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Background: Rapid and sensitive detection of foodborne pathogens in food plays a crucial role in controlling outbreaks of foodborne diseases, of which Listeria monocytogenes and Salmonella typhimurium are representative and notable pathogens. Thus, it's of great importance to achieve the effective detection of these pathogens. However, the most common detection methods (culture-based technique, Polymerase Chain Reaction and immunological methods) have disadvantages that cannot be ignored, such as time-consuming, laborious, complex sample preparation process, and the possibility of cross-reaction. Hence, it is essential to develop a facile detection method for the pathogens with high sensitivity and specificity to avoid the above-mentioned disadvantages., Results: We report a label-free visual platform for the simultaneous capture and detection of Listeria monocytogenes and Salmonella typhimurium. For the first time, we have prepared polydimethylsiloxane-Chromotrope 2R membrane which serves as the substrate for bacterial capture and enrichment through the formation of specific recognition sites. The positively charged Pt-covalent organic framework combines with the pathogens through surface charge interaction, thereby the label-free sandwich platform is formed. Remarkable peroxidase activity of Pt-covalent organic framework converts the conversion of bacterial quantity into amplified color signal by catalyzing 3,3',5,5'-Tetramethylbenzidine to oxidized 3,3',5,5'-Tetramethylbenzidine. The platform demonstrates the capability to identify two representative food-borne pathogens within a time frame of 100 min, exhibiting high sensitivity and excellent specificity without the interference from non-target bacteria. The limit of detection of the visual platform toward Listeria monocytogenes and Salmonella typhimurium was 1.61 CFU mL -1 and 1.31 CFU mL -1 , respectively. And the limit of quantification toward Listeria monocytogenes and Salmonella typhimurium was 4.94 CFU mL -1 and 2.47 CFU mL -1 , respectively. The relative standard derivations of the visual platform for both bacteria were lower than 4.9 %. Furthermore, our proposed platform has obtained reliable and satisfactory results on analyzing diverse food samples., Significance: This research expands the application of a label-free platform combined with unlabeled nanocomponents in the rapid isolation and detection of diverse of food-borne pathogens. The platform possesses the advantages of simple operation and real-time monitoring, without complicated sample pretreatment process. The whole detection process can realize the simultaneous monitoring of Listeria monocytogenes and Salmonella typhimurium within 100 min. Furthermore, it is also of reference significance for the detection of other common pathogens., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Typhimurium melibiose permease (MelB St ) is a prototype of the major facilitator superfamily (MFS) transporters, which play important roles in human health and diseases. MelB St catalyzed the symport of galactosides with Na + , Li + , or H + but prefers the coupling with Na + . Previously, we determined the structures of the inward- and outward-facing conformation of MelB St and the molecular recognition for galactoside and Na + . However, the molecular mechanisms for H + - and Na + -coupled symport remain poorly understood. In this study, we solved two x-ray crystal structures of MelB St , the cation-binding site mutants D59C at an unliganded apo-state and D55C at a ligand-bound state, and both structures display the outward-facing conformations virtually identical as published. We determined the energetic contributions of three major Na + -binding residues for the selection of Na + and H + by free energy simulations. Transport assays showed that the D55C mutant converted MelB St to a solely H + -coupled symporter, and together with the free-energy perturbation calculation, Asp59 is affirmed to be the sole protonation site of MelB St . Unexpectedly, the H + -coupled melibiose transport exhibited poor activities at greater bulky ΔpH and better activities at reversal ΔpH, supporting the novel theory of transmembrane-electrostatically localized protons and the associated membrane potential as the primary driving force for the H + -coupled symport mediated by MelB St . This integrated study of crystal structure, bioenergetics, and free energy simulations, demonstrated the distinct roles of the major binding residues in the cation-binding pocket of MelB St ., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Sensitive and on-site discrimination of live and dead foodborne pathogenic strains remains a significant challenge due to the lack of appropriate assay and signal probes. In this work, a versatile platinum nanoparticle-decorated phage nanozyme (P2@PtNPs) that integrated recognition, bacteriolysis, and catalysis was designed to establish the bioluminescence/pressure dual-mode bioassay for on-site determination of the vitality of foodborne pathogenic strains. Benefiting from the bacterial strain-level specificity of phage, the target Salmonella typhimurium (S.T) was specially captured to form sandwich complexes with P2@PtNPs on another phage-modified glass microbead (GM@P1). As the other part of the P2@PtNPs nanozyme, the introduced PtNPs could not only catalyze the decomposition of hydrogen peroxide to generate a significant oxygen pressure signal but also produce hydroxyl radicals around the target bacteria to enhance the bacteriolysis of phage and adenosine triphosphate release. It significantly improved the bioluminescence signal. The two signals corresponded to the total and live target bacteria counts, so the dead target could be easily calculated from the difference between the total and live target bacteria counts. Meanwhile, the vitality of S.T was realized according to the ratio of live and total S.T. Under optimal conditions, the application range of this proposed bioassay for bacterial vitality was 10 2 -10 7 CFU/mL, with a limit of detections for total and live S.T of 30 CFU/mL and 40 CFU/mL, respectively. This work provides an innovative and versatile nanozyme signal probe for the on-site determination of bacterial vitality for food safety.

Bacterial chemotaxis requires bidirectional flagellar rotation at different rates. Rotation is driven by a flagellar motor, which is a supercomplex containing multiple rings. Architectural uncertainty regarding the cytoplasmic C-ring, or 'switch', limits our understanding of how the motor transmits torque and direction to the flagellar rod. Here we report cryogenic electron microscopy structures for Salmonella enterica serovar typhimurium inner membrane MS-ring and C-ring in a counterclockwise pose (4.0 Å) and isolated C-ring in a clockwise pose alone (4.6 Å) and bound to a regulator (5.9 Å). Conformational differences between rotational poses include a 180° shift in FliF/FliG domains that rotates the outward-facing MotA/B binding site to inward facing. The regulator has specificity for the clockwise pose by bridging elements unique to this conformation. We used these structures to propose how the switch reverses rotation and transmits torque to the flagellum, which advances the understanding of bacterial chemotaxis and bidirectional motor rotation., (© 2024. The Author(s).)

