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Objectives With a fourth of all under-five children affected, stunting remains one of the biggest health challenges worldwide. Even though the main underlying factors are known, the exact pathways to stunting varying in affected regions, and interventions thus need to be tailored to the local contexts. This study aimed assessing and comparing factors associated with stunting in two understudied sub-Saharan urban contexts with some of the highest stunting prevalence globally: Bangui, Central African Republic (~ 36%) and Antananarivo, Madagascar (42%). Methods We performed a case–control study on 175 + 194 stunted and 237 + 230 non-stunted control children aged 2–5 years and matched for age, gender and district of residency. Factors associated with stunting were identified using a standardized, paper questionnaire delivered by trained interviewers. Statistical analysis was done using logistic regression modelling. Results In both sites, formal maternal education lowered the risk of being stunted and restricted access to soap, suffering of anaemia and low birth weight were associated with higher risk of stunting. Short maternal stature, household head different from parents, diarrhoea and coughing were associated with an increased risk and continuing breastfeeding was associated with a lower risk of stunting in Antananarivo. Previous severe undernutrition and dermatitis/ fungal skin infections were associated with higher and changes in diet during pregnancy with lower risk of stunting in Bangui. Conclusions Our results suggest maternal education, antenatal care, iron supplementation and simple WASH interventions such as using soap and infection control as general and breastfeeding (Antananarivo) or better nutrition (Bangui) as area-specified interventions. Supplementary Information The online version contains supplementary material available at 10.1007/s10995-021-03201-8.

BACKGROUND Roux-en-Y gastric bypass (RYGB) results in long-term weight loss and reduced obesity related co-morbidities. However, little is known about how the lengths of the biliopancreatic limb (BPL), the alimentary limb (AL), and the common limb (CL) affect weight loss and glucose metabolism. OBJECTIVES Our aim was to establish a RYGB obese mouse model with defined proportions of the AL and BPL and a constant CL to assess the effects on weight loss,glucose metabolism, and obesity-related co-morbidities. SETTING In vivo mouse study. METHODS Six-week-old male C57BL/6J mice fed with a high-fat diet (HFD) underwent bariatric surgery with defined BPL lengths: a very long, long, and short BPL (35%, 25%, and 15% of total bowel length), or sham surgery. The length of the AL was adjusted to achieve the same CL length. Mice were analyzed for weight loss, glycemic control, and obesity-related co-morbidities. RESULTS Mice undergoing RYGB surgery with a very long BPL had excessive weight loss and mortality and were therefore not further analyzed. Mice with a long BPL showed a significantly increased total weight loss when compared with mice with a short BPL. In addition, a long BPL improved glucose tolerance, particularly early after surgery. A long BPL was also associated with lower triglyceride levels. Resolution of hepatic steatosis and adipose tissue inflammation was, however, not statistically significant. Of note, bariatric surgery dramatically changed gut microbiota, regardless of limb length. CONCLUSION In obese mice, a long BPL results in enhanced weight loss and improved glucose tolerance. These findings could potentially be translated to humans by tailoring the BPL length according to body weight, obesity-related co-morbidities, and total bowel length of an individual patient.

The intestinal microbiota plays a crucial role in health and changes in its composition are linked with major global human diseases. Fully understanding what shapes the human intestinal microbiota composition and knowing ways of modulating the composition are critical for promotion of life-course health, combating diseases, and reducing global health disparities. We aim to provide a foundation for understanding what shapes the human intestinal microbiota on an individual and global scale, and how interventions could utilize this information to promote life-course health and reduce global health disparities. We briefly review experiences within the first 1,000 days of life and how long-term exposures to environmental elements or geographic specific cultures have lasting impacts on the intestinal microbiota. We also discuss major public health threats linked to the intestinal microbiota, including antimicrobial resistance and disappearing microbial diversity due to globalization. In order to promote global health, we argue that the interplay of the larger ecosystem with intestinal microbiota research should be utilized for future research and urge for global efforts to conserve microbial diversity.

Shigellosis is a leading cause of diarrhea and dysentery in young children from low to middle-income countries and adults experiencing traveler's diarrhea worldwide. In addition to acute illness, infection by

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.

