Modulation of Salmonella virulence by a novel SPI-2 injectisome effector that interacts with the dystrophin-associated protein complex.

The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterize the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed among the five subspecies of Salmonella enterica, is found in many clinically relevant serovars, and is co-transcribed with pipB2, a SPI-2 effector gene. The translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely, ß-2-syntrophin, utrophin/dystrophin, α-catulin, α-dystrobrevin, and ß-dystrobrevin. The interaction between SseM and ß-2-syntrophin and α-dystrobrevin was verified in Salmonella Typhimurium-infected cells and relied on the postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain of ß-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A ΔsseM mutant strain had a small competitive advantage over the wild-type strain in the S. Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild-type SseM from S. Typhimurium or S. Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates the systemic growth of bacteria in mice. Furthermore, the ΔsseM mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate Salmonella virulence. IMPORTANCE: In Salmonella enterica, the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for Salmonella virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between Salmonella and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein ß-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC.

Unraveling the role of type 1 fimbriae in Salmonella pathogenesis: insights from a comparative analysis of Salmonella Enteritidis and Salmonella Gallinarum.

Significant differences in pathogenicity between Salmonella Enteritidis and Salmonella Gallinarum exist despite the fact that S. Gallinarum is a direct descendant of S. Enteritidis. It was hypothesized that such various properties may be in part the result of differences in structure and functions of type 1 fimbriae (T1Fs). In S. Enteritidis, T1Fs bind to oligomannosidic structures carried by host cell glycoproteins and are called mannose-sensitive T1Fs (MST1F). In S. Gallinarum, T1Fs lost ability to bind such carbohydrate chains, and were named mannose-resistant MRT1Fs (MRT1F). Therefore, the present study was undertaken to evaluate the role of MST1Fs and MRT1Fs in the adhesion, invasion, intracellular survival and cytotoxicity of S. Enteritidis and S. Gallinarum toward chicken intestinal CHIC8-E11cells and macrophage-like HD11 cells. Using mutant strains: S. Enteritidis fimH::kan and S. Gallinarum fimH::kan devoid of T1Fs and in vitro assays the following observations were made. MST1Fs have a significant impact on the chicken cell invasion by S. Enteritidis as MST1F-mediated adhesion facilitates direct and stable contact of bacteria with host cells, in contrast to MRT1Fs expressed by S. Gallinarum. MST1Fs as well as MRT1Fs did not affected intracellular viability of S. Enteritidis and S. Gallinarum. However, absolute numbers of intracellular viable wild-type S. Enteritidis were significantly higher than S. Enteritidis fimH::kan mutant and wild-type S. Gallinarum and S. Gallinarum fimH::kan mutant. These differences, reflecting the numbers of adherent and invading bacteria, underline the importance of MST1Fs in the pathogenicity of S. Enteritidis infections. The cytotoxicity of wild-type S. Enteritidis and its mutant devoid of MST1Fs to HD11 cells was essentially the same, despite the fact that the number of viable intracellular bacteria was significantly lower in the mutated strain. Using HD11 cells with similar number of intracellular wild-type S. Enteritidis and S. Enteritidis fimH::kan mutant, it was found that the lack of MST1Fs did not affect directly the cytotoxicity, suggesting that the increase in cytotoxicity of S. Enteritidis devoid of MST1Fs may be associated with crosstalk between T1Fs and other virulence factors.

PhoPQ Regulates Quinolone and Cephalosporin Resistance Formation in Salmonella Enteritidis at the Transcriptional Level.

