Comparative transcriptional profiling of the early host response to infection by typhoidal and non-typhoidal Salmonella serovars in human intestinal organoids.

Salmonella enterica represents over 2500 serovars associated with a wide-ranging spectrum of disease; from self-limiting gastroenteritis to invasive infections caused by non-typhoidal serovars (NTS) and typhoidal serovars, respectively. Host factors strongly influence infection outcome as malnourished or immunocompromised individuals can develop invasive infections from NTS, however, comparative analyses of serovar-specific host responses have been constrained by reliance on limited model systems. Here we used human intestinal organoids (HIOs), a three-dimensional "gut-like" in vitro system derived from human embryonic stem cells, to elucidate similarities and differences in host responses to NTS and typhoidal serovars. HIOs discriminated between the two most prevalent NTS, Salmonella enterica serovar Typhimurium (STM) and Salmonella enterica serovar Enteritidis (SE), and typhoidal serovar Salmonella enterica serovar Typhi (ST) in epithelial cell invasion, replication and transcriptional responses. Pro-inflammatory signaling and cytokine output was reduced in ST-infected HIOs compared to NTS infections, consistent with early stages of NTS and typhoidal diseases. While we predicted that ST would induce a distinct transcriptional profile from the NTS strains, more nuanced expression profiles emerged. Notably, pathways involved in cell cycle, metabolism and mitochondrial functions were downregulated in STM-infected HIOs and upregulated in SE-infected HIOs. These results correlated with suppression of cellular proliferation and induction of host cell death in STM-infected HIOs and in contrast, elevated levels of reactive oxygen species production in SE-infected HIOs. Collectively, these results suggest that the HIO model is well suited to reveal host transcriptional programming specific to infection by individual Salmonella serovars, and that individual NTS may provoke unique host epithelial responses during intestinal stages of infection.

Cellular Activity of Salmonella Typhimurium ArtAB Toxin and Its Receptor-Binding Subunit.

Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonellaenterica Typhimurium DT104 phage type, which is associated with multidrug-resistant disease in humans and animals, possesses an ADP-ribosylating toxin called ArtAB. Full-length artAB has been found on a number of broad-host-range non-typhoidal Salmonella species and serovars. ArtAB is also homologous to many AB5 toxins from diverse Gram-negative pathogens, including cholera toxin (CT) and pertussis toxin (PT), and may be involved in Salmonella pathogenesis, however, in vitro cellular toxicity of ArtAB has not been characterized. artAB was cloned into E. coli and initially isolated using a histidine tag (ArtABHIS) and nickel chromatography. ArtABHIS was found to bind to African green monkey kidney epithelial (Vero) cells using confocal microscopy and to interact with glycans present on fetuin and monosialotetrahexosylganglioside (GM1) using ELISA. Untagged, or native, holotoxin (ArtAB), and the pentameric receptor-binding subunit (ArtB) were purified from E. coli using fetuin and d-galactose affinity chromatography. ArtAB and ArtB metabolic and cytotoxic activities were determined using Vero and Chinese hamster ovary (CHO) epithelial cells. Vero cells were more sensitive to ArtAB, however, incubation with both cell types revealed only partial cytotoxicity over 72 h, similar to that induced by CT. ArtAB induced a distinctive clustering phenotype on CHO cells over 72 h, similar to PT, and an elongated phenotype on Vero cells, similar to CT. The ArtB binding subunit alone also had a cytotoxic effect on CHO cells and induced morphological rounding. Results indicate that this toxin induces distinctive cellular outcomes. Continued biological characterization of ArtAB will advance efforts to prevent disease caused by non-typhoidal Salmonella.

How Pathogens Feel and Overcome Magnesium Limitation When in Host Tissues.

