High seroprevalence for SARS-CoV-2 infection in dogs: Age as risk factor for infection in shelter and foster home animals.

SARS-CoV-2 has caused 775 outbreaks in 29 animal species across 36 countries, including dogs, cats, ferrets, minks, non-human primates, white-tailed deer, and lions. Although transmission from owners to dogs has been extensively described, no study to date has also compared sheltered, foster home and owner dogs and associated risk factors. This study aimed to identify SARS-CoV-2 infection and anti-SARS-CoV-2 antibodies from sheltered, fostered, and owned dogs, associated with environmental and management risk factors. Serum samples and swabs were collected from each dog, and an epidemiological questionnaire was completed by the shelter manager, foster care, and owner. A total of 111 dogs, including 222 oropharyngeal and rectal swabs, tested negative by RT-qPCR. Overall, 18/89 (20.22%) dogs presented IgG antibodies against the N protein of SARS-CoV-2 by magnetic ELISA, while none showed a reaction to the Spike protein. SARS-CoV-2 antibodies showed an age-related association, with 4.16 chance of positivity in adult dogs when compared with young ones. High population density among dogs and humans, coupled with repeated COVID-19 exposure, emerged as potential risk factors in canine virus epidemiology. Dogs exhibited higher seropositivity rates in these contexts. Thus, we propose expanded seroepidemiological and molecular studies across species and scenarios, including shelter dogs.

Dynamics of the Immune Response in Hospitalized SARS-CoV-2-infected Cancer Patients.

Seroconversion rates were compared between oncological and nononcological patients infected with SARS-CoV-2 during a 14-day hospitalization time. All COVID-19 non-oncological and solid malignancies patients reached 100% seroconversion at day 14, while less than half of the hematological patients were seroconverted at the same time point. Despite the limited number and variability of the patient's cohort, we conclude that there is a delayed seroconversion in the hematological malignancies group, which may be linked to changes in the hematological parameters, immune suppression and/or oncological treatments that are typically associated with these patients.

SARS-CoV-2 in saliva, viremia and seroprevalence for COVID-19 surveillance at a single hematopoietic stem cell transplantation center: a prospective cohort study.

This prospective cohort study aims to analyze the surveillance of COVID-19 at a single hematopoietic stem cell transplantation (HSCT) center in Brazil, in 29 patients undergoing allogeneic HSCT and 57 healthcare workers (nurses and dentists), through viral shedding of SARS-CoV-2 in saliva and plasma and seroprevalence of anti-SARS-CoV-2 IgG. In addition, we report two cases with prolonged persistent detection of SARS-CoV-2 without seroconversion. The sample collection was performed seven times for patients and five times for healthcare workers. Only two patients tested positive for SARS-CoV-2 in their saliva and plasma samples (6.9%) without seroconversion. All healthcare workers were asymptomatic and none tested positive. Two patients (6.9%) and four nurses (8%) had positive serology. No dentists had positive viral detection or positive serology. Our results reflect a low prevalence of positive RT-PCR and seroprevalence of SARS-CoV-2 in patients and healthcare workers at a single HSCT center. Results have also corroborated how the rigorous protocols adopted in transplant centers were even more strengthened in this pandemic scenario.

A magnetic bead immunoassay to detect high affinity human IgG reactive to SARS-CoV-2 Spike S1 RBD produced in Escherichia coli.

Immunological assays to detect SARS-CoV-2 Spike Receptor Binding Domain (RBD) antigen seroconversion in humans are important tools to monitor the levels of protecting antibodies in the population in response to infection and/or immunization. Here we describe a simple, low cost, and high throughput Ni2+ magnetic bead immunoassay to detect human IgG reactive to Spike S1 RBD Receptor Binding Domain produced in Escherichia coli. A 6xHis-tagged Spike S1 RBD was expressed in E. coli and purified by affinity chromatography. The protein was mobilized on the surface of Ni2+ magnetic beads and used to investigate the presence of reactive IgG in the serum obtained from pre-pandemic and COVID-19 confirmed cases. The method was validated with a cohort of 290 samples and an area under the receiver operating characteristic curve of 0.94 was obtained. The method was operated with > 82% sensitivity at 98% specificity and was also able to track human IgG raised in response to vaccination with Comirnaty at > 85% sensitivity. The IgG signal obtained with the described method was well-correlated with the signal obtained when pre fusion Spike produced in HEK cell lines was used as antigen. This novel low-cost and high throughput immunoassay may act as an important tool to investigate protecting IgG antibodies against SARS-CoV-2 in the human population.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense / Caracterização da glutamina sintetase da estipe excretora de amônio HM053 de Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

SARS-CoV-2 in saliva, viremia and seroprevalence for COVID-19 surveillance at a single hematopoietic stem cell transplantation center: a prospective cohort study

ABSTRACT This prospective cohort study aims to analyze the surveillance of COVID-19 at a single hematopoietic stem cell transplantation (HSCT) center in Brazil, in 29 patients undergoing allogeneic HSCT and 57 healthcare workers (nurses and dentists), through viral shedding of SARS-CoV-2 in saliva and plasma and seroprevalence of anti-SARS-CoV-2 IgG. In addition, we report two cases with prolonged persistent detection of SARS-CoV-2 without seroconversion. The sample collection was performed seven times for patients and five times for healthcare workers. Only two patients tested positive for SARS-CoV-2 in their saliva and plasma samples (6.9%) without seroconversion. All healthcare workers were asymptomatic and none tested positive. Two patients (6.9%) and four nurses (8%) had positive serology. No dentists had positive viral detection or positive serology. Our results reflect a low prevalence of positive RT-PCR and seroprevalence of SARS-CoV-2 in patients and healthcare workers at a single HSCT center. Results have also corroborated how the rigorous protocols adopted in transplant centers were even more strengthened in this pandemic scenario.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense / Caracterização da glutamina sintetase da estipe excretora de amônio HM053 de Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense

Abstract Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

Resumo A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense.

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

NAD+ biosynthesis in bacteria is controlled by global carbon/nitrogen levels via PII signaling.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+ Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+ This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

Regulation of Herbaspirillum seropedicae NifA by the GlnK PII signal transduction protein is mediated by effectors binding to allosteric sites.

Herbaspirillum seropedicae is a plant growth promoting bacterium that is able to fix nitrogen and to colonize the surface and internal tissues of important crops. Nitrogen fixation in H. seropedicae is regulated at the transcriptional level by the prokaryotic enhancer binding protein NifA. The activity of NifA is negatively affected by oxygen and positively stimulated by interaction with GlnK, a PII signaling protein that monitors intracellular levels of the key metabolite 2-oxoglutarate (2-OG) and functions as an indirect sensor of the intracellular nitrogen status. GlnK is also subjected to a cycle of reversible uridylylation in response to intracellular levels of glutamine. Previous studies have established the role of the N-terminal GAF domain of NifA in intramolecular repression of NifA activity and the role of GlnK in relieving this inhibition under nitrogen-limiting conditions. However, the mechanism of this control of NifA activity is not fully understood. Here, we constructed a series of GlnK variants to elucidate the role of uridylylation and effector binding during the process of NifA activation. Our data support a model whereby GlnK uridylylation is not necessary to activate NifA. On the other hand, binding of 2-OG and MgATP to GlnK are very important for NifA activation and constitute the most important signal of cellular nitrogen status to NifA.

Fatty acid biosynthesis is enhanced in Escherichia coli strains with deletion in genes encoding the PII signaling proteins.

The committed and rate-limiting step in fatty acid biosynthesis is catalyzed by acetyl-CoA carboxylase (ACC). In previous studies we showed that ACC activity is inhibited through interactions with the PII signaling proteins in vitro. Here we provide in vivo support for that model; we noted that PII proteins are able to reduce malonyl-CoA levels in vivo in Escherichia coli. Furthermore, we show that fatty acid biosynthesis is strongly enhanced in E. coli strains carrying deletions in PII coding genes. Given that PII proteins act as conserved negative regulators of ACC in Bacteria, our findings may be explored to engineer other prokaryotes to improve fatty acid yields, thereby turning microbial biofuel production economically competitive in the future.

Kinetics and structural features of dimeric glutamine-dependent bacterial NAD+ synthetases suggest evolutionary adaptation to available metabolites.

NADH (NAD+) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD+ also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD+ biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD+ synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadENH3 and octameric glutamine-dependent NadEGln, and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadEGln in bacteria. Substrate preferences and structural analyses suggested that dimeric NadEGln enzymes may constitute evolutionary intermediates between dimeric NadENH3 and octameric NadEGln The characterization of additional NadE isoforms in the diazotrophic bacterium Azospirillum brasilense along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes.

Proteomic profile of hemolymph and detection of induced antimicrobial peptides in response to microbial challenge in Diatraea saccharalis (Lepidoptera: Crambidae).

