A widely conserved protein Rof inhibits transcription termination factor Rho and promotes Salmonella virulence program.

Transcription is crucial for the expression of genetic information and its efficient and accurate termination is required for all living organisms. Rho-dependent termination could rapidly terminate unwanted premature RNAs and play important roles in bacterial adaptation to changing environments. Although Rho has been discovered for about five decades, the regulation mechanisms of Rho-dependent termination are still not fully elucidated. Here we report that Rof is a conserved antiterminator and determine the cryogenic electron microscopy structure of Rho-Rof antitermination complex. Rof binds to the open-ring Rho hexamer and inhibits the initiation of Rho-dependent termination. Rof's N-terminal α-helix undergoes conformational changes upon binding with Rho, and is key in facilitating Rof-Rho interactions. Rof binds to Rho's primary binding site (PBS) and excludes Rho from binding with PBS ligand RNA at the initiation step. Further in vivo analyses in Salmonella Typhimurium show that Rof is required for virulence gene expression and host cell invasion, unveiling a physiological function of Rof and transcription termination in bacterial pathogenesis.

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species.

RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Gram-positive and Gram-negative bacteria. Suitable HBC-Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30-tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.

It takes two to attach - endo-1,3-ß-d-glucanase as a potential receptor of mannose-independent, FimH-dependent Salmonella Typhimurium binding to spinach leaves.

Currently, fresh, unprocessed food has become a relevant element of the chain of transmission of enteropathogenic infections. To survive on a plant surface and further spread the infections, pathogens like Salmonella have to attach stably to the leaf surface. Adhesion, driven by various virulence factors, including the most abundant fim operon encoding type 1 fimbriae, is usually an initial step of infection, preventing physical removal of the pathogen. Adhesion properties of Salmonella's type 1 fimbriae and its FimH adhesin were investigated intensively in the past. However, there is a lack of knowledge regarding its role in interaction with plant cells. Understanding the mechanisms and structures involved in such interaction may facilitate efforts to decrease the risk of contamination and increase fresh food safety. Here, we applied Salmonella genome site-directed mutagenesis, adhesion assays, protein-protein interactions, and biophysics methods based on surface plasmon resonance to unravel the role of FimH adhesin in interaction with spinach leaves. We show that FimH is at least partially responsible for Salmonella binding to spinach leaves, and this interaction occurs in a mannose-independent manner. Importantly, we identified a potential FimH receptor as endo-1,3-ß-d-Glucanase and found that this interaction is strong and specific, with a dissociation constant in the nanomolar range. This research advances our comprehension of Salmonella's interactions with plant surfaces, offering insights that can aid in minimizing contamination risks and improving the safety of fresh, unprocessed foods.

Antibacterial efficiency of apple vinegar marination on beef-borne Salmonella.

Background: Salmonella-related foodborne illnesses are a significant public health concern. Naturally, antibacterial food components have been shown to limit microbial growth proliferation with various degrees of efficacy. Aims: To examine the occurrence, microbial load, and effect of apple vinegar on Salmonella serovars in beef and beef products. Methods: 150 beef and beef products were collected between March and May 2022. Total viable count (TVC), Enterobacteriaceae count (ENT), isolation and identification of Salmonella, and their virulence factors detection by multiplex PCR were determined, and an experimental study of the effect of natural apple vinegar marination on Salmonella spp. Results: TVC was higher in meatballs (3.32 × 106 ± 1.07 × 106) while beef burgers (4.22 × 103 ± 0.71 × 103) had the highest ENT. Concerning the prevalence of Salmonella spp., meatball (46.7%) and beef burger (25.3%) samples were the highest contamination rate. The common serovars detected were Salmonella typhimurium (6%), Salmonella enteritidis (6%), and Salmonella infantis (4%). Based on the results of PCR, 12, 11, and 11 out of 18 samples of Salmonella isolates possess hila, stn, and invA genes. By immersing the inoculated steak meat in apple vinegar at different concentrations (50%, 70%, and 100%), the initial populations of the Salmonella strains after 12 hours were reduced to 0.38 × 102 ± 0.05 × 102 log CFU/ml; however, after 48 hours become the most reduction (0.31 × 102 ± 0.07 × 102 log CFU/ml) at a concentration of 100% apple vinegar. An enhancement in the sensory attributes was noted across all concentrations. Conclusion: The consumed beef and beef products are contaminated with pathogenic bacteria such as Salmonella spp. Marinades made using apple vinegar concentrations of 50%, 75%, and 100% effectively minimized the prevalence of artificially inoculated Salmonella and extended the shelf life of preserved refrigerated beef products to 48 hours.

