RESUMO
In August 2021, the Nebraska Department of Health and Human Services was notified by a local public health department of a cluster of two Lyme disease cases in patients with local exposure to wooded areas in a county located in their jurisdiction. Epidemiological investigations revealed that the two patients had similar symptom onset dates and had likely exposure to ticks at wooded sites located directly adjacent to one another. Two environmental investigations were completed in October 2021 and consisted of tick surveys at the patients' reported sites of tick exposure. 12 ticks were collected across the two surveys and identified the black-legged tick (Ixodes scapularis). During subsequent testing of the collected ticks, spirochete bacteria were isolated, cultured and confirmed as Borrelia burgdorferi sensu stricto by PCR. In total, 7 of 12 (58.3%) I. scapularis ticks tested positive for B. burgdorferi s.s. The results of this study document the fourth known established population of I. scapularis in Nebraska and confirms the first detection of B. burgdorferi s.s. in field collected ticks from Nebraska. The epidemiological and environmental investigation data provide the first evidence for local Lyme disease transmission occurring within Nebraska. These findings highlight the need for continued surveillance of I. scapularis and its associated pathogens in Nebraska to further characterize human risk and monitor emergence into other areas of the state.
Assuntos
Borrelia burgdorferi , Ixodes , Ixodidae , Doença de Lyme , Humanos , Animais , Ixodes/microbiologia , Nebraska/epidemiologia , Doença de Lyme/veterináriaRESUMO
Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between vertebrates and ticks and dramatically remodels its transcriptional profile to survive in disparate environments during infection. A focus of B. burgdorferi studies is to clearly understand how the bacteria responds to its environment through transcriptional changes. In vitro transcription assays allow for the basic mechanisms of transcriptional regulation to be biochemically dissected. Here, we present a detailed protocol describing B. burgdorferi RNA polymerase purification and storage, sigma factor purification, DNA template generation, and in vitro transcription assays. The protocol describes the use of RNA polymerase purified from B. burgdorferi 5A4 RpoC-His (5A4-RpoC). 5A4-RpoC is a previously published strain harboring a 10XHis-tag on the rpoC gene encoding the largest subunit of the RNA polymerase. In vitro transcription assays consist of the RNA polymerase purified from strain 5A4-RpoC, a recombinant version of the housekeeping sigma factor RpoD, and a PCR-generated double-stranded DNA template. While the protein purification techniques and approaches to assembling in vitro transcription assays are conceptually well understood and relatively common, handling considerations for RNA polymerases often differ from organism to organism. The protocol presented here is designed for enzymatic studies on the B. burgdorferi RNA polymerase. The method can be adapted to test the role of transcription factors, promoters, and post-translational modifications on the activity of the RNA polymerase.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Animais , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator sigma/genética , Fator sigma/metabolismoRESUMO
Throughout its enzootic cycle, the Lyme disease spirochete Borreliella (Borrelia) burgdorferi, senses and responds to changes in its environment using a small repertoire of transcription factors that coordinate the expression of genes required for infection of Ixodes ticks and various mammalian hosts. Among these transcription factors, the DnaK suppressor protein (DksA) plays a pivotal role in regulating gene expression in B. burgdorferi during periods of nutrient limitation and is required for mammalian infectivity. In many pathogenic bacteria, the gene regulatory activity of DksA, along with the alarmone guanosine penta- and tetra-phosphate ((p)ppGpp), coordinate the stringent response to various environmental stresses, including nutrient limitation. In this study, we sought to characterize the role of DksA in regulating the transcriptional activity of RNA polymerase and its role in the regulation of RpoS-dependent gene expression required for B. burgdorferi infectivity. Using in vitro transcription assays, we observed recombinant DksA inhibits RpoD-dependent transcription by B. burgdorferi RNA polymerase independent of ppGpp. Additionally, we determined the pH-inducible expression of RpoS-dependent genes relies on DksA, but this relationship is independent of (p)ppGpp produced by Relbbu. Subsequent transcriptomic and western blot assays indicate DksA regulates the expression of BBD18, a protein previously implicated in the post-transcriptional regulation of RpoS. Moreover, we observed DksA was required for infection of mice following intraperitoneal inoculation or for transmission of B. burgdorferi by Ixodes scapularis nymphs. Together, these data suggest DksA plays a central role in coordinating transcriptional responses in B. burgdorferi required for infectivity through DksA's interactions with RNA polymerase and post-transcriptional control of RpoS.
Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ixodes/microbiologia , Doença de Lyme/transmissão , Animais , Proteínas de Bactérias/genética , Feminino , Doença de Lyme/microbiologia , Camundongos , Fator sigma/genética , Fator sigma/metabolismo , Estresse FisiológicoRESUMO
Borrelia (Borreliella) burgdorferi, along with closely related species, is the etiologic agent of Lyme disease. The spirochete subsists in an enzootic cycle that encompasses acquisition from a vertebrate host to a tick vector and transmission from a tick vector to a vertebrate host. To adapt to its environment and persist in each phase of its enzootic cycle, B. burgdorferi wields three systems to regulate the expression of genes: the RpoN-RpoS alternative sigma factor cascade, the Hk1/Rrp1 two-component system and its product c-di-GMP, and the stringent response mediated by RelBbu and DksA. These regulatory systems respond to enzootic phase-specific signals and are controlled or fine- tuned by transcription factors, including BosR and BadR, as well as small RNAs, including DsrABb and Bb6S RNA. In addition, several other DNA-binding and RNA-binding proteins have been identified, although their functions have not all been defined. Global changes in gene expression revealed by high-throughput transcriptomic studies have elucidated various regulons, albeit technical obstacles have mostly limited this experimental approach to cultivated spirochetes. Regardless, we know that the spirochete, which carries a relatively small genome, regulates the expression of a considerable number of genes required for the transitions between the tick vector and the vertebrate host as well as the adaptation to each.
Assuntos
Borrelia burgdorferi/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Doença de Lyme/microbiologia , Transcriptoma , Adaptação Fisiológica , Animais , Vetores Artrópodes/microbiologia , Genes Bacterianos , Interações Hospedeiro-Patógeno , Humanos , Doença de Lyme/transmissão , Carrapatos/microbiologiaRESUMO
Chronic wasting disease (CWD) is an emerging and fatal contagious prion disease that affects cervids, including mule deer, white-tailed deer, black-tailed deer, red deer reindeer, elk, and moose. CWD prions are widely distributed throughout the bodies of CWD-infected animals and are found in the nervous system, lymphoid tissues, muscle, blood, urine, feces, and antler velvet. The mechanism of CWD transmission in natural settings is unknown. Potential mechanisms of transmission include horizontal, maternal, or environmental routes. Due to the presence of prions in the blood of CWD-infected animals, the potential exists for invertebrates that feed on mammalian blood to contribute to the transmission of CWD. The geographic range of the Rocky Mountain Wood tick, Dermancentor andersoni, overlaps with CWD throughout the northwest United States and southwest Canada, raising the possibility that D. andersoni parasitization of cervids may be involved in CWD transmission. We investigated this possibility by examining the blood meal of D. andersoni that fed upon prion-infected hamsters for the presence of prion infectivity by animal bioassay. None of the hamsters inoculated with a D. andersoni blood meal that had been ingested from prion-infected hamsters developed clinical signs of prion disease or had evidence for a subclinical prion infection. Overall, the data do not demonstrate a role for D. andersoni in the transmission of prion disease.IMPORTANCE Chronic wasting disease (CWD) is an emerging prion disease that affects cervids, including mule deer, white-tailed deer, black-tailed deer, red deer reindeer, elk, and moose. The mechanism of CWD transmission in unknown. Due to the presence of prions in the blood of CWD-infected animals, it is possible for invertebrates that feed on cervid blood to contribute to the transmission of CWD. We examined the blood meal of D. andersoni, a tick with a similar geographic range as cervids, that fed upon prion-infected hamsters for the presence of prion infectivity by animal bioassay. None of the D. andersoni blood meals that had been ingested from prion-infected hamsters yielded evidence of prion infection. Overall, the data do not support a role of D. andersoni in the transmission of prion disease.
