[Show abstract][Hide abstract] ABSTRACT: The second messenger nucleotide cyclic diadenylate monophosphate (c-di-AMP) has been identified in several species of Gram positive bacteria and Chlamydia trachomatis. This molecule has been associated with bacterial cell division, cell wall biosynthesis and phosphate metabolism, and with induction of type I interferon responses by host cells. We demonstrate that B. burgdorferi produces a c-di-AMP synthase, which we designated CdaA. Both CdaA and c-di-AMP levels are very low in cultured B. burgdorferi, and no conditions were identified under which cdaA mRNA was differentially expressed. A mutant B. burgdorferi was produced that expresses high levels of CdaA, yet steady state borrelial c-di-AMP levels did not change, apparently due to degradation by the native DhhP phosphodiesterase. The function(s) of c-di-AMP in the Lyme disease spirochete remains enigmatic.
PLoS ONE 04/2015; 10(4):e0125440. DOI:10.1371/journal.pone.0125440 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ticks transmit various human and animal microbial pathogens and may harbour more than one pathogen simultaneously. Both viruses and bacteria can trigger, and may subsequently suppress, vertebrate host and arthropod vector anti-microbial responses. Microbial coinfection of ticks could lead to an advantage or disadvantage for one or more of the microorganisms. In this preliminary study, cell lines derived from the ticks Ixodes scapularis and Ixodes ricinus were infected sequentially with 2 arthropod-borne pathogens, Borrelia burgdorferi s.s., Ehrlichia ruminantium, or Semliki Forest virus (SFV), and the effect of coinfection on the replication of these pathogens was measured. Prior infection of tick cell cultures with the spirochaete B. burgdorferi enhanced subsequent replication of the rickettsial pathogen E. ruminantium whereas addition of spirochaetes to cells infected with E. ruminantium had no effect on growth of the latter. Both prior and subsequent presence of B. burgdorferi also had a positive effect on SFV replication. Presence of E. ruminantium or SFV had no measurable effect on B. burgdorferi growth. In tick cells infected first with E. ruminantium and then with SFV, virus replication was significantly higher across all time points measured (24, 48, 72 h post infection), while presence of the virus had no detectable effect on bacterial growth. When cells were infected first with SFV and then with E. ruminantium, there was no effect on replication of either pathogen. The results of this preliminary study indicate that interplay does occur between different pathogens during infection of tick cells. Further study is needed to determine if this results from direct pathogen–pathogen interaction or from effects on host cell defences, and to determine if these observations also apply in vivo in ticks. If presence of one pathogen in the tick vector results in increased replication of another, this could have implications for disease transmission and incidence.
[Show abstract][Hide abstract] ABSTRACT: The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections.
[Show abstract][Hide abstract] ABSTRACT: Bacteria require explicit control over their proteomes in order to compete and survive in dynamic environments. The Lyme disease
spirochete Borrelia burgdorferi undergoes substantial protein profile changes during its cycling between vector ticks and vertebrate hosts. In an effort
to understand regulation of these transitions, we recently isolated and functionally characterized the borrelial nucleic acid-binding
protein BpuR, a PUR domain-containing protein. We now report that this regulatory protein governs its own synthesis through
direct interactions with bpuR mRNA. In vitro and in vivo techniques indicate that BpuR binds with high affinity and specificity to the 5′ region of its message, thereby inhibiting
translation. This negative feedback could permit the bacteria to fine-tune cellular BpuR concentrations. These data add to
the understanding of this newly described class of prokaryotic DNA- and RNA-binding regulatory proteins.
Journal of bacteriology 08/2013; 195(21). DOI:10.1128/JB.00819-13 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Lyme disease spirochete Borrelia burgdorferi lacks endogenous, surface-exposed proteases. In order to efficiently disseminate throughout the host and penetrate tissue
barriers, borreliae rely on recruitment of host proteases, such as plasmin(ogen). Here we report the identification of a novel
plasminogen-binding protein, BBA70. Binding of plasminogen is dose-dependent and is affected by ionic strength. The BBA70-plasminogen
interaction is mediated by lysine residues, primarily located in a putative C-terminal α-helix of BBA70. These lysine residues
appear to interact with the lysine-binding sites in plasminogen kringle domain 4 because a deletion mutant of plasminogen
lacking that domain was unable to bind to BBA70. Bound to BBA70, plasminogen activated by urokinase-type plasminogen activator
was able to degrade both a synthetic chromogenic substrate and the natural substrate fibrinogen. Furthermore, BBA70-bound
plasmin was able to degrade the central complement proteins C3b and C5 and inhibited the bacteriolytic effects of complement.
Consistent with these functional activities, BBA70 is located on the borrelial outer surface. Additionally, serological evidence
demonstrated that BBA70 is produced during mammalian infection. Taken together, recruitment and activation of plasminogen
could play a beneficial role in dissemination of B. burgdorferi in the human host and may possibly aid the spirochete in escaping the defense mechanisms of innate immunity.
