L. Caetano M. Antunes

Effect of Antibiotic Treatment on the Intestinal Metabolome

ABSTRACT The importance of the mammalian intestinal microbiota to human health has been intensely studied over the past few years. It is now clear that the interactions between human hosts and their associated microbial communities need to be characterized in molecular detail if we are to truly understand human physiology. Additionally, the study of such host-microbe interactions is likely to provide us with new strategies to manipulate these complex systems to maintain or restore homeostasis in order to prevent or cure pathological states. Here, we describe the use of high-throughput metabolomics to shed light on the interactions between the intestinal microbiota and the host. We show that antibiotic treatment disrupts intestinal homeostasis and has a profound impact on the intestinal metabolome, affecting the levels of over 87% of all metabolites detected. Many metabolic pathways that are critical for host physiology were affected, including bile acid, eicosanoid, and steroid hormone synthesis. Dissecting the molecular mechanisms involved in the impact of beneficial microbes on some of these pathways will be instrumental in understanding the interplay between the host and its complex resident microbiota and may aid in the design of new therapeutic strategies that target these interactions.

The Intestinal Microbiota Plays a Role in Salmonella-Induced Colitis Independent of Pathogen Colonization

The intestinal microbiota is composed of hundreds of species of bacteria, fungi and protozoa and is critical for numerous biological processes, such as nutrient acquisition, vitamin production, and colonization resistance against bacterial pathogens. We studied the role of the intestinal microbiota on host resistance to Salmonella enterica serovar Typhimurium-induced colitis. Using multiple antibiotic treatments in 129S1/SvImJ mice, we showed that disruption of the intestinal microbiota alters host susceptibility to infection. Although all antibiotic treatments caused similar increases in pathogen colonization, the development of enterocolitis was seen only when streptomycin or vancomycin was used; no significant pathology was observed with the use of metronidazole. Interestingly, metronidazole-treated and infected C57BL/6 mice developed severe pathology. We hypothesized that the intestinal microbiota confers resistance to infectious colitis without affecting the ability of S. Typhimurium to colonize the intestine. Indeed, different antibiotic treatments caused distinct shifts in the intestinal microbiota prior to infection. Through fluorescence in situ hybridization, terminal restriction fragment length polymorphism, and real-time PCR, we showed that there is a strong correlation between the intestinal microbiota composition before infection and susceptibility to Salmonella-induced colitis. Members of the Bacteroidetes phylum were present at significantly higher levels in mice resistant to colitis. Further analysis revealed that Porphyromonadaceae levels were also increased in these mice. Conversely, there was a positive correlation between the abundance of Lactobacillus sp. and predisposition to colitis. Our data suggests that different members of the microbiota might be associated with S. Typhimurium colonization and colitis. Dissecting the mechanisms involved in resistance to infection and inflammation will be critical for the development of therapeutic and preventative measures against enteric pathogens.

Impact of Salmonella Infection on Host Hormone Metabolism Revealed by Metabolomics

ABSTRACT The interplay between pathogens and their hosts has been studied for decades using targeted approaches, such as the analysis of mutants and host immunological responses. Although much has been learned from such studies, they focus on individual pathways and fail to reveal the global effects of infection on the host. To alleviate this issue, high-throughput methods, such as transcriptomics and proteomics, have been used to study host-pathogen interactions. Recently, metabolomics was established as a new method to study changes in the biochemical composition of host tissues. We report a metabolomic study of Salmonella enterica serovar Typhimurium infection. Our results revealed that dozens of host metabolic pathways are affected by Salmonella in a murine infection model. In particular, multiple host hormone pathways are disrupted. Our results identify unappreciated effects of infection on host metabolism and shed light on mechanisms used by Salmonella to cause disease and by the host to counter infection.

