Antigenic variation occurs via segmental gene conversion from contiguous silent cassettes into the vlsE locus. Because of the high degree of similarity between switch variants and the size of vlsE, short-read NGS technologies have been unsuitable for sequencing vlsE populations. Here we use PacBio sequencing technology coupled with the first fully-automated software pipeline (VAST) to accurately process NGS data by minimizing error frequency, eliminating heteroduplex errors and accurately aligning switch variants. We extend earlier studies by showing use of almost all of the vlsE SNP repertoire. In different tissues of the same mouse, 99.6% of the variants were unique, suggesting that dissemination of Borrelia burgdorferi is predominantly unidirectional with little tissue-to-tissue hematogenous dissemination. We also observed a similar number of variants in SCID and wild-type mice, a heatmap of location and frequency of amino acid changes on the 3D structure and note differences observed in SCID versus wild type mice that hint at possible amino acid function. Our observed selection against diversification of residues at the dimer interface in wild-type mice strongly suggests that dimerization is required for in vivo functionality of vlsE.
  • Thumbnail Image
    ItemOpen Access
    Antigenic variation in the Lyme spirochete: detailed functional assessment of recombinational switching at vlsE in the JD1 strain of Borrelia burgdorferi
    (Wiley, 2019-01) Verhey, Theodore B; Castellanos, Mildred; Chaconas, George
    Borrelia burgdorferi is a causative agent of Lyme disease and establishes long-term infection in mammalian hosts. Persistence is promoted by the VlsE antigenic variation system, which generates combinatorial diversity of VlsE through unidirectional, segmental gene conversion from an array of silent cassettes. Here we explore the variants generated by the vls system of strain JD1, which has divergent sequence and structural elements from the type strain B31, the only B. burgdorferi strain in which recombinational switching at vlsE has been studied in detail. We first completed the sequencing of the vls region in JD1, uncovering a previously unreported 114 bp inverted repeat sequence upstream of vlsE. A five-week infection of WT and SCID mice was used for PacBio long read sequencing along with our recently developed VAST pipeline to analyze recombinational switching at vlsE from 40,000 sequences comprising 226,000 inferred recombination events. We show that antigenic variation in B31 and JD1 is highly similar, despite the lack of 17 bp direct repeats in JD1, a somewhat different arrangement of the silent cassettes, divergent inverted repeat sequences and general divergence in the vls sequences. We also present data that strongly suggest that dimerization is required for in vivo functionality of VlsE.
  • Thumbnail Image
    ItemOpen Access
    A Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat
    (Wilery, 2018-09) Castellanos, Mildred; Verhey, Theodore B; Chaconas, George
    Borrelia burgdorferi evades the host immune system by switching the surface antigen. VlsE, in a process known as antigenic variation. The DNA mechanisms and genetic elements present on the vls locus that participate in the switching process remain to be elucidated. Manipulating the vls locus has been difficult due to its instability on Escherichia coli plasmids. In this study, we generated for the first time a mini-vls system composed of a single silent vlsE variable region (silent cassette 2) through the vlsE gene by performing some cloning steps directly in a highly transformable B. burgdorferi strain. Variants of the mini system were constructed with or without the long inverted repeat (IR) located upstream of vlsE and on both circular and linear plasmids to investigate the importance of the IR and plasmid topology on recombinational switching at vlsE. Amplicon sequencing using PacBio long read technology and analysis of the data with our recently reported pipeline and VAST software showed that the system undergoes switching in mice in both linear and circular versions and that the presence of the hairpin does not seem to be crucial in the linear version, however it is required when the topology is circular.
  • Thumbnail Image
    ItemOpen Access
    Construction and Characterization of a B. burgdorferi strain with Conditional Expression of the Essential Telomere Resolvase, ResT
    (2013-10-09) Bandy, Nick; Chaconas, George
    During DNA replication in Borrelia burgdorferi, replication of linear elements results in an inverted dimer repeat of the telomere which is subsequently resolved by the telomere resolvase, ResT. ResT has been demonstrated as essential and a knock-out strain has never been produced. Using a conditional expression system a resT knock-out strain has been produced and used to investigate the effects of ResT depletion of spirochete viability as well as genome stability. ResT depleted spirochetes ceased to divide after ResT depletion, however, remained viable up to 16 days suggesting an arrested state of the spirochetes. Southern blots of the DNA revealed a shift of linear plasmid DNA to a higher molecular weight representing the circular intermediate. DNA replication did not continue in spirochetes after ResT depletion suggesting ResT may play a larger role in replication than previously thought.