A genome-wide collection of barcoded single-gene deletion mutants in Salmonella enterica serovar Typhimurium.

Genetic screening of pools of mutants can reveal genetic determinants involved in complex biological interactions, processes, and systems. We previously constructed two single-gene deletion resources for Salmonella enterica serovar Typhimurium 14028s in which kanamycin (KanR) and chloramphenicol (CamR) cassettes were used to replace non-essential genes. We have now used lambda-red recombination to convert the antibiotic cassettes in these resources into a tetracycline-resistant (TetR) version where each mutant contains a different 21-base barcode flanked by Illumina Read1 and Read2 primer sequences. A motility assay of a pool of the entire library, followed by a single-tube processing of the bacterial pellet, PCR, and sequencing, was used to verify the performance of the barcoded TetR collection. The new resource is useful for experiments with defined subsets of barcoded mutant strains where biological bottlenecks preclude high numbers of founder bacteria, such as in animal infections. The TetR version of the library will also facilitate the construction of triple mutants by transduction. The resource of 6197 mutants covering 3490 genes is deposited at Biological and Emerging Infections Resources (beiresources.org).

The interaction of InvF-RNAP is mediated by the chaperone SicA in Salmonella sp: an in silico prediction.

In this work we carried out an in silico analysis to understand the interaction between InvF-SicA and RNAP in the bacterium Salmonella Typhimurium strain LT2. Structural analysis of InvF allowed the identification of three possible potential cavities for interaction with SicA. This interaction could occur with the structural motif known as tetratricopeptide repeat (TPR) 1 and 2 in the two cavities located in the interface of the InvF and α-CTD of RNAP. Indeed, molecular dynamics simulations showed that SicA stabilizes the Helix-turn-Helix DNA-binding motifs, i.e., maintaining their proper conformation, mainly in the DNA Binding Domain (DBD). Finally, to evaluate the role of amino acids that contribute to protein-protein affinity, an alanine scanning mutagenesis approach, indicated that R177 and R181, located in the DBD motif, caused the greatest changes in binding affinity with α-CTD, suggesting a central role in the stabilization of the complex. However, it seems that the N-terminal region also plays a key role in the protein-protein interaction, especially the amino acid R40, since we observed conformational flexibility in this region allowing it to interact with interface residues. We consider that this analysis opens the possibility to validate experimentally the amino acids involved in protein-protein interactions and explore other regulatory complexes where chaperones are involved.

Outbreak of Salmonella Typhimurium linked to Swedish pre-washed rocket salad, Sweden, September to November 2022.

In September 2022, the Public Health Agency of Sweden observed an increase in domestic Salmonella Typhimurium cases through the Swedish electronic notification system, and an outbreak strain was identified with whole genome sequencing. Overall, 109 cases with symptom onset between 17 September and 24 November 2022 were reported from 20 of 21 Swedish regions. The median age of cases was 52 years (range 4-87 years) and 62% were female. A case-control study found cases to be associated with consumption of rocket salad (adjusted odds ratio (aOR) = 4.9; 95% confidence interval (CI): 2.4-10, p value < 0.001) and bagged mixed salad (aOR = 4.0; 95% CI: 1.9-8.1, p value < 0.001). Trace-back, supported by Finnish authorities who identified the Swedish outbreak strain in a Finnish cluster during the same time period, identified rocket salad, cultivated, pre-washed and pre-packed in Sweden as the likely source of the outbreak. No microbiological analyses of rocket salad were performed. Our investigation indicates that bagged leafy greens such as rocket salad, regardless of pre-washing procedures in the production chain, may contain Salmonella and cause outbreaks, posing a health risk to consumers. We emphasise the need for primary producers of leafy greens to identify possible contamination points to prevent outbreaks.

Deciphering the Interrelationship of arnT Involved in Lipid-A Alteration with the Virulence of Salmonella Typhimurium.