Insects are organisms extremely well adapted to diverse habitats, primarily due to their innate immune system, which provides them with a range of cellular and humoral responses against microorganisms. Lepidoptera hemolymph proteins involved in humoral responses are well known; however, there is a lack of knowledge about the sugarcane borer Diatraea saccharalis. In this present work, the hemolymph proteins of this pest insect were studied by applying proteomic methodologies. Two-dimensional electrophoresis (2-DE) gels of proteins extracted from naive larvae and larvae challenged with Escherichia coli (ATCC 11224) and Bacillus subtilis (ATCC 6623) showed an average of 300 spots, and 92 of these spots corresponded in all three 2-DE gels. Forty-one spots were excised and digested with trypsin and analyzed using mass spectrometry. After analysis, 10 proteins were identified, including some proteins of the immune system: ß-defensin-like protein, Turandot A-like protein, attacin-like protein, peptidoglycan recognition protein and cyclophilin-like protein. Nine proteins were present in both experimental conditions; however, ß-defensin-like protein was present only in hemolymph challenged by B. subtilis. Notably, attacin-like protein was strongly induced by challenge with E. coli, suggesting an immune response against the infection. However, antimicrobial activity was observed in the test zone of microbial growth inhibition of B. subtilis solely with the hemolymph extract of the larvae challenged with B. subtilis. We made for the first time a proteomic profile of the hemolymph of D. saccharalis in which it was possible to identify the presence of important proteins involved in the immune response.

Mutational analysis of GlnB residues critical for NifA activation in Azospirillum brasilense.

PII proteins are signal transduction that sense cellular nitrogen status and relay this signals to other targets. Azospirillum brasilense is a nitrogen fixing bacterium, which associates with grasses and cereals promoting beneficial effects on plant growth and crop yields. A. brasilense contains two PII encoding genes, named glnB and glnZ. In this paper, glnB was mutagenised in order to identify amino acid residues involved in GlnB signaling. Two variants were obtained by random mutagenesis, GlnBL13P and GlnBV100A and a site directed mutant, GlnBY51F, was obtained. Their ability to complement nitrogenase activity of glnB mutant strains of A. brasilense were determined. The variant proteins were also overexpressed in Escherichia coli, purified and characterized biochemically. None of the GlnB variant forms was able to restore nitrogenase activity in glnB mutant strains of A. brasilense LFH3 and 7628. The purified GlnBY51F and GlnBL13P proteins could not be uridylylated by GlnD, whereas GlnBV100A was uridylylated but at only 20% of the rate for wild type GlnB. Biochemical and computational analyses suggest that residue Leu13, located in the α helix 1 of GlnB, is important to maintain GlnB trimeric structure and function. The substitution V100A led to a lower affinity for ATP binding. Together the results suggest that NifA activation requires uridylylated GlnB bound to ATP.

Rapid identification of bacterial isolates from wheat roots by high resolution whole cell MALDI-TOF MS analysis.

Whole-cell mass spectrometry analysis is a powerful tool to rapidly identify microorganisms. Several studies reported the successful application of this technique to identify a variety of bacterial species with a discriminatory power at the strain level, mainly for bacteria of clinical importance. In this study we used matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to assess the diversity of wheat-associated bacterial isolates. Wheat plants cultivated in non-sterile vermiculite, under greenhouse conditions were used for bacterial isolation. Total cellular extracts of 138 isolates were analyzed by MALDI-TOF MS and the mass spectra were used to cluster the isolates. Taxonomic identification and phylogenetic reconstruction based on 16S rRNA gene sequences showed the presence of Pseudomonas, Pantoea, Acinetobacter, Enterobacter and Curtobacterium. The 16S rRNA gene sequence analyses were congruent with the clusterization from mass spectra profile. Moreover, MALDI-TOF whole cell mass profiling allowed a finer discrimination of the isolates, suggesting that this technique has the potential of differentiating bacterial isolates at the strain level.

Mathematical model of the binding of allosteric effectors to the Escherichia coli PII signal transduction protein GlnB.

PII proteins are important regulators of nitrogen metabolism in a wide variety of organisms: the binding of the allosteric effectors ATP, ADP, and 2-oxoglutarate (2-OG) to PII proteins affects their ability to interact with target proteins. We modeled the simultaneous binding of ATP, ADP, and 2-OG to one PII protein, namely GlnB of Escherichia coli, using a modeling approach that allows the prediction of the proportions of individual binding states. Four models with different binding rules were compared. We selected one of these models (that assumes that the binding of the first nucleotide to GlnB makes it harder for subsequent nucleotides to bind) and used it to explore how physiological concentrations of ATP, ADP, and 2-OG would affect the proportions of those states of GlnB that interact with the target proteins ATase and NtrB. Our simulations indicate that GlnB can, as suggested by previous researchers, act as a sensor of both 2-OG and the ATP:ADP ratio. We conclude that our modeling approach will be an important tool in future studies concerning the PII binding states and their interactions with target proteins.

Proteomic analysis of Herbaspirillum seropedicae cultivated in the presence of sugar cane extract.

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant- H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant.

Draft genome sequence of Herbaspirillum lusitanum P6-12, an endophyte isolated from root nodules of Phaseolus vulgaris.

Herbaspirillum lusitanum strain P6-12 (DSM 17154) is, so far, the only species of Herbaspirillum isolated from plant root nodules. Here we report a draft genome sequence of this organism.