BB0744 Affects Tissue Tropism and Spatial Distribution of Borrelia burgdorferi

Borrelia burgdorferi, the etiologic agent of Lyme disease, produces a variety of proteins that promote survival and colonization in both theIxodesspecies vector and various mammalian hosts. We initially identified BB0744 (also known as p83/100) by screening forB. burgdorferistrain B31 proteins that bind to α1β1integrin and hypothesized that, given the presence of a signal peptide, BB0744 may be a surface-exposed protein. In contrast to this expectation, localization studies suggested that BB0744 resides in the periplasm. Despite its subsurface location, we were interested in testing whether BB0744 is required for borrelial pathogenesis. To this end, abb0744deletion was isolated in aB. burgdorferistrain B31 infectious background, complemented, and queried for the role of BB0744 following experimental infection. A combination of bioluminescent imaging, cultivation of infected tissues, and quantitative PCR (qPCR) demonstrated that Δbb0744mutantB. burgdorferibacteria were attenuated in the ability to colonize heart tissue, as well as skin locations distal to the site of infection. Furthermore, qPCR indicated a significantly reduced spirochetal load in distal skin and joint tissue infected with Δbb0744mutantB. burgdorferi. Complementation withbb0744restored infectivity, indicating that the defect seen in Δbb0744mutantB. burgdorferiwas due to the loss of BB0744. Taken together, these results suggest that BB0744 is necessary for tissue tropism, particularly in heart tissue, alters the ability ofB. burgdorferito disseminate efficiently, or both. Additional studies are warranted to address the mechanism employed by BB0744 that alters the pathogenic potential ofB. burgdorferi.

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Infection of Interleukin 17 Receptor A-Deficient C3H Mice with Borrelia burgdorferi Does Not Affect Their Development of Lyme Arthritis and Carditis

Infection and Immunity ◽

10.1128/iai.00533-15 ◽

2015 ◽

Vol 83 (7) ◽

pp. 2882-2888 ◽

Cited By ~ 8

Author(s):

Carrie E. Lasky ◽

Kara E. Jamison ◽

Darcie R. Sidelinger ◽

Carmela L. Pratt ◽

Guoquan Zhang ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Lyme Borreliosis ◽

Heart Tissue ◽

Lyme Arthritis ◽

Mouse Strains ◽

Interleukin 17 ◽

Content Type ◽

C3h Mice ◽

A Subunit

Recently, a number of studies have reported the presence of interleukin 17 (IL-17) in patients with Lyme disease, and several murine studies have suggested a role for this cytokine in the development of Lyme arthritis. However, the role of IL-17 has not been studied using the experimental Lyme borreliosis model of infection of C3H mice withBorrelia burgdorferi. In the current study, we investigated the role of IL-17 in the development of experimental Lyme borreliosis by infecting C3H mice devoid of the common IL-17 receptor A subunit (IL-17RA) and thus deficient in most IL-17 signaling. Infection of both C3H and C3H IL-17RA−/−mice led to the production of high levels of IL-17 in the serum, low levels in the heart tissue, and no detectable IL-17 in the joint tissue. The development and severity of arthritis and carditis in the C3H IL-17RA−/−mice were similar to what was seen in wild-type C3H mice. In addition, development of antiborrelia antibodies and clearance of spirochetes from tissues were similar for the two mouse strains. These results demonstrate a limited role for IL-17 signaling through IL-17RA in the development of disease following infection of C3H mice withB. burgdorferi.

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CsrA (BB0184) Is Not Involved in Activation of the RpoN-RpoS Regulatory Pathway in Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.01555-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1511-1522 ◽

Cited By ~ 10

Author(s):