The bacterial flagellum is a macromolecular protein complex that harvests energy from uni-directional ion flow across the inner membrane to power bacterial swimming via rotation of the flagellar filament. Rotation is bi-directional, with binding of a cytoplasmic chemotactic response regulator controlling reversal, though the structural and mechanistic bases for rotational switching are not well understood. Here we present cryoelectron microscopy structures of intact Salmonella flagellar basal bodies (3.2-5.5 Å), including the cytoplasmic C-ring complexes required for power transmission, in both counter-clockwise and clockwise rotational conformations. These reveal 180° movements of both the N- and C-terminal domains of the FliG protein, which, when combined with a high-resolution cryoelectron microscopy structure of the MotA 5 B 2 stator, show that the stator shifts from the outside to the inside of the C-ring. This enables rotational switching and reveals how uni-directional ion flow across the inner membrane is used to accomplish bi-directional rotation of the flagellum., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Food borne pathogens threaten food safety and affect human health. The lateral flow immunoassays (LFIAs) are widely concerned because of simplicity, low cost and user friendliness, and have broad application prospects in pathogen detection. However, the sensitivity of LFIAs is limited. Herein, multi-line LFIAs are introduced into pathogen detection for the first time. Compared with traditional single-line LFIAs, the overall signal strength of multi-line LFIAs has been significantly improved. It is particularly noteworthy that multi-line LFIAs detection accuracy of 10 3  CFU/mL pathogen has been improved by about 55%. The proposed multi-line LFIAs reduce the possibility of judging a positive result as a false negative result. The LFIAs strip was validated in real samples of milk and orange juice. This strategy has great potential for rapid detection of pathogens in real samples, and provides new insights for improving the accuracy and sensitivity of LFIAs strips., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The divalent transition metal cation manganese is important for protein function, particularly under conditions of iron limitation, nitrosative stress, and oxidative stress, but can mediate substantial toxicity in excess. Salmonella enterica serovar Typhimurium possesses multiple manganese importers, but the pathways for manganese efflux remain poorly defined. The S . Typhimurium ATCC 14028s genome was analyzed for putative manganese export pathways, which identified a previously uncharacterized homologue of the Escherichia coli manganese exporter mntP , stm1834, and two cation diffusion facilitator family transporters, zitB ( stm0758 ) and yiiP ( stm4061 ). Manganese acquisition by S . Typhimurium has been shown to occur in response to nitric oxide, an important chemical mediator of the mammalian innate immune response. However, cellular manganese can rapidly return to prechallenge levels, strongly suggesting that one or more S . Typhimurium exporters may contribute to this process. Here, we report that mntP and yiiP contribute to manganese resistance and export in S . Typhimurium. YiiP, also known as FieF, has previously been associated with zinc and iron transport, although its physiological role remains ambiguous due to a lack of zinc-sensitive phenotypes in yiiP mutant strains of S . Typhimurium and E. coli. We report that S . Typhimurium Δ mntP Δ yiiP mutants are exquisitely sensitive to manganese and show that both YiiP and MntP contribute to manganese efflux following nitric oxide exposure. IMPORTANCE Transition metal cations are required for the function of many proteins but can mediate toxicity when present in excess. Identifying transporters that facilitate metal ion export, the conditions under which they are expressed, and the role they play in bacterial physiology is an evolving area of interest for environmental and pathogenic organisms. Determining the native targets of metal transporters has proved challenging since bioinformatic predictions, in vitro transport data, and mutant phenotypes do not always agree. This work identifies two transporters that mediate manganese efflux from the Gram-negative pathogen Salmonella enterica serovar Typhimurium in response to manganese overload and nitric oxide stress. While homologues of MntP have been characterized previously, this is the first observation of YiiP contributing to manganese export.

Bacterial toxin-antitoxin modules contribute to the stress adaptation, persistence, and dormancy of bacteria for survival under environmental stresses and are involved in bacterial pathogenesis. In Salmonella Typhimurium, the Gcn5-related N-acetyltransferase toxin TacT reportedly acetylates the α-amino groups of the aminoacyl moieties of several aminoacyl-tRNAs, inhibits protein synthesis, and promotes persister formation during the infection of macrophages. Here, we show that TacT exclusively acetylates Gly-tRNA Gly in vivo and in vitro. The crystal structure of the TacT:acetyl-Gly-tRNA Gly complex and the biochemical analysis reveal that TacT specifically recognizes the discriminator U73 and G71 in tRNA Gly , a combination that is only found in tRNA Gly isoacceptors, and discriminates tRNA Gly from other tRNA species. Thus, TacT is a Gly-tRNA Gly -specific acetyltransferase toxin. The molecular basis of the specific aminoacyl-tRNA acetylation by TacT provides advanced information for the design of drugs targeting Salmonella., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Methylesterase/deamidase CheB is a key component of bacterial chemotaxis systems. It is also a prominent example of a two-component response regulator in which the effector domain is an enzyme. Like other response regulators, CheB is activated by phosphorylation of an aspartyl residue in its regulatory domain, creating an open conformation between its two domains. Studies of CheB in Escherichia coli and related organisms have shown that its enzymatic action is also enhanced by a pentapeptide-binding site for the enzyme at the chemoreceptor carboxyl terminus. Related carboxyl-terminal pentapeptides are found on >25,000 chemoreceptor sequences distributed across 11 bacterial phyla and many bacterial species, in which they presumably play similar roles. Yet, little is known about the interrelationship of CheB phosphorylation, pentapeptide binding, and interactions with its substrate methylesters and amides on the body of the chemoreceptor. We investigated by characterizing the binding kinetics of CheB to Nanodisc-inserted chemoreceptor dimers. The resulting kinetic and thermodynamic constants revealed a synergy between CheB phosphorylation and pentapeptide binding in which a phosphorylation mimic enhanced pentapeptide binding, and the pentapeptide served not only as a high-affinity tether for CheB but also selected the activated conformation of the enzyme. The basis of this selection was revealed by molecular modeling that predicted a pentapeptide-binding site on CheB which existed only in the open, activated enzyme. Recruitment of activated enzyme by selective tethering represents a previously unappreciated strategy for regulating response regulator action, one that may well occur in other two-component systems. IMPORTANCE Two-component signal transduction systems are a primary means by which bacteria sense and respond to their environment. Response regulators are key components of these systems. Phosphorylation of response regulators by cognate histidine kinases generate active conformations which act on specific targets, DNA sequences or proteins. The targets have been considered passive in this process. Our characterization of interaction between response regulator CheB and its target chemoreceptor revealed active participation of the target in response regulator action. We found that a pentapeptide sequence at the carboxyl terminus of Escherichia coli chemoreceptors is a selective tether that binds only phosphorylated CheB, thus selecting the form of this two-component enzyme active for covalent modification of the selecting chemoreceptor. Analogous pentapeptides are found on chemoreceptors in many bacterial species and are presumably also selective tethers. There may well be other, uncharacterized examples of active participation of target molecules in response to regulator action.

A more complete and holistic view on host-microbe interactions is needed to understand the physiological and cellular barriers that affect the efficacy of drug treatments and allow the discovery and development of new therapeutics. Here, we developed a multimodal imaging approach combining histopathology with mass spectrometry imaging (MSI) and same section imaging mass cytometry (IMC) to study the effects of Salmonella Typhimurium infection in the liver of a mouse model using the S. Typhimurium strains SL3261 and SL1344. This approach enables correlation of tissue morphology and specific cell phenotypes with molecular images of tissue metabolism. IMC revealed a marked increase in immune cell markers and localization in immune aggregates in infected tissues. A correlative computational method (network analysis) was deployed to find metabolic features associated with infection and revealed metabolic clusters of acetyl carnitines, as well as phosphatidylcholine and phosphatidylethanolamine plasmalogen species, which could be associated with pro-inflammatory immune cell types. By developing an IMC marker for the detection of Salmonella LPS, we were further able to identify and characterize those cell types which contained S. Typhimurium.

