Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy.

Autophagy plays an essential role in the defense against many microbial pathogens as a regulator of both innate and adaptive immunity. Some pathogens have evolved sophisticated mechanisms that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, motility-associated killing factor A (MakA). pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 (herein referring to MAP1LC3B) lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex, required for LC3 lipidation at the membranous aggregate, resulted in an inhibition of both canonical autophagy and autophagy-related processes, including the unconventional secretion of interleukin-1ß (IL-1ß). These findings identify a novel mechanism of host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.

At-home sampling to meet geographical challenges for serological assessment of SARS-CoV-2 exposure in a rural region of northern Sweden, March to May 2021: a retrospective cohort study.

BackgroundThe current SARS-CoV-2 pandemic has highlighted a need for easy and safe blood sampling in combination with accurate serological methodology. Venipuncture for testing is usually performed by trained staff at healthcare centres. Long travel distances to healthcare centres in rural regions may introduce a bias of testing towards relatively large communities with closer access. Rural regions are therefore often not represented in population-based data.AimThe aim of this retrospective cohort study was to develop and implement a strategy for at-home testing in a rural region of Sweden during spring 2021, and to evaluate its role to provide equal health care for its inhabitants.MethodsWe developed a sensitive method to measure antibodies to the S-protein of SARS-CoV-2 and optimised this assay for clinical use together with a strategy of at-home capillary blood sampling.ResultsWe demonstrated that our ELISA gave comparable results after analysis of capillary blood or serum from SARS-CoV-2-experienced individuals. We demonstrated stability of the assay under conditions that reflected temperature and humidity during winter or summer. By assessment of capillary blood samples from 4,122 individuals, we could show both feasibility of the strategy and that implementation shifted the geographical spread of testing in favour of rural areas.ConclusionImplementation of at-home sampling enabled citizens living in remote rural areas access to centralised and sensitive laboratory antibody tests. The strategy for testing used here could therefore enable disease control authorities to get rapid access to information concerning immunity to infectious diseases, even across vast geographical distance.

A Shigella effector dampens inflammation by regulating epithelial release of danger signal ATP through production of the lipid mediator PtdIns5P.

Upon infection with Shigella flexneri, epithelial cells release ATP through connexin hemichannels. However, the pathophysiological consequence and the regulation of this process are unclear. Here we showed that in intestinal epithelial cell ATP release was an early alert response to infection with enteric pathogens that eventually promoted inflammation of the gut. Shigella evolved to escape this inflammatory reaction by its type III secretion effector IpgD, which blocked hemichannels via the production of the lipid PtdIns5P. Infection with an ipgD mutant resulted in rapid hemichannel-dependent accumulation of extracellular ATP in vitro and in vivo, which preceded the onset of inflammation. At later stages of infection, ipgD-deficient Shigella caused strong intestinal inflammation owing to extracellular ATP. We therefore describe a new paradigm of host-pathogen interaction based on endogenous danger signaling and identify extracellular ATP as key regulator of mucosal inflammation during infection. Our data provide new angles of attack for the development of anti-inflammatory molecules.

Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serotype 5a M90T and genome-wide transcriptional start site determination.

BACKGROUND: Shigella is a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans. Shigella invades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strain S. flexneri serotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches. RESULTS: We have sequenced, assembled, annotated and manually curated the full genome of S. flexneri 5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new hybrid strategy to prepare gapless, highly accurate genome sequences, which also cover AT-rich tracks or repetitive sequences that are transcribed. Furthermore, we have performed genome-wide analysis of transcriptional start sites (TSS) and determined the length of 5' untranslated regions (5'-UTRs) at typical culture conditions for the inoculum of in vitro infection experiments. We identified 6723 primary TSS (pTSS) and 7328 secondary TSS (sTSS). The S. flexneri 5a M90T annotated genome sequence and the transcriptional start sites are integrated into RegulonDB (http://regulondb.ccg.unam.mx) and RSAT (http://embnet.ccg.unam.mx/rsat/) databases to use their analysis tools in the S. flexneri 5a M90T genome. CONCLUSIONS: We provide the first complete genome for S. flexneri serotype 5a, specifically the laboratory reference strain M90T. Our work opens the possibility of employing S. flexneri M90T in high-quality systems biology studies such as transcriptomic and differential expression analyses or in genome evolution studies. Moreover, the catalogue of TSS that we report here can be used in molecular pathogenesis studies as a resource to know which genes are transcribed before infection of host cells. The genome sequence, together with the analysis of transcriptional start sites, is also a valuable tool for precise genetic manipulation of S. flexneri 5a M90T. Further, we present a new hybrid strategy to prepare gapless, highly accurate genome sequences. Unlike currently used hybrid strategies combining long- and short-read DNA sequencing technologies to maximize accuracy, our workflow using long-read DNA sequencing and short-read RNA sequencing provides the added value of using non-redundant technologies, which yield distinct, exploitable datasets.