  • Thumbnail Image
    ItemOpen Access
    CSM murray award lecture - functional studies of the Lyme disease spirochete - from molecules to mice
    (Canadian Science Publishing, 2012-02-17) Chaconas, George
    Lyme borreliosis, also known as Lyme disease, is now the most common vector transmitted disease in the northern hemisphere. It is caused by the spirochete Borrelia burgdorferi and related species. In addition to their clinical importance, these organisms are fascinating to study because of the wide variety of unusual features they possess. Ongoing work in the laboratory in several areas will be described. (1) The segmented genomes contain up to two dozen genetic elements, the majority of which are linear with covalently closed hairpin ends. These linear DNAs also display a very high degree of ongoing genetic rearrangement. Mechanisms for these processes will be described. (2) Persistent infection by Borrelia species requires antigenic variation through a complex DNA rearrangement process at the vlsE locus on the linear plasmid lp28-1. Novel features of this recombination process will be presented. (3) Evidence for a new global regulatory pathway of B. burgdorferi gene expression that is required for pathogenicity will be described. The DEAH box RNA helicase HrpA is involved in this pathway, which may be relevant in other bacteria. (4) The mechanism of B. burgdorferi to effectively disseminate throughout its host is being studied in real time by high resolution intravital imaging in live mice. Recent work will be presented.
  • Thumbnail Image
    ItemOpen Access
    Genetic analysis and the effect upon mouse infection of nucleic acid metabolizing genes in the Lyme disease spirochete
    (2013-04-09) Hardy, Pierre-Olivier; Chaconas, George
    The Lyme spirochete Borrelia burgdorferi causes the most prevalent vector-borne infection in North America. In this study, the importance of DNA metabolizing genes for the infectivity, persistence and survival to DNA damage in B. burgdorferi was determined. During the infection of a vertebrate host, B. burgdorferi undergoes antigenic variation by DNA recombination at vlsE, which encodes for an immunogenic surface lipoprotein required for the persistence of the spirochete. In the present study, eight gene targets were disrupted and only the RuvAB Holiday junction branch migrase subunits affected the switching at vlsE and the persistence of B. burgdorferi in mice. The disruption of these eight genes was part of a wider study aiming to identify nucleic acid metabolizing genes involved in switching at vlsE. Although no other genes were found to strongly affect switching, the disruption of the DEAH-box RNA helicase HrpA abolished the infectivity of B. burgdorferi. Since the complementation of hrpA in trans could not be achieved, the restoration of the wild-type gene by allelic exchange was used as an alternate strategy for complementation. The restoration of the wild-type hrpA did restore infectivity, confirming the importance of hrpA. Point mutations were also introduced by allelic exchange in motifs required for either the RNA helicase or the ATPase activity of HrpA. To avoid an intial screen of a large number of clones by sequencing, a strategy was adapted to confirm by PCR the presence of the mutation in the gene. Infection of mice with these B. burgdorferi hrpA mutants confirmed that the RNA helicase activity, in addition of the ATPase activity, is required for the survival of B. burgdorferi in the mouse. Finally, a strategy was adapted to expediently compare the cell density of multiple cultures. This strategy was used to measure the importance of 25 nucleic acid metabolizing genes for survival of B. burgdorferi to DNA damage. Using this strategy, the nucleotide excision repair pathway was shown to be the sole repair pathway to be significantly involved in repair of UV-induced DNA damage in B. burgdorferi.
  • Thumbnail Image
    ItemOpen Access
    Hairpin Telomere Resolvases
    (American Society for Microbiology, 2014-11-24) Kobryn, Kerri; Chaconas, George
    Covalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from replication intermediates by a process known as telomere resolution. This is a DNA breakage and reunion reaction promoted by hairpin telomere resolvases (also referred to as protelomerases) found in a limited number of phage and bacteria. The reaction promoted by these enzymes is a chemically isoenergetic two-step transesterification without a requirement for divalent metal ions or high-energy cofactors and uses an active site and mechanism similar to that for type IB topoisomerases and tyrosine recombinases. The small number of unrelated telomere resolvases characterized to date all contain a central, catalytic core domain with the active site, but in addition carry variable C- and N-terminal domains with different functions. Similarities and differences in the structure and function of the telomere resolvases are discussed. Of particular interest are the properties of the Borrelia telomere resolvases, which have been studied most extensively at the biochemical level and appear to play a role in shaping the unusual segmented genomes in these organisms and, perhaps, to play a role in recombinational events.
