RESUMO
Lyme disease is on the rise. Caused by a spirochete Borreliella burgdorferi, it affects an estimated 500,000 people in the United States alone. The antibiotics currently used to treat Lyme disease are broad spectrum, damage the microbiome, and select for resistance in non-target bacteria. We therefore sought to identify a compound acting selectively against B. burgdorferi. A screen of soil micro-organisms revealed a compound highly selective against spirochetes, including B. burgdorferi. Unexpectedly, this compound was determined to be hygromycin A, a known antimicrobial produced by Streptomyces hygroscopicus. Hygromycin A targets the ribosomes and is taken up by B. burgdorferi, explaining its selectivity. Hygromycin A cleared the B. burgdorferi infection in mice, including animals that ingested the compound in a bait, and was less disruptive to the fecal microbiome than clinically relevant antibiotics. This selective antibiotic holds the promise of providing a better therapeutic for Lyme disease and eradicating it in the environment.
Assuntos
Antibacterianos/uso terapêutico , Doença de Lyme/tratamento farmacológico , Animais , Borrelia burgdorferi/efeitos dos fármacos , Calibragem , Cinamatos/química , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Fezes/microbiologia , Feminino , Células HEK293 , Células Hep G2 , Humanos , Higromicina B/análogos & derivados , Higromicina B/química , Higromicina B/farmacologia , Higromicina B/uso terapêutico , Doença de Lyme/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Microbiota/efeitos dos fármacosRESUMO
Borrelia burgdorferi (Bb), the causative agent of Lyme disease, establishes a long-term infection and leads to disease manifestations that are the result of host immune responses to the pathogen. Inflammatory manifestations resolve spontaneously despite continued bacterial presence, suggesting inflammatory cells become less responsive over time. This is mimicked by in vitro repeated stimulations, resulting in tolerance, a phenotypic subset of innate immune memory. We performed comparative transcriptional analysis of macrophages in acute and memory states and identified sets of Tolerized, Hyper-Induced, Secondary-Induced and Hyper-Suppressed genes resulting from memory induction, revealing previously unexplored networks of genes affected by cellular re-programming. Tolerized gene families included inflammatory mediators and interferon related genes as would be predicted by the attenuation of inflammation over time. To better understand how cells mediate inflammatory hypo-responsiveness, we focused on genes that could mediate maintenance of suppression, such as Hyper-Induced genes which are up-regulated in memory states. These genes were notably enriched in stress pathways regulated by anti-inflammatory modulators. We examined one of the most highly expressed negative regulators of immune pathways during primary stimulation, Aconitate decarboxylase 1 (Acod1), and tested its effects during in vivo infection with Bb. As predicted by our in vitro model, we show its inflammation-suppressive downstream effects are sustained during in vivo long-term infection with Bb, with a specific role in Lyme carditis.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Humanos , Inflamação , Doença de Lyme/microbiologia , Macrófagos , Anti-InflamatóriosRESUMO
Don't Panic. In the nearly 50 years since the discovery of Lyme disease, Borrelia burgdorferi has emerged as an unlikely workhorse of microbiology. Interest in studying host-pathogen interactions fueled significant progress in making the fastidious microbe approachable in laboratory settings, including the development of culture methods, animal models, and genetic tools. By developing these systems, insight has been gained into how the microbe is able to survive its enzootic cycle and cause human disease. Here, we discuss the discovery of B. burgdorferi and its development as a model organism before diving into the critical lessons we have learned about B. burgdorferi biology at pivotal stages of its lifecycle: gene expression changes during the tick blood meal, colonization of a new vertebrate host, and developing a long-lasting infection in that vertebrate until a new tick feeds. Our goal is to highlight the advancements that have facilitated B. burgdorferi research and identify gaps in our current understanding of the microbe.
