The BB0345 Hypothetical Protein of Borrelia burgdorferi Is Essential for Mammalian Infection

ABSTRACT During the natural enzootic life cycle of Borrelia burgdorferi (also known as Borreliella burgdorferi), the bacteria must sense conditions within the vertebrate and arthropod and appropriately regulate expression of genes necessary to persist within these distinct environments. bb0345 of B. burgdorferi encodes a hypothetical protein of unknown function that is predicted to contain an N-terminal helix-turn-helix (HTH) domain. Because HTH domains can mediate protein-DNA interactions, we hypothesized that BB0345 might represent a previously unidentified borrelial transcriptional regulator with the ability to regulate events critical for the B. burgdorferi enzootic cycle. To study the role of BB0345 within mammals, we generated a bb0345 mutant and assessed its virulence potential in immunocompetent mice. The bb0345 mutant was able to initiate localized infection and disseminate to distal tissues but was cleared from all sites by 14 days postinfection. In vitro growth curve analyses revealed that the bb0345 mutant grew similar to wild-type bacteria in standard Barbour-Stoenner-Kelley II (BSK-II) medium; however, the mutant was not able to grow in dilute BSK-II medium or dialysis membrane chambers (DMCs) implanted in rats. Proteinase K accessibility assays and whole-cell partitioning indicated that BB0345 was intracellular and partially membrane associated. Comparison of protein production profiles between the wild-type parent and the bb0345 mutant revealed no major differences, suggesting BB0345 may not be a global transcriptional regulator. Taken together, these data show that BB0345 is essential for B. burgdorferi survival in the mammalian host, potentially by aiding the spirochete with a physiological function that is required by the bacterium during infection.

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Use of Recombinase-BasedIn VivoExpression Technology To Characterize Enterococcus faecalis Gene Expression during Infection IdentifiesIn Vivo-Expressed Antisense RNAs and Implicates the Protease Eep in Pathogenesis

Infection and Immunity ◽

10.1128/iai.05964-11 ◽

2011 ◽

Vol 80 (2) ◽

pp. 539-549 ◽

Cited By ~ 34

Author(s):

Kristi L. Frank ◽

Aaron M. T. Barnes ◽

Suzanne M. Grindle ◽

Dawn A. Manias ◽

Patrick M. Schlievert ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Rabbit Model ◽

Microscopic Analysis ◽

Mammalian Host ◽

Wild Type ◽

Antisense Transcripts ◽

Content Type ◽

Antisense Rnas

ABSTRACTEnterococcus faecalisis a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades.E. faecalismust be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-basedin vivoexpression technology (RIVET) to identify promoters on theE. faecalisOG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putativein vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected inin vitro- andin vivo-grown cells, providing the first evidence ofin vivo-expressed antisense RNAs inE. faecalis. Deletions in thein vivo-activated genes that encode glutamate 5-kinase (proB[EF0038]), the transcriptional regulator EbrA (ebrA[EF1809]), and the membrane metalloprotease Eep (eep[EF2380]) did not hinder biofilm formation inin vitroassays. In a rabbit model of endocarditis, the ΔebrAstrain was fully virulent, the ΔproBstrain was slightly attenuated, and the Δeepstrain was severely attenuated. The Δeepvirulence defect could be complemented by the expression of the wild-type gene intrans. Microscopic analysis of early Δeepbiofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen.