The FliE component of the bacterial flagellum is the first protein secreted through the flagellar type III secretion system (fT3SS) that is capable of self-assembly into the growing bacterial organelle. The FliE protein plays dual roles in the assembly of the Salmonella flagellum as the final component of the flagellar type III secretion system (fT3SS) and as an adaptor protein that anchors the rod (drive shaft) of the flagellar motor to the membrane-imbedded MS-ring structure. This work has identified the interactions between FliE and other proteins at the inner membrane base of the flagellar machine. The fliE sequence coding for the 104-amino-acid protein was subject to saturating mutagenesis. Single-amino-acid substitutions were generated in fliE , resulting in motility phenotypes. From these mutants, intergenic suppressor mutations were generated, isolated, and characterized. Single-amino-acid mutations defective in FliE function were localized to the N- and C-terminal helices of the protein. Motile suppressors of amino acid mutations in fliE were found in rod protein genes flgB and flgC , the MS ring gene, fliF , and one of the core T3SS genes, fliR . These results support the hypothesis that FliE acts as a linker protein consisting of an N-terminal α-helix that is involved in the interaction with the MS ring with a rotational symmetry and a C-terminal coiled coil that interacts with FliF, FliR, FlgB, and FlgC, and these interactions open the exit gate of the protein export channel of the fT3SS. IMPORTANCE The bacterial flagellum represents one of biology's most complex molecular machines. Its rotary motor spins at speeds of more than 2,000 cycles per second, and its type 3 secretion (T3S) system secretes proteins at rates of tens of thousands of amino acids per second. Within the complex flagellar motility machine resides a unique protein, FliE, which serves as an adaptor to connect a planar, inner membrane-embedded ring structure, the MS-ring, the core T3S secretion complex at the cytoplasmic base, and a rigid, axial structure that spans the periplasmic space, penetrates the outer membrane, and extends 10 to 20 microns from the cell surface. This work combines genetic mutant suppressor analysis with the structural data for the core T3S system, the MS-ring, and the axial drive shaft (rod) that transverses the periplasm to provide insight into the essential adaptor role of FliE in flagellum assembly and function.

The antioxidant and mutagenic/antimutagenic activities of the fixed oils from Nigella sativa (NSO) and Nigella damascena (NDO) seeds, obtained by cold press-extraction from the cultivar samples, were comparatively investigated for the first time. The antimutagenicity test was carried out using classical and modified Ames tests. The fatty acid composition of the fixed oils was characterized by gas chromatography-mass spectrometry (GC-MS) while the quantification of thymoquinone in the fixed oils was determined by UPC 2 . The main components of the NSO and NDO were found to be linoleic acid, oleic acid, and palmitic acid. The results of the Ames test confirmed the safety of NSO and NDO from the viewpoint of mutagenicity. The results of the three antioxidant test methods were correlated with each other, indicating NDO as having a superior antioxidant activity, when compared to the NSO. Both NSO and NDO exhibited a significant protective effect against the mutagenicity induced by aflatoxin B1 in Salmonella typhimurium TA98 and TA100 strains. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, a significant increase in antimutagenic activity was observed, suggesting that the degradation of aflatoxin B1 epoxides by these oils may be a possible antimutagenic mechanism. It is worthy to note that this is the first study to assess the mutagenicity of NSO and NDO according to the OECD 471 guideline and to investigate antimutagenicity of NDO in comparison to NSO against aflatoxin., (© 2021 Wiley-VHCA AG, Zurich, Switzerland.)

SilE and SilB are both proteins involved in the silver efflux pump found in Gram-negative bacteria such as S. typhimurium. Using model peptides along with NMR and CD experiments, we show how SilE may store silver ions prior to delivery and we hypothesize for the first time the interplay between SilB and SilE.

Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonella enterica Typhimurium DT104 phage type, which is associated with multidrug-resistant disease in humans and animals, possesses an ADP-ribosylating toxin called ArtAB. Full-length art AB has been found on a number of broad-host-range non-typhoidal Salmonella species and serovars. ArtAB is also homologous to many AB 5 toxins from diverse Gram-negative pathogens, including cholera toxin (CT) and pertussis toxin (PT), and may be involved in Salmonella pathogenesis, however, in vitro cellular toxicity of ArtAB has not been characterized. art AB was cloned into E. coli and initially isolated using a histidine tag (ArtABHIS) and nickel chromatography. ArtABHIS was found to bind to African green monkey kidney epithelial (Vero) cells using confocal microscopy and to interact with glycans present on fetuin and monosialotetrahexosylganglioside (GM1) using ELISA. Untagged, or native, holotoxin (ArtAB), and the pentameric receptor-binding subunit (ArtB) were purified from E. coli using fetuin and d-galactose affinity chromatography. ArtAB and ArtB metabolic and cytotoxic activities were determined using Vero and Chinese hamster ovary (CHO) epithelial cells. Vero cells were more sensitive to ArtAB, however, incubation with both cell types revealed only partial cytotoxicity over 72 h, similar to that induced by CT. ArtAB induced a distinctive clustering phenotype on CHO cells over 72 h, similar to PT, and an elongated phenotype on Vero cells, similar to CT. The ArtB binding subunit alone also had a cytotoxic effect on CHO cells and induced morphological rounding. Results indicate that this toxin induces distinctive cellular outcomes. Continued biological characterization of ArtAB will advance efforts to prevent disease caused by non-typhoidal Salmonella .

The histone-like nucleoid structuring (H-NS) protein controls the expression of hundreds of genes in Gram-positive bacteria through its capability to coat and condense DNA. This mechanism requires the formation of superhelical H-NS protein filaments that are sensitive to temperature and salinity, allowing H-NS to act as an environment sensor. We use multiscale modeling and simulations to obtain detailed insights into the mechanism of H-NS filament's sensitivity to environmental changes. Through the simulations of the superhelical H-NS filament, we reveal how different environments induce heterogeneity of H-NS monomers. Further, we observe that transient self-association within the H-NS filament creates temperature-inducible strain and might mildly oppose DNA binding. We also probe different H-NS-DNA complex architectures and show that complexation enhances the stability of both DNA and H-NS superhelices. Overall, our results provide unprecedented molecular insights into the environmental sensing and DNA interactions of a prototypical nucleoid-structuring bacterial protein filament.

Salmonella enterica serovar Typhimurium utilizes a series of strategies to evade host innate immune defenses, including the serum complement system. Many microbial pathogens have evolved the ability to bind the complement regulatory protein factor H (FH) through their surface factor H-binding proteins (FHBPs) to circumvent the complement-mediated bactericidal effect. However, the roles of FHBPs in Salmonella pathogenesis are not well understood. In this study, we demonstrated that the survival of S. Typhimurium in human serum was decreased in a time and concentration dependent manner. Pre-incubation with FH attenuated the sensitivity of S. Typhimurium strain χ3761 to complement-mediated serum killing, suggesting FH binding enhance survival in serum. We aimed to identify novel S. Typhimurium FHBPs and characterize their biological functions. Here, six potential FHBPs were identified by two-dimensional (2D)-Far-western blot, and three of them were further confirmed to bind FH by Far-western blot and dot blot. We found that deletion of ompC (ΔompC) significantly inhibited the survival of S. Typhimurium strain χ3761 in human serum. Our results indicated that the ompC mutation does not affect χ3761 adhesion to HeLa cells. Furthermore, a mice infection model showed that deletion of ompC had no significant effect on the histopathological lesions or viability compared with the wild-type strain χ3761. In summary, these results suggested that OmpC is an important FHBP, but not a critical virulence factor of S. Typhimurium., (Copyright © 2021. Published by Elsevier B.V.)

The basal body of the bacterial flagellum is a rotary motor that consists of several rings (C, MS and LP) and a rod. The LP ring acts as a bushing supporting the distal rod for its rapid and stable rotation without much friction. Here, we use electron cryomicroscopy to describe the LP ring structure around the rod, at 3.5 Å resolution, from Salmonella Typhimurium. The structure shows 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners, which explains the structural stability. The inner surface is charged both positively and negatively. Positive charges on the P ring (the part of the LP ring that is embedded within the peptidoglycan layer) presumably play important roles in its initial assembly around the rod with a negatively charged surface., (© 2021. The Author(s).)