The lipopolysaccharide (LPS) that resides on the outermost surface and protects Gram-negative bacteria from host defenses is one of the key components leading to Salmonella infection, particularly the endotoxic lipid A domain of LPS. Lipid A modifications have been associated with several genes such as the arnT that encodes 4-amino-4-deoxy-L-arabinose transferase, which can be critical for bacteria to resist cationic antimicrobial peptides and interfere with host immune recognition. However, the association of arnT with virulence is not completely understood. Thus, this study aimed to elucidate the interrelationship of the major lipid A modification gene arnT with Salmonella Typhimurium virulence. We observed that the arnT-deficient S. Typhimurium (JOL2943), compared to the wild type (JOL401), displayed a significant decrease in several virulence phenotypes such as polymyxin B resistance, intracellular survival, swarming, and biofilm and extracellular polymeric substance (EPS) production. Interestingly, the cell-surface hydrophobicity, adhesion, and invasion characteristics remained unaffected. Additionally, LPS isolated from the mutant induced notably lower levels of endotoxicity-related cytokines in RAW and Hela cells and mice, particularly IL-1ß with a nine-fold decrease, than WT. In terms of in vivo colonization, JOL2943 showed diminished presence in internal organs such as the spleen and liver by more than 60%, while ileal infectivity remained similar to JOL401. Overall, the arnT deletion rendered the strain less virulent, with low endotoxicity, maintained gut infectivity, and reduced colonization in internal organs. With these ideal characteristics, it can be further explored as a potential attenuated Salmonella strain for therapeutics or vaccine delivery systems.

O-Antigen decorations in Salmonella enterica play a key role in eliciting functional immune responses against heterologous serovars in animal models.

Introduction: Different serovars of Salmonella enterica cause systemic diseases in humans including enteric fever, caused by S. Typhi and S. Paratyphi A, and invasive nontyphoidal salmonellosis (iNTS), caused mainly by S. Typhimurium and S. Enteritidis. No vaccines are yet available against paratyphoid fever and iNTS but different strategies, based on the immunodominant O-Antigen component of the lipopolysaccharide, are currently being tested. The O-Antigens of S. enterica serovars share structural features including the backbone comprising mannose, rhamnose and galactose as well as further modifications such as O-acetylation and glucosylation. The importance of these O-Antigen decorations for the induced immunogenicity and cross-reactivity has been poorly characterized. Methods: These immunological aspects were investigated in this study using Generalized Modules for Membrane Antigens (GMMA) as delivery systems for the different O-Antigen variants. This platform allowed the rapid generation and in vivo testing of defined and controlled polysaccharide structures through genetic manipulation of the O-Antigen biosynthetic genes. Results: Results from mice and rabbit immunization experiments highlighted the important role played by secondary O-Antigen decorations in the induced immunogenicity. Moreover, molecular modeling of O-Antigen conformations corroborated the likelihood of cross-protection between S. enterica serovars. Discussion: Such results, if confirmed in humans, could have a great impact on the design of a simplified vaccine composition able to maximize functional immune responses against clinically relevant Salmonella enterica serovars.

Succinate utilisation by Salmonella is inhibited by multiple regulatory systems.

Succinate is a potent immune signalling molecule that is present in the mammalian gut and within macrophages. Both of these infection niches are colonised by the pathogenic bacterium Salmonella enterica serovar Typhimurium during infection. Succinate is a C4-dicarboyxlate that can serve as a source of carbon for bacteria. When succinate is provided as the sole carbon source for in vitro cultivation, Salmonella and other enteric bacteria exhibit a slow growth rate and a long lag phase. This growth inhibition phenomenon was known to involve the sigma factor RpoS, but the genetic basis of the repression of bacterial succinate utilisation was poorly understood. Here, we use an experimental evolution approach to isolate fast-growing mutants during growth of S. Typhimurium on succinate containing minimal medium. Our approach reveals novel RpoS-independent systems that inhibit succinate utilisation. The CspC RNA binding protein restricts succinate utilisation, an inhibition that is antagonised by high levels of the small regulatory RNA (sRNA) OxyS. We discovered that the Fe-S cluster regulatory protein IscR inhibits succinate utilisation by repressing the C4-dicarboyxlate transporter DctA. Furthermore, the ribose operon repressor RbsR is required for the complete RpoS-driven repression of succinate utilisation, suggesting a novel mechanism of RpoS regulation. Our discoveries shed light on the redundant regulatory systems that tightly regulate the utilisation of succinate. We speculate that the control of central carbon metabolism by multiple regulatory systems in Salmonella governs the infection niche-specific utilisation of succinate.