RESUMO
Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Borrelia burgdorferi/metabolismo , Ligantes , Doença de Lyme/genética , Doença de Lyme/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fagossomos/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Proteínas rab de Ligação ao GTP , Animais , CamundongosRESUMO
The Lyme disease bacterial pathogen, Borrelia burgdorferi, establishes a long-term infection inside its mammalian hosts. Despite the continued presence of the bacteria in animal models of disease, inflammation is transitory and resolves spontaneously. T cells with limited effector functions and the inability to become activated by antigen, termed exhausted T cells, are present in many long-term infections. These exhausted T cells mediate a balance between pathogen clearance and preventing tissue damage resulting from excess inflammation. Exhausted T cells express a variety of immunoinhibitory molecules, including the molecule PD-1. Following B. burgdorferi infection, we found that PD-1 and its ligand PD-L1 are significantly upregulated on CD4+ T cells and antigen presenting cell subsets, respectively. Using mice deficient in PD-1, we found that the PD-1/PD-L1 pathway did not impact bacterial clearance but did impact T cell expansion and accumulation in the ankle joint and popliteal lymph nodes without affecting B cell populations or antibody production, suggesting that the PD-1/PD-L1 pathway may play a role in shaping the T cell populations present in affected tissues.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Camundongos , Animais , Receptor de Morte Celular Programada 1 , Antígeno B7-H1 , Doença de Lyme/microbiologia , Linfócitos T CD4-Positivos , Inflamação , MamíferosRESUMO
Borrelia burgdorferi, a Lyme disease spirochete, causes a range of acute and chronic maladies in humans. However, a primary vertebrate reservoir in the United States, the white-footed deermouse Peromyscus leucopus, is reported not to have reduced fitness following infection. Although laboratory strains of Mus musculus mice have successfully been leveraged to model acute human Lyme disease, the ability of these rodents to model B. burgdorferi-P. leucopus interactions remains understudied. Here, we compared infection of P. leucopus with B. burgdorferi B31 with infection of the traditional B. burgdorferi murine models-C57BL/6J and C3H/HeN Mus musculus, which develop signs of inflammation akin to human disease. We find that B. burgdorferi was able to reach much higher burdens (10- to 30-times higher) in multiple M. musculus skin sites and that the overall dynamics of infection differed between the two rodent species. We also found that P. leucopus remained transmissive to larval Ixodes scapularis for a far shorter period than either M. musculus strain. In line with these observations, we found that P. leucopus does launch a modest but sustained inflammatory response against B. burgdorferi in the skin, which we hypothesize leads to reduced bacterial viability and rodent-to-tick transmission in these hosts. Similarly, we also observe evidence of inflammation in infected P. leucopus hearts. These observations provide new insight into reservoir species and the B. burgdorferi enzootic cycle.IMPORTANCEA Lyme disease-causing bacteria, Borrelia burgdorferi, must alternate between infecting a vertebrate host-usually rodents or birds-and ticks. In order to be successful in that endeavor, the bacteria must avoid being killed by the vertebrate host before it can infect a new larval tick. In this work, we examine how B. burgdorferi and one of its primary vertebrate reservoirs, Peromyscus leucopus, interact during an experimental infection. We find that B. burgdorferi appears to colonize its natural host less successfully than conventional laboratory mouse models, which aligns with a sustained seemingly anti-bacterial response by P. leucopus against the microbe. These data enhance our understanding of P. leucopus host-pathogen interactions and could potentially serve as a foundation to uncover ways to disrupt the spread of B. burgdorferi in nature.