Should the human microbiome be considered when developing vaccines

The human microbiome, especially in the intestinal tract has received increased attention in the past few years due to its importance in numerous biological processes. Recent advances in DNA sequencing technology and analysis now allow us to better determine global differences in the composition of the gut microbial population, and ask questions about its role in health and disease. Thus far, roles of these commensal bacteria on nutrient acquisition, vitamin production, and intestinal development have been identified [1]. Furthermore, resistance or susceptibility to a number of diseases, including inflammatory bowel disease, obesity, enteric infections, and most recently ectopic diseases, have been linked to the intestinal microbiota [1], [2]. Data on the mechanisms through which the intestinal microbiota impacts host immune development have also begun to emerge [2]. The impact of the intestinal microbiota on host physiology is undeniable, and experiments using germ-free, mono-, and poly-colonized mice have addressed many aspects of the microbiotas influence on the mammalian immune system. Despite all the increased attention on the interface between the microbiota and host immune responses, it is still unclear whether these commensal bacteria affect the efficacy of vaccines. Due to its impact in the development of immune function, both in the gut and other organs, it is reasonable to consider that the intestinal microbiota will significantly affect how individuals respond to vaccine antigens [3], [4]. For example, segmented filamentous bacteria present in the intestinal microbiota have been shown to induce maturation of intestinal T cell adaptive functions [5]. Moreover, it has been shown that the intestinal microbiota exerts a profound effect on the metabolism of certain drugs and toxins [1], [6], and this may also indicate that oral vaccines could be differentially processed by the body depending on variations in microbial communities between individuals. Hence, the microbiota could be an underappreciated yet important player to consider in the development of vaccines, and also may help explain some of the discrepancies observed in vaccine efficacy in different populations around the world. Clinical trials testing the efficacy of oral vaccines against polio, rotavirus, and cholera have showed a lower immunogenicity of these vaccines in individuals from developing countries when compared to individuals from the developed world [7]–[11]. Clinical trials for a killed oral cholera vaccine in Swedish and Nicaraguan children have also shown blunted antibody responses in Nicaraguan children compared to Swedish children [11]. In a study testing a live cholera oral vaccine, Lagos and colleagues [12] demonstrated that excessive bacterial growth in the small intestine of children in less developed countries might contribute to the low antibody response to the vaccine. Different vaccine strains of Shigella flexneri also showed differential protection on individuals from developing countries. In a study testing Bangladeshi adults and children, no significant immune response to this vaccine was mounted, although the same antigen was reactogenic in North American individuals [13]. Altogether, these data highlight that individuals from different parts of the world can mount different immune responses to the same vaccine. Several hypotheses that may explain this phenomenon exist. For instance, socioeconomic conditions, nutritional status, host genetics, and earlier exposure to related microorganisms are some of the aspects that could contribute to the disparity in the vaccine efficacies in different populations. However, one poorly explored possibility is that the composition of the intestinal microbiota of these individuals may also be a determining factor of vaccine efficacy. In a way analogous to the hygiene hypothesis [14], which states that reduced exposure to microorganisms at an early age may lead to increased susceptibility to allergies, it is possible that the gut microbiota of individuals with increased exposure to microorganisms (and therefore antigens) make them more tolerant to vaccination, being unable to mount a proper response compared to individuals living in better socioeconomic conditions. Discerning the effects of genetic and environmental factors on vaccine efficacy is a challenging task. Large clinical trials involving individuals from different areas of the world will likely be required to shed light on whether the blunt immune responses to some of the oral vaccines mentioned herein are a consequence of genetic factors or environmental variations, such as the gut microbial community. Studies involving immigrant volunteers could be useful in addressing this issue by providing a clear distinction between the effects of genetics and the environment. Although this is still an open question, data in the literature suggest a more direct link between the intestinal microbiota composition and the development of immune responses to certain vaccine antigens. For instance, the use of antibiotics in chickens has been shown to increase the antibody response following immunization [15]. Because antibiotic treatment will have profound effects on the intestinal microbiota, it is tempting to hypothesize that the microbial populations of these animals are important players in their immunological response to the vaccine antigens. Furthermore, certain probiotic strains have been shown to enhance antibody responses to oral vaccines against rotavirus [16], Salmonella [17], polio [18], and cholera [19] in human volunteers, and this effect was observed after a short period (1–5 weeks) of probiotic treatment. The positive effect of probiotics on immune responses was also seen in parenterally administered vaccines against diphtheria, tetanus, Haemophilus influenzae type B, and hepatitis B [20]–[22] in infants after a 6-month period. Because of the number of licensed oral-administered human vaccines available is limited, studies on how the intestinal microbiota affect parenterally administered human vaccines would have a more significant impact on human health. However, in all studies cited above, there was no long-term follow-up on the enhanced effects of the probiotics on vaccine efficacy. Additionally, more detailed studies on the establishment of the probiotic strains within the resident microbiota will be required to establish minimal doses and treatment regimens, important aspects that need to be addressed if the microbiota is to be considered in vaccine development in the future. It has also been suggested that prebiotics, which are compounds that can enhance the proliferation of certain commensals, can enhance the efficacy of oral vaccines. Recently, a well-studied fructo-oligosaccharide prebiotic has been shown to improve the efficacy of a vaccine against Salmonella infection [23]. In this study, administration of the prebiotic prior to vaccination improved host responses and rates of protection against infection in mice. Unfortunately, the authors were unable to show significant changes in microbiota composition, possibly due to the lack of detailed analyses. In another study, Vos et al. [24] showed that a prebiotic mixture containing galacto- and fructo-oligosaccharides enhanced systemic adaptive immune responses in a murine influenza vaccination model. In this case, increased proportions of certain members of the microbiota could be observed, suggesting a role for the microbial community in the increased host immune response. Although some studies indicate that the microbiota may play an important role in vaccine efficacy, this area of research is still in its infancy. For instance, the mechanisms involved in the pro- and prebiotic enhancement of vaccine efficacy mentioned above are largely unknown. Nevertheless, current knowledge of the effect of the intestinal microbiota on the development of not only local but also systemic immune functions provides a direct link between commensal populations in the intestine and immune responses to vaccine antigens [3], [4]. We now have the tools to study and take advantage of what the microbiota has to offer in order to enhance host responses to vaccines, with the use of probiotics or prebiotics as adjuvants. Studies using animal models with defined intestinal microbial communities can be helpful to evaluate the effect of intestinal commensals on the immune response to vaccines. However, animal models can only partially elucidate this issue and, although cumbersome, studies in human volunteers will be essential in defining the effect of commensals in vaccine efficacy. We suggest that the study of the relationships between individual commensal populations of humans and responses to vaccines will be instrumental in our quest to improve general vaccine development. By taking into consideration the microbial populations of geographically diverse groups of individuals, we may be able to develop better-targeted vaccines that will improve protection against multiple pathogens.

