Innate immune defense against intracellular pathogens, like Salmonella, relies heavily on the autophagy machinery of the host. This response is studied intensively in epithelial cells, the target of ...Salmonella during gastrointestinal infections. However, little is known of the role that autophagy plays in macrophages, the predominant carriers of this pathogen during systemic disease. Here we utilize a zebrafish embryo model to study the interaction of S. enterica serovar Typhimurium with the macroautophagy/autophagy machinery of macrophages in vivo. We show that phagocytosis of live but not heat-killed Salmonella triggers recruitment of the autophagy marker GFP-Lc3 in a variety of patterns labeling tight or spacious bacteria-containing compartments, also revealed by electron microscopy. Neutrophils display similar GFP-Lc3 associations, but genetic modulation of the neutrophil/macrophage balance and ablation experiments show that macrophages are critical for the defense response. Deficiency of atg5 reduces GFP-Lc3 recruitment and impairs host resistance, in contrast to atg13 deficiency, indicating that Lc3-Salmonella association at this stage is independent of the autophagy preinitiation complex and that macrophages target Salmonella by Lc3-associated phagocytosis (LAP). In agreement, GFP-Lc3 recruitment and host resistance are impaired by deficiency of Rubcn/Rubicon, known as a negative regulator of canonical autophagy and an inducer of LAP. We also found strict dependency on NADPH oxidase, another essential factor for LAP. Both Rubcn and NADPH oxidase are required to activate a Salmonella biosensor for reactive oxygen species inside infected macrophages. These results identify LAP as the major host protective autophagy-related pathway responsible for macrophage defense against Salmonella during systemic infection.
Abbreviations: ATG: autophagy related gene; BECN1: Beclin 1; CFU: colony forming units; CYBA/P22PHOX: cytochrome b-245, alpha chain; CYBB/NOX2: cytochrome b-245 beta chain; dpf: days post fertilization; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hfp: hours post fertilization; hpi: hours post infection; IRF8: interferon regulatory factor 8; Lcp1/L-plastin: lymphocyte cytosolic protein 1; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1A/1B-light chain 3; mCherry: red fluorescent protein; mpeg1: macrophage expressed gene 1; mpx: myeloid specific peroxidase; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; NCF4/P40PHOX: neutrophil cytosolic factor 4; NTR-mCherry: nitroreductase-mCherry fusion; PTU: phenylthiourea; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB-1 inducible coiled coin 1; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; RUBCN/RUBICON: RUN and cysteine rich domain containing BECN1-interacting protein; SCV: Salmonella-containing vacuole; S. Typhimurium/S.T: Salmonella enterica serovar Typhimurium; TEM: transmission electron microscopy; Tg: transgenic; TSA: tyramide signal amplification; ULK1/2: unc-51-like autophagy activating kinase 1/2; UVRAG: UVRAG: UV radiation resistance associated; wt: wild type
Nitric oxide (NO) generated by inducible NO synthase 2 (NOS2) affects cellular iron homeostasis, but the underlying molecular mechanisms and implications for NOS2-dependent pathogen control are ...incompletely understood. In this study, we found that NO up-regulated the expression of ferroportin-1 (Fpn1), the major cellular iron exporter, in mouse and human cells. Nos2(-/-) macrophages displayed increased iron content due to reduced Fpn1 expression and allowed for an enhanced iron acquisition by the intracellular bacterium Salmonella typhimurium. Nos2 gene disruption or inhibition of NOS2 activity led to an accumulation of iron in the spleen and splenic macrophages. Lack of NO formation resulted in impaired nuclear factor erythroid 2-related factor-2 (Nrf2) expression, resulting in reduced Fpn1 transcription and diminished cellular iron egress. After infection of Nos2(-/-) macrophages or mice with S. typhimurium, the increased iron accumulation was paralleled by a reduced cytokine (TNF, IL-12, and IFN-γ) expression and impaired pathogen control, all of which were restored upon administration of the iron chelator deferasirox or hyperexpression of Fpn1 or Nrf2. Thus, the accumulation of iron in Nos2(-/-) macrophages counteracts a proinflammatory host immune response, and the protective effect of NO appears to partially result from its ability to prevent iron overload in macrophages.