Assuntos
Borrelia burgdorferi , Reservatórios de Doenças , Doença de Lyme , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Peromyscus , Animais , Peromyscus/microbiologia , Camundongos , Doença de Lyme/microbiologia , Doença de Lyme/transmissão , Doença de Lyme/veterinária , Borrelia burgdorferi/fisiologia , Borrelia burgdorferi/genética , Reservatórios de Doenças/microbiologia , Modelos Animais de Doenças , Ixodes/microbiologiaRESUMO
Borrelia burgdorferi is a pathogenic bacterium and the causative agent of Lyme disease. It is exposed to reactive oxygen species (ROS) in both the vertebrate and tick hosts. While some mechanisms by which B. burgdorferi ameliorates the effects of ROS exposure have been studied, there are likely other unknown mechanisms of ROS neutralization that contribute to virulence. Here, we follow up on a three gene cluster of unknown function, bb_0554, bb_0555, and bb_0556, that our prior unbiased transposon insertional sequencing studies implicated in both ROS survival and survival in Ixodes scapularis. We confirmed these findings through genetic knockout and provide evidence that these genes are co-transcribed as an operon to produce a xanthine dehydrogenase. In agreement with these results, we found that B. burgdorferi exposure to either uric acid (a product of xanthine dehydrogenase) or allopurinol (an inhibitor of xanthine dehydrogenase) could modulate sensitivity to ROS in a bb_0554-bb_0556 dependent manner. Together, this study identifies a previously uncharacterized three gene operon in B. burgdorferi as encoding a putative xanthine dehydrogenase critical for virulence. We propose renaming this locus xdhACB.
Assuntos
Borrelia burgdorferi , Ixodes , Doença de Lyme , Animais , Camundongos , Borrelia burgdorferi/genética , Xantina Desidrogenase/genética , Espécies Reativas de Oxigênio , Doença de Lyme/microbiologia , Ixodes/microbiologiaRESUMO
Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted by the bite of an infected tick. Once inoculated into the host dermis, it disseminates to various organs including distant skin sites, the heart, the joint and the nervous system. Most humans will develop an early skin manifestation called erythema migrans at the tick bite site. This can be followed by symptoms such as carditis, neuritis, meningitis, or arthritis if not treated. A specific mouse strain, C3H/HeN develops arthritis with B. burgdorferi infection whereas another strain, C57BL/6, develops minimal to no arthritis. Neither strain of mice show any skin signs of rash or inflammation. Factors that determine the presence of skin inflammation and the joint arthritis susceptibility in the host are only partially characterized. We show in this study that murine fibroblast-like synoviocytes display trained immunity, a program in some cells that results in increased inflammatory responses if the cell has previously come in contact with a stimulus, and that trained immunity in fibroblast-like synoviocytes tested ex vivo correlates with Lyme arthritis susceptibility. Conversely, skin fibroblasts do not exhibit trained immunity, which correlates with the absence of skin symptoms in these mice. Moreover, we demonstrate that the trained phenotype in FLS is affected by the cell environment, which depends on the host genetic background. Future studies expanding this initial report of the role of trained immunity on symptoms of B. burgdorferi infection may provide insight into the pathogenesis of disease in murine models.
Assuntos
Artrite/imunologia , Borrelia burgdorferi/imunologia , Imunidade Inata , Memória Imunológica , Doença de Lyme/imunologia , Sinoviócitos/imunologia , Animais , Artrite/genética , Artrite/patologia , Feminino , Inflamação/imunologia , Inflamação/patologia , Doença de Lyme/genética , Doença de Lyme/patologia , Camundongos , Camundongos Knockout , Sinoviócitos/patologiaRESUMO
Unrecognized immunodeficiency has been proposed as a possible cause of failure of antibiotics to resolve symptoms of Lyme disease. Here, we examined the efficacy of doxycycline in different immunodeficient mice to identify defects that impair antibiotic treatment outcomes. We found that doxycycline had significantly lower efficacy in the absence of adaptive immunity, specifically B cells. This effect was most pronounced in immunodeficient C3H mice compared with C57BL/6 mice, suggesting a role for genetic background beyond immunodeficiency. Addition of a single dose of ceftriaxone to doxycycline treatment effectively cleared infection in C3H mice with severe combined immunodeficiency.