A dual-mode aptasensor using colorimetry and microfluidic chip (MC) together with stir bar sorptive extraction (SBSE) has been developed for firstly qualifying samples contaminated with Vibrio parahaemolyticus (V.P) and Salmonella typhimurium (S.T), then precisely determine both of them in positive samples. For this purpose, the aptamer-streptavidin encoded probes (Apt-SAEs) corresponding to different bacteria were prepared in advance. Then, a stir bar modified with 4-mercaptophenylboronic acid (MPBA) was made to extract bacteria together with Apt-SAE probes. The binding event of aptamer and target triggered the formation of two sandwich structures containing Apt-SAE, V.P or S.T. The concentration of bacteria could be enriched by 1000 times within 15 min to avoid long-time enrichment process. Finally, the stir bar was immersed in the 3,3',5,5'-Tetramethylbenzidine (TMB)-H 2 O 2 solution for color development. The color could be observed by naked eyes to judge whether the analytes were present. The colorless samples were judged to be negative. For the positive samples, the adsorbed encoded probes corresponding to different bacteria would be eluted from the stir bar and rapidly analyzed by the MC. Under the optimized conditions, 100 CFU/mL of V.P or S.T or both of them could be observed by colorimetry and 35 CFU/mL of them could be detected (S/N = 3) by the MC. The assay has significant application value for on-site screening and multiple detection of food-borne pathogenic bacteria.

Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A 'gatekeeper' complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region's structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Salmonella typhimurium (S. typhimurium) and Staphylococcus aureus (S. aureus) are the two most important foodborne pathogens which can easily cause disease infections. Here, the aptamer-facilitated gold/silver nanodimer SERS probes were built for the simultaneous detection of the two bacteria with the help of magnetic separation enrichment. First, two nanodimer SERS signal probes and two magnetic capture probes each connected with the specific aptamer were fabricated. The distance between gold and silver nanoparticles in the dimer can amplify the Raman signal (Cy3 and Rox) at the junction but modified in the aptamer sequence. Then, after the addition of S. typhimurium and S. aureus, the sandwich-like composite structures "SERS signal probes-target-magnetic capture probes" formed because of the high affinity between aptamer sequences and their target bacteria. Under the optimal experimental conditions, the linear correlations between Raman intensity and the logarithm of the concentration of bacteria were y = 876.95x-67.84 (R 2  = 0.9865) for S. typhimurium and y = 1280.43x-1752.6 (R 2  = 0.9883) for S. aureus. The SERS detection showed the nanodimer probe had high selectivity. Besides, the recovery experiment in milk sample indicated good accuracy compared with the traditional plate counting method.

Fluorogenic protein tagging systems have been less developed for prokaryotes than for eukaryotic cell systems. Here, we extend the concept of noncovalent fluorogenic protein tags in bacteria by introducing transcription factor-based tags, namely, LmrR and RamR, for probe binding and fluorescence readout under aerobic and anaerobic conditions. We developed two chemogenetic protein tags that impart fluorogenicity and a longer fluorescence lifetime to reversibly bound organic fluorophores, hence the name Chemogenetic Tags with Probe Exchange (CTPEs). We present an extensive characterization of 30 fluorophores reversibly interacting with the two different CTPEs and conclude that aromatic planar structures bind with high specificity to the hydrophobic pockets of these tags. The reversible binding of organic fluorophores to the CTPEs and the superior photophysical properties of organic fluorophores enable long-term fluorescence microscopy of living bacterial cells. Our protein tags provide a general tool for investigating (sub)cellular protein localization and dynamics, protein-protein interactions, and prolonged live-cell microscopy, even under oxygen-free conditions.

The food-borne bacterial pathogen Salmonella Typhimurium uses a type III protein secretion system to deliver multiple proteins into host cells. These secreted effectors modulate the functions of host cells and activate specific signalling cascades that result in the production of pro-inflammatory cytokines and intestinal inflammation. Some of the Salmonella-encoded effectors counteract this inflammatory response and help to preserve host homeostasis. Here, we demonstrate that the Salmonella effector protein SopD, which is required for pathogenesis, functions to both activate and inhibit the inflammatory response by targeting the Rab8 GTPase, which is a negative regulator of inflammation. We show that SopD has GTPase-activating protein activity for Rab8 and, therefore, inhibits this GTPase and stimulates inflammation. We also show that SopD activates Rab8 by displacing it from its cognate guanosine dissociation inhibitor, resulting in the stimulation of a signalling cascade that suppresses inflammation. We solved the crystal structure of SopD in association with Rab8 to a resolution of 2.3 Å, which reveals a unique contact interface that underlies these complex interactions. These findings show the remarkable evolution of a bacterial effector protein to exert both agonistic and antagonistic activities towards the same host cellular target to modulate the inflammatory response.

Cellular inflammasome activation causes caspase-1 cleavage of the pore-forming protein gasdermin D (GSDMD) with subsequent pyroptotic cell death and cytokine release. Here, we clarify the ambiguous role of the related family member gasdermin E (GSDME) in this process. Inflammasome stimulation in GSDMD-deficient cells led to apoptotic caspase cleavage of GSDME. Endogenous GSDME activation permitted sublytic, continuous interleukin-1β (IL-1β) release and membrane leakage, even in GSDMD-sufficient cells, whereas ectopic expression led to pyroptosis with GSDME oligomerization and complete liberation of IL-1β akin to GSDMD pyroptosis. We find that NLRP3 and NLRP1 inflammasomes ultimately rely concurrently on both gasdermins for IL-1β processing and release separately from their ability to induce cell lysis. Our study thus identifies GSDME as a conduit for IL-1β release independent of its ability to cause cell death., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ultimate goal of bacterial based cancer therapy is to achieve non-toxic penetration and colonisation of the tumour microenvironment. To overcome this efficacy-limiting toxicity of anticancer immunotherapy, we have tested a therapy comprised of systemic delivery of a vascular disrupting agent to induce intratumoral necrotic space, cannabidiol to temporarily inhibit angiogenesis and acute inflammation, and a strain of Salmonella Typhimurium that was engineered for non-toxic colonisation and expression of immunomodulators within the tumour microenvironment. This combination treatment strategy was administered to transgenic mice burdened with autochthonous mammary gland tumours and demonstrated a statistically significant 64% slower tumour growth and a 25% increase in mean survival time compared to control animals without treatment. These experiments were accomplished with minimal toxicity as measured by less than 7% weight loss and a return to normal weight gain within three days following intravenous administration of the bacteria. Thus, non-toxic, robust colonisation of the microenvironment was achieved to produce a significant antitumor effect.

Neutrophils, the first line of defense against pathogens, are critical in the host response to acute and chronic infections. In Gram-negative pathogens, the bacterial outer membrane (OM) is a key mediator of pathogen detection; nonetheless, the effects of variations in its molecular structure on the neutrophil migratory response to bacteria remain largely unknown. Here, we developed a quantitative microfluidic assay that precludes physical contact between bacteria and neutrophils while maintaining chemical communication, thus allowing investigation of both transient and steady-state responses of neutrophils to a library of Salmonella enterica serovar Typhimurium OM-related mutants at single-cell resolution. Using single-cell quantitative metrics, we found that transient neutrophil chemokinesis is highly gradated based upon OM structure, while transient and steady-state chemotaxis responses differ little between mutants. Based on our finding of a lack of correlation between chemokinesis and chemotaxis, we define "stimulation score" as a metric that comprehensively describes the neutrophil response to pathogens. Complemented with a killing assay, our results provide insight into how OM modifications affect neutrophil recruitment and pathogen survival. Altogether, our platform enables the discovery of transient and steady-state migratory responses and provides a new path for quantitative interrogation of cell decision-making processes in a variety of host-pathogen interactions. IMPORTANCE Our findings provide insights into the previously unexplored effects of Salmonella envelope defects on fundamental innate immune cell behavior, which advance the knowledge in pathogen-host cell biology and potentially inspire the rational design of attenuated strains for vaccines or immunotherapeutic strains for cancer therapy. Furthermore, the microfluidic assay platform and analytical tools reported herein enable high-throughput, sensitive, and quantitative screening of microbial strains' immunogenicity in vitro This approach could be particularly beneficial for rapid in vitro screening of engineered microbial strains (e.g., vaccine candidates) as the quantitative ranking of the overall strength of the neutrophil response, reported by "stimulation score," agrees with in vivo cytokine response trends reported in the literature., (Copyright © 2021 Leaman et al.)

