Dual Expression of the Salmonella Effector SrfJ in Mammalian Cells and Plants

SrfJ is an effector of the Salmonella pathogenicity island 2-encoded type III secretion system. Salmonella enterica serovar Typhimurium expresses srfJ under two disparate sets of conditions: media with low Mg2+ and low pH, imitating intravacuolar conditions, and media with myo-inositol (MI), a carbohydrate that can be used by Salmonella as sole carbon source. We investigated the molecular basis for this dual regulation. Here, we provide evidence for the existence of two distinct promoters that control the expression of srfJ. A proximal promoter, PsrfJ, responds to intravacuolar signals and is positively regulated by SsrB and PhoP and negatively regulated by RcsB. A second distant promoter, PiolE, is negatively regulated by the MI island repressor IolR. We also explored the in vivo activity of these promoters in different hosts. Interestingly, our results indicate that the proximal promoter is specifically active inside mammalian cells whereas the distant one is expressed upon Salmonella colonization of plants. Importantly, we also found that inappropriate expression of srfJ leads to reduced proliferation inside macrophages whereas lack of srfJ expression increases survival and decreases activation of defense responses in plants. These observations suggest that SrfJ is a relevant factor in the interplay between Salmonella and hosts of different kingdoms.España, Ministerio de Econonomía, Industria y Competitividad SAF2013-46229-REspaña, Ministerio de Econonomía, Industria y Competitividad SAF2016-75365-

Red light-controlled polymerase chain reaction

A 23-mer DNA “caged” at its 3′-terminus with a 9-anthracenyl moiety was prepared. It can be uncaged in the presence of photosensitizer (In(pyropheophorbide-a)chloride)-containing DNAs (9–12 mers) and upon irradiation with red light. This mixture of DNAs was used to design red-light controlled polymerase chain reaction

Red light activated “caged” reagents for microRNA research

“Caged” reagents for miRNA research (siRNA targeting EGFR, involved in miRNA maturation, and mimics of miR-20a, playing a key role in tumor formation and metastasis) were prepared. It was demonstrated that these reagents can be activated by non-toxic to cells red light both in cells and in cell free settings

All-optical memory based on the injection locking bistability of a two-color laser diode

We study the injection locking bistability of a specially engineered two-color semiconductor Fabry-Perot laser. Oscillation in the uninjected primary mode leads to a bistability of single mode and two-color equilibria. With pulsed modulation of the injected power we demonstrate an all-optical memory element based on this bistability, where the uninjected primary mode is switched with 35 dB intensity contrast. Using experimental and theoretical analysis, we describe the associated bifurcation structure, which is not found in single mode systems with optical injection.Comment: 5 pages, 5 figure

Peptide Mass Spectra from Micrometer-Thick Ice Films Produced with Femtosecond Pulses

We present a cryogenic mass spectrometry protocol with the capability to detect peptides in the attomole dilution range from ice films. Our approach employs femtosecond laser pulses and implements neither substrate modification nor proton donor agents in the aqueous solution, known to facilitate analyte detection in mass spectrometry. In a systematic study, we investigated the impact of temperature, substrate composition, and irradiation wavelength (513 and 1026 nm) on the bradykinin signal onset. Our findings show that substrate choice and irradiation wavelength have a minor impact on signal intensity once the preparation protocol is optimized. However, if the temperature is increased from −140 to 0 °C, which is accompanied by ice film thinning, a somehow complex picture of analyte desorption and ionization is recognizable, which has not been described in the literature yet. Under cryogenic conditions (−140 °C), obtaining a signal is only possible from isolated sweet spots across the film. If the thin ice film is between −100 and −70 °C of temperature, these sweet spots appear more frequently. Ice sublimation triggered by temperatures above −70 °C leads to an intense and robust signal onset that could be maintained for several hours. In addition to the above findings, we notice that a vibrant fragmentation pattern produced is strikingly similar with both wavelengths. Our findings suggest that while following an optimized protocol, femtosecond mass spectrometry has excellent potential to analyze small organic molecules and peptides with a mass range of up to 2.5 kDa in aqueous solution without any matrix, as employed in matrix-assisted laser desorption/ionization (MALDI) or any substrate surface modification, found in surface-assisted laser desorption/ionization (SALDI)