Non-typhoidal Salmonella represents an important human and animal pathogen world-wide. Most human salmonellosis cases are foodborne, but each year infections are also acquired through direct or ...indirect animal contact in homes, veterinary clinics, zoological gardens, farm environments or other public, professional or private settings. Clinically affected animals may exhibit a higher prevalence of shedding than apparently healthy animals, but both can shed Salmonella over long periods of time. In addition, environmental contamination and indirect transmission through contaminated food and water may complicate control efforts. The public health risk varies by animal species, age group, husbandry practice and health status, and certain human subpopulations are at a heightened risk of infection due to biological or behavioral risk factors. Some serotypes such as Salmonella Dublin are adapted to individual host species, while others, for instance Salmonella Typhimurium, readily infect a broad range of host species, but the potential implications for human health are currently unclear. Basic hygiene practices and the implementation of scientifically based management strategies can efficiently mitigate the risks associated with animal contacts. However, the general public is frequently unaware of the specific disease risks involved, and high-risk behaviors are common. Here we describe the epidemiology and serotype distribution of Salmonella in a variety of host species. In addition, we review our current understanding of the public health risks associated with different types of contacts between humans and animals in public, professional or private settings, and, where appropriate, discuss potential risk mitigation strategies.
Salmonella enterica serotype Dublin is a cattle-adapted bacterium that typically causes bloodstream infections in humans. To summarize demographic, clinical, and antimicrobial drug resistance ...characteristics of human infections with this organism in the United States, we analyzed data for 1968-2013 from 5 US surveillance systems. During this period, the incidence rate for infection with Salmonella Dublin increased more than that for infection with other Salmonella. Data from 1 system (FoodNet) showed that a higher percentage of persons with Salmonella Dublin infection were hospitalized and died during 2005-2013 (78% hospitalized, 4.2% died) than during 1996-2004 (68% hospitalized, 2.7% died). Susceptibility data showed that a higher percentage of isolates were resistant to >7 classes of antimicrobial drugs during 2005-2013 (50.8%) than during 1996-2004 (2.4%).
It has been 30 years since the initial emergence and subsequent rapid global spread of multidrug-resistant Salmonella entericaserovar Typhimurium DT104 (MDR DT104). Nonetheless, its origin and ...transmission route have never been revealed. We used whole-genome sequencing (WGS) and temporally structured sequence analysis within a Bayesian framework to reconstruct temporal and spatial phylogenetic trees and estimate the rates of mutation and divergence times of 315S Typhimurium DT104 isolates sampled from 1969 to 2012 from 21 countries on six continents. DT104 was estimated to have emerged initially as antimicrobial susceptible in ∼1948 (95% credible interval CI, 1934 to 1962) and later became MDR DT104 in ∼1972 (95% CI, 1972 to 1988) through horizontal transfer of the 13-kb Salmonella genomic island 1 (SGI1) MDR region into susceptible strains already containing SGI1. This was followed by multiple transmission events, initially from central Europe and later between several European countries. An independent transmission to the United States and another to Japan occurred, and from there MDR DT104 was probably transmitted to Taiwan and Canada. An independent acquisition of resistance genes took place in Thailand in ∼1975 (95% CI, 1975 to 1990). In Denmark, WGS analysis provided evidence for transmission of the organism between herds of animals. Interestingly, the demographic history of Danish MDR DT104 provided evidence for the success of the program to eradicate Salmonellafrom pig herds in Denmark from 1996 to 2000. The results from this study refute several hypotheses on the evolution of DT104 and suggest that WGS may be useful in monitoring emerging clones and devising strategies for prevention of Salmonella infections.
Caspases are either apoptotic or inflammatory. Among inflammatory caspases, caspase-1 and -11 trigger pyroptosis, a form of programmed cell death. Whereas both can be detrimental in inflammatory ...disease, only caspase-1 has an established protective role during infection. Here, we report that caspase-11 is required for innate immunity to cytosolic, but not vacuolar, bacteria. Although Salmonella typhimurium and Legionella pneumophila normally reside in the vacuole, specific mutants (sifA and sdhA, respectively) aberrantly enter the cytosol. These mutants triggered caspase-11, which enhanced clearance of S. typhimurium sifA in vivo. This response did not require NLRP3, NLRC4, or ASC inflammasome pathways. Burkholderia species that naturally invade the cytosol also triggered caspase-11, which protected mice from lethal challenge with B. thailandensis and B. pseudomallei. Thus, caspase-11 is critical for surviving exposure to ubiquitous environmental pathogens.
