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In addition to numerous clinical studies, research using experimental models have contributed extensive evidence to the link between antibiotic exposure and atopic disease. A number of mouse models of allergy have been developed and used to uncover the specific effects of various microbiota members and perturbations on allergy development. Studies in mice that lack microbes entirely have also demonstrated the various components of the immune system that require microbial exposure. The importance of the early-life period and the mechanisms by which atopy “protective” species identified in human cohorts promote immune development have been elucidated in mice. Finally, non-animal models involving human-derived cells shed light on specific effects of bacteria on human epithelial and immune responses. When considered alongside clinical cohort studies, experimental model systems have provided crucial evidence for the link between the neonatal gut microbiota and allergic disease, immensely supporting the stewardship of antibiotic administration in infants. The following review aims to describe the range of experimental models used for studying factors that affect the relationship between the gut microbiota and allergic disease and summarize key findings that have come from research in animal and in vitro models.

The gut houses 70%–80% of the body's immune cells and represents the main point of contact between the immune system and the outside world. Immune maturation occurs largely after birth and is guided by the gut microbiota. In addition to the many human clinical studies that have identified relationships between gut microbiota composition and disease outcomes, experimental research has demonstrated a plethora of mechanisms by which specific microbes and microbial metabolites train the developing immune system. The healthy maturation of the gut microbiota has been well-characterized and discreet stages marked by changes in abundance of specific microbes have been identified. Building on Chapter 8, which discusses experimental models used to study the relationship between the gut microbiota and asthma, the present review aims to dive deeper into the specific microbes and metabolites that drive key processes in immune development. The implications of microbiota maturation patterns in the context of asthma and allergies, as well as the effects of antibiotics on microbe-immune crosstalk, will also be discussed.

Summary: Asthma susceptibility is linked to dysbiosis in early-life gut microbiota, and the antibody secretory immunoglobulin (Ig)A (SIgA) is a key determinant of gut microbiota composition. SIgA is obtained through breast milk during the critical early-life window. We use a mouse model of SIgA deficiency and the house dust mite (HDM) model of asthma to elucidate the role of maternal SIgA in modulating the early-life gut microbiota and asthma protection. Mice that do not receive maternal SIgA display a transient bloom of segmented filamentous bacteria (SFB) in the small intestine during the early post-weaning period. Mice that do not receive maternal SIgA also display elevated T helper type 17 (Th17) cell activation in the intestine, which persists into adulthood and is associated with more severe inflammation in response to the HDM model of asthma. This study demonstrates a mechanism by which breast-milk-derived SIgA influences immune development and asthma susceptibility by modulating the early-life gut microbiota.

With the global prevalence of allergic disease continuing to rise at an alarming rate, the need for effective and safe therapeutics is paramount. Given the critical role of the early-life microbiota on immune development, emerging research suggests the potential use of live biotherapeutic products (LBP) for the prevention and treatment of childhood allergy. However, findings are limited and inconsistent. Therefore, the present review critically evaluates the current animal and human data on the therapeutic value of LBPs in allergy, the underlying immunological mechanisms by which LBPs may mediate allergy susceptibility, limitations of the current research that need to be addressed, and future research directions. Accordingly, LBPs may protect against allergic disease through several immunological and physiological mechanisms during early-life, including regulation of Th1/Th2 balance, SCFA-induced activation of GPR41/43 and HDAC inhibition, and maturation of epithelial barrier integrity. Taken together, current findings indicate powerful immunomodulatory properties of LBPs on allergic immune response, with LBPs offering exciting potential as a novel therapeutic tool for childhood allergy. However, the efficacy of LBPs in allergy is complex and influenced by many population and methodological factors, resulting in varied therapeutic benefits. While research thus far has focused on traditional probiotic strains, greater investigation into microbial consortiums selected from the microbiota of non-allergic infants may provide greater promise as a therapeutic tool for allergic disease. Further investigation, particularly into long-term efficacy, strain-specific effects, optimal supplementation regimes, and use of multi-strain consortiums, is necessary before findings can be translated into clinical applications to tackle childhood allergic disease.