Probiotics in livestock feed supplements are considered to be an alternative to antibiotics. However, effector molecules responsible for the beneficial roles of probiotics in pigs are in general not well known. Thus, this study demonstrated that a well-known virulence factor, flagellin of Salmonella typhimurium, significantly induced IL-8 production in porcine peripheral blood mononuclear cells, whereas lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria Lactobacillus plantarum, L. casei, and L. rhamnosus GG, effectively inhibited flagellin-induced IL-8 production at mRNA and protein levels. However, the lipoproteins of L. plantarum, L. casei, and L. rhamnosus GG did not suppress flagellin-induced IL-8 production. While D-alanine-deficient L. plantarum LTA inhibited flagellin-induced IL-8 production, L. plantarum LTA deficient in both D-alanine and acyl chains failed to inhibit it; this suggests that the acyl moieties of L. plantarum LTA are essential for inhibiting flagellin-induced IL-8 production. Taken together, L. plantarum LTA plays an important role in improving anti-inflammatory responses of porcine peripheral blood mononuclear cells.

Nano-sized Gram-negative bacterial outer membrane vesicles possess unique structural and immunostimulatory effects that could be exploited to regress tumors by alerting the host immune system and reversing the immunosuppressive tumor microenvironment. The current study was conducted to investigate the antitumor activity of the outer membrane vesicles (ST-OMVs) of Salmonella Typhimurium ATCC 14028, in vitro in human colorectal carcinoma (HTC116), breast cancer (MCF-7), and hepatocellular carcinoma (HepG2) cell lines and in vivo in Ehrlich solid carcinoma-bearing mice model either as a mono-immunotherapy or as an adjuvant to a commonly used conventional chemotherapy. In addition, we investigated the safety of ST-OMVs. Adult Swiss albino female mice with transplanted Ehrlich solid carcinoma were treated with either ST-OMVs, paclitaxel or a combination of both. Tumor volume, growth inhibition rate, quantitative RT-PCR of Bax and VEGF genes expression, histopathology and immune-expression of caspase-3, Beclin-1, CD49b and Ki-67 were all analyzed. Our results showed that ST-OMVs significantly decreased tumor volume, significantly increased tumor growth inhibition rate, up-regulated the immunohistochemical expression of caspase-3, Beclin-1, and CD49b (enhanced recruitment of NK cells). Furthermore, ST-OMVs down-regulated the expression of Ki-67, increased Bax gene expression and decreased VEGF gene expression as detected by qRT-PCR analysis. Histologically, ST-OMVs promoted apoptosis, decreased tumor invasion and mitotic activities. Moreover, ST-OMVs showed a remarkable cytotoxic activity in various investigated in vitro cancer cell lines. Our findings demonstrate potential antitumor activity of ST-OMVs that might be used as a promising safe antitumor immunotherapy or an adjuvant to conventional chemotherapeutic drugs, resolving some of their problems., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A water soluble polysaccharide (PGPS) with molecular weight ~ 1.4 × 10 5  Da was isolated by alkali treatment from an edible mushroom Polyporus grammocephalus and purified by gel chromatography using sepharose-6B column. Monosaccharide analysis revealed that PGPS was made up of glucose only. PGPS contained (1 → 3)-α-D-Glcp and (1 → 4)-α-D-Glcp moieties in a molar ratio of nearly 1:2. Through a series of chemical and spectroscopic (1D/2D NMR) investigations, the repeating unit of the glucan was established as: →3)-α-D-Glcp(1 → [4)-α-D-Glcp(1] 2 → This α-glucan was observed to stimulate some prime components of immune system, namely, macrophages, splenocytes, and thymocytes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Structural studies of a Salmonella Typhimurium flagellin protein indicated that four polar or charged C-terminal amino acid residues line the inner channel of the flagellum. The hydrophilic character of these putative channel-lining residues was predicted to be essential to facilitate the transport of unfolded flagellin monomers during flagellar assembly. The structure-function relationship of these putative channel-lining residues was investigated by site-directed mutagenesis to examine effects of side chain polarity and size on flagella assembly and function., Methods: Channel-lining residue variants were generated using site-directed mutagenesis to substitute alanine and other residues to examine the effects of altered side-chain polarity on export and assembly. The export, in vivo motility function, and flagellar structure of variants was characterized by agar motility, video microscopy, immunofluorescence, and SDS-PAGE., Results: Alanine substitution yielded decreased motility and flagellar assembly for three of the four residues. However, alanine substitution of residue Arg 494 did not alter export, although substitution with negatively charged glutamate decreased motility and flagellar filament length. Furthermore, many of the C-terminal mutations affected flagellar filament morphology and stability, often resulting in more tightly coiled and/or more brittle flagella than the wild type., Conclusions: The four channel-lining C-terminal residues may facilitate monomer protein transport but also have structural roles in determining the stability and morphology of the flagellum., General Significance: These results provide further insight into the complex process of bacterial flagellin export and flagellar assembly and provide evidence of previously unknown structural functions for the four putative channel-lining residues., (Copyright © 2020 Elsevier B.V. All rights reserved.)

One of the central systems responsible for bacterial motility is the flagellum. The bacterial flagellum is a macromolecular protein complex that is more than five times the cell length. Flagella-driven motility is coordinated via a chemosensory signal transduction pathway, and so bacterial cells sense changes in the environment and migrate towards more desirable locations. The flagellum of Salmonella enterica serovar Typhimurium is composed of a bi-directional rotary motor, a universal joint and a helical propeller. The flagellar motor, which structurally resembles an artificial motor, is embedded within the cell envelop and spins at several hundred revolutions per second. In contrast to an artificial motor, the energy utilized for high-speed flagellar motor rotation is the inward-directed proton flow through a transmembrane proton channel of the stator unit of the flagellar motor. The flagellar motor realizes efficient chemotaxis while performing high-speed movement by an ingenious directional switching mechanism of the motor rotation. To build the universal joint and helical propeller structures outside the cell body, the flagellar motor contains its own protein transporter called a type III protein export apparatus. In this chapter we summarize the structure and assembly of the Salmonella flagellar motor complex.

Conjugation chemistry is one of the main parameters affecting immunogenicity of glycoconjugate vaccines and a rational approach toward a deeper understanding of their mechanism of action will greatly benefit from highly-defined and well-characterized structures. Herein, different conjugation methods were investigated with the aim of controlling glycosylation site and glycosylation density on the carrier protein. S. Typhimurium lipopolysaccharide O-Antigen and CRM 197 carrier protein were used as models. In particular, thiol and click chemistry were examined, both involving the linkage of the terminal reducing sugar unit of the O-Antigen chain to different amino acids on the carrier protein. Thiol chemistry allowed O-Antigen conjugation only when the carrier protein was activated on the lysines and with a relative high number of linkers, while click chemistry allowed conjugate generation even when just one position on the protein was activated and to both lysine and tyrosine sites. The study highlights click chemistry as a leading approach for the synthesis of well-defined glycoconjugates, useful to investigate the relationship between conjugate design and immune response.

