Borrelia burgdorferi Harbors a Transport System Essential for Purine Salvage and Mammalian Infection

ABSTRACTBorrelia burgdorferiis the tick-borne bacterium that causes the multistage inflammatory disease Lyme disease.B. burgdorferihas a reduced genome and lacks the enzymes required forde novosynthesis of purines for synthesis of RNA and DNA. Therefore, this obligate pathogen is dependent upon the tick vector and mammalian host environments for salvage of purine bases for nucleic acid biosynthesis. This pathway is vital forB. burgdorferisurvival throughout its infectious cycle, as key enzymes in the purine salvage pathway are essential for the ability of the spirochete to infect mice and critical for spirochete replication in the tick. The transport of preformed purines into the spirochete is the first step in the purine salvage pathway and may represent a novel therapeutic target and/or means to deliver antispirochete molecules to the pathogen. However, the transport systems critical for purine salvage byB. burgdorferihave yet to be identified. Herein, we demonstrate that the genesbbb22andbbb23, present onB. burgdorferi's essential plasmid circular plasmid 26 (cp26), encode key purine transport proteins. BBB22 and/or BBB23 is essential for hypoxanthine transport and contributes to the transport of adenine and guanine. Furthermore,B. burgdorferilackingbbb22-23was noninfectious in mice up to a dose of 1 × 107spirochetes. Together, our data establish thatbbb22-23encode purine permeases critical forB. burgdorferimammalian infectivity, suggesting that this transport system may serve as a novel antimicrobial target for the treatment of Lyme disease.

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The Borrelia burgdorferi Glycosaminoglycan Binding Protein Bgp in the B31 Strain Is Not Essential for Infectivity despite Facilitating Adherence and Tissue Colonization

Infection and Immunity ◽

10.1128/iai.00667-17 ◽

2017 ◽

Vol 86 (2) ◽

Cited By ~ 5

Author(s):

Samantha Schlachter ◽

Janakiram Seshu ◽

Tao Lin ◽

Steven Norris ◽

Nikhat Parveen

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Binding Protein ◽

Mammalian Host ◽

Infection Model ◽

Binding Assays ◽

Content Type ◽

Mouse Infection Model ◽

Successful Infection

ABSTRACTThe Lyme disease-causing organismBorrelia burgdorferiis transmitted into the mammalian host by an infected-tick bite. Successful infection relies on the ability of this extracellular pathogen to persist and colonize different tissues.B. burgdorferiencodes a large number of adhesins that are able to interact with host ligands to facilitate adherence and tissue colonization. Multiple glycosaminoglycan binding proteins present inB. burgdorferioffer a degree of redundancy of function during infection, and this highlights the importance of glycosaminoglycans as host cell receptors for spirochete adherence. Of particular interest in this study isBorreliaglycosaminoglycan binding protein (Bgp), which binds to heparin-related glycosaminoglycans. The properties of abgptransposon mutant and atrans-complemented derivative were compared to those of the wild-typeB. burgdorferiin thein vitrobinding assays and in infection studies using a C3H/HeJ mouse infection model. We determined that the loss of Bgp impairs spirochete adherence, infectivity, and tissue colonization, resulting in a reduction of inflammatory manifestations of Lyme disease. Although Bgp is not essential for infectivity, it is an important virulence factor ofB. burgdorferithat allows adherence and tissue colonization and contributes to disease severity.

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Regulatory Protein BBD18 of the Lyme Disease Spirochete: Essential Role During Tick Acquisition?

mBio ◽

10.1128/mbio.01017-14 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 4

Author(s):

Beth M. Hayes ◽

Daniel P. Dulebohn ◽

Amit Sarkar ◽

Kit Tilly ◽

Aaron Bestor ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Critical Role ◽