A pandemic of Salmonella enterica serotype Enteritidis emerged in the 1980s due to contaminated poultry products. How Salmonella Enteritidis rapidly swept through continents remains a historical ...puzzle as the pathogen continues to cause outbreaks and poultry supply becomes globalized. We hypothesize that international trade of infected breeding stocks causes global spread of the pathogen. By integrating over 30,000 Salmonella Enteritidis genomes from 98 countries during 1949-2020 and international trade of live poultry from the 1980s to the late 2010s, we present multifaceted evidence that converges on a high likelihood, global scale, and extended protraction of Salmonella Enteritidis dissemination via centralized sourcing and international trade of breeding stocks. We discovered recent, genetically near-identical isolates from domestically raised poultry in North and South America. We obtained phylodynamic characteristics of global Salmonella Enteritidis populations that lend spatiotemporal support for its dispersal from centralized origins during the pandemic. We identified concordant patterns of international trade of breeding stocks and quantitatively established a driving role of the trade in the geographic dispersal of Salmonella Enteritidis, suggesting that the centralized origins were infected breeding stocks. Here we demonstrate the value of integrative and hypothesis-driven data mining in unravelling otherwise difficult-to-probe pathogen dissemination from hidden origins.
Background & Aims Intraepithelial lymphocytes that express the γδ T-cell receptor (γδ IELs) limit pathogen translocation across the intestinal epithelium by unknown mechanisms. We investigated ...whether γδ IEL migration and interaction with epithelial cells promote mucosal barrier maintenance during enteric infection. Methods Salmonella typhimurium or Toxoplasma gondii were administered to knockout (KO) mice lacking either the T cell receptor δ chain (Tcrd) or CD103, or control TcrdEGFP C57BL/6 reporter mice. Intravital microscopy was used to visualize migration of green fluorescent protein (GFP)-tagged γδ T cells within the small intestinal mucosa of mice infected with DsRed-labeled S typhimurium . Mixed bone marrow chimeras were generated to assess the effects of γδ IEL migration on early pathogen invasion and chronic systemic infection. Results Morphometric analyses of intravital video microscopy data showed that γδ IELs rapidly localized to and remained near epithelial cells in direct contact with bacteria. Within 1 hour, greater numbers of T gondii or S typhimurium were present within mucosae of mice with migration-defective occludin KO γδ T cells, compared with controls. Pathogen invasion in Tcrd KO mice was quantitatively similar to that in mice with occludin-deficient γδ T cells, whereas invasion in CD103 KO mice, which have increased migration of γδ T cells into the lateral intercellular space, was reduced by 63%. Consistent with a role of γδ T-cell migration in early host defense, systemic salmonellosis developed more rapidly and with greater severity in mice with occludin-deficient γδ IELs, relative to those with wild-type or CD103 KO γδ IELs. Conclusions In mice, intraepithelial migration to epithelial cells in contact with pathogens is essential to γδ IEL surveillance and immediate host defense. γδ IEL occludin is required for early surveillance that limits systemic disease.
Conventional wisdom holds that microbes support their growth in vertebrate hosts by exploiting a large variety of nutrients. We show here that use of a specific nutrient (ethanolamine) confers a ...marked growth advantage on Salmonella enterica serovar Typhimurium (S. Typhimurium) in the lumen of the inflamed intestine. In the anaerobic environment of the gut, ethanolamine supports little or no growth by fermentation. However, S. Typhimurium is able to use this carbon source by inducing the gut to produce a respiratory electron acceptor (tetrathionate), which supports anaerobic growth on ethanolamine. The gut normally converts ambient hydrogen sulfide to thiosulfate, which it then oxidizes further to tetrathionate during inflammation. Evidence is provided that S. Typhimurium's growth advantage in an inflamed gut is because of its ability to respire ethanolamine, which is released from host tissue, but is not utilizable by competing bacteria. By inducing intestinal inflammation, S. Typhimurium sidesteps nutritional competition and gains the ability to use an abundant simple substrate, ethanolamine, which is provided by the host.
Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T ...cell homing to the gastrointestinal (GI) tract involves the induction of α4β7 and CCR9 by Peyer's patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid-dependent manner. This paradigm, however, cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration, we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly, we found that lung DCs, like CD103(+) MLN DCs, up-regulate the gut-homing integrin α4β7 in vitro and in vivo, and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses, lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs, which has the potential to inform the design of novel vaccines against mucosal pathogens.
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