The two-component system (TCS) PhoPQ has been demonstrated to be crucial for the formation of resistance to quinolones and cephalosporins in Salmonella Enteritidis (S. Enteritidis). However, the mechanism underlying PhoPQ-mediated antibiotic resistance formation remains poorly understood. Here, it was shown that PhoP transcriptionally regulated an assortment of genes associated with envelope homeostasis, the osmotic stress response, and the redox balance to confer resistance to quinolones and cephalosporins in S. Enteritidis. Specifically, cells lacking the PhoP regulator, under nalidixic acid and ceftazidime stress, bore a severely compromised membrane on the aspects of integrity, fluidity, and permeability, with deficiency to withstand osmolarity stress, an increased accumulation of intracellular reactive oxygen species, and dysregulated redox homeostasis, which are unfavorable for bacterial survival. The phosphorylated PhoP elicited transcriptional alterations of resistance-associated genes, including the outer membrane porin ompF and the aconitate hydratase acnA, by directly binding to their promoters, leading to a limited influx of antibiotics and a well-maintained intracellular metabolism. Importantly, it was demonstrated that the cavity of the PhoQ sensor domain bound to and sensed quinolones/cephalosporins via the crucial surrounding residues, as their mutations abrogated the binding and PhoQ autophosphorylation. This recognition mode promoted signal transduction that activated PhoP, thereby modulating the transcription of downstream genes to accommodate cells to antibiotic stress. These findings have revealed how bacteria employ a specific TCS to sense antibiotics and combat them, suggesting PhoPQ as a potential drug target with which to surmount S. Enteritidis. IMPORTANCE The prevalence of quinolone and cephalosporin-resistant S. Enteritidis is of increasing clinical concern. Thus, it is imperative to identify novel therapeutic targets with which to treat S. Enteritidis-associated infections. The PhoPQ two-component system is conserved across a variety of Gram-negative pathogens, by which bacteria adapt to a range of environmental stimuli. Our earlier work has demonstrated the importance of PhoPQ in the resistance formation in S. Enteritidis to quinolones and cephalosporins. In the current work, we identified a global profile of genes that are regulated by PhoP under antibiotic stresses, with a focus on how PhoP regulated downstream genes, either positively or negatively. Additionally, we established that PhoQ sensed quinolones and cephalosporins in a manner of directly binding to them. These identified genes and pathways that are mediated by PhoPQ represent promising targets for the development of a drug potentiator with which to neutralize antibiotic resistance in S. Enteritidis.

Symbiotic bacteria stabilize the intestinal environment by producing phenylpropanoids.

Salmonella enterica serovar Enteritidis (S. Enteritidis) can colonize in the intestinal tract of chickens and transmit to humans. In order to decrypt the mechanism of avian resistance to S. Enteritidis, we utilized two China local chicken breeds to generate the reciprocal crosses (the Cross and the Reverse-cross). The two lines of hybrids were orally inoculated with S. Enteritidis at 2-day old and sampled at 3 days post-inoculation. Along the analysis direction of multi-omics, differential metabolites, functional pathways and correlated microbes, we found that 12 species of microbes thrived upon S. Enteritidis challenge and probably contributed to the intestinal stability in the Cross by enhancing the production of phenylpropanoids. Our findings can help to understand the symbiotic and resistant mechanisms derived from the intestinal microbiota.

An Improved Real-Time Viability PCR Assay to Detect Salmonella in a Culture-Independent Era.

Viability PCR (vPCR) uses a DNA intercalating dye to irreversibly bind double-stranded DNA from organisms with compromised cell membranes. This allows the selective amplification of DNA from intact cells. An optimized vPCR protocol should minimize false positives (DNA from compromised cells not fully removed) and false negatives (live cell DNA bound by the dye). We aimed to optimize a vPCR protocol using PMAxx™ as the intercalating agent and Salmonella Enteritidis as the target organism. To do this, we studied (1) single vs. sequential PMAxx™ addition; (2) a wash step post-PMAxx™ treatment; (3) a change of tube post-treatment before DNA extraction. The single vs. sequential PMAxx™ addition showed no difference. Results signified that PMAxx™ potentially attached to polypropylene tube walls and bound the released DNA from PMA-treated live cells when lysed in the same tube. A wash step was ineffective but transfer of the treated live cells to a new tube minimized these false-negative results. Our optimized protocol eliminated 108 CFU/mL heat-killed cell DNA in the presence of different live cell dilutions without compromising the amplification of the live cells, minimizing false positives. With further improvements, vPCR has great potential as a culture-independent diagnostic tool.

Evaluation of Microencapsulated Essential Oils in Broilers Challenged with Salmonella Enteritidis: A Focus on the Body's Antioxidant Status, Gut Microbiology, and Morphology.