Regulatory Protein ◽

Site Directed Mutagenesis ◽

Mammalian Host ◽

Content Type ◽

Block Transmission ◽

Infectious Cycle

ABSTRACTThe Lyme disease spirocheteBorrelia burgdorferisenses and responds to environmental cues as it transits between the tick vector and vertebrate host. Failure to properly adapt can block transmission of the spirochete and persistence in either vector or host. We previously identified BBD18, a novel plasmid-encoded protein ofB. burgdorferi, as a putative repressor of the host-essential factor OspC. In this study, we investigate thein vivorole of BBD18 as a regulatory protein, using an experimental mouse-tick model system that closely resembles the natural infectious cycle ofB. burgdorferi. We show that spirochetes that have been engineered to constitutively produce BBD18 can colonize and persist in ticks but do not infect mice when introduced by either tick bite or needle inoculation. Conversely, spirochetes lacking BBD18 can persistently infect mice but are not acquired by feeding ticks. Through site-directed mutagenesis, we have demonstrated that abrogation of spirochete infection in mice by overexpression of BBD18 occurs only withbbd18alleles that can suppress OspC synthesis. Finally, we demonstrate that BBD18-mediated regulation does not utilize a previously describedospCoperator sequence required byB. burgdorferifor persistence in immunocompetent mice. These data lead us to conclude that BBD18 does not represent the putative repressor utilized byB. burgdorferifor the specific downregulation of OspC in the mammalian host. Rather, we suggest that BBD18 exhibits features more consistent with those of a global regulatory protein whose critical role occurs during spirochete acquisition by feeding ticks.IMPORTANCELyme disease, caused byBorrelia burgdorferi, is the most common arthropod-borne disease in North America.B. burgdorferiis transmitted to humans and other vertebrate hosts by ticks as they take a blood meal. Transmission between vectors and hosts requires the bacterium to sense changes in the environment and adapt. However, the mechanisms involved in this process are not well understood. By determining howB. burgdorfericycles between two very different environments, we can potentially establish novel ways to interfere with transmission and limit infection of this vector-borne pathogen. We are studying a regulatory protein called BBD18 that we recently described. We found that too much BBD18 interferes with the spirochete’s ability to establish infection in mice, whereas too little BBD18 appears to prevent colonization in ticks. Our study provides new insight into key elements of the infectious cycle of the Lyme disease spirochete.

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De NovoGuanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus InfectionIn Vivo

Journal of Bacteriology ◽

10.1128/jb.00051-16 ◽

2016 ◽

Vol 198 (14) ◽

pp. 2001-2015 ◽

Cited By ~ 20

Author(s):

Eric M. Kofoed ◽

Donghong Yan ◽

Anand K. Katakam ◽

Mike Reichelt ◽

Baiwei Lin ◽

...

Keyword(s):

Staphylococcus Aureus ◽

De Novo ◽

Alternative Promoter ◽

Antibacterial Drug ◽

Salvage Pathway ◽

Promoter Elements ◽

Purine Salvage ◽

Content Type ◽

Therapeutic Utility

ABSTRACTDe novoguanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement thede novopathway, but the relative importance of either pathway duringStaphylococcus aureusinfection is not known. InS. aureus, genes important for bothde novoand salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5′-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found thatS. aureusguaBandguaAare also transcribed independently of riboswitch control by alternative promoter elements. Deletion ofxpt-pbuX-guaB-guaAgenes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genesxpt-pbuXhad none of these effects. Disruption ofguaBorguaArecapitulates thexpt-pbuX-guaB-guaAdeletionin vivo. In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treatS. aureusinfections but that inhibition ofguaAorguaBcould have therapeutic utility.IMPORTANCEDe novoguanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch inStaphylococcus aureus. We demonstrate here that this is not true, because alternative promoter elements that uncouple thede novopathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient forS. aureussurvival in the absence ofde novoguanine biosynthesis. These data suggest targeting thede novoguanine biosynthesis pathway may have therapeutic utility in the treatment ofS. aureusinfections.

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Spermine and Spermidine Alter Gene Expression and Antigenic Profile of Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.00684-16 ◽

2017 ◽

Vol 85 (3) ◽

Cited By ~ 6

Author(s):

Ying-Han Lin ◽

Jesus A. Romo ◽

Trever C. Smith ◽

Ann N. Reyes ◽

S. L. Rajasekhar Karna ◽

...