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Expression of a luxS Gene Is Not Required for Borrelia burgdorferi Infection of Mice via Needle Inoculation

Infection and Immunity ◽

10.1128/iai.71.5.2892-2896.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2892-2896 ◽

Cited By ~ 44

Author(s):

Anette Hübner ◽

Andrew T. Revel ◽

Dena M. Nolen ◽

Kayla E. Hagman ◽

Michael V. Norgard

Keyword(s):

Quorum Sensing ◽

Borrelia Burgdorferi ◽

Mammalian Host ◽

Wild Type ◽

Sensing System ◽

Autoinducer 2 ◽

Sensing Systems ◽

Quorum Sensing System ◽

Culture Supernatants

ABSTRACT The luxS gene product is an integral component of LuxS/autoinducer-2 (AI-2) quorum-sensing systems in bacteria. A putative luxS gene was expressed at comparable levels by Borrelia burgdorferi strain 297 cultivated either in vitro or in dialysis membrane chambers implanted in rat peritoneal cavities. Although the borrelial luxS gene functionally complemented a LuxS deficiency in Escherichia coli DH5α, AI-2-like activity could not be detected within B. burgdorferi culture supernatants or concentrated cell lysates. Finally, a luxS-deficient mutant of B. burgdorferi was infectious at wild-type levels when it was intradermally needle inoculated into mice, indicating that expression of luxS probably is not required for infectivity but, at the very least, is not essential for mammalian host adaptation. Our findings also challenge the notion that a LuxS/AI-2 quorum-sensing system is operative in B. burgdorferi.

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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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Borrelia burgdorferi bba74 Is Expressed Exclusively during Tick Feeding and Is Regulated by Both Arthropod- and Mammalian Host-Specific Signals

Journal of Bacteriology ◽

10.1128/jb.01802-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2783-2794 ◽

Cited By ~ 42

Author(s):

Vishwaroop B. Mulay ◽

Melissa J. Caimano ◽

Radha Iyer ◽

Star Dunham-Ems ◽

Dionysios Liveris ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Elevated Temperatures ◽

Temperature Shift ◽

Mammalian Host ◽

Wild Type ◽

Tick Feeding ◽

Reciprocal Regulation ◽

Heterogeneous Expression ◽

Membrane Spanning

ABSTRACT Although BBA74 initially was described as a 28-kDa virulence-associated outer-membrane-spanning protein with porin-like function, subsequent studies revealed that it is periplasmic and downregulated in mammalian host-adapted spirochetes. To further elucidate the role of this protein in the Borrelia burgdorferi tick-mammal cycle, we conducted a thorough examination of its expression profile in comparison with the profiles of three well-characterized, differentially expressed borrelial genes (ospA, ospC, and ospE) and their proteins. In vitro, transcripts for bba74 were expressed at 23°C and further enhanced by a temperature shift (37°C), whereas BBA74 protein diminished at elevated temperatures; in contrast, neither transcript nor protein was expressed by spirochetes grown in dialysis membrane chambers (DMCs). Primer extension of wild-type B. burgdorferi grown in vitro, in conjunction with expression analysis of DMC-cultivated wild-type and rpoS mutant spirochetes, revealed that, like ospA, bba74 is transcribed by σ70 and is subject to RpoS-mediated repression within the mammalian host. A series of experiments utilizing wild-type and rpoS mutant spirochetes was conducted to determine the transcriptional and translational profiles of bba74 during the tick-mouse cycle. Results from these studies revealed (i) that bba74 is transcribed by σ70 exclusively during the larval and nymphal blood meals and (ii) that transcription of bba74 is bracketed by RpoS-independent and -dependent forms of repression that are induced by arthropod- and mammalian host-specific signals, respectively. Although loss of BBA74 does not impair the ability of B. burgdorferi to complete its infectious life cycle, the temporal compartmentalization of this gene's transcription suggests that BBA74 facilitates fitness of the spirochete within a narrow window of its tick phase. A reexamination of the paradigm for reciprocal regulation of ospA and ospC, performed herein, revealed that the heterogeneous expression of OspA and OspC displayed by spirochete populations during the nymphal blood meal results from the intricate sequence of transcriptional and translational changes that ensue as B. burgdorferi transitions between its arthropod vector and mammalian host.

