N-acetylcysteine absorption and its potential dual effect improve fitness and fruit yield in Xylella fastidiosa infected plants.

BACKGROUND: Xylella fastidiosa is a multi-host bacterium that can be detected in hundreds of plant species including several crops. Diseases caused by X. fastidiosa are considered a threat to global food production. The primary method for managing diseases caused by X. fastidiosa involves using insecticides to control the vector. Hence, it is necessary to adopt new and sustainable disease management technologies to control not only the insect but also the bacteria and plant health. We demonstrated that N-Acetylcysteine (NAC), a low-cost cysteine analogue, is a sustainable molecule that can be used in agriculture to decrease the damage caused by X. fastidiosa and improve plant health. RESULTS: Using 15N-NAC we proved that it was absorbed by the roots and transported to different parts of the plant. Inside the plant, NAC reduced the bacterial population by 60-fold and the number of xylem vessels blocked by bacterial biofilms. This reflected in a recovery of 0.28-fold of the daily sap flow compared to health plants. In addition, NAC-treated CVC plants decreased the oxidative stress by improving the activity of detoxifying enzymes. Moreover, the use of NAC in field conditions positively contributed for the increase in fruit yield of CVC-diseased plants. CONCLUSION: Our research not only advances the understanding of NAC absorption in plants but also indicates its dual effect as an antimicrobial and antioxidant molecule. This, in turn, negatively affects bacterial survival while improving plant health by decreasing oxidative stress. Overall, the positive field-based evidence supports the viability of NAC as a sustainable agricultural application. This article is protected by copyright. All rights reserved.

Assessment of Nutritional Risk at ICU Admission and Mortality in Patients with Suspected COVID-19.

Background/Objectives: The association between the nutritional risk and mortality in Brazilians with COVID-19 is poorly documented. Therefore, this study, for the first time, aimed at investigating the length of stay in the ICU and the chance of dying in patients with suspected COVID-19, without and with nutritional risk. Subjects/Methods: This retrospective monocentric study enrolled adult, COVID-19-positive patients that were admitted to the ICU at a university hospital. Biochemical analysis and clinical data were collected from medical records and the nutritional risk was assessed according to the Modified-Nutrition Risk in the Critically Ill (mNUTRIC) score. The Cox model was used to assess the chance of mortality in the patients with and without nutritional risk. Results: Out of 71 patients, 63.3% were male and 52% were older (≥60 years). Although no differences were found between groups for the length of stay in ICU, C-reactive protein, alanine aminotransferase and aspartate aminotransferase concentrations, the mNUTRIC ≥ 5 group had higher D-dimer than the mNUTRIC < 5 group. Regarding ICU mortality, most patients (69.5%) in the mNUTRI ≥ 5 group died while in the mNUTRIC < 5 group 33.3% died (p = 0.0001). In addition, patients with mNUTRIC ≥ 5 had (HR: 2.04 [95% CI: 1.02−4.09], p = 0.04) a more likely chance of dying than patients in the mNUTRIC < 5 group, even that adjusted by BMI and D-dimer concentrations (HR: 2.18 [95% CI: 1.04−4.56], p = 0.03). Conclusion: In patients with COVID-19, an mNUTRIC ≥ 5 score at admission leads to a more likely chance of death even after controlling for confounding variables.

High neutrophil-to-lymphocyte ratio at intensive care unit admission is associated with nutrition risk in patients with COVID-19.

BACKGROUND: Inflammation plays a crucial role in nutrition status and can be useful in early nutrition risk screening of patients during the coronavirus disease 2019 (COVID-19) pandemic. Thus, this study aimed to assess the association between systemic inflammatory markers and nutrition risk tools in intensive care unit (ICU) patients with COVID-19. METHODS: Patients with confirmed COVID-19 and ICU admission were enrolled in a retrospective, observational, cross-sectional study. The medians of C-reactive protein (CRP; ≥13.8 mg/dl) and the neutrophil-to-lymphocyte ratio (NLR; ≥12.6) upon admission were used to dichotomize patients. RESULTS: Of the 73 patients, 63% were men; the average age was 56 years, and the median length of hospital stay was 10 (25th: 4; 75th: 17) days. When nutrition risk screening tools were used, 85% were at risk according to Nutritional Risk Screening (≥3 points), whereas 42% had high risk according to the Modified Nutrition Risk in the Critically Ill (mNUTRIC; ≥5 points), and 57% were moderately or severely malnourished according to the Subjective Global Assessment (B or C). Mortality was higher in the group with NLR ≥12.6 than in the group with NLR <12.6, with no difference between CRP groups. A significant association was found only between NLR and mNUTRIC, even when adjusted by sex, age, and body mass index (odds ratio, 1.36; 95% CI, 1.06-1.76; P = 0.016), but not between CRP and nutrition risk. CONCLUSION: Although the inflammatory marker CRP is the most used in hospital clinical practice, we found that only NLR was associated with nutrition risk (NUTRIC score).