Background Increasing antimicrobial resistance (AMR) raises serious health and financial concerns. However, the main drivers of the emergence, spread and subsequent colonisation of resistant bacterial strains between humans, animals and the environment are still poorly understood. Objective The aim of this review was to identify molecular studies on AMR in One Health settings in Africa and to determine the prevalence of antimicrobial resistance genes in humans, animals and the environment. Due to the very low number of studies including environmental samples, the meta‐analysis only includes data obtained from animals and humans. Methods The PubMed, Web of Science and Scopus databases were searched, identifying 10 464 publications on AMR in Africa from January 1st, 2000 until June 1st, 2020. Inclusion criteria were: (i) Integrated studies assessing AMR simultaneously in an animal‐human, animal‐environment, human‐environment or animal‐human‐environment context, (ii) Genotypic characterisation of AMR and (iii) temporal and spatial relationship between samples from humans and animals. Statistical random‐effects model meta‐analysis was performed. Results Overall, 18 studies met our eligibility criteria and were included in this review. Six studies investigated Escherichia coli and Salmonella spp. (N = 6). The most prevalent AMR genes in animals included sul1 (36.2%), sul2 (32.0%), tetA (31.5%), strB (30.8%) and blaTEM (30.0%), whereas sul2 (42.4%), tetA (42.0%), strB (34.9%), blaTEM (28.8%) and sul1 (27.8%) were most prevalent in humans. We observed no clear pattern for a higher prevalence in either the animal or the human reservoir. Conclusion To date, data on AMR in a One Health perspective in Africa are scarce. Prospective and longitudinal studies using an integrated One Health approach assessing the environment, animals and humans at the same time are needed to better understand the main drivers of AMR sharing in Africa.

Invasion and multiplication of the facultative, cytosolic, enteropathogen Shigella flexneri within the colonic epithelial lining leads to an acute inflammatory response, fever and diarrhea. During the inflammatory process, infected cells are subjected to numerous stresses including heat, oxidative stress and genotoxic stress. The evolutionarily conserved pathway of cellular stress management is the formation of stress granules that store translationally inactive cellular mRNAs and interfere with cellular signalling pathways by sequestering signalling components. In this study, we investigated the ability of S. flexneri-infected cells to form stress granules in response to exogenous stresses. We found that S. flexneri infection inhibits movement of the stress granule markers eIF3 and eIF4B into stress granules and prevents the aggregation of G3BP1 and eIF4G-containing stress granules. This inhibition occurred only with invasive, but not with non-invasive bacteria and occurred in response to stresses that induce translational arrest through the phosphorylation of eIF2α and by treating cells with pateamine A, a drug that induces stress granules by inhibiting the eIF4A helicase. The S. flexneri-mediated stress granule inhibition could be largely phenocopied by the microtubule-destabilizing drug nocodazole and while S. flexneri infection did not lead to microtubule depolymerization, infection greatly enhanced acetylation of alpha-tubulin. Our data suggest that qualitative differences in the microtubule network or subversion of the microtubule-transport machinery by S. flexneri may be involved in preventing the full execution of this cellular stress response.

International audience; Linear growth delay (stunting) affects roughly 155 million children under the age of 5 years worldwide. Treatment has been limited by a lack of understanding of the underlying pathophysiological mechanisms. Stunting is most likely associated with changes in the microbial community of the small intestine, a compartment vital for digestion and nutrient absorption. Efforts to better understand the pathophysiology have been hampered by difficulty of access to small intestinal fluids. Here, we describe the microbial community found in the upper gastrointestinal tract of stunted children aged 2-5 y living in sub-Saharan Africa. We studied 46 duodenal and 57 gastric samples from stunted children, as well as 404 fecal samples from stunted and nonstunted children living in Bangui, Central African Republic, and in Antananarivo, Madagascar, using 16S Illumina Amplicon sequencing and semiquantitative culture methods. The vast majority of the stunted children showed small intestinal bacterial overgrowth dominated by bacteria that normally reside in the oropharyngeal cavity. There was an overrepresentation of oral bacteria in fecal samples of stunted children, opening the way for developing noninvasive diagnostic markers. In addition, Escherichia coli/Shigella sp. and Campylobacter sp. were found to be more prevalent in stunted children, while Clostridia, well-known butyrate producers , were reduced. Our data suggest that stunting is associated with a microbiome "decompartmentalization" of the gastrointestinal tract characterized by an increased presence of oropharyngeal bacteria from the stomach to the colon, hence challenging the current view of stunting arising solely as a consequence of small intestine overstimulation through recurrent infections by enteric pathogens.