Restrictions on antibiotic use encourage researchers to seek natural substitutes with the same effects without adverse end effects resulting from antibiotic use. Savory and black pepper have been challenged against Salmonella enteritidis (S. enteritidis) bacterium using the spray dryer method to evaluate growth performance, antioxidant status, immune response, and intestinal health parameters in broilers. In this study, thyme essential oil (50%), savory (25%), peppermint (12.5%), and black pepper seeds (12.5%) were mixed to form essential oil-loaded spherical microcapsules with the particle size of 323 nm and encapsulation efficiency of 96.2%. The main bioactive compounds used in the core of microcapsules included thymol, carvacrol, p-cymene, γ-terpinene, and menthol. Moreover, modified starch (25%) and maltodextrin (55%) were used for the preparation of spherical microcapsules for the enclosed wall with 20% whey protein concentrate. The dietary addition of microcapsules containing essential oil significantly reduced the S. enteritidis population in both ileum and cecum (P<0.05). The results revealed that the dietary inclusion of essential oil-loaded microcapsules significantly (P<0.05) increased the villus height, villus width, V: C ratio, and the number of goblet cells and decreased the crypt depth. Microcapsules have antioxidant and antibacterial activity and their dietary use as feed additive at 0.5, 1, and 2 kg/t concentrations in broilers has been challenged and showed that the final weight, total feed intake, and FCR improved the body's antioxidant status, structure, and inflammation in the ileum tissue.

[Biosynthesis of Salmonella enteritidis O antigen-based glycoproteins].

Salmonella enteritidis (SE) has been recognized as an important zoonotic pathogen, and the prevention and control of salmonellosis has long been a conundrum. However, glycoconjugate vaccines seem to be a promising solution. Glycoproteins are conventionally synthesized by chemical cross-linking which features complex procedure and cost-intensiveness. Therefore, a stable biosynthesis method at lower cost is in urgent need. For the biosynthesis of SE O-antigen-based glycoproteins, we used CRISPR/Cas9 to develop the waaL-deleted SE strain ∆waaL. The synthesis of lipopolysaccharide (LPS) was detected based on silver staining. Circular polymerase extension cloning (CPEC) was employed to construct the plasmids expressing glycosyltransferase PglL, recombinant Pseudomonas aeruginosa exotoxin A (rEPA), and cholera toxin B subunit (CTB). Meanwhile, PilES45-K73 glycosylation motif was added to the N-terminal and C-terminal of rEPA and CTB, respectively. The recombinant plasmids were transformed into SE ∆waaL. After induction, the synthesis of glycoprotein was verified by Western blotting and the synthesized glycoprotein was purified by Ni-NTA column. The results showed that waaL deletion blocked the LPS synthesis of SE, and that rEPA and CTB proteins were expressed in SE ∆waaL. In addition, obvious glycosylation occurred to rEPA and CTB when PglL was expressed, and the glycosylated part was SE O antigen polysaccharide. In summary, after waaL deletion in SE, PglL can transfer its own O antigen polysaccharides (OPS) to the carrier proteins rEPA and CTB, resulting in OPS-rEPA and OPS-CTB glycoproteins. The result lays a basis for the biosynthesis of SE glycoprotein.

Bioinformatic and experimental characterization of SEN1998: a conserved gene carried by the Enterobacteriaceae-associated ROD21-like family of genomic islands.

Genomic islands (GIs) are horizontally transferred elements that shape bacterial genomes and contributes to the adaptation to different environments. Some GIs encode an integrase and a recombination directionality factor (RDF), which are the molecular GI-encoded machinery that promotes the island excision from the chromosome, the first step for the spread of GIs by horizontal transfer. Although less studied, this process can also play a role in the virulence of bacterial pathogens. While the excision of GIs is thought to be similar to that observed in bacteriophages, this mechanism has been only studied in a few families of islands. Here, we aimed to gain a better understanding of the factors involved in the excision of ROD21 a pathogenicity island of the food-borne pathogen Salmonella enterica serovar Enteritidis and the most studied member of the recently described Enterobacteriaceae-associated ROD21-like family of GIs. Using bioinformatic and experimental approaches, we characterized the conserved gene SEN1998, showing that it encodes a protein with the features of an RDF that binds to the regulatory regions involved in the excision of ROD21. While deletion or overexpression of SEN1998 did not alter the expression of the integrase-encoding gene SEN1970, a slight but significant trend was observed in the excision of the island. Surprisingly, we found that the expression of both genes, SEN1998 and SEN1970, were negatively correlated to the excision of ROD21 which showed a growth phase-dependent pattern. Our findings contribute to the growing body of knowledge regarding the excision of GIs, providing insights about ROD21 and the recently described EARL family of genomic islands.

The Evolution of Fluoroquinolone Resistance in Salmonella under Exposure to Sub-Inhibitory Concentration of Enrofloxacin.

The evolution of resistance in Salmonella to fluoroquinolones (FQs) under a broad range of sub-inhibitory concentrations (sub-MICs) has not been systematically studied. This study investigated the mechanism of resistance development in Salmonella enterica serovar Enteritidis (S. Enteritidis) under sub-MICs of 1/128×MIC to 1/2×MIC of enrofloxacin (ENR), a widely used veterinary FQ. It was shown that the resistance rate and resistance level of S. Enteritidis varied with the increase in ENR concentration and duration of selection. qRT-PCR results demonstrated that the expression of outer membrane porin (OMP) genes, ompC, ompD and ompF, were down-regulated first to rapidly adapt and develop the resistance of 4×MIC, and as the resistance level increased (≥8×MIC), the up-regulated expression of efflux pump genes, acrB, emrB amd mdfA, along with mutations in quinolone resistance-determining region (QRDR) gradually played a decisive role. Cytohubba analysis based on transcriptomic profiles demonstrated that purB, purC, purD, purF, purH, purK, purL, purM, purN and purT were the hub genes for the FQs resistance. The 'de novo' IMP biosynthetic process, purine ribonucleoside monophosphate biosynthetic process and purine ribonucleotide biosynthetic process were the top three biological processes screened by MCODE. This study first described the dynamics of FQ resistance evolution in Salmonella under a long-term selection of sub-MICs of ENR in vitro. In addition, this work offers greater insight into the transcriptome changes of S. Enteritidis under the selection of ENR and provides a framework for FQs resistance of Salmonella for further studies.

Novel Insight into the Effects of CpxR on Salmonella enteritidis Cells during the Chlorhexidine Treatment and Non-Stressful Growing Conditions.

The development and spread of antibiotics and biocides resistance is a significant global challenge. To find a solution for this emerging problem, the discovery of novel bacterial cellular targets and the critical pathways associated with antimicrobial resistance is needed. In the present study, we investigated the role of the two most critical envelope stress response regulators, RpoE and CpxR, on the physiology and susceptibility of growing Salmonella enterica serovar enteritidis cells using the polycationic antimicrobial agent, chlorhexidine (CHX). It was shown that deletion of the cpxR gene significantly increased the susceptibility of this organism, whereas deletion of the rpoE gene had no effect on the pathogen's susceptibility to this antiseptic. It has been shown that a lack of the CpxR regulator induces multifaceted stress responses not only in the envelope but also in the cytosol, further affecting the key biomolecules, including DNA, RNA, and proteins. We showed that alterations in cellular trafficking and most of the stress responses are associated with a dysfunctional CpxR regulator during exponential growth phase, indicating that these physiological changes are intrinsically associated with the lack of the CpxR regulator. In contrast, induction of type II toxin-antitoxin systems and decrease of abundances of enzymes and proteins associated with the recycling of muropeptides and resistance to polymixin and cationic antimicrobial peptides were specific responses of the ∆cpxR mutant to the CHX treatment. Overall, our study provides insight into the effects of CpxR on the physiology of S. Enteritidis cells during the exponential growth phase and CHX treatment, which may point to potential cellular targets for the development of an effective antimicrobial agent.

Quantitative proteomics implicates YggT in streptomycin resistance in Salmonella enterica serovar Enteritidis.

OBJECTIVES: To identify proteins that may be associated with antibiotic resistance in the multidrug-resistant Salmonella enterica D14, by constructing proteomic profiles using mass spectrometry-based label-free quantitative proteomics (LFQP). RESULTS: D14 was cultured with four antibiotics (ampicillin, nalidixic acid, streptomycin, and tetracycline) separately. Subsequently, the findings from an equal combination of the four cultures were compared with the profile of sensitive S. enterica 104. 2255 proteins, including 149 differentially up-regulated proteins, were identified. Many of these up-regulated proteins were associated with flagellar assembly and chemotaxis, two-component system, amino acid metabolism, ß-lactam resistance, and transmembrane transport. A subset of 10 genes was evaluated via quantitative real-time PCR (qPCR), followed by the construction of cheR, fliS, fliA, arnA, and yggT deletion mutants. Only the yggT-deleted D14 mutant showed decrease in streptomycin resistance, whereas the other deletions had no effect. Furthermore, complementation of yggT and the overexpression of yggT in S. enterica ATCC 14028 increased the streptomycin resistance. Additionally, spot dilution assay results confirmed that Salmonella strains, harboring yggT, exhibited an advantage in the presence of streptomycin. CONCLUSIONS: The above proteomic and mutagenic analyses revealed that yggT is involved in streptomycin resistance in S. enterica.

WbaP is required for swarm motility and intramacrophage multiplication of Salmonella Enteritidis spiC mutant by glucose use ability.

Salmonella spp. can survive and replicate in macrophage cells to cause persistent infection, SpiC is a necessary T3SS effector, but its pathogenic mechanism is still not known completely. In our study, Salmonella Enteritidis spiC mutant (SEΔspiC) was found to have stronger swarming motility and intramacrophage hyperproliferation which was closely related to glucose metabolism. SEΔspiC wbaP::Tn5 mutant was screened out by transposon mutagenesis, which had weaker swarming motility and intramacrophage replication ability than SEΔspiC in the presence of glucose. Bioinformatics displayed that undecaprenyl-phosphate galactose phosphotransferase (Wbap), encoded by wbaP gene, was a key enzyme for glucose metabolism and Lipopolysaccharide(LPS) synthesis, which confirmed our outcome that Wbap was involved in intramacrophage replication ability by glucose use in addition to swarming motility based on SEΔspiC. This discovery will further promote the understanding of the interaction between wbaP gene and spiC gene and the intracellular Salmonella replication mechanism.

Proteomics study unveils ROS balance in acid-adapted Salmonella Enteritidis.

Salmonella Enteritidis is a major cause of foodborne gastroenteritis and is thus a persistent threat to global public health. The acid adaptation response helps Salmonella survive exposure to gastric environment during ingestion. In a previous study we highlighted the damage caused to cell membrane and the regulation of intracellular reactive oxygen species (ROS) in S. Enteritidis. In this study, we applied both physiologic and iTRAQ analyses to explore the regulatory mechanism of acid resistance in Salmonella. It was found that after S. Enteritidis was subject to a 1 h period of acid adaptation at pH 5.5, an additional 1 h period of acid shock stress at pH 3.0 caused less Salmonella cell death than in non-acid adapted Salmonella cells. Although there were no significant differences between adapted and non-adapted cells in terms of cell membrane damage (e.g., membrane permeability or lipid peroxidation) after 30 min, intracellular ROS level in acid adapted cells was dramatically reduced compared to that in non-acid adapted cells, indicating that acid adaption promoted less ROS generation or increased the ability of ROS scavenging with little reduction in the integrity of the cell membrane. These findings were confirmed via an iTRAQ analysis. The adapted cells were shown to trigger incorporation of exogenous long-chain fatty acids into the cellular membrane, resulting in a different membrane lipid profile and promoting survival rate under acid stress. S. Enteritidis experiences oxidative damage and iron deficiency under acid stress, but after acid adaption S. Enteritidis cells were able to balance their concentrations of intracellular ROS. Specifically, SodAB consumed the free protons responsible for forming reactive oxygen intermediates (ROIs) and KatE protected cells from the toxic effects of ROIs. Additionally, acid-labile proteins released free unbound iron promoting ferroptotic metabolism, and NADH reduced GSSH to G-SH, protecting cells from acid/oxidative stress.

Cellulase-Linked Immunomagnetic Microbial Assay on Electrodes: Specific and Sensitive Detection of a Single Bacterial Cell.

Pathogen-associated infections represent one of the major threats to human health and require reliable methods for immediate and robust identification of pathogenic microorganisms. Here, an inexpensive cellulase-linked immunomagnetic methodology was developed for the specific and ultrasensitive analysis of bacteria at their single-cell levels within a 3 h procedure. Detection of a model bacterium, Escherichia coli, was performed in a sandwich reaction with E. coli-specific either aptamer or antibody (Ab)-modified magnetic beads (MBs) and Ab/aptamer reporter molecules linked to cellulase. The cellulase-labeled immuno-aptamer sandwich applied onto nitrocellulose-film-modified electrodes digested the film and changed its electrical conductivity. Electrode's chronocoulometric responses at 0.3 V, in the absence of any redox indicators, allowed a single E. coli cell detection and from 1 to 4 × 104 CFU mL-1 E. coli quantification. No interference/cross-reactivity from Salmonella enteritidis, Enterobacter agglomerans, Pseudomonas putida, Staphylococcus aureus, and Bacillus subtilis was observed when the assay was performed on Ab-modified MBs, and E. coli could be quantified in tap water and milk. This electrochemically label-free methodology is sufficiently fast, highly specific, and sensitive to be used in direct in-field applications. The assay can be adapted for specific detection of other bacterial strains of either the same or different species and offers new analytical tools for fast, specific, and reliable analysis of bacteria in the clinic, food, and environment.

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation.

BACKGROUND: Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear. RESULTS: A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1, secretion of IL-1ß and phosphorylation of c-Jun N-terminal kinase (Jnk). Transcriptome sequencing (RNA-seq) results showed that most of the differentially expressed genes and enriched KEGG pathways between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups overlapped, which includes multiple signaling pathways related to the inflammasome. C50336Δdam and C50336::dam were both found to be defective in suppressing the expression of several anti-inflammasome factors. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically enhance the activation of NLRP3 inflammasome independently via promoting the Jnk pathway. CONCLUSIONS: These data indicated that Dam was essential for modulating inflammasome activation during SE infection, there were complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

Transcriptome, Spliceosome and Editome Expression Patterns of the Porcine Endometrium in Response to a Single Subclinical Dose of Salmonella Enteritidis Lipopolysaccharide.

Endometrial infections at a young age can lead to fertility issues in adulthood. Bacterial endotoxins, such as lipopolysaccharide (LPS), can participate in long-term molecular changes even at low concentrations. Lipopolysaccharide plays a crucial role in the progression of septic shock, inflammation and auto-immune diseases. The aim of this study was to describe transcriptomic modulations in the porcine endometrium, induced in vivo by a single subclinical dose of LPS from Salmonella Enteritidis. which did not produce clinical symptoms of toxicity. The RNA-seq methodology was applied to reveal 456 differentially expressed regions, including 375 genes, four long noncoding RNAs, and 77 other unclassified transcripts. Two independent methods confirmed 118 alternatively spliced genes that participate i.a., in the formation of the MHC-I complex and the adaptive immune response. Single nucleotide variant-calling algorithms supported the identification of 3730 allele-specific expression variants and 57 canonical A-to-I RNA editing sites. The results demonstrated that the differential expression of genes involved in inflammation, immune response, angiogenesis and endometrial development may be maintained for up to 7 days after exposure to LPS. RNA editing sites and long noncoding RNAs (lncRNAs) play an important role in transcriptional regulatory machinery in the porcine endometrium in response to LPS administration.

Salmonella Enteritidis Effector AvrA Suppresses Autophagy by Reducing Beclin-1 Protein.

Autophagy is a cellular process to clear pathogens. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. However, major studies still focus on Salmonella enterica serovar Typhimurium. Here, we reported that AvrA, a S. Enteritidis effector, inhibited autophagy to promote bacterial survival in the host. We found that AvrA regulates the conversion of LC3 I into LC3 II and the enrichment of lysosomes. Beclin-1, a key molecular regulator of autophagy, was decreased after AvrA expressed strain colonization. In S.E-AvrA--infected cells, we found the increases of protein levels of p-JNK and p-c-Jun and the transcription level of AP-1. AvrA-reduction of Beclin-1 protein expression is through the JNK pathway. The JNK inhibitor abolished the AvrA-reduced Beclin-1 protein expression. Moreover, we identified that the AvrA mutation C186A abolished its regulation of Beclin-1 expression. In addition AvrA protein was found interacted with Beclin-1. In organoids and infected mice, we explored the physiologically related effects and mechanism of AvrA in reducing Beclin-1 through the JNK pathway, thus attenuating autophagic responses. This finding not only indicates an important role of S. Enteritidis effector in reducing host protein as a strategy to suppress autophagy, but also suggests manipulating autophagy as a new strategy to treat infectious diseases.

Inhibition of Salmonella enteritidis biofilms by Salmonella invasion protein-targeting aptamer.