Abstract Allergic diseases affect millions of people worldwide. An increase in their prevalence has been associated with alterations in the gut microbiome, i.e., the microorganisms and their genes within the gastrointestinal tract. Maturation of the infant immune system and gut microbiota occur in parallel; thus, the conformation of the microbiome may determine if tolerant immune programming arises within the infant. Here we show, using deeply phenotyped participants in the CHILD birth cohort (n = 1115), that there are early-life influences and microbiome features which are uniformly associated with four distinct allergic diagnoses at 5 years: atopic dermatitis (AD, n = 367), asthma (As, n = 165), food allergy (FA, n = 136), and allergic rhinitis (AR, n = 187). In a subset with shotgun metagenomic and metabolomic profiling (n = 589), we discover that impaired 1-year microbiota maturation may be universal to pediatric allergies (AD p = 0.000014; As p = 0.0073; FA p = 0.00083; and AR p = 0.0021). Extending this, we find a core set of functional and metabolic imbalances characterized by compromised mucous integrity, elevated oxidative activity, decreased secondary fermentation, and elevated trace amines, to be a significant mediator between microbiota maturation at age 1 year and allergic diagnoses at age 5 years (βindirect = −2.28; p = 0.0020). Microbiota maturation thus provides a focal point to identify deviations from normative development to predict and prevent allergic disease.

ABSTRACTThere is concern that the time taken to publish academic papers in microbiological science has significantly increased in recent years. While the data do not specifically support this, evidence suggests that editors are having to invite more and more reviewers to identify those willing to perform peer review.

ABSTRACTQuorum Sensing (QS) is a form of cell-to-cell communication that enables bacteria to modify behavior according to their population density. While QS has been proposed as a potential intervention against pathogen infection, QS-mediated communication within the mammalian digestive tract remains understudied. Using an LC-MS/MS approach, we discovered that Citrobacter rodentium, a natural murine pathogen used to model human infection by pathogenic Escherichia coli, utilizes the CroIR system to produce three QS-molecules. We then profiled their accumulation both in vitro and across different gastrointestinal sites over the course of infection. Importantly, we found that in the absence of QS capabilities the virulence of C. rodentium is enhanced. This highlights the role of QS as an effective mechanism to regulate virulence according to the pathogen’s spatio-temporal context to optimize colonization and transmission success. These results also demonstrate that inhibiting QS may not always be an effective strategy for the control of virulence.

ABSTRACTEnterohemorrhagic Escherichia coli (EHEC) is a major cause of severe bloody diarrhea, with potentially lethal complications, such as hemolytic uremic syndrome. In humans, EHEC colonizes the colon, which is also home to a diverse community of trillions of microbes known as the gut microbiota. Although these microbes and the metabolites that they produce represent an important component of EHEC’s ecological niche, little is known about how EHEC senses and responds to the presence of gut microbiota metabolites. In this study, we used a combined RNA-Seq and Tn-Seq approach to characterize EHEC’s response to metabolites from an in vitro culture of 33 human gut microbiota isolates (MET-1), previously demonstrated to effectively resolve recurrent Clostridioides difficile infection in human patients. Collectively, the results revealed that EHEC adjusts to growth in the presence of microbiota metabolites in two major ways: by altering its metabolism and by activating stress responses. Metabolic adaptations to the presence of microbiota metabolites included increased expression of systems for maintaining redox balance and decreased expression of biotin biosynthesis genes, reflecting the high levels of biotin released by the microbiota into the culture medium. In addition, numerous genes related to envelope and oxidative stress responses (including cpxP, spy, soxS, yhcN, and bhsA) were upregulated during EHEC growth in a medium containing microbiota metabolites. Together, these results provide insight into the molecular mechanisms by which pathogens adapt to the presence of competing microbes in the host environment, which ultimately may enable the development of therapies to enhance colonization resistance and prevent infection.

IntroductionMicronutrients perform a wide range of physiological functions essential for growth and development. However, most people still need to meet the estimated average requirement worldwide. Globally, 2 billion people suffer from micronutrient deficiency, most of which are co-occurring deficiencies in children under age five. Despite decades of research, animal models studying multiple micronutrient deficiencies within the early-life period are lacking, which hinders our complete understanding of the long-term health implications and may contribute to the inefficacy of some nutritional interventions. Evidence supporting the Developmental Origins of Health and Disease (DOHaD) theory demonstrates that early-life nutritional deficiencies carry life-long consequences mediated through various mechanisms such as abnormal metabolic programming, stunting, altered body composition, and the gut microbiome. However, this is largely unexplored in the multiple micronutrient deficient host.Methodswe developed a preclinical model to examine undernutrition’s metabolic and functional impact on the host and gut microbiome early in life. Three-week-old weanling C57BL/6N male mice were fed a low-micronutrient diet deficient in zinc, folate, iron, vitamin A, and vitamin B12 or a control diet for 4-weeks.ResultsOur results showed that early-life multiple micronutrient deficiencies induced stunting, altered body composition, impaired glucose and insulin tolerance, and altered the levels of other micronutrients not depleted in the diet within the host. In addition, functional metagenomics profiling and a carbohydrate fermentation assay showed an increased microbial preference for simple sugars rather than complex ones, suggestive of a less developed microbiome in the low-micronutrient-fed mice. Moreover, we found that a zinc-only deficient diet was not sufficient to induce these phenotypes, further supporting the importance of studying co-occurring deficiencies.DiscussionTogether, these findings highlight a previously unappreciated role of early-life multiple micronutrient deficiencies in shaping the metabolic phenome of the host and gut microbiome through altered glucose energy metabolism, which may have implications for metabolic disease later in life in micronutrient-deficient survivors.

IntroductionEnteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) belong to a group of pathogens that share the ability to form “attaching and effacing” (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates ler expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood.MethodsTo further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and cat transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis.Results and discussionWe showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR’s alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.

Background. Studies suggest that early-life gut microbiota composition and intestinal short-chain fatty acids (SCFAs) are linked to future asthma susceptibility. Furthermore, infancy offers a critical time window to modulate the microbiota and associated metabolites through diet-microbe interactions to promote infant health. Human milk oligosaccharides (HMOs), nondigestible carbohydrates abundant in breast milk, are prebiotics selectively metabolized by gut microbiota that consequently modify microbiome composition and SCFA production. Methods. Using a house dust mite mouse model of allergy, we investigated the impacts of early oral treatment of pups with biologically relevant doses of 2′-fucosyllactose (2′-FL) and 6′-sialyllactose (6′-SL), two of the most abundant HMOs in human milk, in amelioration of allergic airway disease severity. Results. We found that administration of 2′-FL and 6′-SL during early life reduced lung histopathology scores, circulating IgE, cytokine levels, and inflammatory cell infiltration, all hallmark symptoms of allergic asthma. HMO supplementation also increased the relative abundance of intestinal Bacteroidetes and Clostridia, known SCFA producers within the gut. Indeed, we detected increased SCFA concentrations in both the intestine and blood of adult mice who received HMOs prior to weaning. Conclusion. We propose a model in which orally administered HMOs delivered during early life shift the microbiota toward increased production of SCFAs, which dampens the allergic immune responses behind allergy and asthma. Overall, these data suggest the potential for HMO supplementation to protect infants against asthma development later in life, with possible benefits against additional atopic diseases such as eczema and food allergies.

Abstract Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein–protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.

The practice of rearing cows and calves together is gaining popularity on dairy farms, with different systems currently under assessment in mainland Europe, the United Kingdom, and Oceania. Research into the effects of cow–calf rearing has primarily focused on direct health and welfare implications, and little work has examined the role of different rearing paradigms on calf microbiota. We trialed a cow–calf rearing system on a Canadian dairy farm and compared fecal microbiota of these calves with the microbiota of calves reared according to the conventional practice of the same farm (separated from the dam and fed waste milk). At 4 wk, the conventionally reared calves had reduced relative abundance of Lactobacillus and higher relative abundance of other taxa, including Sutterella, Prevotella, and Bacteroides. We also detected predicted functional differences, such as reduced l-tryptophan biosynthesis in conventionally reared calves. These results suggest that maternal contact may influence the calf microbiota, but the observed differences are also likely related to other aspects of the rearing environment independent of maternal contact (e.g., potential exposure to antibiotic residues in waste milk). These findings provide preliminary evidence of the effects of early rearing environments on the establishment of the dairy calf fecal microbiota. This research is needed, given the critical role of the bovine gut microbiome in behavioral, metabolic, and immune development.