  • Thumbnail Image
    ItemOpen Access
  • Thumbnail Image
    ItemOpen Access
    Investigating the virulence of Treponema phagedenis strains isolated from digital dermatitis lesions in a murine abscess model
    (2023-07) Scott, Colton; De Buck, Jeroen; Chaconas, George; Harrison, Joe
    Digital Dermatitis (DD) is an ulcerative foot lesion in the heel bulbs of dairy cattle. DD is a polymicrobial disease with no precise etiology, although key pathogenic genera have been consistently found disproportional and abundant in diseased tissue as opposed to healthy skin. One such genera is Treponema, a member genus of the phylum Spirochaetes. Within Treponema, many different phylotypes are found in DD, however the species Treponema phagedenis is uniformly found in copious quantities and deep within the skin layers in the active, ulcerative stages of disease. The pathogenic mechanisms these bacteria use to persist in the skin and the role they play in the larger pathology of DD is widely unknown. To explore the pathogenesis and virulence of Treponema phagedenis, isolates of this species were investigated in a subcutaneous murine abscess model. For this purpose, mice were subcutaneously inoculated with T. phagedenis in two infection trials. In the first trial, a dosage study was conducted to elucidate the pathogenicity of strains across three different spirochete per inoculum (SPI) doses, based on abscess volumes. In the second trial, isolates were inoculated with the dose of 5 x 109 SPI selected based on trial 1 results, to determine the expression level of 11 putative virulence genes and gain insight into the virulence of strains. To do this, abscesses were harvested for RNA extraction followed by RT-qPCR. From the RT-qPCR assay, the relative fold change of these genes was surmised by comparing the expression levels of in vitro culture samples against in vivo murine samples. During this analysis, it was determined that genes encoding for two metal-ion import lipoproteins, were found highly upregulated during infection. In addition, two genes involved in the adherence of treponemes to the host were found moderately expressed versus the in vitro samples. Conversely, two genes involved in motility and chemotaxis were found to not be significantly upregulated or utilized during infection. This gene expression analysis highlights the preference in strategy for T. phagedenis to persist and adhere in the host rather than engage motility and disseminate.
  • Thumbnail Image
    ItemOpen Access
  • Thumbnail Image
    ItemOpen Access
    The Lyme disease spirochete can hijack the host immune system for extravasation from the microvasculature
    (Wiley, 2021-04-23) Tan, Xi; Petri, Björn; DeVinney, Rebekah; Jenne, Craig N; Chaconas, George
    Lyme disease is the most common tick-transmitted disease in the northern hemisphere and is caused by the spirochete Borrelia burgdorferi and related Borrelia species. The constellation of symptoms attributable to this malady result from vascular dissemination of B. burgdorferi throughout the body to invade various tissue types. However, little is known about the mechanism by which the spirochetes can breach the blood vessel wall to reach distant tissues. We have studied this process by direct observation of spirochetes in the microvasculature of living mice using multilaser spinning-disk intravital microscopy. Our results show that in our experimental system, instead of phagocytizing B. burgdorferi, host neutrophils are involved in the production of specific cytokines that activate the endothelium and potentiate B. burgdorferi escape into the surrounding tissue. Spirochete escape is not induced by paracellular permeability and appears to occur via a transcellular pathway. Neutrophil repurposing to promote bacterial extravasation represents a new and innovative pathogenic strategy.