Assuntos
Príons/sangue , Príons/patogenicidade , Carrapatos/fisiologia , Doença de Emaciação Crônica/transmissão , Animais , Bioensaio , Sangue , Cricetinae , Cervos/parasitologia , Masculino , Mesocricetus , Príons/isolamento & purificação , Doença de Emaciação Crônica/sangueRESUMO
The Lyme disease spirochete Borrelia burgdorferi exhibits dramatic changes in gene expression as it transits between its tick vector and vertebrate host. A major hurdle to understanding the mechanisms underlying gene regulation in B. burgdorferi has been the lack of a functional assay to test how gene regulatory proteins and sigma factors interact with RNA polymerase to direct transcription. To gain mechanistic insight into transcriptional control in B. burgdorferi, and address sigma factor function and specificity, we developed an in vitro transcription assay using the B. burgdorferi RNA polymerase holoenzyme. We established reaction conditions for maximal RNA polymerase activity by optimizing pH, temperature, and the requirement for divalent metals. Using this assay system, we analyzed the promoter specificity of the housekeeping sigma factor RpoD to promoters encoding previously identified RpoD consensus sequences in B. burgdorferi. Collectively, this study established an in vitro transcription assay that revealed RpoD-dependent promoter selectivity by RNA polymerase and the requirement of specific metal cofactors for maximal RNA polymerase activity. The establishment of this functional assay will facilitate molecular and biochemical studies on how gene regulatory proteins and sigma factors exert control of gene expression in B. burgdorferi required for the completion of its enzootic cycle.
Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Ensaios Enzimáticos/métodos , Ativação Transcricional , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Borrelia burgdorferi/enzimologia , Borrelia burgdorferi/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Estabilidade Enzimática , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Regiões Promotoras Genéticas , Fator sigma/genética , Fator sigma/metabolismoRESUMO
Dermacentor variabilis is the predominant tick species in Nebraska and is presumed to be the primary vector of Rickettsia rickettsii associated with cases of Rocky Mountain spotted fever (RMSF). Interestingly, RMSF cases in Nebraska have increased on a year-to-year basis, yet the prevalence of R. rickettsii in D. variabilis ticks has not been established for Nebraska. Here we sought to set a baseline for the prevalence of R. rickettsii and other spotted fever group (SFG) rickettsiae harbored by D. variabilis ticks. Over a 3-yr period, D. variabilis were collected along the Platte River in south central Nebraska. Individual tick DNA was analyzed using endpoint PCR to identify ticks carrying SFG rickettsiae. In total, 927 D. variabilis were analyzed by PCR and 38 (4.1%) ticks tested positive for SFG rickettsiae. Presumptive positives were sequenced to identify the Rickettsia species, of which 29 (76%) were R. montanensis, 5 (13%) were R. amblyommatis, 4 (11%) were R. bellii, and R. rickettsii was not detected. These data indicate that R. rickettsii is likely at a low prevalence in south central Nebraska and spillover of R. amblyommatis into D. variabilis is likely occurring due to the invasive lone star tick (Amblyomma americanum). In addition, our data suggest that R. montanensis and R. amblyommatis could be associated with the increase in SFG rickettsiae infections in Nebraska. This information will be of value to clinicians and the general public for evaluating diagnosis of disease- and risk-associated environmental exposure, respectively.
