The MqsRA toxin-antitoxin system from xylella fastidiosa plays a key role in bacterial fitness, pathogenicity, and persister cell formation

Through the formation of persister cells, bacteria exhibit tolerance to multidrug and other environmental stresses without undergoing genetic changes. The toxin-antitoxin (TA) systems are involved in the formation of persister cells because they are able to induce cell dormancy. Among the TA systems, the MqsRA system has been observed to be highly induced in persister cells of Xylella fastidiosa (causal agent of citrus variegated chlorosis-CVC) activated by copper stress, and has been described in Escherichia coil as related to the formation of persister cells and biofilms. Thus, we evaluated the role of this TA system in X. fastidiosa by overexpressing the MqsR toxin, and verified that the toxin positively regulated biofilm formation and negatively cell movement, resulting in reduced pathogenicity in citrus plants. The overexpression of MqsR also increased the formation of persister cells under copper stress. Analysis of the gene and protein expression showed that this system likely has an autoregulation mechanism to express the toxin and antitoxin in the most beneficial ratio for the cell to oppose stress. Our results suggest that this TA system plays a key role in the adaptation and survival of X fastidiosa and reveal new insights into the physiology of phytopathogen host interactions7CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão tem2010/50712-9; 2013/17485-7; 2013/02014-

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Different hydrogen-bonded chains in the crystal structures of three alkyl N-[(E )-1-(2-benzylidene-1-methylhydrazinyl)-3- hydroxy-1-oxopropan-2-yl]carbamates

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Analysis of the biofilm proteome of Xylella fastidiosa

<p>Abstract</p> <p>Background</p> <p><it>Xylella fastidiosa </it>is limited to the xylem of the plant host and the foregut of insect vectors (sharpshooters). The mechanism of pathogenicity of this bacterium differs from other plant pathogens, since it does not present typical genes that confer specific interactions between plant and pathogens (avr and/or hrp). The bacterium is injected directly into the xylem vessels where it adheres and colonizes. The whole process leads to the formation of biofilms, which are considered the main mechanism of pathogenicity. Cells in biofilms are metabolically and phenotypically different from their planktonic condition. The mature biofilm stage (phase of higher cell density) presents high virulence and resistance to toxic substances such as antibiotics and detergents. Here we performed proteomic analysis of proteins expressed exclusively in the mature biofilm of <it>X. fastidiosa </it>strain 9a5c, in comparison to planktonic growth condition.</p> <p>Results</p> <p>We found a total of 456 proteins expressed in the biofilm condition, which correspond to approximately 10% of total protein in the genome. The biofilm showed 37% (or 144 proteins) different protein than we found in the planktonic growth condition. The large difference in protein pattern in the biofilm condition may be responsible for the physiological changes of the cells in the biofilm of <it>X. fastidiosa</it>. Mass spectrometry was used to identify these proteins, while real-time quantitative polymerase chain reaction monitored expression of genes encoding them. Most of proteins expressed in the mature biofilm growth were associated with metabolism, adhesion, pathogenicity and stress conditions. Even though the biofilm cells in this work were not submitted to any stress condition, some stress related proteins were expressed only in the biofilm condition, suggesting that the biofilm cells would constitutively express proteins in different adverse environments.</p> <p>Conclusions</p> <p>We observed overexpression of proteins related to quorum sensing, proving the existence of communication between cells, and thus the development of structuring the biofilm (mature biofilm) leading to obstruction of vessels and development of disease. This paper reports a first proteomic analysis of mature biofilm of <it>X. fastidiosa</it>, opening new perspectives for understanding the biochemistry of mature biofilm growth in a plant pathogen.</p

In vitro determination of extracellular proteins from xylella fastidiosa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (330 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components.The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a de7Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2001/07533-7, 2012/51580-4]Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Computational Biology Program)CAPESFAPESP [2011/50268-4]CAPES (Computational Biology Program)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Ex vivo determination of bone tissue strains for an in vivo mouse tibial loading model