Host organisms utilize nutritional immunity to limit the availability of nutrients essential to an invading pathogen. Nutrients may include amino acids, nucleotide bases, and transition metals, the essentiality of which varies among pathogens. The mammalian macrophage protein Slc11a1 (previously Nramp1) mediates resistance to several intracellular pathogens. Slc11a1 is proposed to restrict growth of Salmonella enterica serovar Typhimurium in host tissues by causing magnesium deprivation. This is intriguing because magnesium is the most abundant divalent cation in all living cells. A pathogen's response to factors such as Slc11a1 that promote nutritional immunity may therefore reflect what the pathogen 'feels' in its cytoplasm, rather than the nutrient concentration in host cell compartments.

Endovascular repair combined with staged drainage for the treatment of infectious aortic aneurysm: a case report.

BACKGROUND: Infectious aortic aneurysm, defined as a focal dilation of an infectious arterial wall, is an uncommon life-threatening disease. Compared with open surgery, endovascular repair yields acceptable clinical outcomes. However, residual tissue infection may increase the risk of secondary intervention. Here, we present a successful case of endovascular repair combined with staged drainage for the treatment of infectious aortic aneurysm. CASE PRESENTATION: A 58-year-old man presented to hospital with a 3-day history of lower back pain radiating to the back associated with fever. The dynamic imaging characteristics revealed rapid progress of infectious abdominal aortic aneurysm with negative blood culture. The patient underwent endovascular repair and salmonella enteritidis was identified through drain culture. CONCLUSIONS: Endovascular procedure and staged drainage can be feasible and effective option in selected cases.

Disseminated Salmonella infection.

A 47-year-old Malay man who presented with fever, poor oral intake and loss of weight for 1 month duration. Further work-up revealed evidence of disseminated Salmonella infection that was further complicated with pericardial and pleural empyema. Cultures from pericardial and pleural fluids grew Salmonella species with negative serial blood cultures. Contrast enhanced CT thorax showed pleural effusion with large pericardial effusion. The patient was treated with antibiotics and drainage of pericardial and pleural empyema was done and he was discharged well.

Barcoded Consortium Infections Resolve Cell Type-Dependent Salmonella enterica Serovar Typhimurium Entry Mechanisms.

Bacterial host cell invasion mechanisms depend on the bacterium's virulence factors and the properties of the target cell. The enteropathogen Salmonella enterica serovar Typhimurium (STm) invades epithelial cell types in the gut mucosa and a variety of immune cell types at later infection stages. The molecular mechanism(s) of host cell entry has, however, been studied predominantly in epithelial cell lines. STm uses a type three secretion system (TTSS-1) to translocate effectors into the host cell cytosol, thereby sparking actin ruffle-dependent entry. The ruffles also fuel cooperative invasion by bystander bacteria. In addition, several TTSS-1-independent entry mechanisms exist, involving alternative STm virulence factors, or the passive uptake of bacteria by phagocytosis. However, it remains ill-defined how STm invasion mechanisms vary between host cells. Here, we developed an internally controlled and scalable method to map STm invasion mechanisms across host cell types and conditions. The method relies on host cell infections with consortia of chromosomally tagged wild-type and mutant STm strains, where the abundance of each strain can be quantified by qPCR or amplicon sequencing. Using this methodology, we quantified cooccurring TTSS-1-dependent, cooperative, and TTSS-1-independent invasion events in epithelial, monocyte, and macrophage cells. We found STm invasion of epithelial cells and monocytes to proceed by a similar MOI-dependent mix of TTSS-1-dependent and cooperative mechanisms. TTSS-1-independent entry was more frequent in macrophages. Still, TTSS-1-dependent invasion dominated during the first minutes of interaction also with this cell type. Finally, the combined action of the SopB/SopE/SopE2 effectors was sufficient to explain TTSS-1-dependent invasion across both epithelial and phagocytic cells.IMPORTANCESalmonella enterica serovar Typhimurium (STm) is a widespread and broad-host-spectrum enteropathogen with the capacity to invade diverse cell types. Still, the molecular basis for the host cell invasion process has largely been inferred from studies of a few selected cell lines. Our work resolves the mechanisms that Salmonellae employ to invade prototypical host cell types, i.e., human epithelial, monocyte, and macrophage cells, at a previously unattainable level of temporal and quantitative precision. This highlights efficient bacterium-driven entry into innate immune cells and uncovers a type III secretion system effector module that dominates active bacterial invasion of not only epithelial cells but also monocytes and macrophages. The results are derived from a generalizable method, where we combine barcoding of the bacterial chromosome with mixed consortium infections of cultured host cells. The application of this methodology across bacterial species and infection models will provide a scalable means to address host-pathogen interactions in diverse contexts.