Assuntos
Dermacentor/microbiologia , Rickettsia/isolamento & purificação , Animais , DNA Bacteriano/análise , Dermacentor/crescimento & desenvolvimento , Feminino , Larva/crescimento & desenvolvimento , Larva/microbiologia , Masculino , Nebraska , Ninfa/crescimento & desenvolvimento , Ninfa/microbiologia , Reação em Cadeia da Polimerase , Rickettsiose do Grupo da Febre Maculosa/microbiologiaRESUMO
Tick-borne relapsing fever (TBRF), characterized by recurring febrile episodes, is globally distributed and among the most common bacterial infections in some African countries. Despite the public health concern that this disease represents, little is known regarding the virulence determinants required by TBRF Borrelia during infection. Because the chromosomes of TBRF Borrelia show extensive colinearity with those of Lyme disease (LD) Borrelia, the exceptions represent unique genes encoding proteins that are potentially essential to the disparate enzootic cycles of these two groups of spirochetes. One such exception is a gene encoding an HtrA family protease, BtpA, that is present in TBRF Borrelia, but not in LD spirochetes. Previous work suggested that btpA orthologs may be important for resistance to stresses faced during mammalian infection. Herein, proteomic analyses of the TBRF spirochete, Borrelia turicatae, demonstrated that BtpA, as well as proteins encoded by adjacent genes in the B. turicatae genome, were produced in response to culture at mammalian body temperature, suggesting a role in mammalian infection. Further, transcriptional analyses revealed that btpA was expressed with the genes immediately upstream and downstream as part of an operon. To directly assess if btpA is involved in resistance to environmental stresses, btpA deletion mutants were generated. btpA mutants demonstrated no growth defect in response to heat shock, but were more sensitive to oxidative stress produced by t-butyl peroxide compared to wild-type B. turicatae. Finally, btpA mutants were fully infectious in a murine relapsing fever (RF) infection model. These results indicate that BtpA is either not required for mammalian infection, or that compensatory mechanisms exist in TBRF spirochetes to combat environmental stresses encountered during mammalian infection in the absence of BtpA.
Assuntos
Doenças dos Animais/microbiologia , Proteínas de Bactérias/metabolismo , Borrelia/enzimologia , Febre Recorrente/veterinária , Serina Endopeptidases/metabolismo , Doenças dos Animais/metabolismo , Animais , Proteínas de Bactérias/genética , Temperatura Corporal , Borrelia/genética , Regulação Bacteriana da Expressão Gênica , Temperatura Alta , Mamíferos , Camundongos , Mutação , Óperon , Estresse Oxidativo , Proteômica/métodos , Serina Endopeptidases/genéticaRESUMO
A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.
Assuntos
Reação em Cadeia da Polimerase/métodos , Salmonella/fisiologia , Técnicas Bacteriológicas , Cromossomos Bacterianos , Aptidão Genética , Marcadores Genéticos , Salmonella/genéticaRESUMO
The relapsing fever spirochete Borrelia turicatae possesses a complex life cycle in its soft-bodied tick vector, Ornithodoros turicata. Spirochetes enter the tick midgut during a blood meal, and, during the following weeks, spirochetes disseminate throughout O. turicata. A population persists in the salivary glands allowing for rapid transmission to the mammalian hosts during tick feeding. Little is known about the physiological environment within the salivary glands acini in which B. turicatae persists. In this study, we examined the salivary gland transcriptome of O. turicata ticks and detected the expression of 57 genes involved in oxidant metabolism or antioxidant defences. We confirmed the expression of five of the most highly expressed genes, including glutathione peroxidase (gpx), thioredoxin peroxidase (tpx), manganese superoxide dismutase (sod-1), copper-zinc superoxide dismutase (sod-2), and catalase (cat) by reverse-transcriptase droplet digital polymerase chain reaction (RT-ddPCR). We also found distinct differences in the expression of these genes when comparing the salivary glands and midguts of unfed O. turicata ticks. Our results indicate that the salivary glands of unfed O. turicata nymphs are highly oxidative environments where reactive oxygen species (ROS) predominate, whereas midgut tissues comprise a primarily nitrosative environment where nitric oxide synthase is highly expressed. Additionally, B. turicatae was found to be hyperresistant to ROS compared with the Lyme disease spirochete Borrelia burgdorferi, suggesting it is uniquely adapted to the highly oxidative environment of O. turicata salivary gland acini.