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB40, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB40 a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB40 conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB40 (RI-MUB40) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB40-Cy5 using live cell microscopy. Systemically administered RI-MUB40-Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB40 marker will aid the study of broad ranges of inflammatory diseases. MUB40 is a universal and specific marker of mammalian neutrophils. MUB40 interacts with lactoferrin, stored in specific (β1), tertiary (β2), and secretome (γ) granules secreted upon stimulation. Neutrophil detection in inflamed or infected tissues is facilitated by MUB40 peptides conjugated to fluorophores. Neutrophilic inflammation detection may be envisaged by combining MUB40 with non-invasive imaging.

International audience; Even though tremendous progress has been made in the last decades to elucidate the mechanisms of intestinal homeostasis, dysbiosis and disease, we are only at the beginning of understanding the complexity of the gut ecosystem and the underlying interaction networks. We are also only starting to unravel the mechanisms that pathogens have evolved to overcome the barriers imposed by the microbiota and host to exploit the system to their own benefit. Recent work in these domains clearly indicates that the 'traditional Koch's postulates', which state that a given pathogen leads to a distinct disease, are not valid for all 'infectious' diseases, but that a more complete and complex interpretation of Koch's postulates is needed in order to understand and explain them. This review summarises the current understanding of what defines a healthy gut ecosystem and highlights recent progress in uncovering the interplay between the host, its microbiota and invading intestinal pathogens. Based on these recent findings, we propose a new interpretation of Koch's postulates that we term 'ecological Koch's postulates'.

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

Shigella is a leading cause of dysentery worldwide, with the majority of infections caused by two subgroups, S. flexneri and S. sonnei. Although S. flexneri has been highly prevalent in low-income countries, global development has brought an increase in S. sonnei at the expense of S. flexneri. However, the mechanisms behind this shift are not understood. Here we report that S. sonnei, but not S. flexneri, encodes a type VI secretion system (T6SS) that provides a competitive advantage in the gut. S. sonnei competes against E. coli and S. flexneri in mixed cultures, but this advantage is reduced in T6SS mutant strains. In addition, S. sonnei can persist as well as outcompete E. coli and S. flexneri in mice in a T6SS-dependent manner. These findings suggest that S. sonnei has a competitive advantage over S. flexneri and potentially explain the increasing global prevalence of S. sonnei.

Salmonella enterica serovar Typhimurium (S. Tm) is a facultative intracellular pathogen that induces entry into non-phagocytic cells by a Type III secretion system (TTSS) and cognate effector prote ...

Invasion and multiplication of the facultative, cytosolic, enteropathogen Shigella flexneri within the colonic epithelial lining leads to an acute inflammatory response, fever and diarrhea. During the inflammatory process, infected cells are subjected to numerous stresses including heat, oxidative stress and genotoxic stress. The evolutionarily conserved pathway of cellular stress management is the formation of stress granules that store translationally inactive cellular mRNAs and interfere with cellular signalling pathways by sequestering signalling components. In this study, we investigated the ability of S. flexneri-infected cells to form stress granules in response to exogenous stresses. We found that S. flexneri infection inhibits movement of the stress granule markers eIF3 and eIF4B into stress granules and prevents the aggregation of G3BP1 and eIF4G-containing stress granules. This inhibition occurred only with invasive, but not with non-invasive bacteria and occurred in response to stresses that induce translational arrest through the phosphorylation of eIF2α and by treating cells with pateamine A, a drug that induces stress granules by inhibiting the eIF4A helicase. The S. flexneri-mediated stress granule inhibition could be largely phenocopied by the microtubule-destabilizing drug nocodazole and while S. flexneri infection did not lead to microtubule depolymerization, infection greatly enhanced acetylation of alpha-tubulin. Our data suggest that qualitative differences in the microtubule network or subversion of the microtubule-transport machinery by S. flexneri may be involved in preventing the full execution of this cellular stress response.

Environmental enteropathy (EE) is a subclinical chronic inflammatory disease of the small intestine and has a profound impact on the persistence of childhood malnutrition worldwide. However, the aetiology of the disease remains unknown and no animal model exists to date, the creation of which would aid in understanding this complex disease. Here we demonstrate that early-life consumption of a moderately malnourished diet, in combination with iterative oral exposure to commensal Bacteroidales species and Escherichia coli, remodels the murine small intestine to resemble features of EE observed in humans. We further report the profound changes that malnutrition imparts on the small intestinal microbiota, metabolite and intraepithelial lymphocyte composition, along with the susceptibility to enteric infection. Our findings provide evidence indicating that both diet and microbes combine to contribute to the aetiology of EE, and describe a novel murine model that can be used to elucidate the mechanisms behind this understudied disease., Environmental enteropathy is a disorder of the small intestine that contributes to the persistence of childhood malnutrition worldwide. Here, Brown et al. show in mice that early-life malnourishment, in combination with exposure to commensal bacteria, remodels the small intestine to resemble features of the disease.

International audience; BackgroundSerotype 1 was the most prevalent pneumococcal meningitis serotype encountered in Niger over the period 2003–2011 (pre-vaccination era), accounting for 45.3% of infections.MethodsMultiple locus VNTR analysis (MLVA) was used to create a genotypic snapshot of a representative subset of the pneumococcal population of serotype 1.ResultsMLVA using 16 markers revealed a homogeneous genetic background of pneumococci serotype 1 from Niger, which clustered with few serotype 1 pneumococci from some African countries, while other African countries displayed different clonal complexes. DNA from Niger and from other African countries were different from pneumococci serotype 1 from European countries.ConclusionsMLVA-typing revealed a low genetic diversity among pneumococci serotype 1 from meningitis cases in Niger in the pre-vaccination era.

Salmonella enterica serovar Typhimurium (S. Tm) is a facultative intracellular pathogen that induces entry into non-phagocytic cells by a Type III secretion system (TTSS) and cognate effector proteins. Upon host cell entry, S. Tm expresses a second TTSS and subverts intracellular trafficking to create a replicative niche - the Salmonella-containing vacuole (SCV). SopE, a guanidyl exchange factor (GEF) for Rac1 and Cdc42, is translocated by the TTSS-1 upon host cell contact and promotes entry through triggering of actin-dependent ruffles. After host cell entry, the bulk of SopE undergoes proteasomal degradation. Here we show that a subfraction is however detectable on the nascent SCV membrane up to ∼ 6 h post infection. Membrane localization of SopE and the closely related SopE2 differentially depend on the Rho-GTPase-binding GEF domain, and to some extent involves also the unstructured N-terminus. SopE localizes transiently to the early SCV, dependent on continuous synthesis and secretion by the TTSS-1 during the intracellular state. Mutant strains lacking SopE or SopE2 are attenuated in early intracellular replication, while complementation restores this defect. Hence, the present study reveals an unanticipated role for SopE and SopE2 in establishing the Salmonella replicative niche, and further emphasizes the importance of entry effectors in later stages of host-cell manipulation.

Reorganization of the host cell actin cytoskeleton is crucial during pathogen invasion. We established micropatterned cells as a standardized infection model for cell invasion to quantitatively stu ...