Salmonella heterogeneously expresses flagellin during colonization of plants

Minimally processed or fresh fruits and vegetables are unfortunately linked to an increasing number of food-borne diseases, such as salmonellosis. One of the relevant virulence factors during the initial phases of the infection process is the bacterial flagellum. Although its function is well studied in animal systems, contradictory results have been published regarding its role during plant colonization. In this study, we tested the hypothesis that Salmonella’s flagellin plays a versatile function during the colonization of tomato plants. We have assessed the persistence in plant tissues of a Salmonella enterica wild type strain, and of a strain lacking the two flagellins, FljB and FliC.The work carried out in the C.R.B. and J.R. laboratory has been granted by Ministerio de Ciencia, Innovación y Universidades (MCIU, Spain, RTI2018-095069-B-100) awarded to C.R.B. and J.R., and Proyectos de Excelencia (Junta de Andalucía; PY18-2398) awarded to C.B. This work was co-funded by Fondos Europeos de Desarrollo Regional (FEDER). N.L. received funding for a short training mission (STSM) in the A.S. laboratory from CA 16110 HUPLANT from the EU Cost Action Program. We would like to thank DAAD for scholarship funding of A.A.Z.Peer reviewe

An image classification approach to analyze the suppression of plant immunity by the human pathogen <it>Salmonella</it> Typhimurium

<p>Abstract</p> <p>Background</p> <p>The enteric pathogen <it>Salmonella</it> is the causative agent of the majority of food-borne bacterial poisonings. Resent research revealed that colonization of plants by <it>Salmonella</it> is an active infection process. <it>Salmonella</it> changes the metabolism and adjust the plant host by suppressing the defense mechanisms. In this report we developed an automatic algorithm to quantify the symptoms caused by <it>Salmonella</it> infection on <it>Arabidopsis</it>.</p> <p>Results</p> <p>The algorithm is designed to attribute image pixels into one of the two classes: healthy and unhealthy. The task is solved in three steps. First, we perform segmentation to divide the image into foreground and background. In the second step, a support vector machine (SVM) is applied to predict the class of each pixel belonging to the foreground. And finally, we do refinement by a neighborhood-check in order to omit all falsely classified pixels from the second step. The developed algorithm was tested on infection with the non-pathogenic <it>E. coli</it> and the plant pathogen <it>Pseudomonas syringae</it> and used to study the interaction between plants and <it>Salmonella</it> wild type and T3SS mutants. We proved that T3SS mutants of <it>Salmonella</it> are unable to suppress the plant defenses. Results obtained through the automatic analyses were further verified on biochemical and transcriptome levels.</p> <p>Conclusion</p> <p>This report presents an automatic pixel-based classification method for detecting “unhealthy” regions in leaf images. The proposed method was compared to existing method and showed a higher accuracy. We used this algorithm to study the impact of the human pathogenic bacterium <it>Salmonella</it> Typhimurium on plants immune system. The comparison between wild type bacteria and T3SS mutants showed similarity in the infection process in animals and in plants. Plant epidemiology is only one possible application of the proposed algorithm, it can be easily extended to other detection tasks, which also rely on color information, or even extended to other features.</p

Salmonella Heterogeneously Expresses Flagellin During Colonization of Plants

Minimally processed or fresh fruits and vegetables are unfortunately linked to an increasing number of food-borne diseases, such as salmonellosis. One of the relevant virulence factors during the initial phases of the infection process is the bacterial flagellum. Although its function is well studied in animal systems, contradictory results have been published regarding its role during plant colonization. In this study, we tested the hypothesis that Salmonella's flagellin plays a versatile function during the colonization of tomato plants. We have assessed the persistence in plant tissues of a Salmonella enterica wild type strain, and of a strain lacking the two flagellins, FljB and FliC. We detected no differences between these strains concerning their respective abilities to reach distal, non-inoculated parts of the plant. Analysis of flagellin expression inside the plant, at both the population and single cell levels, shows that the majority of bacteria down-regulate flagellin production, however, a small fraction of the population continues to express flagellin at a very high level inside the plant. This heterogeneous expression of flagellin might be an adaptive strategy to the plant environment. In summary, our study provides new insights on Salmonella adaption to the plant environment through the regulation of flagellin expression.España, Ministerio de Ciencia, Innovación y Universidades (MCIU, Spain, RTI2018-095069-B-100

A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip

Fixed-target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X-ray free-electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high-throughput experiments, which depend on high-quality protein microcrystals. The batch crystallization procedures that are typically applied require time- and sample-intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor-diffusion conditions to in-chip crystallization. Based on conventional hanging-drop vapor-diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high-quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively un­explored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in-chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal-containing cells. These proof-of-principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens