The ancestral stringent response potentiator, DksA has been adapted throughout Salmonella evolution to orchestrate the expression of metabolic, motility, and virulence pathways.

DksA is a conserved RNA polymerase-binding protein known to play a key role in the stringent response of proteobacteria species, including many gastrointestinal pathogens. Here, we used RNA-sequencing of Escherichia coli, Salmonella bongori and Salmonella enterica serovar Typhimurium, together with phenotypic comparison to study changes in the DksA regulon, during Salmonella evolution. Comparative RNA-sequencing showed that under non-starved conditions, DksA controls the expression of 25%, 15%, and 20% of the E. coli, S. bongori, and S. enterica genes, respectively, indicating that DksA is a pleiotropic regulator, expanding its role beyond the canonical stringent response. We demonstrate that DksA is required for the growth of these three enteric bacteria species in minimal medium and controls the expression of the TCA cycle, glycolysis, pyrimidine biosynthesis, and quorum sensing. Interestingly, at multiple steps during Salmonella evolution, the type I fimbriae and various virulence genes encoded within SPIs 1, 2, 4, 5, and 11 have been transcriptionally integrated under the ancestral DksA regulon. Consequently, we show that DksA is necessary for host cells invasion by S. Typhimurium and S. bongori and for intracellular survival of S. Typhimurium in bone marrow-derived macrophages (BMDM). Moreover, we demonstrate regulatory inversion of the conserved motility-chemotaxis regulon by DksA, which acts as a negative regulator in E. coli, but activates this pathway in S. bongori and S. enterica. Overall, this study demonstrates the regulatory assimilation of multiple horizontally acquired virulence genes under the DksA regulon and provides new insights into the evolution of virulence genes regulation in Salmonella spp.

Pore-forming treatments induce aggregation of Salmonella Senftenberg through protein leakage.

Fresh herbs are not commonly associated with foodborne pathogens, due to the production of essential oils with antimicrobial activity. Recalls of contaminated basil, and basil outbreaks caused by Salmonella motivated studies aimed to comprehend the antimicrobial activity of basil essential oils, and to explore the mechanisms in which Salmonella can overcome them. Linalool, a major constituent of basil oil, increases the permeability of Salmonella Senftenberg cells by damaging their membrane. Linalool also induces bacterial aggregation. We hypothesized that the membrane perforation effect triggers cell aggregation through leakage of intracellular substances from live and dead cells. By exposing S. Senftenberg to additional physical (sonication) or chemical (eugenol, Triton-X-100) treatments, we showed that the aggregation is caused by various membrane-targeted treatments. Enzymatic degradation of leaked proteins restricted the bacterial aggregation, and disassembled existing aggregates. Moreover, supplemented proteins such as bacterial intracellular proteins or BSA also caused aggregation, further supporting the hypothesis that non-specific proteins trigger the bacterial aggregation. This study provides a novel understanding of the role of protein leakage in promoting bacterial aggregation. Since aggregation has significant roles in food safety and microbial ecology, this finding may establish future studies about microbial resistance via formation of clusters similar to biofilm development.

Prokaryotic Organelles: Bacterial Microcompartments in E. coli and Salmonella.

Bacterial microcompartments (MCPs) are proteinaceous organelles consisting of a metabolic pathway encapsulated within a selectively permeable protein shell. Hundreds of species of bacteria produce MCPs of at least nine different types, and MCP metabolism is associated with enteric pathogenesis, cancer, and heart disease. This review focuses chiefly on the four types of catabolic MCPs (metabolosomes) found in Escherichia coli and Salmonella: the propanediol utilization (pdu), ethanolamine utilization (eut), choline utilization (cut), and glycyl radical propanediol (grp) MCPs. Although the great majority of work done on catabolic MCPs has been carried out with Salmonella and E. coli, research outside the group is mentioned where necessary for a comprehensive understanding. Salient characteristics found across MCPs are discussed, including enzymatic reactions and shell composition, with particular attention paid to key differences between classes of MCPs. We also highlight relevant research on the dynamic processes of MCP assembly, protein targeting, and the mechanisms that underlie selective permeability. Lastly, we discuss emerging biotechnology applications based on MCP principles and point out challenges, unanswered questions, and future directions.

Poly-L-histidine coated microfluidic devices for bacterial DNA purification without chaotropic solutions.

We present a disposable polymeric microfluidic device capable of reversibly binding and purifying Salmonella DNA through solid phase extraction (SPE). The microfluidic channels are first oxygen plasma treated and simultaneously micro-nanotextured, and then functionalized with amine groups via modification with L-histidine or poly-L-histidine. L-Histidine and poly-L-histidine bind on the plasma treated chip surface, and are not detached when rinsing with DNA purification protocol buffers. A pH-dependent protocol is applied on-chip to purify Salmonella DNA, which is first bound on the protonated amines at a pH (5.0) lower than their pKa of surface amine-groups which is 6.0 and then released at a pH higher than the pKa value (10.5). It was found that modification with poly-L-histidine resulted in higher surface density of amine groups onto microfluidic channel. Using the chip modified with poly-L-histidine, high recovery efficiency of at least 550 ng of isolated Salmonella DNA as well as DNA purification from Salmonella cell lysates corresponding to less than 5000 cells or 0.026 ng of Salmonella DNA was achieved. The protocol developed does not require ethanol or chaotropic solutions typically used in DNA purification, which are known inhibitors for downstream operations such as polymerase chain reactions (PCR) and which can also attack some polymeric microfluidic materials. Therefore, the microfluidic device and the related protocol hold promise for facile incorporation in microfluidics and Lab-on-a-chip (LOC) platforms for pathogen detection or in general for DNA purification.

The flexible linker of the secreted FliK ruler is required for export switching of the flagellar protein export apparatus.

The hook length of the flagellum is controlled to about 55 nm in Salmonella. The flagellar type III protein export apparatus secretes FliK to determine hook length during hook assembly and changes its substrate specificity from the hook protein to the filament protein when the hook length has reached about 55 nm. Salmonella FliK consists of an N-terminal domain (FliKN, residues 1-207), a C-terminal domain (FliKC, residues 268-405) and a flexible linker (FliKL, residues 208-267) connecting these two domains. FliKN is a ruler to measure hook length. FliKC binds to a transmembrane export gate protein FlhB to undergo the export switching. FliKL not only acts as part of the ruler but also contributes to this switching event, but it remains unknown how. Here we report that FliKL is required for efficient interaction of FliKC with FlhB. Deletions in FliKL not only shortened hook length according to the size of deletions but also caused a loose length control. Deletion of residues 206-265 significantly reduced the binding affinity of FliKC for FlhB, thereby producing much longer hooks. We propose that an appropriate length of FliKL is required for efficient interaction of FliKC with FlhB.

Functional protein shells fabricated from the self-assembling protein sheets of prokaryotic organelles.

Fabricating protein compartments from protein units is challenging and limited by the use of external stimuli and crosslinkers. Here we explore the fabrication of all-protein compartments using self-assembled proteins of prokaryotic organelles. These proteins have intrinsic interacting domains which are ionic in nature, and spontaneously self-assemble into sheets when over-expressed. Using a one-step approach, we maneuvered the formation of the protein shells from the sheets without any external stimuli or crosslinker. The spontaneous self-assembly of the native protein sheets into protein shells not only preserves the native functional properties of the protein but also enhances their thermal stability compared to the sheets. We further demonstrate that these compartments can encapsulate macromolecular enzymes and, more interestingly, permit the free exchange of small molecules and substrates through their intrinsic conduit channels. The porous nature of the shell housing active enzymes and allowing movement of small molecules makes them suitable as active bioreactors. Furthermore, to extend the tunability of these protein-compartments with respect to stability, enzyme-encapsulation, and permeability, we fabricated three different compartments using three different sheet proteins, PduA/B/B' and compared their properties. Interestingly we find that all three protein shells show similar behaviour with respect to an encapsulated diol-dehydratase enzyme and vitamin B12, which are native to the Pdu BMC system. Furthermore, for the non-native enzyme CytC, the small molecule R6G dye, doxorubicin, NR and curcumin they behave diversely. Insights from this analysis will allow us to design and develop sheet protein based synthetic active bioreactors requiring meticulous, compartmentalization in process optimization.

Bacteriophage-mediated identification of bacteria using photoacoustic flow cytometry.

Infection with resistant bacteria has become an ever increasing problem in modern medical practice. Currently, broad spectrum antibiotics are prescribed until bacteria can be identified through blood cultures, a process that can take two to three days and is unable to provide quantitative information. To detect and quantify bacteria rapidly in blood samples, we designed a method using labeled bacteriophage in conjunction with photoacoustic flow cytometry (PAFC). PAFC is the generation of ultrasonic waves created by the absorption of laser light in particles under flow. Bacteriophage is a virus that infects bacteria and possesses the ability to discriminate bacterial surface antigens, allowing the bacteriophage to bind only to their target bacteria. Bacteria can be tagged with dyed phage and processed through a photoacoustic flow cytometer where they are detected by the acoustic response. We demonstrate that E. coli; can be detected and discriminated from Salmonella; using this method. Our goal is to develop a method to determine bacterial content in blood samples. We hope to develop this technology into future clinical use and decrease the time required to identify bacterial species from 3 to 4 days to less than 1 hour.

Structure of Salmonella Flagellar Hook Reveals Intermolecular Domain Interactions for the Universal Joint Function.

The bacterial flagellum is a motility organelle consisting of a rotary motor and a long helical filament as a propeller. The flagellar hook is a flexible universal joint that transmits motor torque to the filament in its various orientations that change dynamically between swimming and tumbling of the cell upon switching the motor rotation for chemotaxis. Although the structures of the hook and hook protein FlgE from different bacterial species have been studied, the structure of Salmonella hook, which has been studied most over the years, has not been solved at a high enough resolution to allow building an atomic model of entire FlgE for understanding the mechanisms of self-assembly, stability and the universal joint function. Here we report the structure of Salmonella polyhook at 4.1 Å resolution by electron cryomicroscopy and helical image analysis. The density map clearly revealed folding of the entire FlgE chain forming the three domains D0, D1 and D2 and allowed us to build an atomic model. The model includes domain Dc with a long ß-hairpin structure that connects domains D0 and D1 and contributes to the structural stability of the hook while allowing the flexible bending of the hook as a molecular universal joint.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

Biofilm formation and potential virulence factors of Salmonella strains isolated from ready-to-eat shrimps.

Salmonella species is an important foodborne pathogen with the non-typhoidal serovars such as Enteritidis and Typhimurium as the most predominant strains. This study examines the biofilm formation, phenotypic virulence factors and cell surface characteristics of Salmonella strains from ready-to-eat shrimps. The ready-to-eat shrimps were obtained from open markets between November 2016 and October 2017 in Edo and Delta States, Nigeria. The occurrence of Salmonella strains in this study was 210/1440 (14.58%) of the ready-to-eat shrimp's samples. The identified strains comprise of Salmonella Enteritidis 11, Salmonella Typhimurium 14 and other Salmonella spp. 20. The 45 identified Salmonella strains revealed the following virulence properties: swimming and swarming motility 45(100%); S-layer 39(86.67%); haemolytic activity 40(88.89%); lipase activity 43(95.56%); protease activity 43(95.56%); gelatinase production 43(95.56%); and DNA degrading activity 41(91.11%). The variation in the formation of biofilm-based on the diversity of Salmonella species was observed with higher percentage of Salmonella Typhimurium strains as strong biofilms producers under different environmental conditions. For surface hydrophobicity using bacterial adherence to hydrocarbons, 25(55.56%) were hydrophilic while 20(44.44%) were moderately hydrophobic from the 45 Salmonella isolates. Using salting aggregation test for surface hydrophobicity, all selected isolates 45(100%) was hydrophilic. Autoaggregation index for the 12 selected Salmonella isolates ranged from 15.2-47.2%, while the autoaggragation index for the 12 selected test bacteria ranged from 26.2-71.3%. Coaggragation between the 12 selected test bacteria and 12 Salmonella isolates ranged from 12.5-81.0%. The occurrence of pathogenic species of Salmonella from ready-to-eat shrimps could be detrimental to the consumers. Findings on the physiological conditions of biofilms formed by the foodborne pathogenic Salmonella and the cell surface characteristics therein are crucial for the advancement of methods for controlling Salmonella from ready-to-eat foods.

Multipurpose efficacy of the lyophilized cell-free supernatant of Salmonella bongori isolated from the freshwater fish, Devario aequipinnatus: toxicity against microbial pathogens and mosquito vectors.

Presently, the discovery of effective drugs and pesticides from eco-friendly biological sources is an important challenge in the field of life sciences. The present research was aimed for standardizing an innovative approach in the evaluation of the biological potentiality of the metabolites of fish-associated bacteria. We have identified 17 skin-associated bacteria from the freshwater fish, giant danio, Devario aquipinnatus. They were screened through biofilm forming and extracellular enzyme producing ability. The results of preliminary antibacterial evaluation of the bacterial supernatants underlined the importance of three potential strains (BH8, BH10 and BH11) for further applied research. Hence, such strains were subsequently subjected to a novel extraction procedure to overcome the difficulties found in polar solvents mixed with the supernatant. The lyophilized cell-free supernatant (LCFS) of 3 isolates were individually extracted by using methanol. During the testing of LCFS's methanolic extract (LCFS-ME) of 3 isolates, only the extract of BH11-strain exhibited potent inhibitory activity against the pathogenic bacteria and fungi. Furthermore, the larvicidal and mosquitocidal assays on the filariasis vector, Culex quinquefasciatus also showed its potent toxicity on both the adults and developmental instars of mosquito. Through molecular and phylogenetic analyses, the BH11 strain was identified as Salmonella bongori (KR350635). The present finding emphasized that the S. bongori could be an important novel source of effective antimicrobials and mosquitocidal agents.

Viability and membrane lipid composition under a 57mT static magnetic field in Salmonella Hadar.

The aim of this work is to demonstrate the effects of a static magnetic field (SMF) with an induction 12 equal to 57mT on the viability and membrane lipid composition of Salmonella Hadar. Results showed an increase in the viability of exposed bacteria compared to controls after 9h of exposure. Analysis with gas chromatography of total lipids (TLs) and different fractions of phospholipids: phosphatidylglycerols (PGs), phosphatidylethanolamines (PEs), and cardiolipins (CLs), separated by thin layer chromatography revealed changes in fatty acid levels during exposure. For TLs, the unsaturated fatty acids/saturated fatty acids ratio (UFAs/SFAs) had significantly increased after 9 h of exposure. The variation of this ratio seems to be essentially due to the increase of the proportion of unsaturated fatty acids with 18 carbons, in particular C18:1. The analyses of fatty acid composition carried out on the scale of each fraction of phospholipids showed that CLs contributed significantly to the increase of the proportion of the unsaturated fatty acids between 6 and 9h of exposure thanks to their unsaturated chains with 18 carbons (especially C18:2). CLs appear to be the main phospholipid involved in the adaptation of S. Hadar membranes to the SMF.

Assessment of sediment mutagenicity in areas under the influence of a contaminated site undergoing a remediation process.

Soil contamination enters aquatic ecosystems affecting sediment quality. The region studied is the Taquari River, Brazil, close to a site contaminated by wood preservatives, with a runoff route into the river. The first stage of the remediation process (In this article, the terms intervention and remediation have been used with slightly different meanings. We consider intervention to be the first phase of the remediation process, which aims to remove active sources) was an intervention to remove the main active sources. The Salmonella/microsome assay and polycyclic aromatic hydrocarbons (PAHs) were used to assess sediment quality in organic extracts during different intervention phases. The strains used were TA98, TA97a, and TA100 with and without S9mix (±S9). The results indicated the presence of pro-mutagens at site Ta010 (closest to the contaminated site) in all samplings, and the highest result occurred before intervention for TA100 + S9 (1,672 ± 215.9 rev/g). These values decreased during (83 ± 23.6 rev/g) and after this process (403 ± 105.9 rev/g), although the PAHs concentrations increased. Samples from this site presented PAHs with a carcinogenic potential during the assessed periods. After intervention, Ta006 (4 km downstream from Ta010) showed the most significant mutagenesis for TA100 + S9 (764 ± 230.2 rev/g) and, although the total PAHs values were lower, the species considered carcinogenic had higher concentrations. Mutagenesis predicted values of PAHs confirmed that carcinogenic species were predominantly detected by TA100, and the other PAHs by TA97a strains. Marked contaminant release to the river was observed, mainly in Ta010 at different periods. Mutagenicity and PAHs values in an internal stream, upstream from Ta010, showed a dispersion route of these agents. Thus, contamination in Ta010 and possible contribution to Ta006, after intervention, provides a warning regarding environmental quality in the region. Environ. Mol. Mutagen. 59:625-638, 2018. © 2018 Wiley Periodicals, Inc.

Advanced immunocapture of milk-borne Salmonella by microfluidic magnetically stabilized fluidized bed.

The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (µMSFB), developed for the capture and on-chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti-Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti-Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the µMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.

Antibacterial Mechanism of 405-Nanometer Light-Emitting Diode against Salmonella at Refrigeration Temperature.

The aim of this study was to elucidate the antibacterial mechanism of 405 ± 5-nm light-emitting diode (LED) illumination against Salmonella at 4°C in phosphate-buffered saline (PBS) by determining endogenous coproporphyrin content, DNA oxidation, damage to membrane function, and morphological change. Gene expression levels, including of oxyR, recA, rpoS, sodA, and soxR, were also examined to understand the response of Salmonella to LED illumination. The results showed that Salmonella strains responded differently to LED illumination, revealing that S. enterica serovar Enteritidis (ATCC 13076) and S. enterica subsp. enterica serovar Saintpaul (ATCC 9712) were more susceptible and resistant, respectively, than the 16 other strains tested. There was no difference in the amounts of endogenous coproporphyrin in the two strains. Compared with that in nonilluminated cells, the DNA oxidation levels in illuminated cells increased. In illuminated cells, we observed a loss of efflux pump activity, damage to the glucose uptake system, and changes in membrane potential and integrity. Transmission electron microscopy revealed a disorganization of chromosomes and ribosomes due to LED illumination. The levels of the five genes measured in the nonilluminated and illuminated S Saintpaul cells were upregulated in PBS at a set temperature of 4°C, indicating that increased gene expression levels might be due to a temperature shift and nutrient deficiency rather than to LED illumination. In contrast, only oxyR in S Enteritidis cells was upregulated. Thus, different sensitivities of the two strains to LED illumination were attributed to differences in gene regulation.IMPORTANCE Bacterial inactivation using visible light has recently received attention as a safe and environmentally friendly technology, in contrast with UV light, which has detrimental effects on human health and the environment. This study was designed to understand how 405 ± 5-nm light-emitting diode (LED) illumination kills Salmonella strains at refrigeration temperature. The data clearly demonstrated that the effectiveness of LED illumination on Salmonella strains depended highly on the serotype and strain. Our findings also revealed that its antibacterial mechanism was mainly attributed to DNA oxidation and a loss of membrane functions rather than membrane lipid peroxidation, which has been proposed by other researchers who studied the antibacterial effect of LED illumination by adding exogenous photosensitizers, such as chlorophyllin and hypericin. Therefore, this study suggests that the detailed antibacterial mechanisms of 405-nm LED illumination without additional photosensitizers may differ from that by exogenous photosensitizers. Furthermore, a change in stress-related gene regulation may alter the susceptibility of Salmonella cells to LED illumination at refrigeration temperature. Thus, our study provides new insights into the antibacterial mechanism of 405 ± 5-nm LED illumination on Salmonella cells.

Microfiltration of enzyme treated egg whites for accelerated detection of viable Salmonella.

We report detection of <13 CFU of Salmonella per 25 g egg white within 7 h by concentrating the bacteria using microfiltration through 0.2-µm cutoff polyethersulfone hollow fiber membranes. A combination of enzyme treatment, controlled cross-flow on both sides of the hollow fibers, and media selection were key to controlling membrane fouling so that rapid concentration and the subsequent detection of low numbers of microbial cells were achieved. We leveraged the protective effect of egg white proteins and peptone so that the proteolytic enzymes did not attack the living cells while hydrolyzing the egg white proteins responsible for fouling. The molecular weight of egg white proteins was reduced from about 70 kDa to 15 kDa during hydrolysis. This enabled a 50-fold concentration of the cells when a volume of 525 mL of peptone and egg white, containing 13 CFU of Salmonella, was decreased to a 10 mL volume in 50 min. A 10-min microcentrifugation step further concentrated the viable Salmonella cells by 10×. The final cell recovery exceeded 100%, indicating that microbial growth occurred during the 3-h processing time. The experiments leading to rapid concentration, recovery, and detection provided further insights on the nature of membrane fouling enabling fouling effects to be mitigated. Unlike most membrane processes where protein recovery is the goal, recovery of viable microorganisms for pathogen detection is the key measure of success, with modification of cell-free proteins being both acceptable and required to achieve rapid microfiltration of viable microorganisms. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1464-1471, 2016.

Protective efficacy by various doses of Salmonella ghost vaccine candidate carrying enterotoxigenic Escherichia coli fimbrial antigen against neonatal piglet colibacillosis.