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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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BB0238, a Presumed Tetratricopeptide Repeat-Containing Protein, Is Required during Borrelia burgdorferi Mammalian Infection

Infection and Immunity ◽

10.1128/iai.01977-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4292-4306 ◽

Cited By ~ 9

Author(s):

Ashley M. Groshong ◽

Danielle E. Fortune ◽

Brendan P. Moore ◽

Horace J. Spencer ◽

Robert A. Skinner ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Protein Interactions ◽

Point Mutations ◽

Structural Motif ◽

Tetratricopeptide Repeat ◽

Mammalian Host ◽

Content Type ◽

Mammalian Infection ◽

Tpr Domain

ABSTRACTThe Lyme disease spirochete,Borrelia burgdorferi, occupies both a tick vector and mammalian host in nature. Considering the unique enzootic life cycle ofB. burgdorferi, it is not surprising that a large proportion of its genome is composed of hypothetical proteins not found in other bacterial pathogens.bb0238encodes a conserved hypothetical protein of unknown function that is predicted to contain a tetratricopeptide repeat (TPR) domain, a structural motif responsible for mediating protein-protein interactions. To evaluate the role ofbb0238during mammalian infection, abb0238-deficient mutant was constructed. Thebb0238mutant was attenuated in mice infected via needle inoculation, and complementation ofbb0238expression restored infectivity to wild-type levels.bb0238expression does not change in response to varying culture conditions, and thus, it appears to be constitutively expressed underin vitroconditions.bb0238is expressed in murine tissues during infection, though there was no significant change in expression levels among different tissue types. Localization studies indicate that BB0238 is associated with the inner membrane of the spirochete and is therefore unlikely to promote interaction with host ligands during infection.B. burgdorfericlones containing point mutations in conserved residues of the putative TPR motif of BB0238 demonstrated attenuation in mice that was comparable to that in thebb0238deletion mutant, suggesting that BB0238 may contain a functional TPR domain.

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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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Efficacy of Humanized Carbapenem Exposures against New Delhi Metallo-β-Lactamase (NDM-1)-Producing Enterobacteriaceae in a Murine Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00708-13 ◽

2013 ◽

Vol 57 (8) ◽

pp. 3936-3940 ◽

Cited By ~ 19

Author(s):

Dora E. Wiskirchen ◽

Patrice Nordmann ◽

Jared L. Crandon ◽

David P. Nicolau

Keyword(s):

Maximal Activity ◽

Bacterial Density ◽

Infection Model ◽

High Dose ◽

New Delhi ◽

Wild Type ◽

Content Type ◽

Immunocompetent Mice

ABSTRACTEnterobacteriaceaeproducing the novel carbapenemase New Delhi metallo-β-lactamase (NDM-1) are emerging worldwide. While these organisms often display high levels ofin vitroresistance to multiple antibiotics,in vivoefficacy data are lacking. Here, the activities of humanized ertapenem and doripenem exposures were characterized against a wild-typeK. pneumoniaeand its derived isogenic strains harboring either an NDM-1 or KPC-2 plasmid in immunocompetent mice. In addition, four clinical isolates expressing NDM-1 were evaluated. Human-simulated regimens of ertapenem at 1 g every 24 h and high-dose, prolonged infusion of doripenem at 2 g every 8 h as a 4-h infusion were evaluated over 24 h, and efficacy was determined by the change in bacterial density compared to that in 24-h growth controls. CFU reductions in bacterial density of greater than 1 log unit were observed against the wild-type strain as well as the derived isogenic NDM-1 strain, while no reduction was observed against the derived KPC-2 strain. Postexposure MICs confirmed thein vitromaintenance of the ertapenem resistance marker in both the NDM-1 and KPC-2 strains. Similar to the case for the isogenically derived NDM-1 strain, bacterial density was reduced at 24 h against all four clinical NDM-1 isolates showing variable levels of MICs for carbapenems, with near-maximal activity of both agents occurring when the doripenem MIC was ≤8 μg/ml. While carbapenem monotherapy does not appear to be an option against KPC-based infections, these data suggest that carbapenem monotherapy may be a viable option for treating NDM-1-producingEnterobacteriaceaeunder certain conditions, and this warrants furtherin vivoexploration.