Abstract Background It is estimated that the COVID-19 pandemic will drastically increase all forms of malnutrition. Of particular concern, yet understated, is the potential to increase the double burden of malnutrition (DBM) epidemic. This coexistence of undernutrition together with overweight and obesity, or diet-related non-communicable disease (NCD), within low- to middle-income countries (LMICs) is increasing rapidly. Although multiple factors contribute to the DBM, food insecurity (FI) and gut microbiota dysbiosis play a crucial role. Both under- and overnutrition have been shown to be a consequence of food insecurity. The gut microbiota has also been recently implicated in playing a role in under- and overnutrition, with altered community structure and function common to both. The pandemic has already caused significant shifts in food availability which has immediate effects on the gut microbiome. In this opinion paper, we discuss how COVID-19 may indirectly exacerbate the DBM through food insecurity and the gut microbiome. Main text The World Food Programme (WFP) estimates that 265 million people in LMICs will experience acute hunger in 2020 due to the pandemic, nearly doubling the original projection of 135 million. Global border closures to food trade, loss of food production, and stark decline in household income will exacerbate starvation while simultaneously necessitating that families resort to calorie-dense, nutrient-poor foods, thereby increasing obesity. While food insecurity, which is the persistent lack of consistent access to adequate and nutrient-rich foods, will primarily drive nutrition behavior, the gut microbiome is perhaps a key biological mechanism. Numerous human and animal studies describe low diversity and an increase in inflammatory species as characteristic features of the undernourished and overnourished gut microbiota. Indeed, fecal transplant studies show that microbiota transfer from undernourished and overnourished humans to germ-free mice lacking a microbiome transfers the physical and metabolic phenotype, suggesting a causal role for the microbiota in under- and overnutrition. The observed microbiome dysbiosis within severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coupled with the DBM presents a viscous cycle. Conclusion Low- to mid-income countries will likely see an increase in the DBM epidemic. Providing access to nutritious foods and protecting individuals’ gut microbiome to “flatten the curve” of the DBM trajectory should be prioritized.

Abstract Background Identifying which taxa are targeted by immunoglobulins can uncover important host-microbe interactions. Immunoglobulin binding of commensal taxa can be assayed by sorting bound bacteria from samples and using amplicon sequencing to determine their taxonomy, a technique most widely applied to study Immunoglobulin A (IgA-Seq). Previous experiments have scored taxon binding in IgA-Seq datasets by comparing abundances in the IgA bound and unbound sorted fractions. However, as these are relative abundances, such scores are influenced by the levels of the other taxa present and represent an abstract combination of these effects. Diversity in the practical approaches of prior studies also warrants benchmarking of the individual stages involved. Here, we provide a detailed description of the design strategy for an optimised IgA-Seq protocol. Combined with a novel scoring method for IgA-Seq datasets that accounts for the aforementioned effects, this platform enables accurate identification and quantification of commensal gut microbiota targeted by host immunoglobulins. Results Using germ-free and Rag1 −/− mice as negative controls, and a strain-specific IgA antibody as a positive control, we determine optimal reagents and fluorescence-activated cell sorting (FACS) parameters for IgA-Seq. Using simulated IgA-Seq data, we show that existing IgA-Seq scoring methods are influenced by pre-sort relative abundances. This has consequences for the interpretation of case-control studies where there are inherent differences in microbiota composition between groups. We show that these effects can be addressed using a novel scoring approach based on posterior probabilities. Finally, we demonstrate the utility of both the IgA-Seq protocol and probability-based scores by examining both novel and published data from in vivo disease models. Conclusions We provide a detailed IgA-Seq protocol to accurately isolate IgA-bound taxa from intestinal samples. Using simulated and experimental data, we demonstrate novel probability-based scores that adjust for the compositional nature of relative abundance data to accurately quantify taxon-level IgA binding. All scoring approaches are made available in the IgAScores R package. These methods should improve the generation and interpretation of IgA-Seq datasets and could be applied to study other immunoglobulins and sample types. Video abstract

Abstract Background Child undernutrition is a global health issue that is associated with poor sanitation and an altered intestinal microbiota. Immunoglobulin (Ig) A mediates host-microbial homeostasis in the intestine, and acutely undernourished children have been shown to have altered IgA recognition of the fecal microbiota. We sought to determine whether chronic undernutrition (stunting) or intestinal inflammation were associated with antibody recognition of the microbiota using two geographically distinct populations from the Afribiota project. Fecal bacteria from 200 children between 2 and 5 years old in Antananarivo, Madagascar, and Bangui, Central African Republic (CAR), were sorted into IgA-positive (IgA+) and IgA-negative (IgA−) populations by flow cytometry and subsequently characterized by 16S rRNA gene sequencing to determine IgA-bacterial targeting. We additionally measured IgG+ fecal bacteria by flow cytometry in a subset of 75 children. Results Stunted children (height-for-age z-score ≤ −2) had a greater proportion of IgA+ bacteria in the fecal microbiota compared to non-stunted controls. This trend was consistent in both countries, despite the higher overall IgA-targeting of the microbiota in Madagascar, but lost significance in each country individually. Two of the most highly IgA-recognized bacteria regardless of nutritional status were Campylobacter (in CAR) and Haemophilus (in both countries), both of which were previously shown to be more abundant in stunted children; however, there was no association between IgA-targeting of these bacteria and either stunting or inflammatory markers. IgG-bound intestinal bacteria were rare in both stunted and non-stunted children, similar to levels observed in healthy populations. Conclusions Undernourished children carry a high load of intestinal pathogens and pathobionts. Our data suggest that stunted children have a greater proportion of IgA-recognized fecal bacteria. We moreover identify two putative pathobionts, Haemophilus and Campylobacter, that are broadly targeted by intestinal IgA. This study furthers our understanding of host-microbiota interactions in undernutrition and identifies immune-recognized microbes for future study.