AgsA (aggregation-suppressing protein) is an ATP-independent molecular chaperone machine belonging to the family of small heat shock proteins (sHSP), and it can prevent the aggregation of non-natural proteins. However, the substrate-binding site of AgsA and the functional unit that captures and binds the substrate remain unknown. In this study, different N-terminal and C-terminal deletion mutants of AgsA were constructed and their effects on AgsA oligomer assembly and chaperone activity were investigated. We found that the IXI motif at the C-terminus and the α-helix at the N-terminus affected the oligomerization and molecular chaperone activity of AgsA. In this work, we obtained a 6.8 Å resolution structure of AgsA using Electron cryo-microscopy (cryo-EM), and found that the functional form of AgsA was an 18-mer with D3 symmetry. Through amino acid mutations, disulfide bonds were introduced into two oligomeric interfaces, namely dimeric interface and non-partner interface. Under oxidation and reduction conditions, the chaperone activity of the disulfide-bonded AgsA did not change significantly, indicating that AgsA would not dissociate to achieve chaperone activity. Therefore, we concluded that the oligomer, especially 18-mer, was the primary functional unit., Competing Interests: Declaration of competing interest There is no conflict of interest in this manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cost-effectiveness is an important issue in biotechnological manufacturing industry and using alternative cheap materials with the same benefits has been noticed in most literatures. Isopropyl β-d-1-thiogalactopyranoside (IPTG), a well-known chemical element for induction of protein expression, has several disadvantages such as high expense and toxicity. In this study, we aimed to introduce skimmed milk as an alternative material for protein expression by induction of lac operon. In this way, Escherichia coli BL21 (DE3) bacteria were induced using 1 mM IPTG or 1.0% (w/v) skimmed milk. Protein purification was performed using Ni-NTA (nickel-nitrilotriacetic acid) for His-tagged recombinant proteins and protein purity was evaluated by SDS-PAGE. Results showed high level of recombinant protein expression using skimmed milk, and interestingly, the growth rate of bacteria improved. Our findings suggested that skimmed milk can be a suitable alternative for induction of recombinant protein expression, which has advantages such as more availability and affordability, in comparison to IPTG supplementation., Competing Interests: Declaration of competing interest Authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

An aptamer-based sensitive method was developed here for detection of multiple foodborne pathogens in food matrix by surface-enhanced Raman scattering (SERS) technology. Polydimethylsiloxane (PDMS) film was first prepared and then coated with gold nanoparticles (AuNP) to act as an active substrate for the enhancement of Raman scattering. The as-prepared Au-PDMS film was functionalized with specific pathogen aptamers (Apt) to capture the targets. In addition, aptamers functionalized AuNP integrated with Raman reporters (4-Mercaptobenzoic acid (4-MBA)/Nile blue A (NBA)) were fabricated as pathogen-specific SERS probes. In this scheme, pathogens were first captured by Apt-Au-PDMS film and then bind with SERS probes to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens. With Vibrio parahaemolyticus and Salmonella typhimurium as model targets, this protocol can selectively detect 18 cfu/mL and 27 cfu/mL, respectively. Furthermore, this platform can be successfully applied to detect pathogens in seafood samples with recoveries ranging from 82.9% to 95.1%., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Salmonella enterica subsp. enterica serovar Typhimurium PhoPQ two-component system is activated within the intracellular phagosome environment, where it promotes remodeling of the outer membrane and resistance to innate immune antimicrobial peptides. Maintenance of the PhoPQ-regulated outer membrane barrier requires PbgA, an inner membrane protein with a transmembrane domain essential for growth, and a periplasmic domain required for PhoPQ-activated increases in outer membrane cardiolipin. Here, we report the crystal structure of cardiolipin-bound PbgA, adopting a novel transmembrane fold that features a cardiolipin binding site in close proximity to a long and deep cleft spanning the lipid bilayer. The end of the cleft extends into the periplasmic domain of the protein, which is structurally coupled to the transmembrane domain via a functionally critical C-terminal helix. In conjunction with a conserved putative catalytic dyad situated at the middle of the cleft, our structural and mutational analyses suggest that PbgA is a multifunction membrane protein that mediates cardiolipin transport, a function essential for growth, and perhaps catalysis of an unknown enzymatic reaction. IMPORTANCE Gram-negative bacteria cause many types of infections and have become increasingly resistant to available antibiotic drugs. The outer membrane serves as an important barrier that protects bacteria against antibiotics and other toxic compounds. This outer membrane barrier function is regulated when bacteria are in host environments, and the protein PbgA contributes significantly to this increased barrier function by transporting cardiolipin to the outer membrane. We determined the crystal structure of PbgA in complex with cardiolipin and propose a model for its function. Knowledge of the mechanisms of outer membrane assembly and integrity can greatly contribute to the development of new and effective antibiotics, and this structural information may be useful in this regard., (Copyright © 2020 Fan et al.)

The bacterial flagellum is a motility organelle consisting of a long helical filament as a propeller and a rotary motor that drives rapid filament rotation to produce thrust. Salmonella enterica serovar Typhimurium has two genes of flagellin, fljB and fliC , for flagellar filament formation and autonomously switches their expression at a frequency of 10 -3 -10 -4 per cell per generation. We report here differences in their structures and motility functions under high-viscosity conditions. A Salmonella strain expressing FljB showed a higher motility than one expressing FliC under high viscosity. To examine the reasons for this motility difference, we carried out structural analyses of the FljB filament by electron cryomicroscopy and found that the structure was nearly identical to that of the FliC filament except for the position and orientation of the outermost domain D3 of flagellin. The density of domain D3 was much lower in FljB than FliC, suggesting that domain D3 of FljB is more flexible and mobile than that of FliC. These differences suggest that domain D3 plays an important role not only in changing antigenicity of the filament but also in optimizing motility function of the filament as a propeller under different conditions.

During serial transplantation of bone marrow derived from young and aged donor CBA mice to 5-month-old recipients, the counts of multipotent stromal cells (MSC) in transplants from young donors assessed at each passage surpassed those of aged donors by 3.2, 7.8, 3.0, and 2.2 times attesting to the age-related decrease of active pool of bone marrow MSC. The medullary curettage in mouse femur increased the total number of MSC and the number of osteogenic MSC both in the contralateral femur and in the bone marrow transplants attesting to spread of the effects of osteogenic factors after bone injury onto the bone tissue of the body even if this tissue if not topographically related to the skeleton. Combined and simultaneous administration of antigenic complex of S. typhimurium (or LPS) with BMP-2 markedly increased the count of osteogenic medullary MSC by 3.6 or 4.6 times in comparison with intact control or by 2.1 and 2.7 times in comparison with administration of BMP-2 alone, which probably resulted from enlargement of the pool of osteogenesis-inducible MSC due to inflammation. Addition of BMP-2 to the culture of splenic stromal cells where osteogenesis does not occur under normal conditions provoked appearance of MSC colonies with alkaline phosphatase activity attesting to involvement of inducible osteogenic MSC in vascular calcification. It can be hypothesized that the reaction to the age-related changes in the bone tissue and osteoporosis is similar to the reaction to bone marrow injury and includes initiation of systemic inflammation and elevation of blood BMP-2, both of which are prerequisite for vascular calcification.