Persister Cells Form in the Plant Pathogen Xanthomonas citri subsp. citri under Different Stress Conditions.

Citrus canker disease, caused by the bacterium Xanthomonas citri subsp. citri is a constant threat to citrus-producing areas. Since it has no cure, agricultural practices to restrain its dissemination are essential to reduce the economic damage. Hence, increased knowledge of the basic aspects of X. citri biology could lead to more efficient management practices that can eliminate dormant bacteria in the field. The dormant cells, also referred to as persisters, are phenotypic variants with lowered metabolism, which in turn leads to tolerance to antimicrobials and undermines existing control approaches. We show here that X. citri forms persisters, identifying triggers for this phenotype, including antibiotics, high temperature, and metals (copper and zinc), which increase persistence rates by 10-100 times. The antioxidant N-acetylcysteine reduced copper and zinc-induced persisters, but not those induced by tetracycline, indicating that oxidative stress may be an important inducer of X. citri persistence. In addition, we found that metabolism-independent drugs like cisplatin and mitomycin C are able to eliminate X. citri persistent cells, as well as copper, at high concentrations. Specific amino acids like proline and isoleucine interfered with the physiological balance of the dormancy in X. citri, stimulating or preventing persister resuscitation. Taken together, we discover chemicals that can induce, wake, and kill X. citri persister cells; these results provide insights that should be considered for more efficient integrated control management in the field.

Riboswitch theo/metE as a Transcription Regulation Tool for Xanthomonas citri subsp. citri.

: Xanthomonas citri subsp. citri (X. citri) is the causal agent of Asiatic Citrus Canker (ACC), a disease that affects citrus. ACC has no cure, and growers must rely on special agricultural practices to prevent bacterial spreading. Understanding X. citri basic biology is essential to foresee potential genetic targets to control ACC. Traditionally, microbial genetics use gene deletion/disruption to investigate gene function. However, essential genes are difficult to study this way. Techniques based on small-RNAs and antisense-RNAs are powerful for gene characterization, but not yet fully explored in prokaryotes. One alternative is riboswitches, which derive from bacteria, and can control transcription/translation. Riboswitches are non-coding RNAs able to modulate gene expression in the presence of specific ligands. Here we demonstrate that the riboswitch theo/metE decreases parB expression in X. citri in a platform responsive to theophylline. By monitoring cell respiration, we showed that higher concentrations of the ligand interfered with bacterial viability. Therefore, we determined the safe dose of theophylline to be used with X. citri. Finally, in downstream investigations of parB transcription modulation, we show evidence for the fact that ParB is stable, remains functional throughout the cell cycle, and is inherited by the daughter cells upon cell division.

Persistence in Phytopathogenic Bacteria: Do We Know Enough?

Phytopathogenic bacteria affect a wide range of crops worldwide and have a negative impact in agriculture due to their associated economic losses and environmental impacts. Together with other biotic and abiotic stress factors, they pose a threat to global food production. Therefore, understanding bacterial survival strategies is an essential step toward the development of new strategies to control plant diseases. One mechanism used by bacteria to survive under stress conditions is the formation of persister cells. Persisters are a small fraction of phenotypic variants within an isogenic population that exhibits multidrug tolerance without undergoing genetic changes. They are dormant cells that survive treatment with antimicrobials by inactivating the metabolic functions that are disrupted by these compounds. They are thus responsible for the recalcitrance of many human diseases, and in the same way, they are thought to contribute to the survival of bacterial phytopathogens under a range of stresses they face in the environment. It is believed that persister cells of bacterial phytopathogens may lead to the reoccurrence of disease by recovering growth and recolonizing the host plant after the end of stress. However, compared to human pathogens, little is known about persister cells in phytopathogens, especially about their genetic regulation. In this review, we describe the overall knowledge on persister cells and their regulation in bacterial phytopathogens, focusing on their ability to survive stress conditions, to recover from dormancy and to maintain virulence.

Turbo-extraction of glycosides from Stevia rebaudiana using a fractional factorial design

