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Dysregulation of gut homeostasis may drive a variety of pathological conditions like inflammatory bowel disease (IBD). One of the first events associated with induction of a proinflammatory state is the recruitment of neutrophils to the intestinal lumen. Neutrophils play a critical role in the maintenance homeostasis by eliminating pathogens and contributing to mucosal healing for the resolution of inflammation. However, an excessive recruitment and accumulation of activated neutrophils in the intestine under pathological conditions, such as IBD, is associated with mucosal injury. While the basolateral release of interleukin-8 by intestinal epithelial cells stimulates the recruitment of neutrophils from the vasculature to the submucosa, secretion of the bioactive lipid hepoxilin A3 (HxA3) from the apical surface is required to draw neutrophils across the epithelial barrier. HxA3 is a potent neutrophil chemoattractant, derived from arachidonic acid by the 12/15-lipoxygenase (12/15-LO) pathway, that is secreted to the intestinal lumen by the multidrug resistance protein 2 (MRP2) to form a gradient required for neutrophil trans-epithelial migration (TEM). Previous research developed in our laboratory demonstrated that another multidrug resistance protein, P-glycoprotein (P-gp), was responsible for the apical secretion of endocannabinoids (eCBs) to the intestinal lumen. Moreover, these eCBs were shown to inhibit HxA3-driven neutrophil TEM. Given that neutrophil TEM is critical to the development of inflammatory pathology and that current therapies for IBD suffer from damaging sequelae and an inability to prevent relapses, the main goal of this project is to characterize the mechanisms by which cannabinoids (CB) modulate the TEM of neutrophils induced by HxA3 and to develop CB-based therapies to treat IBD. We used collagen-coated transwells to screen a panel of eCBs, phytocannabinoids (pCBs), and synthetic cannabinoids (SC) for their ability to inhibit HxA3-driven migration. These CB showed a different efficiency to inhibit neutrophil migration, with anandamide (100%), cannabidiol (90%), and AM1241 (90%), exhibiting the highest inhibition. Moreover, migration experiments using an agonist (CP55940) and an antagonist (SR144528) specific for the cannabinoid receptor 2 (CB2), revealed that although this receptor participates in the modulation of neutrophil TEM mediated by CB, there are other still unidentified receptors involved in this process. Finally, to understand the fine resolution decision-making process neutrophils formulate between activation and inhibitory signals, we are using a novel microfluidic device coupled to an on-demand chemotaxis gradient that allows us to interrogate neutrophil responses to various combinations of HxA3 and CB gradients. Together, our results will greatly help define neutrophil biology in health and disease and should allow us to uncover new therapeutic strategies for controlling intestinal inflammation. This research was funded by grant R01 DK109677 from National Institutes of Health This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BackgroundThe gastrointestinal (GI) tract is the reservoir for multidrug-resistant (MDR) pathogens, specifically carbapenem-resistant (CR) Klebsiella pneumoniae and other Enterobacteriaceae, which often lead to the spread of antimicrobial resistance genes, severe extraintestinal infections, and lethal outcomes. Selective GI decolonization has been proposed as a new strategy for preventing transmission to other body sites and minimizing spreading to susceptible individuals.ResultsHere, we purify the to-date uncharacterized class IIb microcin I47 (MccI47) and demonstrate potent inhibition of numerous Enterobacteriaceae, including multidrug-resistant clinical isolates, in vitro at concentrations resembling those of commonly prescribed antibiotics. We then genetically modify the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to produce MccI47 from a stable multicopy plasmid by using MccI47 toxin production in a counterselection mechanism to engineer one of the native EcN plasmids, which renders provisions for inducible expression and plasmid selection unnecessary. We then test the clinical relevance of the MccI47-producing engineered EcN in a murine CR K. pneumoniae colonization model and demonstrate significant MccI47-dependent reduction of CR K. pneumoniae abundance after seven days of daily oral live biotherapeutic administration without disruption of the resident microbiota.ConclusionsThis study provides the first demonstration of MccI47 as a potent antimicrobial against certain Enterobacteriaceae, and its ability to significantly reduce the abundance of CR K. pneumoniae in a preclinical animal model, when delivered from an engineered live biotherapeutic product. This study serves as the foundational step towards the use of engineered live biotherapeutic products aimed at the selective removal of MDR pathogens from the GI tract