Aptamer-mediated double strand displacement amplification with microchip electrophoresis for ultrasensitive detection of Salmonella typhimurium.

Rapid and quantitative detection of foodborne bacteria is of great significance to public health. In this work, an aptamer-mediated double strand displacement amplification (SDA) strategy was first explored to couple with microchip electrophoresis (MCE) for rapid and ultrasensitive detection of Salmonella typhimurium (S. Typhimurium). In double-SDA, a bacteria-identified probe consisting of the aptamer (Apt) and trigger sequence (Tr) was ingeniously designed. The aptamer showed high affinity to the S. Typhimurium, releasing the Tr sequence from the probe. The released Tr hybridized with template C1 chain, initiating the first SDA to produce numerous output strands (OS). The second SDA process was induced with the hybridization of the liberated OS and template C2 sequence, generating a large number of reporter strands (RS), which were separated and quantified through MCE. Cascade signal amplification and rapid separation of nucleic acids could be realized by the proposed double-SDA method with MCE, achieving the limit of detection for S. typhimurium down to 6 CFU/mL under the optimal conditions. Based on the elaborate design of the probes, the double-SDA assisted MCE strategy achieved better amplification performance, showing high separation efficiency and simple operation, which has satisfactory expectation for bacterial disease diagnosis.

Assessing microbiome population dynamics using wild-type isogenic standardized hybrid (WISH)-tags.

Microbiomes feature recurrent compositional structures under given environmental conditions. However, these patterns may conceal diverse underlying population dynamics that require intrastrain resolution. Here we developed a genomic tagging system, termed wild-type isogenic standardized hybrid (WISH)-tags, that can be combined with quantitative polymerase chain reaction and next-generation sequencing for microbial strain enumeration. We experimentally validated the performance of 62 tags and showed that they can be differentiated with high precision. WISH-tags were introduced into model and non-model bacterial members of the mouse and plant microbiota. Intrastrain priority effects were tested using one species of isogenic barcoded bacteria in the murine gut and the Arabidopsis phyllosphere, both with and without microbiota context. We observed colonization resistance against late-arriving strains of Salmonella Typhimurium in the mouse gut, whereas the phyllosphere accommodated Sphingomonas latecomers in a manner proportional to their presence at the late inoculation timepoint. This demonstrates that WISH-tags are a resource for deciphering population dynamics underlying microbiome assembly across biological systems.

A machine vision-assisted Argonaute-mediated fluorescence biosensor for the detection of viable Salmonella in food without convoluted DNA extraction and amplification procedures.

The precise identification viable pathogens hold paramount significance in the prevention of foodborne diseases outbreaks. In this study, we integrated machine vision and learning with single microsphere to develop a phage and Clostridium butyricum Argonaute (CbAgo)-mediated fluorescence biosensor for detecting viable Salmonella typhimurium (S. typhimurium) without convoluted DNA extraction and amplification procedures. Phage and lysis buffer was utilized to capture and lyse viable S. typhimurium, respectively. Subsequently, CbAgo can cleave the bacterial DNA to obtain target DNA that guides a newly targeted cleavage of fluorescent probes. After that, the resulting fluorescent signal accumulates on the streptavidin-modified single microsphere. The overall detection process is then analyzed and interpreted by machine vision and learning algorithms, achieving highly sensitive detection of S. typhimurium with a limit of detection at 40.5 CFU/mL and a linear range of 50-107 CFU/mL. Furthermore, the proposed biosensor demonstrates standard recovery rates and coefficients of variation at 93.22% - 106.02% and 1.47% - 12.75%, respectively. This biosensor exhibits exceptional sensitivity and selectivity, presenting a promising method for the rapid and effective detection of foodborne pathogens. ENVIRONMENTAL IMPLICATION: Bacterial pathogens exist widely in the environment and seriously threaten the safety of human life. In this study, we developed a phage and Clostridium butyricum Argonaute-mediated fluorescence biosensor for the detection of viable Salmonella typhimurium in environmental water and food samples. Compared with other Salmonella detection methods, this method does not need complex DNA extraction and amplification steps, which reduces the use of chemical reagents and experimental consumables in classic DNA extraction kit methods.

Epidemiology and antimicrobial resistance of Salmonella serovars Typhimurium and 4,[5],12:i- recovered from hospitalized patients in China.