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Molecular Mechanisms ◽

Transcriptional Analysis ◽

Transport Systems ◽

Mammalian Host ◽

Polyamine Biosynthesis ◽

Serum Factors ◽

Content Type ◽

Adaptive Capabilities

ABSTRACT Borrelia burgdorferi, the agent of Lyme disease, responds to numerous host-derived signals to alter adaptive capabilities during its enzootic cycle in an arthropod vector and mammalian host. Molecular mechanisms that enable B. burgdorferi to detect, channel, and respond to these signals have become an intense area of study for developing strategies to limit transmission/infection. Bioinformatic analysis of the borrelial genome revealed the presence of polyamine transport components (PotA, PotB, PotC, and PotD), while homologs for polyamine biosynthesis were conspicuously absent. Although potABCD is cotranscribed, the level of PotA was elevated under in vitro growth conditions mimicking unfed ticks compared to the level in fed ticks, while the levels of PotD were similar under the aforementioned conditions in B. burgdorferi. Among several polyamines and polyamine precursors, supplementation of spermine or spermidine in the borrelial growth medium induced synthesis of major regulators of gene expression in B. burgdorferi, such as RpoS and BosR, with a concomitant increase in proteins that contribute to colonization and survival of B. burgdorferi in the mammalian host. Short transcripts of rpoS were elevated in response to spermidine, which was correlated with increased protein levels of RpoS. Transcriptional analysis of rpoZ and B. burgdorferi rel (relBbu ; bb0198) in the presence of spermidine revealed the interplay of multiple regulatory factors in B. burgdorferi gene expression. The effect of spermidine on the levels of select borrelial proteins was also influenced by serum factors. These studies suggest that multiple host-derived signals/nutrients and their transport systems contribute to B. burgdorferi adaptation during the vector and vertebrate host phases of infection.

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Manganese and Zinc Regulate Virulence Determinants in Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.00507-13 ◽

2013 ◽

Vol 81 (8) ◽

pp. 2743-2752 ◽

Cited By ~ 23

Author(s):

Bryan Troxell ◽

Meiping Ye ◽

Youyun Yang ◽

Sebastian E. Carrasco ◽

Yongliang Lou ◽

...

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Critical Role ◽

Transition Metal Ions ◽

Mammalian Host ◽

Virulence Determinants ◽

Temperature Dependent ◽

Content Type ◽

Mammalian Infection

ABSTRACTBorrelia burgdorferi, the causative agent of Lyme disease, must adapt to two diverse niches, an arthropod vector and a mammalian host. RpoS, an alternative sigma factor, plays a central role in spirochetal adaptation to the mammalian host by governing expression of many genes important for mammalian infection.B. burgdorferiis known to be unique in metal utilization, and little is known of the role of biologically available metals inB. burgdorferi. Here, we identified two transition metal ions, manganese (Mn2+) and zinc (Zn2+), that influenced regulation of RpoS. The intracellular Mn2+level fluctuated approximately 20-fold under different conditions and inversely correlated with levels of RpoS and the major virulence factor OspC. Furthermore, an increase in intracellular Mn2+repressed temperature-dependent induction of RpoS and OspC; this repression was overcome by an excess of Zn2+. Conversely, a decrease of intracellular Mn2+by deletion of the Mn2+transporter gene,bmtA, resulted in elevated levels of RpoS and OspC. Mn2+affected RpoS through BosR, a Fur family homolog that is required forrpoSexpression: elevated intracellular Mn2+levels greatly reduced the level of BosR protein but not the level ofbosRmRNA. Thus, Mn2+and Zn2+appeared to be important in modulation of the RpoS pathway that is essential to the life cycle of the Lyme disease spirochete. This finding supports the emerging notion that transition metals such as Mn2+and Zn2+play a critical role in regulation of virulence in bacteria.

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Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance

Journal of Bacteriology ◽

10.1128/jb.00813-15 ◽

2016 ◽

Vol 198 (7) ◽

pp. 1087-1100 ◽

Cited By ~ 14

Author(s):

Gursonika Binepal ◽

Kamal Gill ◽

Paula Crowley ◽

Martha Cordova ◽

L. Jeannine Brady ◽

...

Keyword(s):