Assuntos
Borrelia/crescimento & desenvolvimento , Borrelia/fisiologia , Ornithodoros/microbiologia , Febre Recorrente/transmissão , Glândulas Salivares/metabolismo , Animais , Catalase/biossíntese , Catalase/genética , Regulação da Expressão Gênica/genética , Glutationa Peroxidase/biossíntese , Glutationa Peroxidase/genética , Estresse Oxidativo/fisiologia , Peroxirredoxinas/biossíntese , Peroxirredoxinas/genética , Espécies Reativas de Oxigênio/metabolismo , Febre Recorrente/microbiologia , Glândulas Salivares/microbiologia , Superóxido Dismutase-1/biossíntese , Superóxido Dismutase-1/genéticaRESUMO
The pathogenic spirochete Borrelia burgdorferi senses and responds to changes in the environment, including changes in nutrient availability, throughout its enzootic cycle in Ixodes ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) in the transcriptional response of B. burgdorferi to starvation. Wild-type and dksA mutant B. burgdorferi strains were subjected to starvation by shifting cultures grown in rich complete medium, Barbour-Stoenner-Kelly II (BSK II) medium, to a defined mammalian tissue culture medium, RPMI 1640, for 6 h under microaerobic conditions (5% CO2, 3% O2). Microarray analyses of wild-type B. burgdorferi revealed that genes encoding flagellar components, ribosomal proteins, and DNA replication machinery were downregulated in response to starvation. DksA mediated transcriptomic responses to starvation in B. burgdorferi, as the dksA-deficient strain differentially expressed only 47 genes in response to starvation compared to the 500 genes differentially expressed in wild-type strains. Consistent with a role for DksA in the starvation response of B. burgdorferi, fewer CFU of dksA mutants were observed after prolonged starvation in RPMI 1640 medium than CFU of wild-type B. burgdorferi spirochetes. Transcriptomic analyses revealed a partial overlap between the DksA regulon and the regulon of RelBbu, the guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase that controls the stringent response; the DksA regulon also included many plasmid-borne genes. Additionally, the dksA mutant exhibited constitutively elevated (p)ppGpp levels compared to those of the wild-type strain, implying a regulatory relationship between DksA and (p)ppGpp. Together, these data indicate that DksA, along with (p)ppGpp, directs the stringent response to effect B. burgdorferi adaptation to its environment.IMPORTANCE The Lyme disease bacterium Borrelia burgdorferi survives diverse environmental challenges as it cycles between its tick vectors and various vertebrate hosts. B. burgdorferi must withstand prolonged periods of starvation while it resides in unfed Ixodes ticks. In this study, the regulatory protein DksA is shown to play a pivotal role controlling the transcriptional responses of B. burgdorferi to starvation. The results suggest that DksA gene regulatory activity impacts B. burgdorferi metabolism, virulence gene expression, and the ability of this bacterium to complete its natural life cycle.
Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Regulação Bacteriana da Expressão Gênica , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Adaptação Fisiológica , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/crescimento & desenvolvimento , Contagem de Colônia Microbiana , Meios de Cultura/química , Deleção de Genes , Perfilação da Expressão Gênica , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Análise em Microsséries , Viabilidade Microbiana , Regulon , Fatores de Transcrição/genéticaRESUMO
The genus Salmonella is responsible for many illnesses in humans and other vertebrate animals. We report here that Salmonella enterica serovar Typhimurium harbors three transketolases that support the non-oxidative branch of the pentose phosphate pathway. BLAST analysis identified two genes, STM14_2885 and STM14_2886, that together encode a putative transketolase (TktC) with 46-47% similarity to the known TktA and TktB isoforms. Assessing the mRNA and protein expression for each of the three transketolases, we determined that all are expressed in WT cells and regulated to varying extents by the alternative sigma factor RpoS. Enzyme assays with lysates from WT and transketolase-knockout strains established that TktA is responsible for >88% of the