Assuntos
Antibacterianos , Patrimônio Genético , Síndromes de Imunodeficiência , Doença de Lyme , Animais , Antibacterianos/uso terapêutico , Borrelia burgdorferi , Doxiciclina , Síndromes de Imunodeficiência/genética , Doença de Lyme/tratamento farmacológico , Doença de Lyme/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BLRESUMO
Lyme disease Borrelia are obligately parasitic, tick- transmitted, invasive, persistent bacterial pathogens that cause disease in humans and non-reservoir vertebrates primarily through the induction of inflammation. During transmission from the infected tick, the bacteria undergo significant changes in gene expression, resulting in adaptation to the mammalian environment. The organisms multiply and spread locally and induce inflammatory responses that, in humans, result in clinical signs and symptoms. Borrelia virulence involves a multiplicity of mechanisms for dissemination and colonization of multiple tissues and evasion of host immune responses. Most of the tissue damage, which is seen in non-reservoir hosts, appears to result from host inflammatory reactions, despite the low numbers of bacteria in affected sites. This host response to the Lyme disease Borrelia can cause neurologic, cardiovascular, arthritic, and dermatologic manifestations during the disseminated and persistent stages of infection. The mechanisms by which a paucity of organisms (in comparison to many other infectious diseases) can cause varied and in some cases profound inflammation and symptoms remains mysterious but are the subjects of diverse ongoing investigations. In this review, we provide an overview of virulence mechanisms and determinants for which roles have been demonstrated in vivo, primarily in mouse models of infection.
Assuntos
Borrelia , Suscetibilidade a Doenças , Doença de Lyme/microbiologia , Animais , Vetores Artrópodes/microbiologia , Borrelia/genética , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Doença de Lyme/transmissão , Carrapatos/microbiologia , Virulência , Fatores de Virulência/genéticaRESUMO
Borrelia burgdorferi, the causative agent of Lyme disease in humans, is maintained in a complex biphasic life cycle, which alternates between tick and vertebrate hosts. To successfully survive and complete its enzootic cycle, B. burgdorferi adapts to diverse hosts by regulating genes required for survival in specific environments. Here we describe the first ever use of transposon insertion sequencing (Tn-seq) to identify genes required for B. burgdorferi survival in its tick host. We found that insertions into 46 genes resulted in a complete loss of recovery of mutants from larval Ixodes ticks. Insertions in an additional 56 genes resulted in a >90% decrease in fitness. The screen identified both previously known and new genes important for larval tick survival. Almost half of the genes required for survival in the tick encode proteins of unknown function, while a significant portion (over 20%) encode membrane-associated proteins or lipoproteins. We validated the results of the screen for five Tn mutants by performing individual competition assays using mutant and complemented strains. To better understand the role of one of these genes in tick survival, we conducted mechanistic studies of bb0017, a gene previously shown to be required for resistance against oxidative stress. In this study we show that BB0017 affects the regulation of key borrelial virulence determinants. The application of Tn-seq to in vivo screening of B. burgdorferi in its natural vector is a powerful tool that can be used to address many different aspects of the host pathogen interaction.
Assuntos
Proteínas de Bactérias/genética , Borrelia burgdorferi/crescimento & desenvolvimento , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Doença de Lyme/microbiologia , Carrapatos/crescimento & desenvolvimento , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/genética , Borrelia burgdorferi/imunologia , Modelos Animais de Doenças , Vetores de Doenças , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno , Doença de Lyme/imunologia , Camundongos , Carrapatos/microbiologia , Fatores de Virulência/metabolismoRESUMO
The bacterial pathogen Borrelia burgdorferi is the causative agent of Lyme disease and is transmitted to humans through an Ixodes tick vector. B. burgdorferi is able to survive in both mammalian and tick hosts through careful modulation of its gene expression. This allows B. burgdorferi to adapt to the environmental and nutritional changes that occur when it is transmitted between the two hosts. Distinct interactions between the spirochete and its host occur at every step of the enzootic cycle and dictate the ability of the spirochete to survive until the next stage of the cycle. Studying the interface between B. burgdorferi, the Ixodes tick vector and the natural mammalian reservoirs has been made significantly more feasible through the complete genome sequences of the organisms and the advent of high throughput screening technologies. Ultimately, a thorough investigation of the interplay between the two domains (and two phyla within one domain) is necessary in order to completely understand how the pathogen is transmitted.