The impact of intestinal eukaryotes on the prokaryotic microbiome composition of asymptomatic carriers has not been extensively explored, especially in infants and mothers with multiple parasitic infections. In this work, we studied the relationship between protist and helminth parasite colonization and the intestinal microbiota structure in an asymptomatic population of mother-child binomials from a semirural community in Mexico.

Recent evidence suggests an immunomodulatory role for commensal fungi (mycobiota) in the gut, yet little is known about the composition and dynamics of early-life gut fungal communities. In this work, we show for the first time that the composition of the gut mycobiota of Canadian infants changes dramatically over the course of the first year of life, is associated with environmental factors such as geographical location, diet, and season of birth, and can be used in conjunction with knowledge of a small number of key early-life factors to predict inhalant atopy status at age 5 years.

Summary: Microbiota maturation and immune development occur in parallel with, and are implicated in, allergic diseases, and research has begun to demonstrate the importance of prenatal influencers on both. Here, we investigate the meconium metabolome, a critical link between prenatal exposures and both early microbiota and immune development, to identify components of the neonatal gut niche that contribute to allergic sensitization. Our analysis reveals that newborns who develop immunoglobulin E (IgE)-mediated allergic sensitization (atopy) by 1 year of age have a less-diverse gut metabolome at birth, and specific metabolic clusters are associated with both protection against atopy and the abundance of key taxa driving microbiota maturation. These metabolic signatures, when coupled with early-life microbiota and clinical factors, increase our ability to accurately predict whether or not infants will develop atopy. Thus, the trajectory of both microbiota colonization and immune development are significantly affected by metabolites present in the neonatal gut at birth.

Secretory immunoglobulin A (IgA) interacts with intestinal microbiota and promotes mucosal homeostasis. IgA-bacteria interactions are altered during inflammatory diseases, but how these interactions are shaped by bacterial, host, and environmental factors remains unclear. In this study, we utilized IgA-SEQ to profile IgA-bound fecal bacteria in 48 recurrent Clostridioides difficile patients before and after successful fecal microbiota transplantation (FMT) to gain further insight. Prior to FMT, Escherichia coli was the most highly IgA-targeted taxon; following restoration of the microbiota by FMT, highly IgA-targeted taxa included multiple Firmicutes species. Post-FMT IgA-targeting was unaffected by the route of FMT delivery (colonoscopy versus capsule), suggesting that both methods lead to the establishment of healthy immune–bacterial interactions in the gut. Interestingly, IgA-targeting in FMT recipients closely resembled the IgA-targeting patterns of the donors, and fecal donor identity was significantly associated with IgA-targeting of the recipient microbiota. These data support the concept that intrinsic bacterial properties drive IgA recognition across genetically distinct human hosts. Together, this study suggests that IgA-bacterial interactions are reestablished in human FMT recipients to resemble that of the healthy fecal donor.