[Show abstract][Hide abstract] ABSTRACT: The PUR domain is a nucleic acid-binding motif found in critical regulatory proteins of higher eukaryotes and in certain species
of bacteria. During investigations into mechanisms by which the Lyme disease spirochete controls synthesis of its Erp surface
proteins, it was discovered that the borrelial PUR domain protein, Bpur, binds with high affinity to double-stranded DNA adjacent
to the erp transcriptional promoter. Bpur was found to enhance the effects of the erp repressor protein, BpaB. Bpur also bound single-stranded DNA and RNA, with relative affinities RNA > double-stranded DNA
> single-stranded DNA. Rational site-directed mutagenesis of Bpur identified amino acid residues and domains critical for
interactions with nucleic acids, and it revealed that the PUR domain has a distinct mechanism of interaction with each type
of nucleic acid ligand. These data shed light on both gene regulation in the Lyme spirochete and functional mechanisms of
the widely distributed PUR domain.
[Show abstract][Hide abstract] ABSTRACT: A site-specific DNA-binding protein was purified from Borrelia burgdorferi cytoplasmic extracts, and determined to be a member of the highly conserved SpoVG family. This is the first time a function has been attributed to any of these ubiquitous bacterial proteins. Further investigations into SpoVG orthologues indicated that the Staphylococcus aureus protein also binds DNA, but interacts preferentially with a distinct nucleic acid sequence. Site-directed mutagenesis and domain swapping between the S. aureus and B. burgdorferi proteins identified that a 6-residue stretch of the SpoVG α-helix contributes to DNA sequence specificity. Two additional, highly conserved amino acid residues on an adjacent β-sheet are essential for DNA-binding, apparently by contacts with the DNA phosphate backbone. Results of these studies thus identified a novel family of bacterial DNA-binding proteins, developed a model of SpoVG-DNA interactions, and provide direction for future functional studies on these wide-spread proteins.
PLoS ONE 06/2013; 8(6):e66683. DOI:10.1371/journal.pone.0066683 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lyme disease spirochetes possess complex genomes, consisting of a main chromosome and 20 or more smaller replicons. Among
those small DNAs are the cp32 elements, a family of prophages that replicate as circular episomes. All complete cp32s contain
an erp locus, which encodes surface-exposed proteins. Sequences were compared for all 193 erp alleles carried by 22 different strains of Lyme disease-causing spirochete to investigate their natural diversity and evolutionary
histories. These included multiple isolates from a focus where Lyme disease is endemic in the northeastern United States and
isolates from across North America and Europe. Bacteria were derived from diseased humans and from vector ticks and included
members of 5 different Borrelia genospecies. All erp operon 5′-noncoding regions were found to be highly conserved, as were the initial 70 to 80 bp of all erp open reading frames, traits indicative of a common evolutionary origin. However, the majority of the protein-coding regions
are highly diverse, due to numerous intra- and intergenic recombination events. Most erp alleles are chimeras derived from sequences of closely related and distantly related erp sequences and from unknown origins. Since known functions of Erp surface proteins involve interactions with various host
tissue components, this diversity may reflect both their multiple functions and the abilities of Lyme disease-causing spirochetes
to successfully infect a wide variety of vertebrate host species.
[Show abstract][Hide abstract] ABSTRACT: Leptospirosis in horses has been considered a relatively uncommon infection. However, recent data suggest that the infection is widespread, with the incidence and infecting serovars varying considerably in different geographical regions. The majority of infections remain asymptomatic. Clinical signs in equine leptospirosis resemble those seen in other animal species. However, leptospirosis as a cause of acute respiratory distress is becoming more frequently recognised. A particular feature of equine leptospirosis is post infection recurrent uveitis (moon blindness or periodic ophthalmia), which appears to be mediated by autoimmune mechanisms involving cross reactivity between ocular tissues and leptospiral membrane proteins. There are no leptospiral vaccines licensed for use in horses, with no prospect for any becoming available in the foreseeable future. Accordingly, prevention of equine leptospirosis must rely on good hygiene practices, minimisation of rodent contact, and vaccination of other species of production and companion animals.
[Show abstract][Hide abstract] ABSTRACT: The Lyme disease spirochete controls production of its OspC and Erp outer surface proteins, repressing protein synthesis during colonization of vector ticks but increasing expression when those ticks feed on vertebrate hosts. Early studies found that synthesis of OspC and Erps can be stimulated in culture by shifting temperature from 23 to 34°C, leading to a hypothesis that B. burgdorferi senses environmental temperature to determine its location in the tick-mammal infectious cycle. However, borreliae cultured at 34°C divide several times faster than do those cultured at 23°C. We developed methods that disassociate bacterial growth rate and temperature, allowing separate evaluation of each factor's impacts on B. burgdorferi gene and protein expression. Altogether, the data support a new paradigm that B. burgdorferi actually responds to changes in its own replication rate, not temperature per se, as the impetus to increase expression of the OspC and Erp infection-associated proteins.
Journal of bacteriology 12/2012; 195(4). DOI:10.1128/JB.01956-12 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Borrelia burgdorferi, the etiological agent of Lyme disease, exploits an array of strategies to establish infection and to overcome host innate and adaptive immune responses. One key borrelial immune escape mechanism involves the inactivation of host complement attack through acquisition of human immune regulators factor H (CFH), factor H-like protein 1 (FHL1), factor H-related protein 1 (CFHR1), CFHR2, and/or CFHR5. Binding of these host proteins is primarily mediated by bacterial surface-exposed proteins that have been collectively referred to as complement regulator-acquiring surface proteins, or CRASPs. Different strains of B. burgdorferi produce as many as 5 different CRASP molecules that comprise 3 distinct, genetically unrelated groups. Depending on bacterial genetic composition, different combinations of these proteins can be found on the borrelial outer surface. The 3 groups differ in their gene location, gene regulatory mechanisms, expression patterns during the tick-mammal infection cycle, protein sequence and structure as well as binding affinity for complement regulators and other serum proteins. These attributes influence the proteins' abilities to contribute to complement resistance of this emerging human pathogen. In this review, we focus on the current knowledge on structure, function, and gene regulation of these B. burgdorferi infection-associated proteins.
[Show abstract][Hide abstract] ABSTRACT: Leptospirosis, caused by pathogenic species of genus Leptospira, is a highly prevalent zoonotic disease throughout many parts of the world, and an important emerging disease within the United States. Uveitis is a common complication of systemic infection in humans. A similar condition in horses is characterized by recurrent bouts of inflammation. In this article, we review advances in our understanding of leptospiral uveitis and its pathogenic mechanisms.
Zoonoses and Public Health 09/2012; Suppl 2:132-41. DOI:10.1111/j.1863-2378.2011.01445.x · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Borrelia burgdorferi BpaB proteins of the spirochete's ubiquitous cp32 prophages are DNA-binding proteins, required both for maintenance of the bacteriophage episomes and for transcriptional regulation of the cp32 erp operons. Through use of DNase I footprinting, we demonstrate that BpaB binds the erp operator initially at the sequence 5'-TTATA-3'. Electrophoretic mobility shift assays indicated that BpaB also binds with high affinity to sites located in the 5' noncoding regions of two additional cp32 genes. Characterization of the proteins encoded by those genes indicated that they are a single-stranded DNA-binding protein and a nuclease, which we named SsbP and NucP, respectively. Chromatin immunoprecipitation indicated that BpaB binds erp, ssbP, and nucP in live B. burgdorferi. A mutant bacterium that overexpressed BpaB produced significantly higher levels of ssbP and nucP transcript than did the wild-type parent.
Journal of bacteriology 06/2012; 194(17):4570-8. DOI:10.1128/JB.00661-12 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nearly every known species of Eubacteria encodes a homolog of the Borrelia burgdorferi EbfC DNA-binding protein. We now demonstrate that fluorescently tagged EbfC associates with B. burgdorferi nucleoids in vivo and that chromatin immunoprecipitation (ChIP) of wild-type EbfC showed it to bind in vivo to sites throughout the genome, two hallmarks of nucleoid-associated proteins. Comparative RNA sequencing (RNA-Seq) of a mutant B. burgdorferi strain that overexpresses EbfC indicated that approximately 4.5% of borrelial genes are significantly impacted by EbfC. The ebfC gene was highly expressed in rapidly growing bacteria, but ebfC mRNA was undetectable in stationary phase. Combined with previous data showing that EbfC induces bends in DNA, these results demonstrate that EbfC is a nucleoid-associated protein and lead to the hypothesis that B. burgdorferi utilizes cellular fluctuations in EbfC levels to globally control transcription of numerous genes. The ubiquity of EbfC proteins in Eubacteria suggests that these results apply to a wide range of pathogens and other bacteria.
Journal of bacteriology 04/2012; 194(13):3395-406. DOI:10.1128/JB.00252-12 · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This units presents methods through which one may isolate and identify novel bacterial DNA-binding proteins. Briefly, the DNA sequence of interest is affixed to beads, and then incubated with bacterial cytoplasmic extract. Washes with buffers containing nonspecific DNA and low-salt concentrations will remove non-adhering and low-specificity DNA-binding proteins, while subsequent washes with higher salt concentrations will elute more specific DNA-binding proteins. Eluted proteins may then be identified by standard proteomic techniques.
Current protocols in microbiology 02/2012; Chapter 1:Unit1F.1. DOI:10.1002/9780471729259.mc01f01s24
[Show abstract][Hide abstract] ABSTRACT: In little more than 30 years, Lyme disease, which is caused by the spirochaete Borrelia burgdorferi, has risen from relative obscurity to become a global public health problem and a prototype of an emerging infection. During this period, there has been an extraordinary accumulation of knowledge on the phylogenetic diversity, molecular biology, genetics and host interactions of B. burgdorferi. In this Review, we integrate this large body of information into a cohesive picture of the molecular and cellular events that transpire as Lyme disease spirochaetes transit between their arthropod and vertebrate hosts during the enzootic cycle.
[Show abstract][Hide abstract] ABSTRACT: Borrelia burgdorferi
evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of
and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a
strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of