CRISPR-Cas-Guided Mutagenesis of Chromosome and Virulence Plasmid in Shigella flexneri by Cytosine Base Editing.

Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrheal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large-scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas-dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by single guide RNA (sgRNA) to introduce targeted C→T transitions, resulting in internal stop codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter, we were able to generate loss-of-function mutants in both Escherichia coli and Shigella flexneri with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion was in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Furthermore, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella flexneri genes and found the mutant phenotype to be similar to those of the reported gene deletion mutants, with no apparent polar effects at the phenotype level. This method can be used in a 96-well-plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days. IMPORTANCE Loss-of-function mutagenesis is critical in understanding the physiological role of genes. Therefore, high-throughput techniques to generate such mutants are important for facilitating the assessment of gene function at a pace that matches systems biology approaches. However, to our knowledge, no such method was available for generating an array of single gene mutants in an important enteropathogen-Shigella. This pathogen causes high morbidity and mortality in children, and antibiotic-resistant strains are quickly emerging. Therefore, determination of the function of unknown Shigella genes is of the utmost importance to develop effective strategies to control infections. Our present work will bridge this gap by providing a rapid method for generating loss-of-function mutants. The highly effective and specific method has the potential to be programmed to generate multiple mutants in a single, massively parallel reaction. By virtue of plasmid compatibility, this method can be extended to other members of Enterobacteriaceae.

Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages.

Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. The precise mechanisms and the bacterial factors allowing B. cereus to circumvent host immune responses remain to be elucidated. We have previously shown that B. cereus induces macrophage cell death by an unknown mechanism. Here we identified the toxic component from the B. cereus supernatant. We report that Haemolysin II (HlyII) provokes macrophage cell death by apoptosis through its pore-forming activity. The HlyII-induced apoptotic pathway is caspase 3 and 8 dependent, thus most likely mediated by the death receptor pathway. Using insects and mice as in vivo models, we show that deletion of hlyII strongly reduces virulence. In addition, we show that after infection of Bombyx mori larvae, the immune cells are apoptotic, demonstrating that HlyII induces apoptosis of phagocytic cells in vivo. Altogether, our results clearly unravel HlyII as a novel virulence protein that induces apoptosis in phagocytic cells in vitro and in vivo.

Infection-induced membrane ruffling initiates danger and immune signaling via the mechanosensor PIEZO1.

Microorganisms are generally sensed by receptors recognizing microbial molecules, which evoke changes in cellular activities and gene expression. Bacterial pathogens induce secretion of the danger signal ATP as an early alert response of intestinal epithelial cells, initiating overt inflammation. However, what triggers ATP secretion during infection is unclear. Here we show that the inherently mechanosensitive plasma membrane channel PIEZO1 acts as a sensor for bacterial entry. PIEZO1 is mechanically activated by invasion-induced membrane ruffles upstream of Ca2+ influx and ATP secretion. Mimicking mechanical stimuli of pathogen uptake with sterile beads equally elicits ATP secretion. Chemical or genetic PIEZO1 inactivation inhibits mechanically induced ATP secretion. Moreover, chemical or mechanical PIEZO1 activation evokes gene expression in immune and barrier pathways. Thus, mechanosensation of invasion-induced plasma membrane distortion initiates immune signaling upon infection, independently of detection of microbial molecules. Hence, PIEZO1-dependent detection of infection is driven by physical signals instead of chemical ligands.

RpuS/R Is a Novel Two-Component Signal Transduction System That Regulates the Expression of the Pyruvate Symporter MctP in Sinorhizobium fredii NGR234.

The SLC5/STAC histidine kinases comprise a recently identified family of sensor proteins in two-component signal transduction systems (TCSTS), in which the signaling domain is fused to an SLC5 solute symporter domain through a STAC domain. Only two members of this family have been characterized experimentally, the CrbS/R system that regulates acetate utilization in Vibrio and Pseudomonas, and the CbrA/B system that regulates the utilization of histidine in Pseudomonas and glucose in Azotobacter. In an attempt to expand the characterized members of this family beyond the Gammaproteobacteria, we identified two putative TCSTS in the Alphaproteobacterium Sinorhizobium fredii NGR234 whose sensor histidine kinases belong to the SLC5/STAC family. Using reverse genetics, we were able to identify the first TCSTS as a CrbS/R homolog that is also needed for growth on acetate, while the second TCSTS, RpuS/R, is a novel system required for optimal growth on pyruvate. Using RNAseq and transcriptional fusions, we determined that in S. fredii the RpuS/R system upregulates the expression of an operon coding for the pyruvate symporter MctP when pyruvate is the sole carbon source. In addition, we identified a conserved DNA sequence motif in the putative promoter region of the mctP operon that is essential for the RpuR-mediated transcriptional activation of genes under pyruvate-utilizing conditions. Finally, we show that S. fredii mutants lacking these TCSTS are affected in nodulation, producing fewer nodules than the parent strain and at a slower rate.