Targeting of permissive entry sites is crucial for bacterial infection. The targeting mechanisms are incompletely understood. We have analyzed target-site selection by S. Typhimurium. This enteropathogenic bacterium employs adhesins (e.g. fim) and the type III secretion system 1 (TTSS-1) for host cell binding, the triggering of ruffles and invasion. Typically, S. Typhimurium invasion is focused on a subset of cells and multiple bacteria invade via the same ruffle. It has remained unclear how this is achieved. We have studied target-site selection in tissue culture by time lapse microscopy, movement pattern analysis and modeling. Flagellar motility (but not chemotaxis) was required for reaching the host cell surface in vitro. Subsequently, physical forces trapped the pathogen for ∼1.5–3 s in “near surface swimming”. This increased the local pathogen density and facilitated “scanning” of the host surface topology. We observed transient TTSS-1 and fim-independent “stopping” and irreversible TTSS-1-mediated docking, in particular at sites of prominent topology, i.e. the base of rounded-up cells and membrane ruffles. Our data indicate that target site selection and the cooperative infection of membrane ruffles are attributable to near surface swimming. This mechanism might be of general importance for understanding infection by flagellated bacteria., PLoS Pathogens, 8 (7), ISSN:1553-7374, ISSN:1553-7366

A genome wide RNAi screen identifies 72 host cell genes affecting S. Typhimurium entry, including actin regulators and COPI. This study implicates COPI-dependent cholesterol and sphingolipid localization as a common mechanism of infection by bacterial and viral pathogens., Genome-scale RNAi screen identifies 72 host genes affecting S. Typhimurium host cell invasion. Step-specific follow-up assays assign the phenotypes to specific steps of the invasion process. COPI effects on host cell binding, ruffling and invasion were traced to a key role of COPI in membrane targeting of cholesterol, sphingolipids, Rac1 and Cdc42. This new role of COPI explains why COPI is required for host cell infection by numerous bacterial and viral pathogens., Pathogens are not only a menace to public health, but they also provide excellent tools for probing host cell function. Thus, studying infection mechanisms has fueled progress in cell biology (Ridley et al, 1992; Welch et al, 1997). In the presented study, we have performed an RNAi screen to identify host cell genes required for Salmonella host cell invasion. This screen identified proteins known to contribute to Salmonella-induced actin rearrangements (e.g., Cdc42 and the Arp2/3 complex; reviewed in Schlumberger and Hardt, 2006) and vesicular traffic (e.g., Rab7) as well as unexpected hits, such as the COPI complex. COPI is a known organizer of Golgi-to-ER vesicle transport (Bethune et al, 2006; Beck et al, 2009). Here, we show that COPI is also involved in plasma membrane targeting of cholesterol, sphingolipids and the Rho GTPases Cdc42 and Rac1, essential host cell factors required for Salmonella invasion. This explains why COPI depletion inhibits infection by S. Typhimurium and illustrates how combining bacterial pathogenesis and systems approaches can promote cell biology. Salmonella Typhimurium is a common food-borne pathogen and worldwide a major public health problem causing severe diarrhea. The pathogen uses the host's gut mucosa as a portal of entry and gut tissue invasion is a key event leading to the disease. This explains the intense interest from medicine and basic biology in the mechanism of Salmonella host cell invasion. Tissue culture infection models have delineated a sequence of events leading host cell invasion (Figure 1; Schlumberger and Hardt, 2006): (i) pathogen binding to the host cell surface; (ii) activation of a syringe-like apparatus (‘Type III secretion system 1', T1) of the bacterium and injection of a bacterial toxin cocktail into the host cell. These toxins include SopE, a key virulence factor triggering invasion (Hardt et al, 1998), which was analyzed in our study; (iii) toxin-triggered membrane ruffling. To a significant extent, this is facilitated by SopE-triggered activation of Cdc42 and Rac1 and subsequent actin polymerization at the site of infection; (iv) engulfment of the pathogen within a vesicular compartment (SCV) and (v) maturation of the SCV, a process driven by a second Type III secretion system (T2), which is expressed by the pathogen upon bacterial entry (Figure 1). This sequence of events mediates Salmonella invasion into the gut epithelium and illustrates that this pathogen can be used for probing