Stress Tolerance ◽

Streptococcus Mutans ◽

Transport System ◽

Biofilm Formation ◽

Cellular Response ◽

Pathogenic Bacteria ◽

Transport Systems ◽

Content Type ◽

Growth And Survival ◽

Dental Biofilm

ABSTRACTPotassium (K+) is the most abundant cation in the fluids of dental biofilm. The biochemical and biophysical functions of K+and a variety of K+transport systems have been studied for most pathogenic bacteria but not for oral pathogens. In this study, we establish the modes of K+acquisition inStreptococcus mutansand the importance of K+homeostasis for its virulence attributes. TheS. mutansgenome harbors four putative K+transport systems that included two Trk-like transporters (designated Trk1 and Trk2), one glutamate/K+cotransporter (GlnQHMP), and a channel-like K+transport system (Kch). Mutants lacking Trk2 had significantly impaired growth, acidogenicity, aciduricity, and biofilm formation. [K+] less than 5 mM eliminated biofilm formation inS. mutans. The functionality of the Trk2 system was confirmed by complementing anEscherichia coliTK2420 mutant strain, which resulted in significant K+accumulation, improved growth, and survival under stress. Taken together, these results suggest that Trk2 is the main facet of the K+-dependent cellular response ofS. mutansto environment stresses.IMPORTANCEBiofilm formation and stress tolerance are important virulence properties of caries-causingStreptococcus mutans. To limit these properties of this bacterium, it is imperative to understand its survival mechanisms. Potassium is the most abundant cation in dental plaque, the natural environment ofS. mutans. K+is known to function in stress tolerance, and bacteria have specialized mechanisms for its uptake. However, there are no reports to identify or characterize specific K+transporters inS. mutans. We identified the most important system for K+homeostasis and its role in the biofilm formation, stress tolerance, and growth. We also show the requirement of environmental K+for the activity of biofilm-forming enzymes, which explains why such high levels of K+would favor biofilm formation.

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Structural and Biomolecular Analyses of Borrelia burgdorferi BmpD Reveal a Substrate-Binding Protein of an ABC-Type Nucleoside Transporter Family

Infection and Immunity ◽

10.1128/iai.00962-19 ◽

2020 ◽

Vol 88 (4) ◽

Author(s):

Julia Cuellar ◽

Mia Åstrand ◽

Heli Elovaara ◽

Annukka Pietikäinen ◽

Saija Sirén ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Cellular Localization ◽

De Novo ◽

Binding Protein ◽

Substrate Binding ◽

Nucleoside Transporter ◽

Bacterial Survival ◽

Content Type ◽

Transporter Family ◽

Substrate Binding Protein

ABSTRACT Borrelia burgdorferi sensu lato, the causative agent of tick-borne Lyme borreliosis (LB), has a limited metabolic capacity and needs to acquire nutrients, such as amino acids, fatty acids, and nucleic acids, from the host environment. Using X-ray crystallography, liquid chromatography-mass spectrometry, microscale thermophoresis, and cellular localization studies, we show that basic membrane protein D (BmpD) is a periplasmic substrate-binding protein of an ABC transporter system binding to purine nucleosides. Nucleosides are essential for bacterial survival in the host organism, and these studies suggest a key role for BmpD in the purine salvage pathway of B. burgdorferi sensu lato. Because B. burgdorferi sensu lato lacks the enzymes required for de novo purine synthesis, BmpD may play a vital role in ensuring access to the purines needed to sustain an infection in the host. Furthermore, we show that, although human LB patients develop anti-BmpD antibodies, immunization of mice with BmpD does not confer protection against B. burgdorferi sensu lato infection.

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Role of the BBA64 Locus of Borrelia burgdorferi in Early Stages of Infectivity in a Murine Model of Lyme Disease

Infection and Immunity ◽

10.1128/iai.01118-07 ◽

2007 ◽

Vol 76 (1) ◽

pp. 391-402 ◽

Cited By ~ 37

Author(s):

Mahulena Maruskova ◽

M. Dolores Esteve-Gassent ◽

Valerie L. Sexton ◽

J. Seshu

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Immunoblot Analysis ◽

Open Reading Frames ◽

Mammalian Host ◽

Environmental Signals ◽

Significant Difference ◽

Linear Plasmid 54

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, undergoes rapid adaptive gene expression in response to environmental signals encountered during different stages of its life cycle in the arthropod vector or the mammalian host. Among all the plasmid-encoded genes of B. burgdorferi, several linear plasmid 54 (lp54)-encoded open reading frames (ORFs) exhibit the greatest differential expression in response to mammalian host-specific temperature, pH, and other uncharacterized signals. These ORFs include members of the paralogous gene family 54 (pgf 54), such as BBA64, BBA65, and BBA66, present on lp54. In an attempt to correlate transcriptional up-regulation of these pgf 54 members to their role in infectivity, we inactivated BBA64 and characterized the phenotype of this mutant both in vitro and in vivo. There were no major differences in the protein profiles between the BBA64 mutant and the control strains, while immunoblot analysis indicated that inactivation of BBA64 resulted in increased levels of BBA65. Moreover, there was no significant difference in the ability of the BBA64 mutant to infect C3H/HeN mice compared to that of its parental or complemented control strains as determined by culturing of viable spirochetes from infected tissues. However, enumeration of spirochetes using quantitative real-time PCR revealed tissue-specific differences, suggesting a minimal role for BBA64 in the survival of B. burgdorferi in select tissues. Infectivity analysis of the BBA64 mutant suggests that B. burgdorferi may utilize multiple determinants to establish infection in mammalian hosts.