During the past several decades, new diagnostic tools, interventional approaches, and population-wide changes in the major coronary risk factors have taken place. However, few studies have examined relatively recent trends in the demographic characteristics, clinical profile, and the short-term outcomes of patients hospitalized for acute myocardial infarction (AMI) from the more generalizable perspective of a population-based investigation. We examined decade long trends (2001 to 2011) in patient's demographic and clinical characteristics, treatment practices, and hospital outcomes among residents of the Worcester metropolitan area hospitalized with an initial AMI (n = 3,730) at all 11 greater Worcester medical centers during 2001, 2003, 2005, 2007, 2009, and 2011. The average age of the study population was 68.5 years and 56.9% were men. Patients hospitalized with a first AMI during the most recent study years were significantly younger (mean age = 69.9 years in 2001/2003; 65.2 years in 2009/2011), had lower serum troponin levels, and experienced a shorter hospital stay compared with patients hospitalized during the earliest study years. Hospitalized patients were more likely to received evidence-based medical management practices over the decade long period under study. Multivariable-adjusted regression models showed a considerable decline over time in the hospital death rate and a significant reduction in the proportion of patients who developed atrial fibrillation, heart failure, and ventricular fibrillation during their acute hospitalization. These results highlight the changing nature of patients hospitalized with an incident AMI, and reinforce the need for surveillance of AMI at the community level.

The epithelial surfaces of the skin and intestinal, respiratory, and reproductive tracts are persistently exposed to the myriad of microorganisms present in the external environment. Because this constitutes a limited access border, the cells of these epithelia must be able to establish barriers against microbial intruders and to raise the alarm if the barriers are breached. The gastrointestinal epithelium, in particular, forms a critical interface between the internal milieu and the lumen. The barrier formed by the gastrointestinal tract is essential in preventing noxious luminal contents from accessing the interstitial tissues, and must also support digestion and active vectorial transport of nutrients, electrolytes, and water. Therefore, the barrier formed by gastrointestinal epithelium must be highly regulated and selectively permeable.

Different Escherichia coli strains generally have the same metabolic capacity for growth on sugars in vitro, but they appear to use different sugars in the streptomycin-treated mouse intestine (Fabich et al., Infect. Immun. 76:1143-1152, 2008). Here, mice were precolonized with any of three human commensal strains ( E. coli MG1655, E. coli HS, or E. coli Nissle 1917) and 10 days later were fed 10 5 CFU of the same strains. While each precolonized strain nearly eliminated its isogenic strain, confirming that colonization resistance can be modeled in mice, each allowed growth of the other commensal strains to higher numbers, consistent with different commensal E. coli strains using different nutrients in the intestine. Mice were also precolonized with any of five commensal E. coli strains for 10 days and then were fed 10 5 CFU of E. coli EDL933, an O157:H7 pathogen. E. coli Nissle 1917 and E. coli EFC1 limited growth of E. coli EDL933 in the intestine (10 3 to 10 4 CFU/gram of feces), whereas E. coli MG1655, E. coli HS, and E. coli EFC2 allowed growth to higher numbers (10 6 to 10 7 CFU/gram of feces). Importantly, when E. coli EDL933 was fed to mice previously co-colonized with three E. coli strains (MG1655, HS, and Nissle 1917), it was eliminated from the intestine (E. coli strains can provide a barrier to infection and suggest that it may be possible to construct E. coli probiotic strains that prevent growth of pathogenic E. coli strains in the intestine.

