digital-pcr-based-competitive-index-for-high-throughput-analysis

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

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 ...

essential-components-borreliella-borrelia-burgdorferi-vitro

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between ...

Creighton University School of Medicine

Creighton University

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. 

The Nucleotide Excision Repair Pathway Protects Borrelia burgdorferi from Nitrosative Stress in Ixodes scapularis Ticks.

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.

The Magnaporthe oryzae nitrooxidative stress response suppresses rice innate immunity during blast disease.

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 Mg 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 O 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 (O) produced in the electron transport chain. The mechanism underlying PhoPQ-mediated resistance to RNS is linked to the coordination of Mg homeostasis through the PhoPQ-regulated MgtA transporter. Collectively, our investigations are consistent with a model in which PhoPQ-dependent Mg homeostasis protects Salmonella against nitrooxidative stress.

Magnesium homeostasis protects Salmonella against nitrooxidative stress.

The genus  is responsible for many illnesses in humans and other vertebrate animals. We report here that  serovar Typhimurium harbors three transketolases that support the non-oxidative branch of the pentose phosphate pathway. BLAST analysis identified two genes, _ and _, 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  (539-1362 μm NADH consumed/s),  (80-739 μm), and catalytic efficiency (1.02 × 10-1.06 × 10 m/s) for each substrate tested. The recombinant form of TktC had lower  values (23-120 μm), whereas the  (7.8-16 μm NADH consumed/s) and catalytic efficiency (5.58 × 10 to 6.07 × 10 m/s) were 10-100-fold lower. Using a murine model of  infection, we showed that a strain lacking all three transketolases is avirulent in C57BL/6 mice. These data provide evidence that  Typhimurium possesses three transketolases that contribute to pathogenesis.

 serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway.

The pathogenic spirochete  senses and responds to changes in the environment, including changes in nutrient availability, throughout its enzootic cycle in  ticks and vertebrate hosts. This study examined the role of DnaK suppressor protein (DksA) in the transcriptional response of  to starvation. Wild-type and  mutant  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% CO, 3% O). Microarray analyses of wild-type  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 , as the -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 , fewer CFU of  mutants were observed after prolonged starvation in RPMI 1640 medium than CFU of wild-type  spirochetes. Transcriptomic analyses revealed a partial overlap between the DksA regulon and the regulon of Rel, 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  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  adaptation to its environment. The Lyme disease bacterium  survives diverse environmental challenges as it cycles between its tick vectors and various vertebrate hosts.  must withstand prolonged periods of starvation while it resides in unfed  ticks. In this study, the regulatory protein DksA is shown to play a pivotal role controlling the transcriptional responses of  to starvation. The results suggest that DksA gene regulatory activity impacts  metabolism, virulence gene expression, and the ability of this bacterium to complete its natural life cycle.

DksA Controls the Response of the Lyme Disease Spirochete  to Starvation.

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.

The relapsing fever spirochete Borrelia turicatae persists in the highly oxidative environment of its soft-bodied tick vector.

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  during infection. Because the chromosomes of TBRF  show extensive colinearity with those of Lyme disease (LD) , 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 , but not in LD spirochetes. Previous work suggested that  orthologs may be important for resistance to stresses faced during mammalian infection. Herein, proteomic analyses of the TBRF spirochete, , demonstrated that BtpA, as well as proteins encoded by adjacent genes in the  genome, were produced in response to culture at mammalian body temperature, suggesting a role in mammalian infection. Further, transcriptional analyses revealed that  was expressed with the genes immediately upstream and downstream as part of an operon. To directly assess if  is involved in resistance to environmental stresses,  deletion mutants were generated.  mutants demonstrated no growth defect in response to heat shock, but were more sensitive to oxidative stress produced by butyl peroxide compared to wild-type . Finally,  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.

Assessing the Contribution of an HtrA Family Serine Protease During  Mammalian Infection.

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.

Molecular Detection of Spotted Fever Group Rickettsiae (Rickettsiales: Rickettsiaceae) in Dermacentor variabilis (Acari: Ixodidae) Collected Along the Platte River in South Central Nebraska.

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.

Establishment of an in vitro RNA polymerase transcription system: a new tool to study transcriptional activation in Borrelia burgdorferi.

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, , overlaps with CWD throughout the northwest United States and southwest Canada, raising the possibility that  parasitization of cervids may be involved in CWD transmission. We investigated this possibility by examining the blood meal of  that fed upon prion-infected hamsters for the presence of prion infectivity by animal bioassay. None of the hamsters inoculated with a  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  in the transmission of prion disease. 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  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  blood meals that had been ingested from prion-infected hamsters yielded evidence of prion infection. Overall, the data do not support a role of  in the transmission of prion disease.

Failure To Detect Prion Infectivity in Ticks following Prion-Infected Blood Meal.

 () , 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,  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 Rel 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.

Gene Regulation and Transcriptomics.

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.

Travis J. Bourret

Department of Medical Microbiology and Immunology

Creighton University School of Medicine

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

Travis J. Bourret

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medical Microbiology and Immunology

Creighton University School of Medicine

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

Department of Medical Microbiology and Immunology