Zhiming Ouyang ◽

Jianli Zhou ◽

Michael V. Norgard

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Animal Studies ◽

Expression System ◽

Regulatory Pathway ◽

Content Type ◽

Mouse Tissues ◽

Differential Gene ◽

Carbon Storage Regulator

ABSTRACTBorrelia burgdorferiencodes a homologue of the bacterial carbon storage regulator A (CsrA). Recently, it was reported that CsrA contributes toB. burgdorferiinfectivity and is required for the activation of the central RpoN-RpoS regulatory pathway. However, many questions concerning the function of CsrA inB. burgdorferigene regulation remain unanswered. In particular, there are conflicting reports concerning the molecular details of how CsrA may modulaterpoSexpression and, thus, how CsrA may influence the RpoN-RpoS pathway inB. burgdorferi. To address these key discrepancies, we examined the role of CsrA in differential gene expression in the Lyme disease spirochete. Upon engineering an induciblecsrAexpression system inB. burgdorferi, controlled hyperexpression of CsrA in a merodiploid strain did not significantly alter the protein and transcript levels ofbosR,rpoS, and RpoS-dependent genes (such asospCanddbpA). In addition, we constructed isogeniccsrAmutants in two widely used infectiousB. burgdorferistrains. When expression ofbosR,rpoS,ospC, anddbpAwas compared between thecsrAmutants and their wild-type counterparts, no detectable differences were observed. Finally, animal studies indicated that thecsrAmutants remained infectious for and virulent in mice. Analyses ofB. burgdorferigene expression in mouse tissues showed comparable levels ofrpoStranscripts by thecsrAmutants and the parental strains. Taken together, these results constitute compelling evidence that CsrA is not involved in activation of the RpoN-RpoS pathway and is dispensable for mammalian infectious processes carried out byB. burgdorferi.

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Effect of Complement Component C3 Deficiency on Experimental Lyme Borreliosis in Mice

Infection and Immunity ◽

10.1128/iai.71.8.4432-4440.2003 ◽

2003 ◽

Vol 71 (8) ◽

pp. 4432-4440 ◽

Cited By ~ 39

Author(s):

Matthew B. Lawrenz ◽

R. Mark Wooten ◽

James F. Zachary ◽

Scott M. Drouin ◽

Janis J. Weis ◽

...

Keyword(s):

Lyme Borreliosis ◽

Early Time ◽

Heart Tissue ◽

Complement Component ◽

Joint Tissue ◽

Complement Component C3 ◽

In The Wild ◽

Spirochete Load ◽

The Complement System

ABSTRACT Mice deficient in complement component C3 (C3−/−) and syngeneic C57BL/6 control mice were challenged with Borrelia burgdorferi to determine the role of complement in immune clearance and joint histopathology during experimental Lyme borreliosis. Tibiotarsal joint, ear, and heart tissues were monitored for spirochete numbers at 2, 4, 8, and 12 weeks postinoculation with 105 B. burgdorferi B31 clone 5A4 by using quantitative real-time PCR. The spirochete load in joint and ear tissue remained higher in the C3−/− mice than in the wild-type counterparts throughout the 12-week study, whereas the numbers in heart tissue of both groups of mice decreased substantially at 8 to 12 weeks postinfection. Histopathology scores for joint tissue were generally higher in the C3−/− mice compared to C57BL/6 controls at 2 and 4 weeks postinfection, which may reflect the presence of higher numbers of bacteria in the joints at these early time points. Levels of anti-B. burgdorferi immunoglobulin G tended to be reduced in the C3−/− mice compared to control mice. Furthermore, a 5.5-fold-lower number of the complement-sensitive Borrelia garinii was needed to infect C3−/− mice compared to C57BL/6 mice, indicating that its sensitivity to complement is one barrier to infection of the mouse model by B. garinii. These results indicate that the complement system may be important in controlling the early dissemination and progression of B. burgdorferi infection.

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Analysis of the HD-GYP Domain Cyclic Dimeric GMP Phosphodiesterase Reveals a Role in Motility and the Enzootic Life Cycle of Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.05153-11 ◽

2011 ◽

Vol 79 (8) ◽

pp. 3273-3283 ◽

Cited By ~ 58

Author(s):

Syed Z. Sultan ◽

Joshua E. Pitzer ◽

Tristan Boquoi ◽

Gerry Hobbs ◽

Michael R. Miller ◽

...

Keyword(s):

Life Cycle ◽

Borrelia Burgdorferi ◽

Double Mutant ◽

Ixodes Scapularis ◽

Metabolizing Enzymes ◽

Content Type ◽

Motility Regulation ◽

Mutant Cells ◽

Single Set

ABSTRACTHD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria.Borrelia burgdorferipossesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation inpdeAresulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with aKmof 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing thepdeA pdeBdouble mutant, we demonstrate that no additional phosphodiesterases are present inB. burgdorferi.pdeBsingle mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing apilZpdeBdouble mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly inpdeBmutant cells, these cells exhibited a reduced ability to survive inIxodes scapularisticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role ofpdeBincreases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle ofB. burgdorferi.