transketolase activity in WT cells. We purified recombinant forms of each isoenzyme to assess the kinetics for canonical transketolase reactions. TktA and TktB had comparable values for Vmax (539-1362 µm NADH consumed/s), Km (80-739 µm), and catalytic efficiency (1.02 × 108-1.06 × 109 m-1/s) for each substrate tested. The recombinant form of TktC had lower Km values (23-120 µm), whereas the Vmax (7.8-16 µm NADH consumed/s) and catalytic efficiency (5.58 × 106 to 6.07 × 108 m-1/s) were 10-100-fold lower. Using a murine model of Salmonella infection, we showed that a strain lacking all three transketolases is avirulent in C57BL/6 mice. These data provide evidence that S Typhimurium possesses three transketolases that contribute to pathogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Via de Pentose Fosfato , Salmonella typhimurium/metabolismo , Transcetolase/metabolismo , Animais , Proteínas de Bactérias/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos Endogâmicos C57BL , Oxirredução , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/patogenicidade , Transcetolase/genética , VirulênciaRESUMO
The PhoPQ two-component regulatory system coordinates the response of Salmonella enterica serovar Typhimurium to diverse environmental challenges encountered during infection of hosts, including changes in Mg2+ concentrations, pH, and antimicrobial peptides. Moreover, PhoPQ-dependent regulation of gene expression promotes intracellular survival of Salmonella in macrophages, and contributes to the resistance of this pathogen to reactive nitrogen species (RNS) generated from the nitric oxide produced by the inducible nitric oxide (NO) synthase of macrophages. We report here that Salmonella strains with mutations of phoPQ are hypersensitive to killing by RNS generated in vitro. The increased susceptibility of ∆phoQ Salmonella to RNS requires molecular O2 and coincides with the nitrotyrosine formation, the oxidation of [4Fe-4S] clusters of dehydratases, and DNA damage. Mutations of respiratory NADH dehydrogenases prevent nitrotyrosine formation and abrogate the cytotoxicity of RNS against ∆phoQ Salmonella, presumably by limiting the formation of peroxynitrite (ONOO-) arising from the diffusion-limited reaction of exogenous NO and endogenous superoxide (O2â¢-) produced in the electron transport chain. The mechanism underlying PhoPQ-mediated resistance to RNS is linked to the coordination of Mg2+ homeostasis through the PhoPQ-regulated MgtA transporter. Collectively, our investigations are consistent with a model in which PhoPQ-dependent Mg2+ homeostasis protects Salmonella against nitrooxidative stress.
Assuntos
Homeostase , Magnésio/metabolismo , Estresse Oxidativo , Espécies Reativas de Nitrogênio/metabolismo , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Macrófagos/microbiologia , Viabilidade Microbiana/genética , Mutação , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/fisiologiaRESUMO
Understanding how microorganisms manipulate plant innate immunity and colonize host cells is a major goal of plant pathology. Here, we report that the fungal nitrooxidative stress response suppresses host defences to facilitate the growth and development of the important rice pathogen Magnaporthe oryzae in leaf cells. Nitronate monooxygenases encoded by NMO genes catalyse the oxidative denitrification of nitroalkanes. We show that the M. oryzae NMO2 gene is required for mitigating damaging lipid nitration under nitrooxidative stress conditions and, consequently, for using nitrate and nitrite as nitrogen sources. On plants, the Δnmo2 mutant strain penetrated host cuticles like wild type, but invasive hyphal growth in rice cells was restricted and elicited plant immune responses that included the formation of cellular deposits and a host reactive oxygen species burst. Development of the M. oryzae effector-secreting biotrophic interfacial complex (BIC) was misregulated in the Δnmo2 mutant. Inhibiting or quenching host reactive oxygen species suppressed rice innate immune responses and allowed the Δnmo2 mutant to grow and develop normally in infected cells. NMO2 is thus essential for mitigating nitrooxidative cellular damage and, in rice cells, maintaining redox balance to avoid triggering plant defences that impact M. oryzae growth and BIC development.