Assuntos
Vetores Aracnídeos/microbiologia , Borrelia burgdorferi/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Ixodes/microbiologia , Doença de Lyme/microbiologia , Mamíferos/microbiologia , Animais , Vetores Aracnídeos/imunologia , Borrelia burgdorferi/genética , Expressão Gênica , Humanos , Ixodes/imunologia , Doença de Lyme/epidemiologia , Doença de Lyme/transmissão , Mamíferos/sangue , Mamíferos/parasitologia , Microbiota , Ninfa/microbiologia , Glândulas Salivares/microbiologiaRESUMO
BACKGROUND: Pyruvate oxidase (Pox) is an important enzyme in bacterial metabolism for increasing ATP production and providing a fitness advantage via hydrogen peroxide production. However, few Pox enzymes have been characterized from bacterial species. The tetrameric non-hydrogen-peroxide producing Pox from E. coli is activated by phospholipids, which is important for its function in vivo. RESULTS: We characterized the hydrogenperoxide-producing Pox from L. delbrueckii strain STYM1 and showed it is specifically activated by phosphotidylethanolamine (16:0-18:1), but not by phosphotidylcholine or phosphotidylglycerol. This activation is a mixture of K- and V-type activation as both km and enzyme turnover are altered. Furthermore, we demonstrated that the L. delbrueckii Pox forms pentamers and either decamers or dimers of pentamers in solution, which is different from other characterized Pox enzymes. Lastly, we generated a C-terminal truncation mutant that was only weakly activated by phosphotidylethanolamine, which suggests the C-terminus is important for lipid activation. CONCLUSIONS: To our knowledge this is the first known hydrogenperoxide-producing Pox enzyme that is activated by phospholipids. Our results suggest that there are substantial differences between Pox enzymes from different bacterial species, which could be important for their role in biological systems as well as in the development of Pox-based biosensors.
Assuntos
Lactobacillus delbrueckii/enzimologia , Fosfatidiletanolaminas/metabolismo , Piruvato Oxidase/genética , Piruvato Oxidase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ativação Enzimática , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Lactobacillus delbrueckii/genética , Mutação , Multimerização Proteica , Piruvato Oxidase/químicaRESUMO
Borrelia burgdorferi, the causative agent of Lyme disease in humans, is exposed to reactive oxygen and nitrogen species (ROS and RNS) in both the tick vector and vertebrate reservoir hosts. B. burgdorferi contains a limited repertoire of canonical oxidative stress response genes, suggesting that novel gene functions may be important for protection of B. burgdorferi against ROS or RNS exposure. Here, we use transposon insertion sequencing (Tn-seq) to conduct an unbiased search for genes involved in resistance to nitric oxide, hydrogen peroxide, and tertiary-butyl hydroperoxide in vitro. The screens identified 66 genes whose disruption resulted in increased susceptibility to at least one of the stressors. These genes include previously characterized mediators of ROS and RNS resistance (including components of the nucleotide excision repair pathway and a subunit of a riboflavin transporter), as well as novel putative resistance candidates. DNA repair mutants were among the most sensitive to RNS in the Tn-seq screen, and survival assays with individual Tn mutants confirmed that the putative ribonuclease BB0839 is involved in resistance to nitric oxide. In contrast, mutants lacking predicted inner membrane proteins or transporters were among the most sensitive to ROS, and the contribution of three such membrane proteins (BB0017, BB0164, and BB0202) to ROS sensitivity was confirmed using individual Tn mutants and complemented strains. Further analysis showed that levels of intracellular manganese are significantly reduced in the Tn::bb0164 mutant, identifying a novel role for BB0164 in B. burgdorferi manganese homeostasis. Infection of C57BL/6 and gp91phox-/- mice with a mini-library of 39 Tn mutants showed that many of the genes identified in the in vitro screens are required for infectivity in mice. Collectively, our data provide insight into how B. burgdorferi responds to ROS and RNS and suggests that this response is relevant to the in vivo success of the organism.