Humoral immune responses and protective efficacy by various doses of Salmonella ghost cells carrying enterotoxigenic Escherichia coli (ETEC) fimbrial antigens for protection against piglet colibacillosis were studied. All groups were orally primed and boosted at 11 and 14 wk of pregnancy, respectively. Group A sows were inoculated with phosphate-buffered saline (PBS), and groups B, C, and D sows were immunized with 2 × 10(9), 2 × 10(10), and 2 × 10(11) ghost cells, respectively. Serum immunoglobulin (Ig) G, and colostrum IgG and IgA levels of groups C and D sows were significantly higher than those of group A sows. In addition, serum IgG and IgA levels in group C and D piglets were significantly increased compared to those of group A piglets. After challenge with wild-type ETEC, diarrhea and mortality were not observed in group C and D piglets, while diarrhea was observed in 88.9% and 58.8% of groups A and B piglets, respectively, and 16.7% mortality was observed in group A piglets. These findings indicate that oral immunization of sows with 2 × 10(10) or 10(11) ghost cells can effectively protect their offspring from colibacillosis.

Les réponses immunitaires humorales et l'efficacité protectrice de doses variées de cellules fantômes de Salmonella transportant des antigènes fimbriaires d'Escherichia coli entérotoxinogène (ETEC) pour protéger des porcelets contre la colibacillose ont été étudiées. Tous les groupes ont reçu une dose initiale et un rappel par voie orale à 11 et 14 sem de gestation, respectivement. Les truies du groupe A furent inoculées avec de la saline tamponnée (PBS), et les groupes B, C, et D immunisés avec 2 × 109, 2 × 1010, et 2 × 1011 cellules fantômes, respectivement. Les niveaux d'immunoglobulines (Ig) G sériques, ainsi que des IgG et IgA du colostrum des truies des groupes C et D étaient significativement supérieurs à ceux des truies du groupe A. De plus, les niveaux des IgG et IgA sériques des porcelets des groupes C et D étaient significativement augmentés comparativement à ceux des porcelets du groupe A. Après infection défi avec une souche sauvage d'ETEC, aucune diarrhée et mortalité ne furent observées chez les porcelets des groupes C et D, alors que de la diarrhée fut observée chez 88,9 % et 58,8 % des porcelets des groupes A et B, respectivement, et 16,7 % de mortalité fut notée chez les porcelets du groupe A. Ces résultats indiquent que l'immunisation des truies avec 2 × 1010 ou 1011 de cellules fantômes peut protéger efficacement les petits de la portée contre la colibacillose.(Traduit par Docteur Serge Messier).

A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes.

The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. Although RNA sequencing (RNA-seq) has greatly advanced our ability to study the transcriptomes of prokaryotes and eukaryotes separately, limitations in existing protocols for the generation and analysis of RNA-seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low-yield sample inputs and a computational pipeline for analysis of both mammalian and microbial reads from mixed host-pathogen RNA-seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach that is suitable for large-scale studies and will enable the field to obtain in-depth analysis of host-pathogen interactions in infection models.

Bacterial Outer Membrane Vesicles as a Delivery System for Virulence Regulation.

Outer membrane vesicles (OMVs) are spherical nanostructures that are ubiquitously shed from gram-negative bacteria both in vitro and in vivo. Recent findings revealed that OMVs, which contain diverse components derived from the parent bacterium, play an important role in communication with neighboring bacteria and the environment. Furthermore, nanoscale proteoliposomes decorated with pathogen-associated molecules attract considerable attention as a non-replicative carrier for vaccines and drug materials. This review introduces recent advances in OMV biogenesis and discusses the roles of OMVs in the context of bacterial communication and virulence regulation. It also describes the remarkable accomplishments in OMV engineering for diverse therapeutic applications.

Using MHC Molecules to Define a Chlamydia T Cell Vaccine.

Vaccines based on humoral immunity alone are unlikely to protect against infections caused by intracellular pathogens and today's most pressing infectious diseases of public health importance are caused by intracellular infections that include tuberculosis, malaria, HIV/AIDS, and others such as Chlamydia trachomatis. For these infections, vaccines that induce cellular immune responses are essential. Major impediments in developing such vaccines include difficulty in identifying relevant T cell antigens and delivering them in ways that elicit protective cellular immunity. Genomics and proteomics now provide tools to allow unbiased empirical identification of candidate T cell antigens. This approach represents an advance on bioinformatic searches for candidate T cell antigens. This chapter discusses an immunoproteomic approach we have used to identify Chlamydia T cell antigens. We further discuss how these T cell antigens can be developed into a human vaccine.