Infection-generated electric field in gut epithelium drives bidirectional migration of macrophages.

Many bacterial pathogens hijack macrophages to egress from the port of entry to the lymphatic drainage and/or bloodstream, causing dissemination of life-threatening infections. However, the underlying mechanisms are not well understood. Here, we report that Salmonella infection generates directional electric fields (EFs) in the follicle-associated epithelium of mouse cecum. In vitro application of an EF, mimicking the infection-generated electric field (IGEF), induces directional migration of primary mouse macrophages to the anode, which is reversed to the cathode upon Salmonella infection. This infection-dependent directional switch is independent of the Salmonella pathogenicity island 1 (SPI-1) type III secretion system. The switch is accompanied by a reduction of sialic acids on glycosylated surface components during phagocytosis of bacteria, which is absent in macrophages challenged by microspheres. Moreover, enzymatic cleavage of terminally exposed sialic acids reduces macrophage surface negativity and severely impairs directional migration of macrophages in response to an EF. Based on these findings, we propose that macrophages are attracted to the site of infection by a combination of chemotaxis and galvanotaxis; after phagocytosis of bacteria, surface electrical properties of the macrophage change, and galvanotaxis directs the cells away from the site of infection.

Invasive non-typhoidal Salmonella in sickle cell disease in Africa: is increased gut permeability the missing link?

Non-typhoidal Salmonella usually induces self-limiting gastroenteritis. However, in many parts of Africa, especially in individuals who are malnourished, infected with malaria, or have sickle cell disease, the organism causes serious and potentially fatal systemic infections. Since the portal of entry of non-typhoidal Salmonella into the systemic circulation is by way of the intestine, we argue that an increased gut permeability plays a vital role in the initiation of invasive non-typhoidal Salmonella in these patients. Here, we will appraise the evidence supporting a breach in the intestinal barrier and propose the mechanisms for the increased risks for invasive non-typhoidal Salmonella infections in these individuals.

Autophagy and Ubiquitination in Salmonella Infection and the Related Inflammatory Responses.

Salmonellae are facultative intracellular pathogens that cause globally distributed diseases with massive morbidity and mortality in humans and animals. In the past decades, numerous studies were focused on host defenses against Salmonella infection. Autophagy has been demonstrated to be an important defense mechanism to clear intracellular pathogenic organisms, as well as a regulator of immune responses. Ubiquitin modification also has multiple effects on the host immune system against bacterial infection. It has been indicated that ubiquitination plays critical roles in recognition and clearance of some invading bacteria by autophagy. Additionally, the ubiquitination of autophagy proteins in autophagy flux and inflammation-related substance determines the outcomes of infection. However, many intracellular pathogens manipulate the ubiquitination system to counteract the host immunity. Salmonellae interfere with host responses via the delivery of ~30 effector proteins into cytosol to promote their survival and proliferation. Among them, some could link the ubiquitin-proteasome system with autophagy during infection and affect the host inflammatory responses. In this review, novel findings on the issue of ubiquitination and autophagy connection as the mechanisms of host defenses against Salmonella infection and the subverted processes are introduced.

A sex-dependent change in behavioral temperature regulation in African house snakes (Lamprophis fuliginosus) challenged with different pathogens.