Assuntos
Borrelia burgdorferi/genética , Borrelia burgdorferi/imunologia , Genes Bacterianos/imunologia , Doença de Lyme/microbiologia , Animais , Modelos Animais de Doenças , Sequenciamento de Nucleotídeos em Larga Escala , Doença de Lyme/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Despite a growing interest in using probiotic microorganisms to prevent disease, the mechanisms by which probiotics exert their action require further investigation. Porphyromonas gingivalis is an important pathogen implicated in the development of periodontitis. We isolated several strains of Lactobacillus delbrueckii from dairy products and examined their ability to inhibit P. gingivalis growth in vitro We observed strain-specific inhibition of P. gingivalis growth in vitro Whole-genome sequencing of inhibitory and noninhibitory strains of L. delbrueckii revealed significant genetic differences supporting the strain specificity of the interaction. Extracts of the L. delbrueckii STYM1 inhibitory strain contain inhibitory activity that is abolished by treatment with heat, proteinase K, catalase, and sodium sulfite. We purified the inhibitory protein(s) from L. delbrueckii STYM1 extracts using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration chromatography. Pyruvate oxidase was highly enriched in the purified samples. Lastly, we showed that purified, catalytically active, recombinant pyruvate oxidase is sufficient to inhibit P. gingivalis growth in vitro without the addition of cofactors. Further, using a saturated transposon library, we isolated transposon mutants of P. gingivalis in the feoB2 (PG_1294) gene that are resistant to killing by inhibitory L. delbrueckii, consistent with a mechanism of hydrogen peroxide production by pyruvate oxidase. Our results support the current understanding of the importance of strain selection, not simply species selection, in microbial interactions. Specific L. delbrueckii strains or their products may be effective in the treatment and prevention of P. gingivalis-associated periodontal disease.IMPORTANCEP. gingivalis is implicated in the onset and progression of periodontal disease and associated with some systemic diseases. Probiotic bacteria represent an attractive preventative therapy for periodontal disease. However, the efficacy of probiotic bacteria can be variable between studies. Our data support the known importance of selecting particular strains of bacteria for probiotic use, not simply a single species. Specifically, in the context of probiotic intervention of periodontitis, our data suggest that high-level expression of pyruvate oxidase with hydrogen peroxide production in L. delbrueckii could be an important characteristic for the design of a probiotic supplement or a microbial therapeutic.
Assuntos
Proteínas de Bactérias/metabolismo , Lactobacillus delbrueckii/fisiologia , Porphyromonas gingivalis/fisiologia , Piruvato Oxidase/metabolismo , Lactobacillus delbrueckii/enzimologia , Probióticos/química , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Iogurte/microbiologiaRESUMO
Borrelia burgdorferi is the causative agent of Lyme borreliosis. Antibiotic therapy of early acute infection is effective for most patients, but 10 to 20% go on to develop posttreatment Lyme disease syndrome (PTLDS). The nature of PTLDS remains unknown, but currently approved antibiotics for the treatment of Lyme disease do not appear to impact these symptoms after they have developed. We reason that minimizing the time the pathogen interacts with the host will diminish the probability of developing PTLDS, irrespective of its nature. This calls for an efficient eradication of the pathogen during acute infection. In search of a superior killing antibiotic, we examined approved antibiotics for their ability to kill B. burgdorferi Vancomycin proved more effective in killing the pathogen in vitro than ceftriaxone, the standard of care for disseminated B. burgdorferi infection. Both compounds were also the most effective in killing stationary-phase cells. This is surprising, given that inhibitors of cell wall biosynthesis are known to only kill growing bacteria. We found that peptidoglycan synthesis continues in stationary-phase cells of B. burgdorferi, explaining this paradox. A combination of vancomycin and gemifloxacin sterilized a stationary-phase culture of B. burgdorferi Examination of the action of antibiotics in severe combined immunodeficient (SCID) mice showed that doxycycline, a standard of care for uncomplicated acute infection, did not clear the pathogen. In contrast, both ceftriaxone and vancomycin cleared the infection. A trial examining the early use of more potent antibiotics on the development of PTLDS may be warranted.