Global emergence of multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium is a continuing challenge for modern healthcare. However, the knowledge, regarding the epidemiology of salmonellosis caused by the monophasic variant S. 4,[5],12:i:- in hospitalized patients, is limited in China. To bridge this gap, we carried out a retrospective study to determine the antimicrobial resistance, trends, and risk factors of S. Typhimurium and S. 4,[5],12:i:- (n = 329) recovered from patients in Zhejiang province between 2011 and 2019. The results showed that 90.57% (298/329) of the isolates were MDR; among them, 48.94% (161/329) and 12.46% (41/329) were phenotypically resistant to cephalosporins and fluoroquinolones, respectively, which are the drugs of choice used to treat salmonellosis in clinics. Additionally, we observed a higher incidence of infections among the young population (<5 years old). Notably, the higher prevalence of ST34 (sequence type 34) isolates, especially after 2014, with MDR (57.05%, 170/298) phenotype, and incidence of ST34 isolates co-harbouring mcr-1 (mobile colistin resistance gene) and blaCTX-M-14 (ß-lactamase gene) suggest an association between STs and drug resistance. Together, the increasing prevalence of MDR ST34 calls for enhanced monitoring strategies to mitigate the spread and dissemination of MDR clones of S. Typhimurium and S. 4,[5],12:i-. Our study provides improved knowledge about non-typhoid Salmonella (NTS) infections, which could help in the effective recommendation of antimicrobials in hospitalized patients.

Evolutionary trade-off between heat shock resistance, growth at high temperature, and virulence expression in Salmonella Typhimurium.

Understanding the evolutionary dynamics of foodborne pathogens throughout our food production chain is of utmost importance. In this study, we reveal that Salmonella Typhimurium can readily and reproducibly acquire vastly increased heat shock resistance upon repeated exposure to heat shock. Counterintuitively, this boost in heat shock resistance was invariantly acquired through loss-of-function mutations in the dnaJ gene, encoding a heat shock protein that acts as a molecular co-chaperone of DnaK and enables its role in protein folding and disaggregation. As a trade-off, however, the acquisition of heat shock resistance inevitably led to attenuated growth at 37°C and higher temperatures. Interestingly, loss of DnaJ also downregulated the activity of the master virulence regulator HilD, thereby lowering the fraction of virulence-expressing cells within the population and attenuating virulence in mice. By connecting heat shock resistance evolution to attenuation of HilD activity, our results confirm the complex interplay between stress resistance and virulence in Salmonella Typhimurium. IMPORTANCE: Bacterial pathogens such as Salmonella Typhimurium are equipped with both stress response and virulence features in order to navigate across a variety of complex inhospitable environments that range from food-processing plants up to the gastrointestinal tract of its animal host. In this context, however, it remains obscure whether and how adaptation to one environment would obstruct fitness in another. In this study, we reveal that severe heat stress counterintuitively, but invariantly, led to the selection of S. Typhimurium mutants that are compromised in the activity of the DnaJ heat shock protein. While these mutants obtained massively increased heat resistance, their virulence became greatly attenuated. Our observations, therefore, reveal a delicate balance between optimal tuning of stress response and virulence features in bacterial pathogens.

Nutrient limitation and oxidative stress induce the promoter of acetate operon in Salmonella Typhimurium.

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the ß-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in ß galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments.

Lack of correlation between growth, stress, and virulence phenotypes in strains of Salmonella enterica serovar Enteritidis, S. Typhimurium DT104, S. 4,12, b:- and S. Liverpool.

Strains of Salmonella Enteritidis (SEnt, n = 10) and S. Typhimurium (STm, n = 11), representing clones with high impact on human health, and strains of S. 4,12: b:- (S412B n = 11) and S. Liverpool (SLiv, n = 4), representing clones with minor impact on human health were characterized for 16 growth, stress, and virulence phenotypes to investigate whether systematic differences exist in their performance in these phenotypes and whether there was correlation between performance in different phenotypes. The term serotype was not found to be predictive of a certain type of performance in any phenotype, and surprisingly, on average, strains of SEnt and STm were not significantly better in adhering to and invading cultured intestinal cells than the less pathogenic types. Forest analysis identified desiccation tolerance and the ability to grow at 42°C with high salt as the characters that separated serovars with low human health impact (S412B/SLiv) from serovars with high human health impact (SEnt/STm). The study showed that variation in phenotypes was high even within serovars and correlation between phenotypes was low, i.e. the way that a strain performed phenotypically in one of the tested conditions had a low predictive value for the performance of the strain in other conditions.