ABSTRACT Stevia rebaudiana (Bertoni) Bertoni, Asteraceae, leaf extract has recently called the attention of food industry as a proposal for natural sweetener. The sweet flavor is attributed to the glycosides, in especial stevioside and rebaudioside A, which are the plant main chemical markers. The aim of the work reported here was to optimize the turbo-extraction of stevia leaves using water, ethanol 70% and 90% (w/w) as green solvents. A 25-2 factorial design was applied to study the linear effects of the drug size, solvent to drug ratio, temperature, time and also the turbolysis speed on the extraction of glycosides. The glycosides exhaustive extraction showed that ethanol 70% gave better results and was used for turbo-extraction. The stevioside and rebaudioside A contents were quantified by a validated method by high performance liquid chromatographic with photodiode array detector. The contents of stevioside and rebaudioside A in fluid extract increased with the drug size, but decreased at high shearing speeds and solvent to drug ratio, while their yields decreased at higher temperature and were not affected by turbo speed. An increase in solvent to drug ratio reduced significantly the glycosides percent in dried extract. Optimal solution for S. rebaudiana leaves turbo-extraction was determined by desirability functions. The optimal extraction condition corresponded to drug size of 780 µm, solvent to drug ratio of 10, extraction time of 18 min; temperature of 23 ºC and turbo speed of 20,000 rpm, resulting in yields of 4.98% and 2.70%, for stevioside and rebaudioside A, respectively. These yields are comparable to the ones recently published for dynamic maceration, but with the advantage of shorter extraction times. This work demonstrates that turbolysis is promising for S. rebaudiana glycosides extraction and stimulate new research on the purification of these extracts, which may become an interesting source of income for developing countries such as India and Brazil.

Protein depletion using the arabinose promoter in Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (X. citri) is a plant pathogen and the etiological agent of citrus canker, a severe disease that affects all the commercially important citrus varieties, and has worldwide distribution. Citrus canker cannot be healed, and the best method known to control the spread of X. citri in the orchards is the eradication of symptomatic and asymptomatic plants in the field. However, in the state of São Paulo, Brazil, the main orange producing area in the world, control is evolving to an integrated management system (IMS) in which growers have to use less susceptible plants, windshields to prevent bacterial spread out and sprays of cupric bactericidal formulations. Our group has recently proposed alternative methods to control citrus canker, which are based on the use of chemical compounds able to disrupt vital cellular processes of X. citri. An important step in this approach is the genetic and biochemical characterization of genes/proteins that are the possible targets to be perturbed, a task not always simple when the gene/protein under investigation is essential for the organism. Here, we describe vectors carrying the arabinose promoter that enable controllable protein expression in X. citri. These vectors were used as complementation tools for the clean deletion of parB in X. citri, a widespread and conserved gene involved in the essential process of bacterial chromosome segregation. Overexpression or depletion of ParB led to increased cell size, which is probably a resultant of delayed chromosome segregation with subsequent retard of cell division. However, ParB is not essential in X. citri, and in its absence the bacterium was fully competent to colonize the host citrus and cause disease. The arabinose expression vectors described here are valuable tools for protein expression, and especially, to assist in the deletion of essential genes in X. citri.

PAMPs, PRRs, effectors and R-genes associated with citrus-pathogen interactions.

BACKGROUND: Recent application of molecular-based technologies has considerably advanced our understanding of complex processes in plant-pathogen interactions and their key components such as PAMPs, PRRs, effectors and R-genes. To develop novel control strategies for disease prevention in citrus, it is essential to expand and consolidate our knowledge of the molecular interaction of citrus plants with their pathogens. SCOPE: This review provides an overview of our understanding of citrus plant immunity, focusing on the molecular mechanisms involved in the interactions with viruses, bacteria, fungi, oomycetes and vectors related to the following diseases: tristeza, psorosis, citrus variegated chlorosis, citrus canker, huanglongbing, brown spot, post-bloom, anthracnose, gummosis and citrus root rot.

Type II Toxin-Antitoxin Distribution and Adaptive Aspects on Xanthomonas Genomes: Focus on Xanthomonas citri.

Prokaryotic toxin-antitoxin (TA) systems were first described as being designed to prevent plasmid loss in bacteria. However, with the increase in prokaryotic genome sequencing, recently many TAs have been found in bacterial chromosomes, having other biological functions, such as environmental stress response. To date, only few studies have focused on TA systems in phytopathogens, and their possible impact on the bacterial fitness. This may be especially important for pathogens like Xanthomonas spp., which live epiphytically before entering the host. In this study, we looked for TA systems in the genomes of 10 Xanthomonas strains. We verified that citrus-infecting pathovars have, on average, 50% more TAs than other Xanthomonas spp. and no genome harbors classical toxins such as MqsR, RelB, and HicA. Only one TA system (PIN_VapC-FitB-like/SpoVT_AbrB) was conserved among the Xanthomonas genomes, suggesting adaptive aspects concerning its broad occurrence. We also detected a trend of toxin gene loss in this genus, while the antitoxin gene was preferably maintained. This study discovers the quantitative and qualitative differences among the type II TA systems present in Xanthomonas spp., especially concerning the citrus-infecting strains. In addition, the antitoxin retention in the genomes is possibly related with the resistance mechanism of further TA infections as an anti-addiction system or might also be involved in regulation of certain specific genes.

A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri

Abstract Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.

A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri.

Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.

Temperature stress promotes cell division arrest in Xanthomonas citri subsp. citri.

Citrus canker is an economically important disease that affects orange production in some of the most important producing areas around the world. It represents a great threat to the Brazilian and North American citriculture, particularly to the states of São Paulo and Florida, which together correspond to the biggest orange juice producers in the world. The etiological agent of this disease is the Gram-negative bacterium Xanthomonas citri subsp. citri (Xcc), which grows optimally in laboratory cultures at ~30 °C. To investigate how temperatures differing from 30 °C influence the development of Xcc, we subjected the bacterium to thermal stresses, and afterward scored its recovery capability. In addition, we analyzed cell morphology and some markers of essential cellular processes that could indicate the extent of the heat-induced damage. We found that the exposure of Xcc to 37 °C for a period of 6 h led to a cell cycle arrest at the division stage. Thermal stress might have also interfered with the DNA replication and/or the chromosome segregation apparatuses, since cells displayed an increased number of sister origins side-by-side within rods. Additionally, Xcc treated at 37 °C was still able to induce citrus canker symptoms, showing that thermal stress did not affect the ability of Xcc to colonize the host citrus. At 40-42 °C, Xcc lost viability and became unable to induce disease symptoms in citrus. Our results provide evidence about essential cellular mechanisms perturbed by temperature, and can be potentially explored as a new method for Xanthomonas citri synchronization in cell cycle studies, as well as for the sanitation of plant material.

Asymmetric chromosome segregation in Xanthomonas citri ssp. citri.

This study was intended to characterize the chromosome segregation process of Xanthomonas citri ssp. citri (Xac) by investigating the functionality of the ParB factor encoded on its chromosome, and its requirement for cell viability and virulence. Using TAP tagging we show that ParB is expressed in Xac. Disruption of parB increased the cell doubling time and precluded the ability of Xac to colonize the host citrus. Moreover, Xac mutant cells expressing only truncated forms of ParB exhibited the classical phenotype of aberrant chromosome organization, and seemed affected in cell division judged by their reduced growth rate and the propensity to form filaments. The ParB-GFP localization pattern in Xac was suggestive of an asymmetric mode of replicon partitioning, which together with the filamentation phenotype support the idea that Xac may control septum placement using mechanisms probably analogous to Caulobacter crescentus, and perhaps Vibrio cholerae, and Corynebacterium glutamicum. Xac exhibits asymmetric chromosome segregation, and the perturbation of this process leads to an inability to colonize the host plant.

Influence of different substrates on the production of a mutant thermostable glucoamylase in submerged fermentation.

Three mutations, Ser54→Pro, Thr314→Ala, and His415→Tyr, were identified in Aspergillus awamori glucoamylase gene expressed by Saccharomyces cerevisiae. The mutant glucoamylase (GA) was substantially more thermostable than a wild-type GA at 70 °C, with a 3.0 KJ mol(-1) increase in the free energy of thermo-inactivation. The effect of starch from different botanical sources on the production of this GA was measured in liquid fermentation using commercial soluble starch, cassava, potato, and corn as the carbon source. The best substrate for GA production was the potato starch showing an enzymatic activity of 6.6 U/mL. The commercial soluble starch was also a good substrate for the enzyme production with 6.3 U/mL, followed by cassava starch and corn starch with 5.9 and 3.0 U/mL, respectively. These results showed a significant difference on GA production related to the carbon source employed. The mutant GA was purified by acarbose-Sepharose affinity chromatography; the estimated molecular mass was 100 kDa. The mutant GA exhibited optimum activity at pH 4.5 and an optimum temperature of 65 °C.

Subcellular localization of proteins labeled with GFP in Xanthomonas citri ssp. citri: targeting the division septum.

Xanthomonas citri ssp. citri (Xac) is the causal agent of citrus canker, an economically important disease that affects citrus worldwide. To initiate the characterization of essential biological processes of Xac, we constructed integrative plasmids for the ectopic expression of green fluorescent protein (GFP)-labeled proteins within this bacterium. Here, we show that the disruption of the alpha-amylase gene (amy), the site of plasmid integration into the bacterial chromosome, does not alter its pathogenesis while abolishing completely the ability of Xac to degrade starch. Furthermore, our GFP expression system was used to characterize ORF XAC3408, a hypothetical protein encoded by Xac that shares significant homology to the FtsZ-stabilizing factor ZapA from Bacillus subtilis (ZapA(Bsu)). GFP-XAC3408 expressed in Xac exhibited a septal localization pattern typical of GFP-ZapA(Bsu), which indicates that XAC3408 is the Xac orthologue of the cell division protein ZapA(Bsu). The results demonstrate the potential of GFP labeling for protein functional characterizations in Xac, and, in addition, the Xac mutant strain labeled at the septum constitutes a biological model for the exploration of antibacterial compounds able to inhibit cell division in this plant pathogen.