Assuntos
Antibacterianos/farmacologia , Borrelia burgdorferi/efeitos dos fármacos , Doença de Lyme/tratamento farmacológico , Vancomicina/farmacologia , Aminoglicosídeos/farmacologia , Animais , Ceftriaxona/farmacologia , Doxiciclina/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos C3H , Camundongos SCID , Testes de Sensibilidade Microbiana/métodos , Peptídeos/farmacologiaRESUMO
Cellular pigmentation is an important virulence factor of the oral pathogen Porphyromonas gingivalis Pigmentation has been associated with many bacterial functions, including but not limited to colonization, maintaining a local anaerobic environment by binding oxygen molecules, and defense against reactive oxygen species (ROS) produced by immune cells. Pigmentation-associated loci identified to date have involved lipopolysaccharide, fimbriae, and heme acquisition and processing. We utilized a transposon mutant library of P. gingivalis strain ATCC 33277 and screened for pigmentation-defective colonies using massively parallel sequencing of the transposon junctions (Tn-seq) to identify genes involved in pigmentation. Transposon insertions at 235 separate sites, located in 67 genes and 15 intergenic regions, resulted in altered pigmentation: 7 of the genes had previously been shown to be involved in pigmentation, while 75 genes and intergenic regions had not. To further confirm identification, we generated a smaller transposon mutant library in P. gingivalis strain W83 and identified pigment mutations in several of the same loci as those identified in the screen in ATCC 33277 but also eight that were not identified in the ATCC 33277 screen. PGN_0361/PG_0264, a putative glycosyltransferase gene located between two tRNA synthetase genes and adjacent to a miniature inverted-repeat transposable element, was identified in the Tn-seq screen and then verified through targeted deletion and complementation. Deletion mutations in PGN_0361/PG_0264 glycosyltransferase abolish pigmentation, modulate gingipain protease activity, and alter lipopolysaccharide. The mechanisms of involvement in pigmentation for other loci identified in this study remain to be determined, but our screen provides the most complete survey of genes involved in pigmentation to date.IMPORTANCEP. gingivalis has been implicated in the onset and progression of periodontal disease. One important virulence factor is the bacterium's ability to produce pigment. Using a transposon library, we were able to identify both known and novel genes involved in pigmentation of P. gingivalis We identified a glycosyltransferase, previously not associated with pigmentation, that is required for pigmentation and determined its mechanism of involvement. A better understanding of the genes involved in pigmentation may lead to new insights into the complex mechanisms involved in this important virulence characteristic and could facilitate development of novel therapeutics.
Assuntos
Elementos de DNA Transponíveis/genética , Glicosiltransferases/metabolismo , Pigmentos Biológicos/metabolismo , Porphyromonas gingivalis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , Regulação para Baixo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes Bacterianos , Glicosiltransferases/genética , Mutação , Técnicas de Amplificação de Ácido Nucleico , Pigmentos Biológicos/genética , Porphyromonas gingivalis/genéticaRESUMO
Phagocytosis of the Lyme disease-causing pathogen Borrelia burgdorferi has been shown to be important for generating an inflammatory response to the pathogen. As a result, understanding the mechanisms of phagocytosis has been an area of great interest in the field of Lyme disease. Several cell surface receptors that participate in B. burgdorferi phagocytosis have been reported, including the scavenger receptor MARCO and integrin α3ß1. We sought to define the mechanisms by which these receptors mediate phagocytosis and to identify signaling pathways activated downstream of these receptors upon contact with B. burgdorferi We identified both Syk and Src signaling pathways as ones that participate in B. burgdorferi phagocytosis and the resulting cytokine activation. In our studies, we found that both MARCO and integrin ß1 play a role in the activation of the Src kinase pathway. However, only integrin ß1 participates in the activation of Syk. Interestingly, the integrin activates Syk without the help of the signaling adaptor Dap12 or FcRγ. Thus, we report that multiple pathways participate in B. burgdorferi internalization and that different cell surface receptors act simultaneously in cooperation and independently to mediate phagocytosis.