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Competitive Advantage of Borrelia burgdorferi with Outer Surface Protein BBA03 during Tick-Mediated Infection of the Mammalian Host

Infection and Immunity ◽

10.1128/iai.00521-12 ◽

2012 ◽

Vol 80 (10) ◽

pp. 3501-3511 ◽

Cited By ~ 22

Author(s):

Aaron Bestor ◽

Ryan O. M. Rego ◽

Kit Tilly ◽

Patricia A. Rosa

Keyword(s):

Borrelia Burgdorferi ◽

Competitive Advantage ◽

Surface Protein ◽

Culture Conditions ◽

Mammalian Host ◽

Wild Type ◽

Tick Feeding ◽

Transmitted Infection ◽

Content Type ◽

Tick Transmission

ABSTRACTLinear plasmid lp54 is one of the most highly conserved and differentially expressed elements of the segmented genome of the Lyme disease spirocheteBorrelia burgdorferi. We previously reported that deletion of a 4.1-kb region of lp54 (bba01tobba07[bba01-bba07]) led to a slight attenuation of tick-transmitted infection in mice following challenge with a large number of infected ticks. In the current study, we reduced the number of ticks in the challenge to more closely mimic the natural dose and found a profound defect in tick-transmitted infection of thebba01-bba07mutant relative to wild-typeB. burgdorferi. We next focused on deletion ofbba03as the most likely cause of this mutant phenotype, as previous studies have shown that expression ofbba03is increased by culture conditions that simulate tick feeding. Consistent with this hypothesis, we demonstrated increased expression ofbba03by spirochetes in fed relative to unfed ticks. We also observed that abba03deletion mutant, although fully competent by itself, did not efficiently infect mice when transmitted by ticks that were simultaneously coinfected with wild-typeB. burgdorferi. These results suggest that BBA03 provides a competitive advantage to spirochetes carrying this protein during tick transmission to a mammalian host in the natural infectious cycle.

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Bacterial Heterogeneity Is a Requirement for Host Superinfection by the Lyme Disease Spirochete

Infection and Immunity ◽

10.1128/iai.01817-14 ◽

2014 ◽

Vol 82 (11) ◽

pp. 4542-4552 ◽

Cited By ~ 10

Author(s):

Artem S. Rogovskyy ◽

Troy Bankhead

Keyword(s):

Secondary Infection ◽

Wild Type ◽

Tissue Samples ◽

Antibiotic Resistant ◽

Major Barrier ◽

Content Type ◽

Immunodeficient Mice ◽

Immunocompetent Mice ◽

Immune Barriers

ABSTRACTIn nature, mixedBorrelia burgdorferiinfections are common and possibly can be acquired by either superinfection or coinfection. Superinfection by heterologousB. burgdorferistrains has been established experimentally, although the ability of homologousB. burgdorfericlones to superinfect a host has not been studied in detail. Information regarding any potential immune barriers to secondary infection also currently is unavailable. In the present study, the ability to superinfect various mouse models by homologous wild-type clones was examined and compared to superinfection by heterologous strains. To assess the ability of homologousB. burgdorfericlones to successfully superinfect a mouse host, primary- and secondary-infecting spirochetes were recovered viain vitrocultivation of collected blood or tissue samples. This was accomplished by generating two different antibiotic-resistant versions of the wild-type B31-A3 clone in order to distinguish superinfectingB. burgdorferifrom primary-infecting spirochetes. The data demonstrate an inability of homologousB. burgdorferito superinfect immunocompetent mice as opposed to heterologous strains. Attempts to superinfect different types of immunodeficient mice with homologousB. burgdorferiindicate that the murine innate immune system represents a major barrier to intrastrain superinfection. Consequently, the possibility of innate immunity as a driving force forB. burgdorferiheterogeneity during the enzootic cycle is discussed.

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