Previously we showed that the tricarboxylic acid (TCA) cycle operates as a full cycle during Salmonella enterica serovar Typhimurium SR-11 peroral infection of BALB/c mice (M. Tchawa Yimga et al., Infect. Immun. 74:1130-1140, 2006). The evidence was that a Δ sucCD mutant (succinyl coenzyme A [succinyl-CoA] synthetase), which prevents the conversion of succinyl-CoA to succinate, and a Δ sdhCDA mutant (succinate dehydrogenase), which blocks the conversion of succinate to fumarate, were both attenuated, whereas an SR-11 Δ aspA mutant (aspartase) and an SR-11 Δ frdABCD mutant (fumarate reductase), deficient in the ability to run the reductive branch of the TCA cycle, were fully virulent. In the present study, evidence is presented that a serovar Typhimurium SR-11 Δ frdABCD Δ sdhCDA double mutant is avirulent in BALB/c mice and protective against subsequent infection with the virulent serovar Typhimurium SR-11 wild-type strain via the peroral route and is highly attenuated via the intraperitoneal route. These results suggest that fumarate reductase, which normally runs in the reductive pathway in the opposite direction of succinate dehydrogenase, can replace it during infection by running in the same direction as succinate dehydrogenase in order to run a full TCA cycle in an SR-11 Δ sdhCDA mutant. The data also suggest that the conversion of succinate to fumarate plays a key role in serovar Typhimurium virulence. Moreover, the data raise the possibility that S. enterica Δ frdABCD Δ sdhCDA double mutants and Δ frdABCD Δ sdhCDA double mutants of other intracellular bacterial pathogens with complete TCA cycles may prove to be effective live vaccine strains for animals and humans.

Salmonella enterica serotype Typhimurium is a food-borne pathogen that also selectively grows in tumours and functionally decreases P-glycoprotein (P-gp), a multidrug resistance transporter. Here we report that the Salmonella type III secretion effector, SipA, is responsible for P-gp modulation through a pathway involving caspase-3. Mimicking the ability of Salmonella to reverse multidrug resistance, we constructed a gold nanoparticle system packaged with a SipA corona, and found this bacterial mimic not only accumulates in tumours but also reduces P-gp at a SipA dose significantly lower than free SipA. Moreover, the Salmonella nanoparticle mimic suppresses tumour growth with a concomitant reduction in P-gp when used with an existing chemotherapeutic drug (that is, doxorubicin). On the basis of our finding that the SipA Salmonella effector is fundamental for functionally decreasing P-gp, we engineered a nanoparticle mimic that both overcomes multidrug resistance in cancer cells and increases tumour sensitivity to conventional chemotherapeutics., S. Typhimurium can grow selectively on tumours and decreases the cellular levels of the multidrug resistance transporter Pgp. Here, the authors reveal the SipA-dependent mechanism of Pgp down-regulation and produce a SipA-based gold nanoparticle that increases sensitivity to the anticancer drug doxorubicin.

Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73: 8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS 1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD 4 C 2 . Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD 4 C 2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

Acute gastroenteritis caused by Salmonella enterica serovar typhimurium is a significant public health problem. This pathogen has very sophisticated molecular machinery encoded by the two pathogenicity islands, namely Salmonella Pathogenicity Island 1 and 2 (SPI-1 and SPI-2). Remarkably, both SPI-1 and SPI-2 are very tightly regulated in terms of timing of expression and spatial localization of the encoded effectors during the infection process within the host cell. This regulation is governed at several levels, including transcription and translation, and by post-translational modifications. In the context of a finely tuned regulatory system, we will highlight how these effector proteins co-opt host signaling pathways that control the ability of the organism to infect and survive within the host, as well as elicit host pro-inflammatory responses.

ATP binding cassette (ABC) transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, secretion and toxicity of xenobiotics. In addition to their essential function in drug resistance, there is also emerging evidence documenting the important role ABC transporters play in tissue defense. In this respect, the gastrointestinal tract represents a critical vanguard of defense against oral exposure of drugs while at the same time functions as a physical barrier between the lumenal contents (including bacteria) and the intestinal epithelium. Given emerging evidence suggesting that multidrug resistance protein (MDR) plays an important role in host-bacterial interactions in the gastrointestinal tract, this review will discuss the interplay between MDR of the intestinal epithelial cell barrier and gut microbes in health and disease. In particular, we will explore host-microbe interactions involving three apically restricted ABC transporters of the intestinal epithelium; P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2) and cystic fibrosis transmembrane regulator (CFTR).