Assuntos
Borrelia burgdorferi/imunologia , Cadeias beta de Integrinas/metabolismo , Fagocitose , Receptores Imunológicos/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo , Quinases da Família src/metabolismo , Animais , Borrelia burgdorferi/fisiologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Camundongos , Receptores Imunológicos/imunologia , Receptores Depuradores/metabolismoRESUMO
Borrelia burgdorferi maintains a complex life cycle between tick and vertebrate hosts. Although some genes have been identified as contributing to bacterial adaptation in the different hosts, the list is incomplete. In this manuscript, we report the first use of transposon mutagenesis combined with high-throughput sequencing (Tn-seq) in B. burgdorferi. We utilize the technique to investigate mechanisms of carbohydrate utilization in B. burgdorferi and the role of carbohydrate metabolism during mouse infection. We performed genetic fitness analyses to identify genes encoding factors contributing to growth on glucose, maltose, mannose, trehalose and N-acetyl-glucosamine. We obtained insight into the potential functions of proteins predicted to be involved in carbohydrate utilization and identified additional factors previously unrecognized as contributing to the metabolism of the tested carbohydrates. Strong phenotypes were observed for the putative carbohydrate phosphotransferase transporters BB0408 and BBB29 as well as the response regulator Rrp1. We further validated Tn-seq for use in mouse studies and were able to correctly identify known infectivity factors as well as additional transporters and genes on lp54 that may contribute to optimal mouse infection. As such, this study establishes Tn-seq as a powerful method for both in vitro and in vivo studies of B. burgdorferi.
Assuntos
Borrelia burgdorferi/metabolismo , Borrelia burgdorferi/patogenicidade , Doença de Lyme/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/genética , Metabolismo dos Carboidratos/genética , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Aptidão Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional , Fosfotransferases/genética , Fosfotransferases/metabolismo , Carrapatos/microbiologia , Fatores de Virulência/metabolismoRESUMO
Innate immune engagement results in the activation of host defenses that produce microbe-specific inflammatory responses. A long-standing interest in the field of innate immunity is to understand how varied host responses are generated through the signaling of just a limited number of receptors. Recently, intracellular trafficking and compartmental partitioning have been identified as mechanisms that provide signaling specificity for receptors by regulating signaling platform assembly. We show that cytokine activation as a result of TLR2 stimulation occurs at different intracellular locations and is mediated by the phagosomal trafficking molecule adaptor protein-3 (AP-3). AP-3 is required for trafficking TLR2 purified ligands or the Lyme disease causing bacterium, Borrelia burgdorferi, to LAMP-1 lysosomal compartments. The presence of AP-3 is necessary for the activation of cytokines such as IL-6 but not TNF-α or type I IFNs, suggesting induction of these cytokines occurs from a different compartment. Lack of AP-3 does not interfere with the recruitment of TLR signaling adaptors TRAM and MyD88 to the phagosome, indicating that the TLR-MyD88 signaling complex is assembled at a prelysosomal stage and that IL-6 activation depends on proper localization of signaling molecules downstream of MyD88. Finally, infection of AP-3-deficient mice with B. burgdorferi resulted in altered joint inflammation during murine Lyme arthritis. Our studies further elucidate the effects of phagosomal trafficking on tailoring immune responses in vitro and in vivo.