Assuntos
Imunidade Inata , Magnaporthe/fisiologia , Oryza/imunologia , Oryza/microbiologia , Estresse Oxidativo , Estresse Fisiológico , Proteínas Fúngicas/genética , Hifas/crescimento & desenvolvimento , Magnaporthe/genética , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/imunologia , Mutação , Oryza/genética , Oryza/metabolismo , Oxirredução , Doenças das Plantas/genética , Folhas de Planta/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The Lyme disease spirochete Borrelia burgdorferi encounters a wide range of environmental conditions as it cycles between ticks of the genus Ixodes and its various mammalian hosts. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are potent antimicrobial molecules generated during the innate immune response to infection, however, it is unclear whether ROS and RNS pose a significant challenge to B. burgdorferi in vivo. In this study, we screened a library of B. burgdorferi strains with mutations in DNA repair genes for increased susceptibility to ROS or RNS in vitro. Strains with mutations in the methyl-directed mismatch repair gene mutS1 are hypersensitive to killing by ROS, while strains lacking the nucleotide excision repair (NER) gene uvrB show increased susceptibility to both ROS and RNS. Therefore, mutS1-deficient and uvrB-deficient strains were compared for their ability to complete their infectious cycle in Swiss Webster mice and I. scapularis ticks to help identify sites of oxidative and nitrosative stresses encountered by B. burgdorferi in vivo. Both mutS1 and uvrB were dispensable for infection of mice, while uvrB promoted the survival of spirochetes in I. scapularis ticks. The decreased survival of uvrB-deficient B. burgdorferi was associated with the generation of RNS in I. scapularis midguts and salivary glands during feeding. Collectively, these data suggest that B. burgdorferi must withstand cytotoxic levels of RNS produced during infection of I. scapularis ticks.
RESUMO
Pathogenic spirochetes cause clinically relevant diseases in humans and animals, such as Lyme disease and leptospirosis. The causative agent of Lyme disease, Borrelia burgdorferi, and the causative agent of leptospirosis, Leptospria interrogans, encounter reactive oxygen species (ROS) during their enzootic cycles. This report demonstrated that physiologically relevant concentrations of pyruvate, a potent H2O2 scavenger, and provided passive protection to B. burgdorferi and L. interrogans against H2O2. When extracellular pyruvate was absent, both spirochetes were sensitive to a low dose of H2O2 (≈0.6 µM per h) generated by glucose oxidase (GOX). Despite encoding a functional catalase, L. interrogans was more sensitive than B. burgdorferi to H2O2 generated by GOX, which may be due to the inherent resistance of B. burgdorferi because of the virtual absence of intracellular iron. In B. burgdorferi, the nucleotide excision repair (NER) and the DNA mismatch repair (MMR) pathways were important for survival during H2O2 challenge since deletion of the uvrB or the mutS genes enhanced its sensitivity to H2O2 killing; however, the presence of pyruvate fully protected ΔuvrB and ΔmutS from H2O2 killing further demonstrating the importance of pyruvate in protection. These findings demonstrated that pyruvate, in addition to its classical role in central carbon metabolism, serves as an important H2O2 scavenger for pathogenic spirochetes. Furthermore, pyruvate reduced ROS generated by human neutrophils in response to the Toll-like receptor 2 (TLR2) agonist zymosan. In addition, pyruvate reduced neutrophil-derived ROS in response to B. burgdorferi, which also activates host expression through TLR2 signaling. Thus, pathogenic spirochetes may exploit the metabolite pyruvate, present in blood and tissues, to survive H2O2 generated by the host antibacterial response generated during infection.
Assuntos
Peróxido de Hidrogênio/toxicidade , Ácido Pirúvico/metabolismo , Spirochaetales/efeitos dos fármacos , Spirochaetales/metabolismo , Acetatos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/efeitos dos fármacos , Borrelia burgdorferi/genética , Borrelia burgdorferi/metabolismo , Reparo do DNA/efeitos dos fármacos , Deleção de Genes , Glucose Oxidase/metabolismo , Humanos , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Pirúvico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Spirochaetales/genéticaRESUMO
Borrelia burgdorferi encounters potentially harmful reactive nitrogen species (RNS) throughout its infective cycle. In this study, diethylamine NONOate (DEA/NO) was used to characterize the lethal effects of RNS on B. burgdorferi. RNS produce a variety of DNA lesions in a broad spectrum of microbial pathogens; however, levels of the DNA deamination product, deoxyinosine, and the numbers of apurinic/apyrimidinic (AP) sites were identical in DNA isolated from untreated and DEA/NO-treated B. burgdorferi cells. Strains with mutations in the nucleotide excision repair (NER) pathway genes uvrC or uvrB treated with DEA/NO had significantly higher spontaneous mutation frequencies, increased numbers of AP sites in DNA and reduced survival compared with wild-type controls. Polyunsaturated fatty acids in B. burgdorferi cell membranes, which are susceptible to peroxidation by reactive oxygen species (ROS), were not sensitive to RNS-mediated lipid peroxidation. However, treatment of B. burgdorferi cells with DEA/NO resulted in nitrosative damage to several proteins, including the zinc-dependent glycolytic enzyme fructose-1,6-bisphosphate aldolase (BB0445), the Borrelia oxidative stress regulator (BosR) and neutrophil-activating protein (NapA). Collectively, these data suggested that nitrosative damage to proteins harbouring free or zinc-bound cysteine thiols, rather than DNA or membrane lipids underlies RNS toxicity in wild-type B. burgdorferi.