ABSTRACT Enteropathogenic Escherichia coli (EPEC) causes severe diarrheal disease and is present globally. EPEC virulence requires a bacterial type III secretion system to inject >20 effector proteins into human intestinal cells. Three effectors travel to mitochondria and modulate apoptosis; however, the mechanisms by which effectors control apoptosis from within mitochondria are unknown. To identify and quantify global changes in mitochondrial proteolysis during infection, we applied the mitochondrial terminal proteomics technique mitochondrial stable isotope labeling by amino acids in cell culture-terminal amine isotopic labeling of substrates (MS-TAILS). MS-TAILS identified 1,695 amino N-terminal peptides from 1,060 unique proteins and 390 N-terminal peptides from 215 mitochondrial proteins at a false discovery rate of 0.01. Infection modified 230 cellular and 40 mitochondrial proteins, generating 27 cleaved mitochondrial neo-N termini, demonstrating altered proteolytic processing within mitochondria. To distinguish proteolytic events specific to EPEC from those of canonical apoptosis, we compared mitochondrial changes during infection with those reported from chemically induced apoptosis. During infection, fewer than half of all mitochondrial cleavages were previously described for canonical apoptosis, and we identified nine mitochondrial proteolytic sites not previously reported, including several in proteins with an annotated role in apoptosis, although none occurred at canonical Asp-Glu-Val-Asp (DEVD) sites associated with caspase cleavage. The identification and quantification of novel neo-N termini evidences the involvement of noncaspase human or EPEC protease(s) resulting from mitochondrial-targeting effectors that modulate cell death upon infection. All proteomics data are available via ProteomeXchange with identifier PXD016994. IMPORTANCE To our knowledge, this is the first study of the mitochondrial proteome or N-terminome during bacterial infection. Identified cleavage sites that had not been previously reported in the mitochondrial N-terminome and that were not generated in canonical apoptosis revealed a pathogen-specific strategy to control human cell apoptosis. These data inform new mechanisms of virulence factors targeting mitochondria and apoptosis during infection and highlight how enteropathogenic Escherichia coli (EPEC) manipulates human cell death pathways during infection, including candidate substrates of an EPEC protease within mitochondria. This understanding informs the development of new antivirulence strategies against the many human pathogens that target mitochondria during infection. Therefore, mitochondrial stable isotope labeling by amino acids in cell culture-terminal amine isotopic labeling of substrates (MS-TAILS) is useful for studying other pathogens targeting human cell compartments.

The redox balance in a variety of Gram-negative bacteria was explored using redox sensitive GFP (roGFP2), J. van der Heijden et al. doi:10.1016/j.freeradbiomed.2015.11.029 [1]. This data article provides Supporting material to further investigate the relationship between Salmonella typhimurium survival and oxidative stress. The first set of data presented in this article, shows the percentage of surviving bacteria after exposure to hydrogen peroxide. The second set of data shows the concentration of hydrogen peroxide that was produced by S. Typhimurium in different growth phases. The last set of data shows the percentage of surviving S. Typhimurium bacteria after exposure to different antibiotics. Keywords: Oxidative stress, Reactive oxygen species, Hydrogen peroxide, Bacterial survival, Antibiotics

ABSTRACT Many Gram-negative bacterial pathogens utilize a specialized protein delivery system, called the type III secretion system (T3SS), to translocate effector proteins into the host cells. The translocated effectors are crucial for bacterial infection and survival. The base of the T3SS transverses both bacterial membranes and contains an export apparatus that comprises five membrane proteins. Here, we study the export apparatus of enteropathogenic Escherichia coli (EPEC) and characterize its central component, called the EscR protein. We found that the third transmembrane domain (TMD) of EscR mediates strong self-oligomerization in an isolated genetic reporter system. Replacing this TMD sequence with an alternative hydrophobic sequence within the full-length protein resulted in a complete loss of function of the T3SS, further suggesting that the EscR TMD3 sequence has another functional role in addition to its role as a membrane anchor. Moreover, we found that an aspartic acid residue, located at the core of EscR TMD3, is important for the oligomerization propensity of TMD3 and that a point mutation of this residue within the full-length protein abolishes the T3SS activity and the ability of the bacteria to translocate effectors into host cells. IMPORTANCE Many Gram-negative bacterial pathogens that cause life-threatening diseases employ a type III secretion system (T3SS) for their virulence. The T3SS comprises several proteins that assemble into a syringe-like structure dedicated to the injection of bacterial virulence factors into the host cells. Although many T3SS proteins are transmembrane proteins, our knowledge of these proteins is limited mostly to their soluble domains. In this study, we found that the third transmembrane domain (TMD) of EscR, a central protein of the T3SS in enteropathogenic E. coli, contributes to protein self-oligomerization. Moreover, we demonstrated that a single aspartic acid residue, located at the core of this TMD, is critical for the activity of the full-length protein and the function of the entire T3SS, possibly due to its involvement in mediating TMD-TMD interactions. Our findings should encourage the mapping of the entire interactome of the T3SS components, including interactions mediated through their TMDs.

A common strategy among intracellular bacterial pathogens is to enter into a vacuolar environment upon host cell invasion. One such pathogen, Salmonella enterica, resides within the Salmonella-containing vacuole (SCV) inside epithelial cells and macrophages. Salmonella hijacks the host endosomal system to establish this unique intracellular replicative niche, forming a highly complex and dynamic network of Salmonella-induced filaments (SIFs). SIFs radiate outwards from the SCV upon onset of bacterial replication. SIF biogenesis is dependent on the activity of bacterial effector proteins secreted by the Salmonella-pathogenicity island-2 (SPI-2) encoded type III secretion system. While the presence of SIFs has been known for almost 25 years, their precise role during infection remains elusive. This review summarizes our current knowledge of SCV maturation and SIF biogenesis, and recent advances in our understanding of the role of SIFs inside cells.