Salmonella is a facultative intracellular pathogen that invades epithelial cells of the intestine using the SPI-1 Type 3 secretion System (T3SS). Insertion of the SPI-1 T3SS translocon is facilitated by acylation of the translocator SipB, which involves a protein-protein interaction with the acyl carrier protein IacP. Using nuclear magnetic resonance and biological tests, we identified the residues of IacP that are involved in the interaction with SipB. Our results suggest that the 4'-phosphopantetheine group that functionalizes IacP participates in the interaction. Its solvent exposition may rely on two residues highly conserved in acyl carrier proteins associated with T3SS. This study is the first to address the specificity of acyl carrier proteins associated with T3SS., (© 2019 Federation of European Biochemical Societies.)

Among the Gram-negative bacterial secretion systems, type III secretion systems (T3SS) possess a unique extracellular molecular apparatus called the needle. This macromolecular protein assembly is a nanometre-size filament formed by the helical arrangement of hundreds of copies of a single, small protein, which is highly conserved between T3SSs from animal to plant bacterial pathogens. The needle filament forms a hollow tube with a channel ~20 Å in diameter that serves as a conduit for proteins secreted into the targeted host cell. In the past ten years, technical breakthroughs in biophysical techniques such as cryo-electron microscopy (cryo-EM) and solid-state NMR (SSNMR) spectroscopy have uncovered atomic resolution details about the T3SS needle assembly. Several high-resolution structures of Salmonella typhimurium and Shigella flexneri T3SS needles have been reported demonstrating a common structural fold. These structural models have been used to explain the active role of the needle in transmitting the host-cell contact signal from the tip to the base of the T3SS through conformational changes as well as during the injection of effector proteins. In this chapter, we summarize the current knowledge about the structure and the role of the T3SS needle during T3SS assembly and effector secretion.

Background: Colon cancer is one of the major causes of morbidity and mortality worldwide. Cycle inhibiting factors (Cifs) have been shown to deamidate Nedd8, resulting in cell cycle arrest., Objective: To determine the antitumor effect of Cifs on colon cancer by using attenuated Salmonella typhimurium VNP20009., Methods: The VNP-SOPE2-cif and VNP-SOPE2-cif-C/A plasmids were transfected into attenuated Salmonella typhimurium VNP20009. The efficiency and specificity of the Cif promoter were validated in colon cancer SW480 cell lines. Western blotting was subsequently performed to evaluate cell cycle regulators, including P21, P27 and Wee1. In vivo, the antitumor effect of VNP20009 was evaluated in a colon cancer xenograft model., Results: Firstly, VNP-SOPE2-cif and VNP-SOPE2-cif-C/A were selectively expressed both in the bacterial and colon cancer cells. Cif expression in SW480 cells via the VNP tumor-targeted expression system induced the accumulation of Wee1, p21 and p27 expression. Moreover, tumor growth was significantly inhibited in the mice with VNP-SOPE2-cif compared to the mice with VNP with the empty construct., Conclusion: These results suggest that Cif gene delivered by VNP20009 is a promising approach for the treatment of colon cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Aims: To investigate the effects of ohmic heating (OH) and water bath heating (WB) on the membrane permeability, membrane structure, intracellular organization and leakage of intracellular substances of Escherichia coli O157:H7 at the same inactivation level and at a heating temperature of 72°C., Methods and Results: Flow cytometry analysis indicated that membrane permeability of E. coli O157:H7 by OH was comparable to WB at 72°C. Scanning electron microscopy analysis showed that the OH-treated E. coli O157:H7 had greater morphological changes than those of WB-treated ones both at the same inactivation level and the same heating temperature. Transmission electron microscopy analysis showed that both OH and WB caused severe damage on the intracellular organization of E. coli O157:H7 at 72°C. Moreover, OH-treated E. coli O157:H7 had more leakage of intracellular substances than those treated with WB due to the electroporation caused by OH., Conclusion: OH presents considerable potential in inactivation of E. coli O157:H7, especially OH at 10 V cm -1 with a much shorter heating time., Significance and Impact of the Study: The nonthermal effect of OH had a greater effect on the cell membrane of E. coli O157:H7, resulting in more pores and more leakage of intracellular substances out of the E. coli O157:H7 cells. These results could promote the application of OH in food processing., (© 2019 The Society for Applied Microbiology.)

The bacterial injectisome is a syringe-shaped macromolecular nanomachine utilized by many pathogenic Gram-negative bacteria, including the causative agents of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. Bacterial proteins destined for self-assembly and host-cell targeting are translocated by the injectisome in a process known as type III secretion (T3S). The core structure is the ~4 MDa needle complex (NC), built on a foundation of three highly oligomerized ring-forming proteins that create a hollow scaffold spanning the bacterial inner membrane (IM) (24-mer ring-forming proteins PrgH and PrgK in the Salmonella enterica serovar Typhimurium Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS)) and outer membrane (OM) (15-mer InvG, a member of the broadly conserved secretin pore family). An internalized helical needle projects from the NC and bacterium, ultimately forming a continuous passage to the host, for delivery of virulence effectors. Here, we have captured snapshots of the entire prototypical SPI-1 NC in four distinct needle assembly states, including near-atomic resolution, and local reconstructions in the absence and presence of the needle. These structures reveal the precise localization and molecular interactions of the internalized SpaPQR 'export apparatus' complex, which is intimately encapsulated and stabilized within the IM rings in the manner of a nanodisc, and to which the PrgJ rod directly binds and functions as an initiator and anchor of needle polymerization. We also describe the molecular details of the extensive and continuous coupling interface between the OM secretin and IM rings, which is remarkably facilitated by a localized 16-mer stoichiometry in the periplasmic-most coupling domain of the otherwise 15-mer InvG oligomer.

Salmonella enterica serovar typhimurium (S. typhimurium) causes food poisoning in human and animals. Its infection rate is the highest among all salmonella serotypes. Metabolomics is a potential way to study the pathogenesis of S. typhimurium via analysis of various small molecular substances. Due to the lack of a uniform protocol for the extraction of metabolites, we evaluated five commonly used extraction methods including cold methanol (CM), hot ethanol (HE), chloroform-methanol cocktail (CMC), perchloric acid (PCA), and alkali (AL) for their efficacy in extracting the intracellular metabolites of S. typhimurium. Samples were quenched in 60% methanol at - 40 °C, and then the five methods were used to extract the metabolites. After derivatization, all samples were analyzed on a gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS). Our results suggest that CM and HE extraction methods provide the best compromise allowing identification of 98 and 95 metabolites in a single analysis. For targeted metabolome analysis, the optimal extraction method for alcohols and organic acids is HE. CMC preferentially extracted lipid metabolites. PCA is suitable for extraction of small molecular carbohydrates. The optimal extraction method for macromolecular carbohydrates is the CM method.