AbstractPrevious studies introduced the digital image correlation (DIC) as a viable technique for measuring bone strain during loading. In this study, we investigated the sensitivity of a DIC system in determining surface strains in a mouse tibia while loaded in compression through the knee joint. Specifically, we examined the effect of speckle distribution, facet size and overlap, initial vertical alignment of the bone into the loading cups, rotation with respect to cameras, and ex vivo loading configurations on the strain contour maps measured with a DIC system.We loaded tibiae of C57BL/6 mice (12 and 18 weeks old male) up to 12N at 8N/min. Images of speckles on the bone surface were recorded at 1N intervals and DIC was used to compute strains. Results showed that speckles must have the correct size and density with respect to the facet size of choice for the strain distribution to be computed and reproducible. Initial alignment of the bone within the loading cups does not influence the strain distribution measured during peak loading, but bones must be placed in front of the camera with the same orientation in order for strains to be comparable. Finally, the ex vivo loading configurations with the tibia attached to the entire mouse, or to the femur and foot, or only to the foot, showed different strain contour maps.This work provides a better understanding of parameters affecting full field strain measurements from DIC in ex vivo murine tibial loading tests

The association of diabetes, subclinical hypothyroidism and carotid intima-media thickness: results from the Brazilian Longitudinal Study of Adult Health (ELSA-Brazil)

Introduction: The association of diabetes with subclinical thyroid diseases may increase the risk of cardiovascular diseases. We analyzed the association of subclinical hypothyroidism, diabetes, and both diseases with carotid Intima-Media Thickness (cIMT) as a surrogate maker for early cardiovascular disease in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). Methods: Cross-sectional analysis with data from the 3rd visit (2017‒2019). Linear regression models were used to evaluate the association of subclinical hypothyroidism, diabetes and of both diseases with a cIMT presented as Beta (95% Confidence Interval ‒ 95% CI) without adjustment, with adjustment for sociodemographic variables (Model 1) and multivariable adjustment (Model 1 more cardiovascular risk factors). We also used logistic regression models to analyze the Odds Ratio (OR) and 95% CI for the association of both diseases using cIMT &gt; P75%. Results: After the exclusion of patients with previous cardiovascular disease, 5,077 participants with no diseases, 1578 with diabetes, 662 with subclinical hypothyroidism, and 234 with both diseases were included in the analysis. Linear regression models showed an association of cIMT with only diabetes (β&nbsp;=&nbsp;0.019; 95% CI 0.012 to 0.027; p &lt; 0.0001) and subclinical hypothyroidism more diabetes (β&nbsp;=&nbsp;0.03; 95% CI 0.010‒0.047, p &lt; 0.0001). The logistic regression model reported an association between diabetes and CIMT higher than P75% (OR&nbsp;=&nbsp;1.49, 95% CI 1.30‒1.71). No interaction between diabetes and subclinical hypothyroidism was detected using cIMT respectively as a continuous (p&nbsp;=&nbsp;0.29) or as a categorical variable (p&nbsp;=&nbsp;0.92). Discussion: Diabetes was associated with higher cIMT values. However, no additive effect of subclinical hypothyroidism associated with diabetes over cIMT was detected

Surface Physicochemical Properties At The Micro And Nano Length Scales: Role On Bacterial Adhesion And Xylella Fastidiosa Biofilm Development.

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.8e7524

Adherence to the EAT-Lancet sustainable reference diet and cardiometabolic risk profile: cross-sectional results from the ELSA-Brasil cohort study

Purpose The EAT-Lancet Commission released a reference sustainable diet to improve human health and respect the planetary boundaries. The Planetary Health Diet Index (PHDI) was developed with the purpose of evaluate the adherence to this reference diet. The aim of the present study was to evaluate the association between adherence to the EAT-Lancet diet with cardiometabolic risk profile. Methods We used the cross-sectional baseline data from 14,155 participants of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil), a multicenter ongoing cohort study. Dietary data were collected using a 114-item validated food frequency questionnaire. The PHDI was used to assess the adherence to the EAT-Lancet diet. It consists of 16 components and the total score can range from 0 to 150 points. Linear, logistic and quasi-Poisson regression models were built to evaluate the associations between PHDI and the outcomes. Results Individuals with higher adherence to EAT-Lancet diet (PHDI, 5th quintile) had lower values for systolic blood pressure (β − 0.84; 95% CI − 1.66: − 0.01), diastolic blood pressure (β − 0.70; 95% CI − 1.24: − 0.15), total cholesterol (β − 3.15; 95% CI − 5.30: − 1.01), LDL-c (β − 4.10; 95% CI − 5.97: − 2.23), and non-HDL-cholesterol (β − 2.57; 95% CI − 4.62: − 0.52). No association was observed for HDL-c, triglycerides and HOMA-IR. Conclusions Our results indicate that higher adherence to the EAT-Lancet diet is associated with lower levels of blood pressure, total cholesterol, LDL-c, and non-HDL-c