STnc1280, a trans-coding sRNA is involved in virulence modulation via targeting gldA mRNA in Salmonella Typhimurium.

Introduction. Salmonella Typhimurium (STM) is a food-borne Gram-negative bacterium, which can infect humans and a wide range of livestock and poultry, causing a variety of diseases such as septicaemia, enteritis and abortion.Hypothesis/Gap Statement. We will decipher the impacts of sRNA STnc1280 on STM virulence and provide a theoretical basis to reveal the regulatory role and molecular mechanism of STnc1280.Aim. The main objective of this study was to clarify whether sRNA STnc1280 exerts regulatory roles on STM pathogenicity.Methodology. The STnc1280 gene was amplified and its molecular characteristics were analysed in this study. Then, STnc1280 gene deletion strain (STM-ΔSTnc1280) and the complementary strain (ΔSTnc1280/STnc1280) were constructed by λ-Red homologous recombination method, respectively, to analyse of adhesion and invasive ability and pathogenicity of different strains. Subsequently, the potential target gene regulated by STnc1280 was predicted using target RNA2 software, followed by the verification of the interaction between STnc1280 and target mRNA using the dual plasmid reporter system (DPRS). Furthermore, the mRNA and protein level of target gene was determined using qRT-PCR and Western blot, respectively.Results. The results revealed that the cell adhesion and invasive ability and pathogenicity of STM-ΔSTnc1280 were significantly reduced compared to STM-SL1344 strain, indicating that the deficiency of STnc1280 gene significantly influenced STM pathogenicity. The DPRS results showed that STnc1280 can interact with the mRNA of target gene gldA, thus suppressing the expression of lacZ gene. Furthermore, the level of gldA mRNA was not influenced in STM-ΔSTnc1280, but the expression of GldA protein decreased significantly.Conclusion. Combining the bioinformatic analysis, these findings suggested that STnc1280 may bind to the SD sequence of gldA mRNA, hindering the binding of ribosomes to gldA mRNA, thereby inhibiting the expression of GldA protein to modulate the virulence of STM.

Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium expresses two type III secretion systems, T3SS1 and T3SS2, which are encoded in Salmonella pathogenicity island 1 (SPI1) and SPI2, respectively. These are essential virulent factors that secrete more than 40 effectors that are translocated into host animal cells. This study focuses on three of these effectors, SlrP, SspH1, and SspH2, which are members of the NEL family of E3 ubiquitin ligases. We compared their expression, regulation, and translocation patterns, their role in cell invasion and intracellular proliferation, their ability to interact and ubiquitinate specific host partners, and their effect on cytokine secretion. We found that transcription of the three genes encoding these effectors depends on the virulence regulator PhoP. Although the three effectors have the potential to be secreted through T3SS1 and T3SS2, the secretion of SspH1 and SspH2 is largely restricted to T3SS2 due to their expression pattern. We detected a role for these effectors in proliferation inside fibroblasts that is masked by redundancy. The generation of chimeric proteins allowed us to demonstrate that the N-terminal part of these proteins, containing the leucine-rich repeat motifs, confers specificity towards ubiquitination targets. Furthermore, the polyubiquitination patterns generated were different for each effector, with Lys48 linkages being predominant for SspH1 and SspH2. Finally, our experiments support an anti-inflammatory role for SspH1 and SspH2.

Assessment of the performance of the Ames MPF™ assay: A multicenter collaborative study with six coded chemicals.

The Ames MPF™ is a miniaturized, microplate fluctuation format of the Ames test. It is a standardized, commercially available product which can be used to assess mutagenicity in Salmonella and E. coli strains in 384-well plates using a color change-based readout. Several peer-reviewed comparisons of the Ames MPF™ to the Ames test in Petri dishes confirmed its suitability to evaluate the mutagenic potential of a variety of test items. An international multicenter study involving seven laboratories tested six coded chemicals with this assay using five bacterial strains, as recommended by the OECD test guideline 471. The data generated by the participating laboratories was in excellent agreement (93%), and the similarity of their dose response curves, as analyzed with sophisticated statistical approaches further confirmed the suitability of the Ames MPF™ assay as an alternative to the Ames test on agar plates, but with advantages with respect to significantly reduced amount of test substance and S9 requirements, speed, hands-on time and, potentially automation.