Behavioral fever in reptiles is often considered an adaptive response used to eliminate pathogens, yet empirical data showing the wide-spread use of this response is mixed. This behavioral change can be beneficial by enhancing the host's immune response and increasing the animal's chance of survival, but it can also be detrimental in terms of host energetic requirements and enzymatic performance. Thus, we examined whether captive-bred African house snakes (Lamprophis fuliginosus) employed behavioral fever in response to pathogen stimulus. Twenty-one African house snakes were injected separately with three different strains of ultraviolet (UV) light-killed bacteria (Escherichia coli, Staphylococcus aureus, Salmonella enterica). We found an increased variance of hourly cloacal temperatures following exposure to pathogens in male but not female house snakes. We did not, however, find a significant febrile response to pathogen exposure as measured via mean cloacal temperature. This research adds critical information to the field of reptilian physiology as this field remains understudied. Reptilian immune function and its relationship with thermal biology is ever more pertinent as new challenges arise, such as novel pathogens and changing climate.

Probiotic/prebiotic correction for adverse effects of iron fortification on intestinal resistance to Salmonella infection in weaning mice.

Iron fortification has been associated with a modest increase in diarrhea risk among children. Herein, we investigate the correction for this unwanted side effect with probiotic/prebiotic supplementation in weaning mice. Iron fortification with 250 ppm and 500 ppm ferrous sulfate for 30 days significantly increased the species richness of the mouse gut microbiota compared to controls. The 500 ppm-FeSO4 diet caused a significantly decreased abundance of potentially beneficial Lactobacillus. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), mice on the 500 ppm-FeSO4 diet showed earlier appearance of poisoning symptoms, higher rates of weight and appetite loss, and lower survival rates, all of which were effectively reversed by supplementation with a probiotic (Lactobacillus acidophilus) or a prebiotic (inulin) for 7 days before infection. Iron fortification with 500 ppm ferrous sulfate also increased fecal shedding and spleen and liver load of viable S. Typhimurium, suggesting its promoting effect on pathogen colonization and translocation, and this negative effect was found to be well corrected by supplementation with Lactobacillus acidophilus or inulin. Light and transmission electron microscopic observation on the ileal villus structure revealed the histopathological impairment of the intestine by iron fortification with both 250 ppm and 500 ppm ferrous sulfate, and the intestinal lesions were markedly alleviated by supplementation with Lactobacillus acidophilus or inulin. These results provide experimental evidence for the increased diarrhea risk upon iron fortification with high pathogen load, and demonstrate that probiotic or prebiotic supplementation can be used to eliminate the potential harm of iron fortification on gut health.

Chronic Disseminated Salmonellosis in a Patient With Interleukin-12p40 Deficiency.

Interleukin (IL)-12 is composed of p35 and p40 subunits; in this case, IL-12p40 deficiency is a rare genetic etiology of Mendelian susceptibility to mycobacterial disease. Salmonellosis has been reported in almost half of these patients and mostly present in recurrent extraintestinal form. In this report, we described an 18-month-old boy with absence of IL-12p40 production suffering from chronic disseminated nontyphoidal salmonellosis. To the best of our knowledge, this is the first-reported case.

Salmonella Intracellular Lifestyles and Their Impact on Host-to-Host Transmission.

More than a century ago, infections by Salmonella were already associated with foodborne enteric diseases with high morbidity in humans and cattle. Intestinal inflammation and diarrhea are hallmarks of infections caused by nontyphoidal Salmonella serovars, and these pathologies facilitate pathogen transmission to the environment. In those early times, physicians and microbiologists also realized that typhoid and paratyphoid fever caused by some Salmonella serovars could be transmitted by "carriers," individuals outwardly healthy or at most suffering from some minor chronic complaint. In his pioneering study of the nontyphoidal serovar Typhimurium in 1967, Takeuchi published the first images of intracellular bacteria enclosed by membrane-bound vacuoles in the initial stages of the intestinal epithelium penetration. These compartments, called Salmonella-containing vacuoles, are highly dynamic phagosomes with differing biogenesis depending on the host cell type. Single-cell studies involving real-time imaging and gene expression profiling, together with new approaches based on genetic reporters sensitive to growth rate, have uncovered unprecedented heterogeneous responses in intracellular bacteria. Subpopulations of intracellular bacteria displaying fast, reduced, or no growth, as well as cytosolic and intravacuolar bacteria, have been reported in both in vitro and in vivo infection models. Recent investigations, most of them focused on the serovar Typhimurium, point to the selection of persisting bacteria inside macrophages or following an autophagy attack in fibroblasts. Here, we discuss these heterogeneous intracellular lifestyles and speculate on how these disparate behaviors may impact host-to-host transmissibility of Salmonella serovars.