Assuntos
Borrelia burgdorferi/efeitos dos fármacos , Cisteína/análogos & derivados , Óxido Nítrico/toxicidade , Compostos de Sulfidrila/metabolismo , Proteínas de Bactérias/metabolismo , Quimiocinas CXC/metabolismo , Cisteína/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Nitrosação , Transativadores/metabolismo , Zinco/metabolismoRESUMO
We report herein a critical role for the stringent response regulatory DnaK suppressor protein (DksA) in the coordination of antioxidant defenses. DksA helps fine-tune the expression of glutathione biosynthetic genes and discrete steps in the pentose phosphate pathway and tricarboxylic acid cycle that are associated with the generation of reducing power. Control of NAD(P)H/NAD(P)(+) redox balance by DksA fuels downstream antioxidant enzymatic systems in nutritionally starving Salmonella. Conditional expression of the glucose-6-phosphate dehydrogenase-encoding gene zwf, shown here to be under DksA control, increases both the NADPH pool and antioxidant defenses of dksA mutant Salmonella. The DksA-mediated coordination of redox balance boosts the antioxidant defenses of stationary phase bacteria. Not only does DksA increase resistance of Salmonella against hydrogen peroxide (H(2)O(2)), but it also promotes fitness of this intracellular pathogen when exposed to oxyradicals produced by the NADPH phagocyte oxidase in an acute model of infection. Given the role of DksA in the adjustment of gene expression in most bacteria undergoing nutritional deprivation, our findings raise the possibility that the control of central metabolic pathways by this regulatory protein maintains redox homeostasis essential for antioxidant defenses in phylogenetically diverse bacterial species.
Assuntos
Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Salmonella/imunologia , Infecções por Salmonella/virologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Virulência , Animais , Genes Supressores , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , NADPH Oxidases/fisiologia , Oxirredução , Estresse Oxidativo , Fagócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções por Salmonella/metabolismo , beta-Galactosidase/metabolismoRESUMO
We show herein that the Salmonella pathogenicity island 2 (SPI2) response regulator SsrB undergoes S-nitrosylation upon exposure of Salmonella to acidified nitrite, a signal encountered by this enteropathogen in phagosomes of macrophages. Mutational analysis has identified Cys(203) in the C-terminal dimerization domain of SsrB as the redox-active residue responding to nitric oxide (NO) congeners generated in the acidification of nitrite. Peroxynitrite and products of the autooxidation of NO in the presence of oxygen, but not hydrogen peroxide, inhibit the DNA-binding capacity of SsrB, demonstrating the selectivity of the reaction of Cys(203) with reactive nitrogen species (RNS). These findings identify the two-component response regulator SsrB Cys(203) as a thiol-based redox sensor. A C203S substitution protects SsrB against the attack of RNS while preserving its DNA-binding capacity. When exposed to SPI2-inducing conditions, Salmonella expressing the wild-type ssrB allele or the ssrB C203S variant sustain transcription of the sifA, sspH2, and srfJ effector genes. Nonetheless, compared with the strain expressing a redox-resistant SsrB C203S variant, wild-type Salmonella bearing the NO-responsive allele exhibit increased fitness when exposed to RNS in an NRAMP(R), C3H/HeN murine model of acute oral infection. Given the widespread occurrence of the wild-type allele in Salmonella enterica, these findings indicate that SsrB Cys(203) increases Salmonella virulence by serving as a redox sensor of NO resulting from the host immune response to oral infection.