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Histoplasma capsulatum Depends onDe NovoVitamin Biosynthesis for Intraphagosomal Proliferation

Infection and Immunity ◽

10.1128/iai.00824-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 393-404 ◽

Cited By ~ 26

Author(s):

Andrew L. Garfoot ◽

Olga Zemska ◽

Chad A. Rappleye

Keyword(s):

Minimal Medium ◽

Histoplasma Capsulatum ◽

De Novo ◽

Mammalian Host ◽

Riboflavin Biosynthesis ◽

Yeast Growth ◽

Content Type ◽

Druggable Targets ◽

Survival Mechanisms

ABSTRACTDuring infection of the mammalian host,Histoplasma capsulatumyeasts survive and reside within macrophages of the immune system. Whereas some intracellular pathogens escape into the host cytosol,Histoplasmayeasts remain within the macrophage phagosome. This intracellularHistoplasma-containing compartment imposes nutritional challenges for yeast growth and replication. We identified and annotated vitamin synthesis pathways encoded in theHistoplasmagenome and confirmed by growth in minimal medium thatHistoplasmayeasts can synthesize all essential vitamins with the exception of thiamine. Riboflavin, pantothenate, and biotin auxotrophs ofHistoplasmawere generated to probe whether these vitamins are available to intracellular yeasts. Disruption of theRIB2gene (riboflavin biosynthesis) prevented growth and proliferation of yeasts in macrophages and severely attenuatedHistoplasmavirulence in a murine model of respiratory histoplasmosis. Rib2-deficient yeasts were not cleared from lung tissue but persisted, consistent with functional survival mechanisms but inability to replicatein vivo. In addition, depletion of Pan6 (pantothenate biosynthesis) but not Bio2 function (biotin synthesis) also impairedHistoplasmavirulence. These results indicate that theHistoplasma-containing phagosome is limiting for riboflavin and pantothenate and thatHistoplasmavirulence requiresde novosynthesis of these cofactor precursors. Since mammalian hosts do not rely on vitamin synthesis but instead acquire essential vitamins through diet, vitamin synthesis pathways represent druggable targets for therapeutics.

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Evaluation of RevA, a Fibronectin-Binding Protein of Borrelia burgdorferi, as a Potential Vaccine Candidate for Lyme Disease

Clinical and Vaccine Immunology ◽

10.1128/cvi.00758-12 ◽

2013 ◽

Vol 20 (6) ◽

pp. 892-899 ◽

Cited By ~ 12

Author(s):

Angela M. Floden ◽

Tammy Gonzalez ◽

Robert A. Gaultney ◽

Catherine A. Brissette

Keyword(s):

Lyme Disease ◽

Borrelia Burgdorferi ◽

Passive Immunization ◽

Surface Protein ◽

Surface Expression ◽

Content Type ◽

Good Target ◽

Potential Vaccine ◽

Mammalian Infection

ABSTRACTPrevious studies indicated that the Lyme disease spirocheteBorrelia burgdorferiexpresses the RevA outer surface protein during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA appears to be a good target for preventive therapies. RevA proteins are highly conserved across all Lyme borreliae, and antibodies against RevA protein are cross-reactive among RevA proteins from diverse strains. Mice infected withB. burgdorferimounted a rapid IgM response to RevA, followed by a strong IgG response that generally remained elevated for more than 12 months, suggesting continued exposure of RevA protein to the immune system. RevA antibodies were bactericidalin vitro. To evaluate the RevA antigen as a potential vaccine, mice were vaccinated with recombinant RevA and challenged withB. burgdorferiby inoculation with a needle or by a tick bite. Cultured tissues from all treatment groups were positive forB. burgdorferi. Vaccinated animals also appeared to have similar levels ofB. burgdorferiDNA compared to nonvaccinated controls. Despite its antigenicity, surface expression, and the production of bactericidal antibodies against it, RevA does not protect againstBorrelia burgdorferiinfection in a mouse model. However, passive immunization with anti-RevA antibodies did prevent infection, suggesting the possible utility of RevA-based immunotherapeutics or vaccine.

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