Neutrophil elastase alters the murine gut microbiota resulting in enhanced Salmonella colonization

The intestinal microbiota has been found to play a central role in the colonization of Salmonella enterica serovar Typhimurium in the gastrointestinal tract. In this study, we present a novel process through which Salmonella benefit from inflammatory induced changes in the microbiota in order to facilitate disease. We show that Salmonella infection in mice causes recruitment of neutrophils to the gut lumen, resulting in significant changes in the composition of the intestinal microbiota. This occurs through the production of the enzyme elastase by neutrophils. Administration of recombinant neutrophil elastase to infected animals under conditions that do not elicit neutrophil recruitment caused shifts in microbiota composition that favored Salmonella colonization, while inhibition of neutrophil elastase reduced colonization. This study reveals a new relationship between the microbiota and the host during infection.

Metabolomics: towards understanding host–microbe interactions

Metabolomics employs an array of analytical techniques, including high-resolution nuclear magnetic resonance spectroscopy and mass spectrometry, to simultaneously analyze hundreds to thousands of small-molecule metabolites in biological samples. In conjunction with chemoinformatics and bioinformatics tools, metabolomics enables comprehensive characterization of the metabolic phenotypes (metabotypes) of the human, and other mammalian, hosts that have co-evolved with a large number of diverse commensal microbes, especially in the intestinal tract. Correlation of the metabotypes with the microbial profiles derived from culture-independent molecular techniques is increasingly helping to decipher inherent and intimate host-microbe relationships. This integrated, systems biology approach is improving our understanding of the molecular mechanisms underlying multilevel host-microbe interactions, and promises to elucidate the etiologies of human disorders resulting from unfavorable human-microbial associations, including enteric infections.