  • Thumbnail Image
    ItemOpen Access
    Next-Generation Sequencing of vlsE Recombinational Switching in the Lyme Spirochete
    (2017) Verhey, Theodore; Chaconas, George; Schriemer, David; Samuels, Scott; Zimmerly, Steven; DeVinney, Rebekah; de Koning, Jason; McGhee, James
    Borrelia burgdorferi and other spirochetes that cause Lyme disease effectively evade the acquired immune response through antigenic variation. The VlsE antigen is expressed on the spirochete surface during mammalian infection. Virtually unlimited numbers of variants are generated through segmental gene conversion events at the vlsE gene from a series of nearby silent cassette sequences that are homologous to the variable region of vlsE. In contrast to other antigenic variation systems, the molecular mechanism for this switching is unknown. Switching at vlsE is dependent on mammalian host factors and the only known requirement is the RuvAB branch migrase; RecA and many of the other homologous recombination proteins in B. burgdorferi are not required. In this study, we developed a new assay for switching at vlsE based on PacBio sequencing and an analytical pipeline that allows the analysis of tens of thousands of full-length variants. We developed the first fully-automated and unbiased method to accurately identify switch events and non-templated mutations from sequence data. This software also contains a large suite of dataset management and analysis tools to quantify many aspects of switch events and the switching process in the dataset. Following a time-course of B. burgdorferi infection in immunocompetent and immunocompromised mice in a variety of tissues, we uncover a series of new insights into recombinational switching at vlsE. We demonstrate that although switching requires mammalian host factors, the rate of vlsE switching is unaffected by the presence or absence of the required immune system. We identify residues that undergo diversifying and stabilizing selection in the VlsE protein in the presence of acquired immunity. We also report accurate rates of recombination and nontemplated mutation, the size and origin of switch events, a role for local sequence homology in promoting switching, and that switch events accumulate in a clustered rather than uniform fashion. We also quantify a secondary mechanism of sequence variation by demonstrating that polymerase slippage generates in-frame, surface-localized insertions and deletions that contribute to VlsE variability.
  • Thumbnail Image
    ItemOpen Access
    Structure, function, and evolution of linear replicons in Borrelia
    (Annual Reviews, 2010-06-10) Chaconas, George; Kobryn, Kerri
    Spirochetes of the genus Borrelia include important human pathogens that cause Lyme borreliosis and relapsing fever. The genomes of Borrelia species can be composed of up to 24 DNA molecules, most of which are linear. The plasmid content and linear replicon sequence arrangement vary widely between isolates. The linear replicons are terminated by covalently closed DNA hairpins or hairpin telomeres. Replication of these elements involves a unique reaction, called telomere resolution, to produce hairpin telomeres from replicative intermediates. The telomere resolvase, ResT, is thought to contribute to the genetic flux of the linear molecules by promoting stabilized telomere fusions. Telomere resolvases are related to the tyrosine recombinases and ResT can generate the crucial reaction intermediate of this class of enzyme, the Holliday junction. This observation has led to the proposal that telomere resolvases evolved from tyrosine recombinases inducing DNA linearization in the genomes that acquired them.
  • Thumbnail Image
    ItemOpen Access
    Studies on Hematogenous Dissemination of Lyme Disease Spirochetes
    (2021-08-19) Tan, Xi; Chaconas, George; DeVinney, Rebekah; Jenne, Craig
    Lyme disease (LD), caused by various members of the genus Borrelia, is the most prevalent tick-transmitted illness in North America and Europe (Groshong & Blevins, 2014, Stanek et al., 2011). There is an increasing risk of LD in Canada. In 2014, the Government of Canada launched a national communication campaign to raise social awareness and promote individual preventive behaviors toward LD. Hematogenous dissemination is important for infection by Borrelia burgdorferi (B. burgdorferi). B. burgdorferi can attach to the vascular endothelium and disseminate into a variety of tissue types. B. burgdorferi hematogenous dissemination is a multistep process and consists of several successive stages: transient (tethering plus dragging) interactions, followed by stationary adhesion and/or extravasation (Norman et al., 2008, Coburn et al., 2013). However, the mechanism of borrelial vascular adhesion and extravasation remained unclear. We are using intravital microscopy (IVM) (Kumar et al., 2015, Secklehner et al., 2017, Stolp & Melican, 2016) to identify and characterize B. burgdorferi adhesins, which are believed to be involved in vascular adhesion, transmigration as well as tissue tropism (Coburn et al., 2013, Caine & Coburn, 2016). From the B. burgdorferi side, we have shown that OspC is a dermatan sulfate- (DS-) and fibronectin- (FN-) binding adhesin that is required for vascular transmigration and joint colonization in mice (Lin et al., 2020). We also identified that VlsE, a well-known antigenically variable outer surface lipoprotein, is a DS-binding adhesin that efficiently promotes transient adhesion to the microvasculature via its DS binding activity in vivo. Moreover, on the host side, we have shown that instead of phagocytizing B. burgdorferi, host neutrophils are involved in producing specific cytokines (TNF-α, MCP-1, IL-10) that activate the endothelium and potentiate B. burgdorferi escape into the surrounding tissue. Spirochete escape is not induced by paracellular permeability and appears to occur via a transcellular pathway. Neutrophil repurposing to promote bacterial extravasation represents a new and innovative pathogenic strategy (Tan et al., 2021). Our findings provide insight into the mechanism of Borrelia hematogenous dissemination and how LD spirochetes employ host immune cells for vascular extravasation.
  • Thumbnail Image
    ItemOpen Access
    The Effect of B. burgdorferi Infection on Cytokine Expression of the Mammalian Host
    (2023-04-26) Abolhosseini, Shiva; Chaconas, George; Devinney, Rebekah; Savchenko, Alexei; Harrison, Joe
    Lyme disease, or Lyme borreliosis, is a multi-organ, zoonotic disease predominant in the northern hemisphere. Lyme borreliosis is caused by different members of the genus Borrelia, most notably Borrelia burgdorferi. The spirochete is transmitted to humans by a tick vector during its blood meal. Lyme disease results in a wide range of symptoms making the disease difficult to diagnose. These various clinical manifestations are due to the spirochetes’ ability to extravasate into different tissues all over the body. Previous studies have shown that treatment with IL-10, TNF-α, and MCP-1 can potentiate the vasculature and accelerate the vascular transmigration process. The direct assessment of cytokine transcript levels from mouse leukocytes that can contribute to extravasation had not previously been reported. In this study, we analyzed the effects of infection with various strains of B. burgdorferi at different time points before vascular transmigration on the cytokine transcript levels of host leukocytes. Additionally, we further characterized the differences between BALB/c and Cd1d -/- mice in their inflammatory responses.
  • Thumbnail Image
    ItemOpen Access
    The Metabolism of Uropathogenic Bacteria in in Vitro Human Urine Cultures
    (2024-10-07) Chan, Carly C. Y.; Lewis, Ian A.; Turner, Ray J.; Harrison, Joe; Chaconas, George; Montenegro-Burke, Rafael
    Urinary tract infections (UTIs) are common infections primarily caused by bacterial colonization of the host’s bladder and/or kidney. Research into the molecular underpinnings behind UTIs primarily focused on the various macromolecular virulence factors that enable uropathogens to invade and colonize the host’s urinary tract. As such, there is an extensive body of literature characterizing these UTI-associated virulence factors. However, one important aspect that remains relatively unexplored is pathogen metabolism. Pathogens must be able to metabolize the nutrients available in its microenvironment to survive and grow within their host. Human urine is a chemically complex medium with a diverse range of amino acids and nucleic acids, but generally lacks carbohydrates, the preferred carbon source for most microbes and thus, urine is often considered a nutrient-poor substance. Recent technological advancement in metabolomic tools can allow researchers to discover new insights into uropathogen metabolism and further our understanding of how uropathogens overcome nutritional adversity and survive in human urine. To address this gap, I used liquid chromatography-mass spectrometry (LC-MS) to analyze in vitro human urine cultures of uropathogenic bacteria. My initial metabolomics survey of eight common uropathogenic species found that these species can be divided into four distinct metabolic clades: serine consumers, glutamine consumers, amino acid abstainers, and amino acid minimalists. There were also several other metabolic phenotypes exclusive to a single or a few species. Metabolites found to be secreted by uropathogens could be candidate UTI biomarkers. Previous work discovered that agmatine was a robust UTI biomarker for several bacterial species in the Enterobacterales order including Escherichia coli. Investigation into bacterial agmatine production revealed that E. coli utilizes several different decarboxylase-based acid resistance systems in urine, induced at different pH ranges. Meanwhile, a few non-Enterobacterales species, like Staphylococcus spp., were found to secrete N6-methyladenine, which was also identified as a UTI biomarker. Advances to metabolomics methods can greatly enhance the efficiency of these metabolomics analyses, and so we developed a new LC-MS strategy for monitoring microbial metabolic activity in real-time. By investigating uropathogen metabolism with current metabolomics tools, we can better understand how these pathogens may persist and thrive during UTIs.
  • Thumbnail Image
    ItemOpen Access
    The use of T4 polynucleotide kinase to probe protein-DNA interactions
    (1978) Chaconas, George; Church, Robert B.