Previously reported photothermal strip test methods generally used a membrane as the antibody carrier and a thermal imaging camera or sensor as detector. To further simplify the detection device, a modified mercury head of the glass thermometer can act as the antibody carrier. Meanwhile, the temperature variation signal (ΔT) generated by the photothermal effect of labeled nanomaterials can be detected by the thermometer directly. Thus, the antibody carrier and detector can be integrated on a portable and cheap thermometer, which greatly simplified the device and detection steps of the photothermal method. The excellent photothermal effect of graphene oxides was used to improve the detection sensitivity. The main parameters of the performance of immune-thermometer assay were optimized and the Salmonella typhimurium was chosen as the representative target. Under the optimized conditions, the ΔT and the different number of Salmonella typhimurium were plotted to establish the standard curve. The detection limit was estimated to be 10 3  CFU·mL -1 . The entire detection operation was consistently finished in 15 min. Overall, the proposed immune-thermometer exhibited good precision, selectivity and acceptable stability. The immune-thermometer assay was also successfully implemented and validated in different foodstuffs, which showed that it can be used as a novel and promising technique for rapid, simple and on-site screening of hazards in food, biological, clinical or environmental samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

An aptamer-based assay is described for the determination of Salmonella typhimurium (S. typh). Carboxymethyl chitosan was loaded with amino-modified aptamer against S. typh, and then adsorbed on gold nanoparticles by electrostatic interaction to form a composite that acts as the molecular recognition element. In the presence of S. typh, it will be bound by the aptamer, and this changes the structure of the recognition element. On addition of salt solution, the gold nanoparticles agglomerate so that the color of the solution changes from red to blue. S. typh can be detected via measurement of the absorbance at 550 nm. Absorbance increases linearly with the logarithm of the S. typh concentration in the range from 100 to 10 9  cfu·mL -1 . The limit of detection is 16 cfu·mL -1 . The specificity and practicability of the assay were evaluated. The recoveries of S. typh from spiked milk samples are between 92.4 and 97.2%. The analytical results are basically consistent with those of a plate counting method. Graphical abstract Schematic representation of the colorimetric assay for Salmonella typhimuium (S. typh) using carboxymethyl chitosan (CMCS)-aptamer (Apt)-gold nanoparticles (AuNPs) composites.

Bacterial locomotion by rotating flagella is achieved through the hook, which transmits torque from the motor to the filament. The hook is a tubular structure composed of a single type of protein, yet it adopts a curved shape. To perform its function, it must be simultaneously flexible and torsionally rigid. The molecular mechanism by which chemically identical subunits form such a dynamic structure is unknown. Here, we show the complete structure of the hook from Salmonella enterica in its supercoiled 'curved' state, at 2.9 Å resolution. Subunits in the curved hook are grouped into 11 distinctive conformations, each shared along 11 protofilaments. The domains of the elongated hook subunit behave as rigid bodies connected by two hinge regions. The reconstituted model demonstrates how identical subunits can dynamically change conformation by physical interactions while bending. These multiple subunit states contradict the two-state model, which is a key feature of flagellar polymorphism.

Type III secretion injectisomes are essential virulence factors for many pathogenic bacteria by mediating the transport of effector proteins into eukaryotic host cells. The secretion conduit of injectisomes is formed by a helical assembly of three hydrophobic proteins (SctR, SctS and SctT), an inner rod (SctI) and a needle filament (SctF). SctI is thought to play a role in switching between the secretion of different substrate classes and assembly of the inner rod has been implicated in regulating the length of the needle filament. While high-resolution structures of the hydrophobic components and of the needle filament have been solved, little is known about the structure and the assembly of the inner rod, which impedes the deeper assessment of its function. Here we show by exhaustive in vivo photocrosslinking that SctI engages in extensive interactions with SctR and SctT throughout its entire length. Our data imply that the inner rod serves as an adapter between the export apparatus and the needle filament by forming one helical turn. We show that assembly of the inner rod does not play a role in needle length control nor in substrate specificity switching. Instead, our findings imply that inner rod assembly must precede assembly of the needle filament., (© 2019 John Wiley & Sons Ltd.)

Lipopolysaccharide (LPS) is one of the major components in the outer membrane of Gram-negative bacteria. However, its heterogeneity and variability in different bacteria and differentiation conditions make it difficult to extract all of the structural variants. We designed a solution to improve quality and biological activity of LPS extracted from various bacteria with different types of LPS, as compared to conventional methods. We introduced a quality index as a simple measure of LPS purity in terms of a degree of polysaccharide content detected by absorbance at 204 nm. Further experiments using gel electrophoresis, endotoxin test, and macrophage activation test were performed to evaluate the performance and reliability of a proposed 'T-sol' method and the biological effectiveness and character of the LPS products. We presented that the T-sol method had differential effects on extraction of a RAW 264.7 cell-activating LPS, which was effective in the macrophage activation with similar effects in stimulating the production of TNF-alpha. In conclusion, the T-sol method provides a simple way to improve quality and biological activity of LPS with high yield.

Background: Engineered vaccine proteins incorporating both antigen and adjuvant components are constructed with the aim of combining functions to induce effective protective immunity. Bacterial flagellin is a strong candidate for an engineered vaccine scaffold as it is known to provide adjuvant activity through its TLR5 and inflammasome activation. Moreover, polymerized flagellin filaments can elicit a more robust immunoglobulin response than monomeric flagellin, and the multimeric antigen form can also promote T cell-independent antibody responses. Here, we aim to produce and test a covalently stabilized polymerized flagellar filament, providing additional immune efficacy through stabilization of its polymeric filament structure, as well as stabilization for long-term storage., Results: Computational modeling of monomer packing in flagellin filaments helped identify amino acids with proximity to neighboring flagella protofilaments. Paired cysteine substitutions were made at amino acids predicted to form inter-monomer disulfide cross-links, and these substitutions were capable of forming flagella when transfected into a flagellin-negative strain of Salmonella enterica subspecies Typhimurium. Interestingly, each paired substitution stabilized different helical conformational polymorphisms; the stabilized filaments lost the ability to transition between conformations, reducing bacterial motility. More importantly, the paired substitutions enabled extensive disulfide cross links and intra-filament multimer formation, and in one of the three variants, permitted filament stability in high acidic and temperature conditions where wild-type filaments would normally rapidly depolymerize. In addition, with regard to potential adjuvant activity, all crosslinked flagella filaments were able to induce wild-type levels of epithelial NF-κB in a cell reporter system. Finally, bacterial virulence was unimpaired in epithelial adherence and invasion, and the cysteine substitutions also appeared to increase bacterial resistance to oxidizing and reducing conditions., Conclusions: We identified amino acid pairs, with cysteine substitutions, were able to form intermolecular disulfide bonds that stabilized the resulting flagellar filaments in detergent, hydrochloric acid, and high temperatures while retaining its immunostimulatory function. Flagellar filaments with disulfide-stabilized protofilaments introduce new possibilities for the application of flagella as a vaccine adjuvant. Specifically, increased stability and heat tolerance permits long-term storage in a range of temperature environments, as well as delivery under a range of clinical conditions.

One of the strategies adopted for the development of a bivalent conjugate vaccine against invasive nontyphoidal Salmonella consists of linking the O-antigen component of S. Typhimurium and S. Entertidis lipopolysaccharides to the carrier protein CRM 197 , a non-toxic variant of diphtheria toxin. The conjugation reaction uses the reducing end residue 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core to which the O-antigen chain is bound (OAg-core). OAg-core chains are cleaved from the lipid A directly in the fermentation broth by mild acid treatment. Kdo has been reported to undergo structural changes under these conditions and therefore the Kdo at the reducing end was thoroughly analysed to verify its structural integrity. For this purpose, low molecular mass OAg-core chains extracted from S. Typhimurium wild type bacteria and core oligosaccharides extracted from S. Typhimurium bacteria mutated not to produce O-antigen repeats were characterized by GLC-MS, MALDI-TOF-MS and NMR spectroscopy. Moreover, a combination of 1 H- 1 H and 1 H- 13 C experiments confirmed the linkage positions, sequence and structure of the octasaccharide core with 5-linked Kdo present at the reducing end in its native structure: α-GlcpNAc-(1→2)-α-Glcp-(1→2)-α-Galp-(1→3)-[α-Galp-(1→6)]-α-Glcp-(1→3)-[α-Hepp-(1→7)]-α-Hepp-(1→3)-α-Hepp-(1→5)-Kdo., (Copyright © 2019 Elsevier Ltd. All rights reserved.)