Endogenous and exogenous control of gastrointestinal epithelial function: building on the legacy of Bayliss and Starling.

Transport of fluid and electrolytes in the intestine allows for appropriate adjustments in luminal fluidity while reclaiming water used in digesting and absorbing a meal, and is closely regulated. This article discusses various endogenous and exogenous mechanisms whereby transport is controlled in the gut, placing these in the context of the ideas about the neurohumoral control of alimentary physiology that were promulgated by William Bayliss and Ernest Starling. The article considers three themes. First, mechanisms that intrinsically regulate chloride secretion, centred on the epidermal growth factor receptor (EGFr), are discussed. These may be important in ensuring that excessive chloride secretion, with the accompanying loss of fluid, is not normally stimulated by intestinal distension as the meal passes through the gastrointestinal tract. Second, mechanisms whereby probiotic microorganisms can impart beneficial effects on the gut are described, with a focus on targets at the level of the epithelium. These findings imply that the commensal microbiota exert important influences on the epithelium in health and disease. Finally, mechanisms that lead to diarrhoea in patients infected with an invasive pathogen, Salmonella, are considered, based on recent studies in a novel mouse model. Diarrhoea is most likely attributable to reduced expression of absorptive transporters and may not require the influx of neutrophils that accompanies infection. Overall, the goal of the article is to highlight the many ways in which critical functions of the intestinal epithelium are regulated under physiological and pathophysiological conditions, and to suggest possible targets for new therapies for digestive disease states.

Interferon-γ in Salmonella pathogenesis: New tricks for an old dog.

Salmonella enterica is a facultative intracellular bacterium that is the leading cause of food borne illnesses in humans. The cytokine IFN-γ has well-established antibacterial properties against Salmonella and other intracellular microbes, for example its capacity to activate macrophages, promote phagocytosis, and destroy phagocytosed microbes by free radical-driven toxification of phagosomes. But IFN-γ induces the expression of hundreds of uncharacterized genes, suggesting that this cytokine deploys additional antimicrobial strategies that await discovery. Recently, one such mechanism, mediated by a family of IFN-inducible small GTPases called Guanylate Binding Proteins (GBPs) has been uncovered. GBPs were shown to facilitate the pyroptotic clearance of Salmonella from infected macrophages by rupturing the protective intracellular vacuole this microbe forms around itself. Once this protective vacuole is lost, exposed Salmonella activates pyroptosis, which destroys the infected cell. In this review, we summarize such emerging roles for IFN-γ in restricting Salmonella pathogenesis.

Free-floating epithelial micro-tissue arrays: a low cost and versatile technique.

Three-dimensional (3D) tissue models are invaluable tools that can closely reflect the in vivo physiological environment. However, they are usually difficult to develop, have a low throughput and are often costly; limiting their utility to most laboratories. The recent availability of inexpensive additive manufacturing printers and open source 3D design software offers us the possibility to easily create affordable 3D cell culture platforms. To demonstrate this, we established a simple, inexpensive and robust method for producing arrays of free-floating epithelial micro-tissues. Using a combination of 3D computer aided design and 3D printing, hydrogel micro-moulding and collagen cell encapsulation we engineered microenvironments that consistently direct the growth of micro-tissue arrays. We described the adaptability of this technique by testing several immortalised epithelial cell lines (MDCK, A549, Caco-2) and by generating branching morphology and micron to millimetre scaled micro-tissues. We established by fluorescence and electron microscopy that micro-tissues are polarised, have cell type specific differentiated phenotypes and regain native in vivo tissue qualities. Finally, using Salmonella typhimurium we show micro-tissues display a more physiologically relevant infection response compared to epithelial monolayers grown on permeable filter supports. In summary, we have developed a robust and adaptable technique for producing arrays of epithelial micro-tissues. This in vitro model has the potential to be a valuable tool for studying epithelial cell and tissue function/architecture in a physiologically relevant context.