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Investigating the Function of Ddr48p in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00107-12 ◽

2012 ◽

Vol 11 (6) ◽

pp. 718-724 ◽

Cited By ~ 4

Author(s):

I. A. Cleary ◽

N. B. MacGregor ◽

S. P. Saville ◽

D. P. Thomas

Keyword(s):

Candida Albicans ◽

Biofilm Formation ◽

The United States ◽

Pathogenic Potential ◽

Content Type ◽

General Stress Response ◽

Damage Response ◽

Sense And Respond ◽

Immune Defects

ABSTRACTCandidiasis now represents the fourth most frequent nosocomial infection both in the United States and worldwide.Candida albicansis an increasingly common threat to human health as a consequence of AIDS, steroid therapy, organ and tissue transplantation, cancer therapy, broad-spectrum antibiotics, and other immune defects. The pathogenic potential ofC. albicansis intimately related to certain key processes, including biofilm formation and filamentation. Ddr48p is a damage response protein that is significantly upregulated during both biofilm formation and filamentation, but its actual function is unknown. Previous studies have indicated that this protein may be essential inC. albicansbut notSaccharomyces cerevisiae. Here we examined the function of Ddr48p and investigated the role of this protein in biofilm formation and filamentation. We demonstrated that this protein is not essential inC. albicansand appears to be dispensable for filamentation. However,DDR48is required for the flocculation response stimulated by 3-aminotriazole-induced amino acid starvation. Furthermore, we examined the response of this deletion strain to a wide variety of environmental stressors and antifungal compounds. We observed several mild sensitivity or resistance phenotypes and also found that Ddr48p contributes to the DNA damage response ofC. albicans. The results of this study reveal that the role of this highly expressed protein goes beyond a general stress response and impinges on a key facet of pathogenesis, namely, the ability to sense and respond to changes in the host environment.

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Characterization of Stress and Innate Immunity Resistance of Wild-Type and Δp66 Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.00186-17 ◽

2017 ◽

Vol 86 (2) ◽

Cited By ~ 5

Author(s):

Michael W. Curtis ◽

Beth L. Hahn ◽

Kai Zhang ◽

Chunhao Li ◽

Richard T. Robinson ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Antimicrobial Peptide ◽

Membrane Integrity ◽

The United States ◽

Laboratory Model ◽

Wild Type ◽

Content Type ◽

Inoculation Site

ABSTRACTBorrelia burgdorferiis a causative agent of Lyme disease, the most common arthropod-borne disease in the United States.B. burgdorferievades host immune defenses to establish a persistent, disseminated infection. Previous work showed that P66-deficientB. burgdorferi(Δp66) is cleared quickly after inoculation in mice. We demonstrate that the Δp66strain is rapidly cleared from the skin inoculation site prior to dissemination. The rapid clearance of Δp66bacteria is not due to inherent defects in multiple properties that might affect infectivity: bacterial outer membrane integrity, motility, chemotactic response, or nutrient acquisition. This led us to the hypothesis that P66 has a role in mouse cathelicidin-related antimicrobial peptide (mCRAMP; a major skin antimicrobial peptide) and/or neutrophil evasion. Neither wild-type (WT) nor Δp66 B. burgdorferiwas susceptible to mCRAMP. To examine the role of neutrophil evasion, we administered neutrophil-depleting antibody anti-Ly6G (1A8) to C3H/HeN mice and subsequently monitored the course ofB. burgdorferiinfection. Δp66mutants were unable to establish infection in neutrophil-depleted mice, suggesting that the important role of P66 during early infection is through another mechanism. Neutrophil depletion did not affect WTB. burgdorferibacterial burdens in the skin (inoculation site), ear, heart, or tibiotarsal joint at early time points postinoculation. This was unexpected given that priorin vitrostudies demonstrated neutrophils phagocytose and killB. burgdorferi. These data, together with our previous work, suggest that despite thein vitroability of host innate defenses to killB. burgdorferi, individual innate immune mechanisms have limited contributions to controlling earlyB. burgdorferiinfection in the laboratory model used.

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The need to unravel the twisted nature of the Borrelia burgdorferi sensu lato complex across Europe

Microbiology ◽

10.1099/mic.0.000899 ◽

2020 ◽

Vol 166 (5) ◽

pp. 428-435

Author(s):

Martin Strnad ◽

Ryan O.M. Rego

Keyword(s):

Borrelia Burgdorferi ◽

Single Species ◽

Sensu Stricto ◽

Borrelia Burgdorferi Sensu Lato ◽

Pathogenic Potential ◽

Content Type ◽

Differential Gene ◽

Vector Borne ◽

Genetic Techniques ◽

Infectious Cycle

Lyme borreliosis is a vector-borne infection caused by bacteria under the Borrelia burgdorferi sensu lato complex, both in Europe and North America. Differential gene expression at different times throughout its infectious cycle allows the spirochete to survive very diverse environments within different mammalian hosts as well as the tick vector. To date, the vast majority of data about spirochetal proteins and their functions are from genetic studies carried out on North American strains of a single species, i.e. B. burgdorferi sensu stricto. The whole-genome sequences recently obtained for several European species/strains make it feasible to adapt and use genetic techniques to study inherent differences between them. This review highlights the crucial need to undertake independent studies of genospecies within Europe, given their varying genetic content and pathogenic potential, and differences in clinical manifestation.

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DhhP, a Cyclic di-AMP Phosphodiesterase of Borrelia burgdorferi, Is Essential for Cell Growth and Virulence

Infection and Immunity ◽

10.1128/iai.00030-14 ◽

2014 ◽

Vol 82 (5) ◽

pp. 1840-1849 ◽

Cited By ~ 48

Author(s):

Meiping Ye ◽

Jun-Jie Zhang ◽

Xin Fang ◽

Gavin B. Lawlis ◽

Bryan Troxell ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Wild Type Strain ◽

Wall Structure ◽

Mammalian Host ◽

Dependent Manner ◽

Gram Positive ◽

Content Type ◽

Gram Positive Bacteria

ABSTRACTCyclic di-AMP (c-di-AMP) is a recently discovered second messenger in bacteria. Most of work on c-di-AMP signaling has been done in Gram-positive bacteria, firmicutes, and actinobacteria, where c-di-AMP signaling pathways affect potassium transport, cell wall structure, and antibiotic resistance. Little is known about c-di-AMP signaling in other bacteria.Borrelia burgdorferi, the causative agent of Lyme disease, is a spirochete that has a Gram-negative dual membrane. In this study, we demonstrated thatB. burgdorferiBB0619, aDHH-DHHA1 domainprotein (herein designated DhhP), functions as c-di-AMP phosphodiesterase. Recombinant DhhP hydrolyzed c-di-AMP to pApA in a Mn2+- or Mg2+-dependent manner. In contrast to c-di-AMP phosphodiesterases reported thus far, DhhP appears to be essential forB. burgdorferigrowth bothin vitroand in the mammalian host. Inactivation of the chromosomaldhhPgene could be achieved only in the presence of a plasmid-encoded inducibledhhPgene. The conditionaldhhPmutant had a dramatic increase in intracellular c-di-AMP level in comparison to the isogenic wild-type strain. Unlike what has been observed in Gram-positive bacteria, elevated cellular c-di-AMP inB. burgdorferidid not result in an increased resistance to β-lactamase antibiotics, suggesting that c-di-AMP's functions in spirochetes differ from those in Gram-positive bacteria. In addition, thedhhPmutant was defective in induction of the σSfactor, RpoS, and the RpoS-dependent outer membrane virulence factor OspC, which uncovers an important role of c-di-AMP inB. burgdorferivirulence.

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An Invasive Mammal (the Gray Squirrel, Sciurus carolinensis) Commonly Hosts Diverse and Atypical Genotypes of the Zoonotic Pathogen Borrelia burgdorferi Sensu Lato

Applied and Environmental Microbiology ◽

10.1128/aem.00109-15 ◽

2015 ◽

Vol 81 (13) ◽

pp. 4236-4245 ◽

Cited By ~ 12

Author(s):

Caroline Millins ◽

Agnieszka Magierecka ◽

Lucy Gilbert ◽

Alissa Edoff ◽

Amelia Brereton ◽

...