The Deubiquitinase Activity of the Salmonella Pathogenicity Island 2 Effector, SseL, Prevents Accumulation of Cellular Lipid Droplets

ABSTRACT To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets.

Inhibition of Salmonella Host Cell Invasion by Dimethyl Sulfide

ABSTRACT We show that dimethyl sulfoxide (DMSO) inhibits Salmonella hilA expression and that this inhibition is stronger under anaerobiosis. Because DMSO can be reduced to dimethyl sulfide (DMS) during anaerobic growth, we hypothesized that DMS was responsible for hilA inhibition. Indeed, DMS strongly inhibited the expression of hilA and multiple Salmonella pathogenicity island 1 (SPI-1)-associated genes as well as the invasion of cultured epithelial cells. Because DMSO and DMS are widespread in nature, we hypothesize that this phenomenon may contribute to environmental sensing by Salmonella.

15-Deoxy-Δ12,14-prostaglandin J2 inhibits macrophage colonization by Salmonella enterica serovar Typhimurium.

15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) is an anti-inflammatory downstream product of the cyclooxygenase enzymes. It has been implicated to play a protective role in a variety of inflammatory mediated diseases, including rheumatoid arthritis, neural damage, and myocardial infarctions. Here we show that 15d-PGJ2 also plays a role in Salmonella infection. Salmonella enterica Typhimurium is a Gram-negative facultative intracellular pathogen that is able to survive and replicate inside phagocytic immune cells, allowing for bacterial dissemination to systemic sites. Salmonella species cause a wide range of morbidity and mortality due to gastroenteritis and typhoid fever. Previously we have shown that in mouse models of typhoid fever, Salmonella infection causes a major perturbation in the prostaglandin pathway. Specifically, we saw that 15d-PGJ2 production was significantly increased in both liver and feces. In this work we show that 15d-PGJ2 production is also significantly increased in macrophages infected with Salmonella. Furthermore, we show that the addition of 15d-PGJ2 to Salmonella infected RAW264.7, J774, and bone marrow derived macrophages is sufficient to significantly reduce bacterial colonization. We also show evidence that 15d-PGJ2 is reducing bacterial uptake by macrophages. 15d-PGJ2 reduces the inflammatory response of these infected macrophages, as evidenced by a reduction in the production of cytokines and reactive nitrogen species. The inflammatory response of the macrophage is important for full Salmonella virulence, as it can give the bacteria cues for virulence. The reduction in bacterial colonization is independent of the expression of Salmonella virulence genes SPI1 and SPI2, and is independent of the 15d-PGJ2 ligand PPAR-γ. 15d-PGJ2 also causes an increase in ERK1/2 phosphorylation in infected macrophages. In conclusion, we show here that 15d-PGJ2 mediates the outcome of bacterial infection, a previously unidentified role for this prostaglandin.

Harvesting the biological potential of the human gut microbiome.

There is a tremendous history of capitalizing on the biodiversity of our natural environment for molecules to treat diseases and regulate host physiology (e.g. penicillin, acetylsalicylic acid). However, until recently we have failed to probe the therapeutic potential of our microbial symbionts: a biodiverse population of bacteria, archaea, viruses, protists, and fungi that reside on and within us. Recently there has been an increased realization that the microbiota significantly impact many of our physiological and immunological processes (Fig. 1), suggesting that there is great opportunity for therapeutic discoveries. Figure 1 Some of the many mechanisms through which gut microbes and their signals can affect host health. SFB, segmented filamentous bacteria. The potential of the microbiota to affect the host is vast, considering its impressive genetic composition. The collective genomes of our resident microbes, referred to as the metagenome, represent a gene set which is estimated to be 150-fold greater than the human genetic complement [1]. Many of these genes are the product of co-evolution and have been selected on the basis of improving host fitness rather than the fitness of the microbe itself. As we begin to understand previously unidentified properties and molecules of the microbiota, we will be able to “mine” these for potential therapeutic benefit. Here, we discuss some of the avenues of discovery that present opportunities for further exploitation. Possibilities discussed include strategies for excluding pathogens, manipulation of the immune system and regulation of non-intestinal sites.