ABSTRACT The type III secretion system (T3SS) is a multiprotein complex that plays a central role in the virulence of many Gram-negative bacterial pathogens. To ensure that effector proteins are efficiently translocated into the host cell, bacteria must be able to sense their contact with the host cell. In this study, we found that EscP, which was previously shown to function as the ruler protein of the enteropathogenic Escherichia coli T3SS, is also involved in the switch from the secretion of translocator proteins to the secretion of effector proteins. In addition, we demonstrated that EscP can interact with the gatekeeper protein SepL and that the EscP-SepL complex dissociates upon a calcium concentration drop. We suggest a model in which bacterial contact with the host cell is accompanied by a drop in the calcium concentration that causes SepL-EscP complex dissociation and triggers the secretion of effector proteins. IMPORTANCE The emergence of multidrug-resistant bacterial strains, especially those of pathogenic bacteria, has serious medical and clinical implications. At the same time, the development and approval of new antibiotics have been limited for years. Recently, antivirulence drugs have received considerable attention as a novel antibiotic strategy that specifically targets bacterial virulence rather than growth, an approach that applies milder evolutionary pressure on the bacteria to develop resistance. A highly attractive target for the development of antivirulence compounds is the type III secretion system, a specialized secretory system possessed by many Gram-negative bacterial pathogens for injecting virulence factors (effectors) into host cells. In this study, we shed light on the molecular mechanism that allows bacteria to sense their contact with the host cell and to respond with the timed secretion of effector proteins. Understanding this critical step for bacterial virulence may provide a new therapeutic strategy.

ABSTRACT The outer membrane (OM) of Gram-negative bacteria provides protection against toxic molecules, including reactive oxygen species (ROS). Decreased OM permeability can promote bacterial survival under harsh circumstances and protects against antibiotics. To better understand the regulation of OM permeability, we studied the real-time influx of hydrogen peroxide in Salmonella bacteria and discovered two novel mechanisms by which they rapidly control OM permeability. We found that pores in two major OM proteins, OmpA and OmpC, could be rapidly opened or closed when oxidative stress is encountered and that the underlying mechanisms rely on the formation of disulfide bonds in the periplasmic domain of OmpA and TrxA, respectively. Additionally, we found that a Salmonella mutant showing increased OM permeability was killed more effectively by treatment with antibiotics. Together, these results demonstrate that Gram-negative bacteria regulate the influx of ROS for defense against oxidative stress and reveal novel targets that can be therapeutically targeted to increase bacterial killing by conventional antibiotics. IMPORTANCE Pathogenic bacteria have evolved ways to circumvent inflammatory immune responses. A decrease in bacterial outer membrane permeability during infection helps protect bacteria from toxic molecules produced by the host immune system and allows for effective colonization of the host. In this report, we reveal molecular mechanisms that rapidly alter outer membrane pores and their permeability in response to hydrogen peroxide and oxidative stress. These mechanisms are the first examples of pores that are rapidly opened or closed in response to reactive oxygen species. Moreover, one of these mechanisms can be targeted to artificially increase membrane permeability and thereby increase bacterial killing by the antibiotic cefotaxime during in vitro experiments and in a mouse model of infection. We envision that a better understanding of the regulation of membrane permeability will lead to new targets and treatment options for multidrug-resistant infections.

Abstract In response to the 2011 European health alert caused by a pathogenic Escherichia coli O104:H4 outbreak, the European Academy of Microbiology (EAM), established by the Federation of European Microbiological Societies (FEMS), convened a meeting in Paris on November 30th, 2011 on ‘EHEC infection and control’ attended by world renowned experts in pathogenic E. coli. The major aims of this group were to review the scientific issues raised by the outbreak, to assess the handling of the crisis at the scientific and political levels, and to propose future actions. Several conclusions, which will have impact on future potential E. coli outbreaks, are outlined here.

ABSTRACT Nontyphoidal Salmonella enterica (NTS) infections are a major burden to global public health, as they lead to diseases ranging from gastroenteritis to systemic infections and there is currently no vaccine available. Here, we describe a highly effective component vaccine against S. enterica serovar Typhimurium in both gastroenteritis and systemic murine infection models. We devised an approach to generate supernatants of S. enterica serovar Typhimurium, an organism that is highly abundant in virulence factors. Immunization of mice with this supernatant resulted in dramatic protection against a challenge with serovar Typhimurium, showing increased survival in the systemic model and decreased intestinal pathology in the gastrointestinal model. Protection correlated with specific IgA and IgG levels in the serum and specific secretory IgA levels in the feces of immunized mice. Initial characterization of the protective antigens in the bacterial culture supernatants revealed a subset of antigens that exhibited remarkable stability, a highly desirable characteristic of an effective vaccine to be used under suboptimal environmental conditions in developing countries. We were able to purify a subset of the peptides present in the supernatants and show their potential for immunization of mice against serovar Typhimurium resulting in a decreased level of colonization. This component vaccine shows promise with regard to protecting against NTS, and further work should significantly help to establish vaccines against these prevalent infections. IMPORTANCE Salmonella enterica infections other than typhoid and paratyphoid fever are a major global health burden, as they cause high morbidity and mortality worldwide. Strategies that prevent Salmonella-related diseases are greatly needed, and there is a significant push for the development of vaccines against nontyphoidal Salmonella enterica serovars. In this work, we describe an S. Typhimurium supernatant-derived vaccine that is effective in reducing bacterial colonization in mouse models of gastroenteritis as well as invasive disease. This is a component vaccine that shows high stability to heat, a feature that is important for use under suboptimal conditions, such as those found in sub-Saharan Africa.

ABSTRACT The mammalian gut contains a complex assembly of commensal microbes termed microbiota. Although much has been learned about the role of these microbes in health, the mechanisms underlying these functions are ill defined. We have recently shown that the mammalian gut contains thousands of small molecules, most of which are currently unidentified. Therefore, we hypothesized that these molecules function as chemical cues used by hosts and microbes during their interactions in health and disease. Thus, a search was initiated to identify molecules produced by the microbiota that are sensed by pathogens. We found that a secreted molecule produced by clostridia acts as a strong repressor of Salmonella virulence, obliterating expression of the Salmonella pathogenicity island 1 as well as host cell invasion. It has been known for decades that the microbiota protects its hosts from invading pathogens, and these data suggest that chemical sensing may be involved in this phenomenon. Further investigations should reveal the exact biological role of this molecule as well as its therapeutic potential. IMPORTANCE Microbes can communicate through the production and sensing of small molecules. Within the complex ecosystem formed by commensal microbes living in and on the human body, it is likely that these molecular messages are used extensively during the interactions between different microbial species as well as with host cells. Deciphering such a molecular dialect will be fundamental to our understanding of host-microbe interactions in health and disease and may prove useful for the design of new therapeutic strategies that target these mechanisms of communication.

ABSTRACT Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. IMPORTANCE As a host defense system, autophagy is known to target a population of Salmonella for degradation and hence restricting Salmonella replication. In contrast to this concept, a recent report showed that knockdown of Rab1, a GTPase required for autophagy of Salmonella, decreases Salmonella replication in HeLa cells. Here, we have reexamined the fate of Salmonella targeted by autophagy by various cell biology-based assays. We found that the association of autophagy components with cytosolic Salmonella increases shortly after initiation of intracellular bacterial replication. Furthermore, through a live-cell imaging method, a subset of cytosolic Salmonella was found to be extensively associated with autophagy components p62 and/or LC3, and they replicated quickly. Most importantly, depletion of autophagy components significantly reduced the replication of cytosolic Salmonella in HeLa cells. Hence, in contrast to previous reports, we propose that autophagy facilitates Salmonella replication in the cytosol of HeLa cells.

Bacterial pathogens have evolved numerous strategies to exploit their host's cellular processes so that they can survive and persist. Often, a bacterium must adhere very tightly to the cells and mediate its effects extracellularly, or it must find a way to invade the host's cells and survive intracellularly. In either case, the pathogen hijacks the host's cytoskeleton. The cytoskeleton provides a flexible framework for the cell and is involved in mediating numerous cellular functions, from cell shape and structure to programmed cell death. Altering the host cytoskeleton is crucial for mediating pathogen adherence, invasion, and intracellular locomotion. We highlight recent advances in the pathogenesis of enteropathogenic Escherichia coli, Salmonella Typhimurium, and Shigella flexneri. Each illustrates how bacterial pathogens can exert dramatic effects on the host cytoskeleton.