Keyword(s):

United Kingdom ◽

Borrelia Burgdorferi ◽

Early Summer ◽

Borrelia Burgdorferi Sensu Lato ◽

Sciurus Carolinensis ◽

Borrelia Garinii ◽

Content Type ◽

Zoonotic Pathogen ◽

The United Kingdom

ABSTRACTInvasive vertebrate species can act as hosts for endemic pathogens and may alter pathogen community composition and dynamics. For the zoonotic pathogenBorrelia burgdorferisensu lato, the agent of Lyme borreliosis, recent work shows invasive rodent species can be of high epidemiological importance and may support host-specific strains. This study examined the role of gray squirrels (Sciurus carolinensis) (n= 679), an invasive species in the United Kingdom, asB. burgdorferi sensu latohosts. We found that gray squirrels were frequently infested withIxodes ricinus, the main vector ofB. burgdorferi sensu latoin the United Kingdom, and 11.9% were infected withB. burgdorferi sensu lato. All four genospecies that occur in the United Kingdom were detected in gray squirrels, and unexpectedly, the bird-associated genospeciesBorrelia gariniiwas most common. The second most frequent infection was withBorrelia afzelii. Genotyping ofB. gariniiandB. afzeliiproduced no evidence for strains associated with gray squirrels. Generalized linear mixed models (GLMM) identified tick infestation and date of capture as significant factors associated withB. burgdorferi sensu latoinfection in gray squirrels, with infection elevated in early summer in squirrels infested with ticks. Invasive gray squirrels appear to become infected with locally circulating strains ofB. burgdorferi sensu lato, and further studies are required to determine their role in community disease dynamics. Our findings highlight the fact that the role of introduced host species inB. burgdorferi sensu latoepidemiology can be highly variable and thus difficult to predict.

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Borrelia burgdorferi CheD Promotes Various Functions in Chemotaxis and the Pathogenic Life Cycle of the Spirochete

Infection and Immunity ◽

10.1128/iai.01347-15 ◽

2016 ◽

Vol 84 (6) ◽

pp. 1743-1752 ◽

Cited By ~ 15

Author(s):

Ki Hwan Moon ◽

Gerry Hobbs ◽

M. A. Motaleb

Keyword(s):

Life Cycle ◽

Borrelia Burgdorferi ◽

Phosphatase Activity ◽

Response Regulator ◽

Wild Type ◽

Content Type ◽

Mouse Infection ◽

Protein Receptors ◽

Chemotaxis Proteins

Borrelia burgdorferipossesses a sophisticated chemotaxis signaling system; however, the roles of the majority of the chemotaxis proteins in the infectious life cycle have not yet been demonstrated. Specifically, the role of CheD during host colonization has not been demonstrated in any bacterium. Here, we systematically characterized theB. burgdorferiCheD homolog using genetics and biochemical and mouse-tick-mouse infection cycle studies.Bacillus subtilisCheD plays an important role in chemotaxis by deamidation of methyl-accepting chemotaxis protein receptors (MCPs) and by increasing the receptor kinase activity or enhancing CheC phosphatase activity, thereby regulating the levels of the CheY response regulator. Our biochemical analysis indicates thatB. burgdorferiCheD significantly enhances CheX phosphatase activity by specifically interacting with the phosphatase. Moreover, CheD specifically binds two of the six MCPs, indicating that CheD may also modulate the receptor proteins. Although the motility of thecheDmutant cells was indistinguishable from that of the wild-type cells, the mutant did exhibit reduced chemotaxis. Importantly, the mutant showed significantly reduced infectivity in C3H/HeN mice via needle inoculation. Mouse-tick-mouse infection assays indicated that CheD is dispensable for acquisition or transmission of spirochetes; however, the viability ofcheDmutants in ticks is marginally reduced compared to that of the wild-type or complementedcheDspirochetes. These data suggest that CheD plays an important role in the chemotaxis and pathogenesis ofB. burgdorferi. We propose potential connections between CheD, CheX, and MCPs and discuss how these interactions play critical roles during the infectious life cycle of the spirochete.

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