The current study aimed to assess the inhibitory effect of a DNA aptamer (Apt17) which targeted Salmonella invasion proteinA (SipA). The effect of Apt17, on biofilm formation by two Salmonella enteritidis strains, was tested either separately or in combination with ampicillin at different Sub MIC concentrations. Maximum inhibitory effect equivalent to 24.34% and 26.81% was recorded when Apt17 was co-incubated with S. enteritidis TM 6 and S. enteritidis TM 68 respectively for 13 h. The inhibitory effect of Apt17 was also confirmed with Triphenyl Tetrazolium Chloride. Under Scanning Electron Microscope, the presence of Apt17 resulted in altered three dimensional structure. While the treated cells of S. enteritidis TM 6 were arranged as monolayers, the sessile aggregates of S. enteritidis TM 68 appeared thinner and exhibited less surface coverage when compared to control. Moreover, the treated cells lost their exopolysaccharide matrix. The co-incubation of Apt17 with ampicillin MIC/10 for 24 h, inhibited the biofilms of S. enteritidis TM 6 and S. enteritidis TM 68 by 12.5 and 20.9% respectively. This study demonstrated quantitative and qualitative antibiofilm effect of Apt17 against the biofilms of two Salmonella enteritidis strains. According to our knowledge, this is the first study employing an aptamer that targets SipA protein to inhibit biofilm formation in Salmonella.

Pathogen Concentration Combined Solid-Phase PCR on Supercritical Angle Fluorescence Microlens Array for Multiplexed Detection of Invasive Nontyphoidal Salmonella Serovars.

Bloodstream infections and invasive nontyphoidal Salmonellosis in particular remain a major health and economic burden worldwide. The complexity of blood matrixes along with extremely low concentration of pathogens in blood poses a great challenge for rapid and ultrasensitive detection. Sample preparation has been the critical step that should provide blood-matrix-free sample with the targeted pathogen in the highest possible concentration. In this work, we addressed this challenge by combining magnetic-bead-based pathogen concentration and solid-phase PCR (SP-PCR). The SP-PCR performed on a supercritical angle fluorescence (SAF) microlens array embedded in a microchip enabled quick and accurate detection of low levels of Salmonella enterica serovar typhimurium and enteritidis in blood samples without culture enrichment. Protein AG-magnetic beads immobilized with antisalmonella antibody could efficiently concentrate both Salmonella serovars with a capturing efficiency >95%. Higher tolerance of Phusion hot start DNA polymerase to PCR inhibitors and its compatibility with protein AG-magnetic beads allowed the integration of SP-PCR. Analysis of Salmonella-spiked blood samples with the SP-PCR resulted in a limit of detection (LoD) as low as 86 CFU/mL and 94 CFU/mL for S. typhimurium and S. enteritidis, respectively, that could be attributed to the high fluorescence collection efficiency of the SAF microlens array. These combinations reduced the duration of analysis to less than 3 h including sample preparation. This platform has the potential for wide application as a high-throughput biosensor to analyze pathogens in clinical, food, and environmental samples.

Chaperones Promote Remarkable Solubilization of Salmonella enterica serovar Enteritidis Flagellin Expressed in Escherichia coli.

BACKGROUND: Flagellin of Salmonella enterica serovar Enteritidis (SEF) stimulates immune responses to both itself and coapplied antigens. It is therefore used in vaccine development and immunotherapy. Removal of pathogenic S. enterica ser. Enteritidis from SEF production process is advantageous due to the process safety improvement. The protein solubility analysis using SDS-PAGE indicated that 53.49% of SEF expressed in Escherichia coli formed inclusion bodies. However, the protein recovery from inclusion bodies requires a complex process with a low yield. OBJECTIVE: We thus aim to study possibility of enhancing SEF expression in E. coli in soluble form using chemical and molecular chaperones. METHODS: Chemical chaperones including arginine, sorbitol, trehalose, sodium chloride and benzyl alcohol were used as cultivation medium additives during SEF expression. SEF solubilization by coexpression of molecular chaperones DnaK, DnaJ, and GrpE was also investigated. RESULTS: All of the chemical chaperones were effective in improving SEF solubility. However, sorbitol showed the most profound effect. SEF solubilization by molecular chaperones was slightly better than that using sorbitol and this approach enhanced noticeably SEF soluble concentration and SEF solubility percentage to almost two folds and 96.37% respectively. Results of limited proteolysis assay and native PAGE indicated similar conformational states and proper folding for SEF obtained without using chaperones and for those obtained using sorbitol and the molecular chaperones. However, the molecular chaperones based system was less costly than the sorbitol based system. CONCLUSION: The coexpression of molecular chaperones was then considered as the most appropriate approach for soluble SEF production. Therefore, SEF production for medical purposes is expected to be facilitated.