Salmonella spv locus suppresses host innate immune responses to bacterial infection.

Salmonella enterica serovar typhimurium (S. typhimurium) is globally distributed and causes massive morbidity and mortality in humans and animals. S. typhimurium carries Salmonella plasmid virulence (spv) locus, which is highly conserved and closely related to bacterial pathogenicity, while its exact role in host immune responses during infection remains to be elucidated. To counteract the invaders, the host has evolved numerous strategies, among which the innate immunity and autophagy act as the first defense. Recently, zebrafish has been universally accepted as a valuable and powerful vertebrate model in analyzing bacteria-host interactions. To investigate whether spv locus enhances the virulence of Salmonella by exerting an effect on the host early defense, zebrafish larvae were employed in this study. LD50 of S. typhimurium to zebrafish larvae and bacterial dissemination were analyzed. Sudan black B and neutral red staining were performed to detect the responses of neutrophils and macrophages to Salmonella infection. Autophagy agonist Torin1 and inhibitor Chloroquine were used to interfere in autophagic flux, and the protein level of Lc3 and p62 were measured by western blotting. Results indicated that spv locus could decrease the LD50 of S. typhimurium to zebrafish larvae, accelerate the reproduction and dissemination of bacteria by inhibiting the function of neutrophils and macrophages. Moreover, spv locus restrained the formation of autophagosomes in the earlier stage of autophagy. These findings suggested the virulence of spv locus involving in suppressing host innate immune responses for the first time, which shed new light on the role of spv operon in Salmonella pathogenicity.

Salmosan, a ß-Galactomannan-Rich Product, Protects Epithelial Barrier Function in Caco-2 Cells Infected by Salmonella enterica Serovar Enteritidis.

BACKGROUND: One promising strategy for reducing human salmonellosis induced by Salmonella Enteritidis is to supplement animal diets with natural feed additives such as mannan oligosaccharides (MOSs). OBJECTIVE: We sought to investigate the potential role of Salmosan (S-ßGM), an MOS product extremely rich in ß-galactomannan, in preventing epithelial barrier function disruption induced by S. Enteritidis colonization in an in vitro model of intestinal Caco-2 cells in culture. METHODS: Differentiated Caco-2 cells were incubated for 3 h with S. Enteritidis at a multiplicity of infection of 10 in the absence or presence of 500 µg S-ßGM/mL. Paracellular permeability (PP) was assessed by transepithelial electrical resistance (TER), d-mannitol, and fluorescein isothiocyanate-dextran (FD-4) flux. Tight junction proteins and cytoskeletal actin were also localized by confocal microscopy. Reactive oxygen species (ROS) and lipid peroxidation products were evaluated. Scanning and transmission electron microscopy were used to visualize S. Enteritidis adhesion to, and invasion of, the Caco-2 cell cultures. RESULTS: Compared with controls, TER was significantly reduced by 30%, and d-mannitol and FD-4 flux were significantly increased by 374% and 54% in S. Enteritidis-infected cultures, respectively. The presence of S-ßGM in infected cultures induced total recoveries of TER and FD-4 flux to values that did not differ from the control and a partial recovery of d-mannitol flux. These effects were confirmed by immunolocalization of actin, zonula occludens protein 1, and occludin. Similar results were obtained for Salmonella Dublin. The protection of S-ßGM on PP in infected cultures may be associated with a total recovery of ROS production to values that did not differ from the control. Moreover, S-ßGM has the capacity to agglutinate bacteria, leading to a significant reduction of 32% in intracellular S Enteritidis. CONCLUSION: The results demonstrate that S-ßGM contributes to protecting epithelial barrier function in a Caco-2 cell model disrupted by S. Enteritidis.