Serological assessment of SARS-CoV-2 exposure in northern Sweden by the use of at-home sampling to meet geographical challenges in rural regions.

The current SARS-CoV-2 pandemic has highlighted a need for easy and safe blood sampling in combination with accurate serological methodology. Venipuncture is usually performed by trained staff at health care centers. Long travel distances may introduce a bias of testing towards relatively large communities with close access to health care centers. Rural regions may thus be overlooked. Here, we demonstrate a sensitive method to measure antibodies to the S-protein of SARS-CoV-2. We adapted and optimized this assay for clinical use together with capillary blood sampling to meet the geographical challenges of serosurveillance. Finally, we tested remote at-home capillary blood sampling together with centralized assessment of S-specific IgG in a rural region of northern Scandinavia that encompasses 55,185 sq kilometers. We conclude that serological assessment from capillary blood sampling gives comparable results as analysis of venous blood. Importantly, at-home sampling enabled citizens living in remote rural areas access to centralized and sensitive laboratory antibody tests.

Generation of plasma cells and CD27-IgD- B cells during hantavirus infection is associated with distinct pathological findings.

OBJECTIVE: Human hantavirus infections can cause haemorrhagic fever with renal syndrome (HFRS). The pathogenic mechanisms are not fully understood, nor if they affect the humoral immune system. The objective of this study was to investigate humoral immune responses to hantavirus infection and to correlate them to the typical features of HFRS: thrombocytopenia and transient kidney dysfunction. METHODS: We performed a comprehensive characterisation of longitudinal antiviral B-cell responses of 26 hantavirus patients and combined this with paired clinical data. In addition, we measured extracellular adenosine triphosphate (ATP) and its breakdown products in circulation and performed in vitro stimulations to address its effect on B cells. RESULTS: We found that thrombocytopenia was correlated to an elevated frequency of plasmablasts in circulation. In contrast, kidney dysfunction was indicative of an accumulation of CD27-IgD- B cells and CD27-/low plasmablasts. Finally, we provide evidence that high levels of extracellular ATP and matrix metalloproteinase 8 can contribute to shedding of CD27 during human hantavirus infection. CONCLUSION: Our findings demonstrate that thrombocytopenia and kidney dysfunction associate with distinctly different effects on the humoral immune system. Moreover, hantavirus-infected individuals have significantly elevated levels of extracellular ATP in circulation.

Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria.

Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence. Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) using the enteric pathogen Shigella flexneri serotype 5a strain M90T as model. However, this method can be employed in any other bacterial species of choice. In the first steps, total RNA is purified from bacterial cultures using the hot phenol method. Ribosomal RNA (rRNA) is specifically depleted via hybridization probes using a commercial kit. A 5'-monophosphate-dependent exonuclease (TEX)-treated RNA library enriched in primary transcripts is then prepared for comparison with a library that has not undergone TEX-treatment, followed by ligation of an RNA linker adaptor of known sequence allowing the determination of TSS with single nucleotide precision. Finally, the RNA is processed for Illumina sequencing library preparation and sequenced as purchased service. TSS are identified by in-house bioinformatic analysis. Our protocol is cost-effective as it minimizes the use of commercial kits and employs freely available software.

Gentamicin Protection Assay to Determine the Number of Intracellular Bacteria during Infection of Human TC7 Intestinal Epithelial Cells by Shigella flexneri.

Shigella flexneri is an intracellular bacterial pathogen that gains access to the gut epithelium using a specialized Type III Secretion System (T3SS). Various determinants mediating this invasive infection have been experimentally verified using the classical gentamicin protection assay presented here. In this assay epithelial cell lines are infected by bacteria in vitro and the extracellular bacteria are killed by gentamicin. The internalized bacteria, which are protected from the bactericidal action of gentamicin, are recovered by lysing the epithelial cells and enumerated by determining the colonies formed on solid medium. Various techniques based on light microscopy, such as immunofluorescence and bacteria expressing fluorescent proteins, are also used for studying intracellular bacteria. However, these techniques are not only labor intensive and require sophisticated equipment, but mostly are also not quantitative. Despite being an easy quantitative method to study invasiveness of bacteria, the gentamicin protection assay cannot distinguish between the survival and multiplication of the internalized bacteria over longer incubation periods. To alleviate the complications created by multiplication and dissemination of internalized bacteria, complementary assays like plaque formation assays are required. This protocol presents an easy and cost-effective method to determine the invasiveness and the capacity to establish an infection of Shigella under different conditions.

Plaque Assay to Determine Invasion and Intercellular Dissemination of Shigella flexneri in TC7 Human Intestinal Epithelial Cells.

Shigella flexneri invades the epithelial cells lining the gut lumen and replicates intracellularly. The specialized Type III Secretion System (T3SS) and its effector proteins, encoded on a large virulence plasmid, assist the bacterium to gain access to the cytosol. Thereafter Shigella disseminates to neighboring cells in an epithelial layer without further extracellular steps. Host cell lysis occurs when these bacteria have extensively replicated in the target cell cytosol. Here we describe a simple method to qualitatively as well as quantitatively study the capacity of Shigella to invade and disseminate within an epithelium by assessing the number and size of plaques representing the dead cells in a monolayer of TC7 cells. This classical protocol follows a simple approach of infecting the monolayers of epithelial cell lines with Shigella and visualizing the dead cells as plaques formed against a stained background.

Where and how do anthrax toxins exit endosomes to intoxicate host cells?

The role of Bacillus anthracis virulence factors in its pathogenesis has been subjected to intense investigation with the aim of finding novel preventive and therapeutic protocols. Toxins that are endocytosed and act in the cytosol of host cells have a central role in B. anthracis infection. Understanding of anthrax toxin cell entry has increased during the past few years and a composite picture is emerging. Nevertheless, unanswered and controversial questions remain, particularly concerning the site and mode of anthrax toxin cell entry, the role of anthrax toxin receptors in the process and the possible involvement of cytosolic chaperones, which might affect entry efficiency. Here, the current model of anthrax toxin cell entry, an alternative model and experimental approaches for clarifying unanswered questions will be discussed.

cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin.

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.

NADH oxidation drives respiratory Na+ transport in mitochondria from Yarrowia lipolytica.

It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na+ uptake, indicating that a redox-driven, primary Na+ pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4 micromol min(-1) mg(-1)) to Na+ translocation (2.0 micromol min(-1) mg(-1)). NADH-driven Na+ transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na+ pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na+ gradient established by complex I.

PI5P Triggers ICAM-1 Degradation in Shigella Infected Cells, Thus Dampening Immune Cell Recruitment.

Shigella flexneri, the pathogen responsible for bacillary dysentery, has evolved multiple strategies to control the inflammatory response. Here, we show that Shigella subverts the subcellular trafficking of the intercellular adhesion molecule-1 (ICAM-1), a key molecule in immune cell recruitment, in a mechanism dependent on the injected bacterial enzyme IpgD and its product, the lipid mediator PI5P. Overexpression of IpgD, but not a phosphatase dead mutant, induced the internalization and the degradation of ICAM-1 in intestinal epithelial cells. Remarkably, addition of permeant PI5P reproduced IpgD effects and led to the inhibition of neutrophil recruitment. Finally, these results were confirmed in an in vivo model of Shigella infection where IpgD-dependent ICAM-1 internalization reduced neutrophil adhesion. In conclusion, we describe here an immune evasion mechanism used by the pathogen Shigella to divert the host cell trafficking machinery in order to reduce immune cell recruitment.

Distinct mutations led to inactivation of type 1 fimbriae expression in Shigella spp.

Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events.

Type III secretion system.

The type III secretion system (T3SS) is a membrane-embedded nanomachine found in several Gram-negative bacteria. Upon contact between bacteria and host cells, the syringe-like T3SS (Figure 1) transfers proteins termed effectors from the bacterial cytosol to the cytoplasm or the plasma membrane of a single target cell. This is a major difference from secretion systems that merely release molecules into the extracellular milieu, where they act on potentially distant target cells expressing the relevant surface receptors. The syringe architecture is conserved at the structural and functional level and supports injection into a great variety of hosts and tissues. However, the pool of effectors is species specific and determines the outcome of the interaction, via modulation of target-cell function.

Preventing acute gut wall damage in infectious diarrhoeas with glycosylated dendrimers.

Intestinal pathogens use the host's excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non-antibiotic-based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4-MD-2-LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)-6 by Gram-negative bacteria. In a rabbit model of shigellosis, PAMAM-DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)-DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM-DG potently inhibited Shigella Lipid A-induced IL-6 expression. In rabbits, PETIM-DG prevented Shigella-induced epithelial gut wall damage, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. There was no change in ß-defensin, IL-10, interferon-ß, transforming growth factor-ß, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases.