Opioid receptor antagonists increase hyperalgesia in humans and animals, indicating that endogenous activation of opioid receptors provides relief from acute pain; however, the mechanisms of long-term opioid inhibition of pathological pain have remained elusive. We found that tissue injury produced μ-opioid receptor constitutive activity (MORCA) that repressed spinal nociceptive signaling for months. Pharmacological blockade during the post-hyperalgesia state with MOR inverse agonists reinstated central pain sensitization, and precipitated hallmarks of opioid withdrawal (including cAMP overshoot and hyperalgesia) that required N-methyl-D-aspartate receptor activation of adenylyl cyclase type 1 (AC1). Thus, MORCA initiates both analgesic signaling as well as a compensatory opponent process that generates endogenous opioid dependence. Tonic MORCA suppression of withdrawal hyperalgesia may prevent the transition from acute to chronic pain.

Arthropods transmit diverse infectious agents; however, the ways microbes influence their vector to enhance colonization are poorly understood.Ixodes scapularisticks harbor numerous human pathogens, includingAnaplasma phagocytophilum,the agent of human granulocytic anaplasmosis. We now demonstrate thatA. phagocytophilummodifies theI. scapularismicrobiota to more efficiently infect the tick.A. phagocytophiluminduces ticks to expressIxodes scapularisantifreeze glycoprotein (iafgp), which encodes a protein with several properties, including the ability to alter bacterial biofilm formation. IAFGP thereby perturbs the tick gut microbiota, which influences the integrity of the peritrophic matrix and gut barrier—critical obstacles forAnaplasmacolonization. Mechanistically, IAFGP binds the terminald-alanine residue of the pentapeptide chain of bacterial peptidoglycan, resulting in altered permeability and the capacity of bacteria to form biofilms. These data elucidate the molecular mechanisms by which a human pathogen appropriates an arthropod antibacterial protein to alter the gut microbiota and more effectively colonize the vector.

Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi. A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ∼10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PGBb) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb, which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb. In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations.

Horizontal gene transfer (HGT) allows organisms to rapidly acquire adaptive traits1. Though documented instances of HGT from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option2. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce anti-bacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system (T6SS)3. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years via purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the etiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for facile co-option by eukaryotic innate immune systems.

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.

Successful infection of a host requires that the invading pathogen control its production of virulence determinants. The infectious agent must sense its environment and respond by increasing production of appropriate factors and repressing production of unnecessary ones. These features are especially critical for vector-borne pathogens, which must not only efficiently infect two extremely different host types but also be transmitted back and forth between hosts. Deciphering the regulatory pathways used by pathogens to control production of infection-associated proteins provides significant insight into the infectious nature of those organisms. Moreover, regulatory factors are attractive candidates for development of novel preventative and curative therapies.The spirochetal bacterium Borrelia burgdorferi, the agent of Lyme disease, is an excellent model organism for the study of gene regulation by a vector-borne pathogen. B. burgdorferi is genetically tractable, and its natural mammal-tick infectious cycle can be replicated in the laboratory. In addition, infection by B. burgdorferi is a significant cause of human morbidity, being the most commonly reported vector-borne disease in the United States and many other parts of the world (51, 55, 56).B. burgdorferi Erp lipoproteins are produced throughout mammalian infection but are largely repressed during colonization of vector ticks (10,31,48,49). Erp synthesis is greatly enhanced when B. burgdorferi is transmitted from a feeding tick into a warm-blooded host. Regulation of Erp protein production is controlled at the level of transcription (6). Erp proteins are located in the bacterial outer membrane and are exposed to the external environment (25,32,41). Known functions of Erp proteins include binding of host plasmin(ogen), laminin, and the complement regulators factor H and factor H-related proteins 1, 2, and 5 (2,3,11,12,34,37,40,45,59). These functions indicate roles for Erp proteins in host adherence, dissemination, and resistance to the alternative pathway of complement-mediated killing. Borrelial erp genes are located in mono-or bicistronic operons on extrachromosomal cp32 prophages, most of which replicate autonomously as circular episomes (24,60,63,64,72). Individual Lyme spirochetes naturally contain numerous different cp32 elements, each with a unique erp locus, and therefore produce multiple, distinct Erp surface proteins. A bacterium simultaneously expresses its entire repertoire of Erp proteins (26).A highly conserved DNA region immediately 5= of all erp promoters, the erp operator, is required for regulation of erp transcription (see Fig. 1) (6,10,64). Two erp operator-binding proteins have been identified, and their binding sites have been characterized: BpaB (borrelial plasmid ParB analogue) and EbfC (erp-binding factor, chromosomal) (4, 13, 52). BpaB binds with high affinity to a 5-bp sequence within the erp operator (13; C. A. Adams, unpublished). Binding of one BpaB protein to that sequence then facilitates binding of additional BpaB molecules al...

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 DNAbinding 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.

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 the synthesis of OspC and Erps can be stimulated in culture by shifting the temperature from 23°C to 34°C, leading to a hypothesis that Borrelia 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 a 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 the expression of the OspC and Erp infection-associated proteins.V ector-borne pathogens, such as the Lyme disease spirochete, Borrelia burgdorferi, have overcome the challenges of persisting within two very different animal environments and possess mechanisms to efficiently transmit back and forth between vertebrate hosts and arthropod vectors. To facilitate this complex lifestyle, the bacterium produces specific proteins appropriate for each step in the infectious cycle. Considerable effort has been expended to identify the mechanisms by which B. burgdorferi senses its environment and accordingly regulates gene expression (1, 2). Such information both provides insight into pathogenic mechanisms and identifies new targets for preventative and curative therapies.One of the first studies to delve into the mechanisms of B. burgdorferi gene regulation focused on OspC (outer surface protein C). The exact function of that protein remains to be elucidated, but it is necessary for the establishment of mammalian infection (3)(4)(5)(6)(7)(8)(9)(10). In a landmark study in 1995, Schwan et al. demonstrated that synthesis of OspC is repressed in bacteria within unfed ticks, yet OspC production is induced as those ticks begin to feed (11). Furthermore, they showed that regulation of OspC synthesis can be recapitulated in culture: the protein is poorly expressed by bacteria cultured at 23°C but is abundantly expressed by bacteria that are first grown at 23°C, diluted into fresh medium, and then cultured at 34°C to 37°C (11).Shortly thereafter, it was demonstrated that increasing the culture temperature from 23°C to 34°C enhances the production of several other antigenic proteins (12). Among these are a paralogous family of outer surface lipoproteins designated Erp (OspE/ OspF-related proteins) (12-15). Erp proteins are expressed throughout vertebrate infection, adhere to various host factors, and appear to play roles in dissemination and colonization (16)(17)(18)(19)(20)(21)(22)(23)(24)(25). As with ospC, erp transcription is repressed during tick colonization and induced during tick feedin...