mechanisms of host cell actin control, membrane biogenesis, vesicle formation and vesicular trafficking. SopE is a key virulence factor of invasion and triggers the activation of Cdc42 and Rac1 and subsequent actin polymerization at the site of infection. We have employed a SopE-expressing S. Typhimurium strain and RNAi screening technology to identify host cell factors affecting invasion. First, we developed an automated fluorescence microscopy assay to quantify S. Typhimurium entry in a high-throughput format (Figure 1C). This assay was based on a GFP reporter expressed by the pathogen after invasion and maturation of the SCV. Using this assay, we screened a ‘druggable genome' siRNA library (6978 genes, 3 oligos each, 1 oligo per well) and identified 72 invasion hits. These included established regulators of the actin cytoskeleton (Cdc42, Arp2/3, Nap1; Schlumberger and Hardt, 2006), some of which have not been implicated so far in Salmonella entry (Pfn1, Cap1), as well as proteins not previously thought to influence infection (Atp1a1, Rbx1, COPI complex). Potentially, these hits could affect any step of the invasion process (Figure 1A). In the second stage of the study, we have assigned each ‘invasion hit' to particular steps of the invasion process. For this purpose, we developed step-specific assays for Salmonella binding, injection, ruffling and membrane engulfment and re-screened the genes found as hits in the first screen (four siRNAs per gene). As expected, a significant number of ‘hits' affected binding to the host cell, others affected binding and ruffling (e.g., Pfn1, Itgβ5, Cap1), a few were specific for the ruffling step (e.g., Cdc42) and some affected SCV maturation, namely Rab7a, the trafficking protein Vps39 and the vacuolar proton pump Atp6ap2. Thus, our experimental strategy allowed mechanistic interpretation and linked novel hits to particular phenotypes, thus providing a basis for further studies (Figure 1). COPI depletion impaired effector injection and ruffling. This was surprising, as the COPI complex was known to regulate retrogade Golgi-to-ER transport, but was not expected to affect pathogen interactions at the plasma membrane. Therefore, we have investigated the underlying mechanism. We have observed that COPI depletion entailed dramatic changes in the plasma membrane composition (Figure 6). Cholesterol and sphingolipids, which form domains (‘lipid rafts') in the plasma membrane, were depleted from the cell surface and redirected into a large vesicular compartment. The same was true for the Rho GTPases Rac1 and Cdc42. This strong decrease in the amount of cholesterol-enriched microdomains and Rho GTPases in the plasma membrane explained the observed defects in S. Typhimurium host cell invasion and assigned a novel role for COPI in controlling mammalian plasma membrane composition. It should be noted that other viral and bacterial pathogens do show a similar dependency on host cellular COPI and plasma membrane lipids. This includes notorious pathogens such as Staphylococcus aureus (Ramet et al, 2002; Potrich et al, 2009), Listeria monocytogenes (Seveau et al, 2004; Agaisse et al, 2005; Cheng et al, 2005; Gekara et al, 2005), Mycobacterium tuberculosis (Munoz et al, 2009), Chlamydia trachomatis (Elwell et al, 2008), influenza virus (Hao et al, 2008; Konig et al, 2010), hepatitis C virus (Tai et al, 2009; Popescu and Dubuisson, 2010) and the vesicular stomatitis virus (presented study) and suggests that COPI-mediated control of host cell plasma membrane composition might be of broad importance for pathogenesis. Future work will have to address whether this might offer starting points for developing anti-infective therapeutics with a very broad spectrum of activity., The pathogen Salmonella Typhimurium is a common cause of diarrhea and invades the gut tissue by injecting a cocktail of virulence factors into epithelial cells, triggering actin rearrangements, membrane ruffling and pathogen entry. One of these factors is SopE, a G-nucleotide exchange factor for the host cellular Rho GTPases Rac1 and Cdc42. How SopE mediates cellular invasion is incompletely understood. Using genome-scale RNAi screening we identified 72 known and novel host cell proteins affecting SopE-mediated entry. Follow-up assays assigned these ‘hits' to particular steps of the invasion process; i.e., binding, effector injection, membrane ruffling, membrane closure and maturation of the Salmonella-containing vacuole. Depletion of the COPI complex revealed a unique effect on virulence factor injection and membrane ruffling. Both effects are attributable to mislocalization of cholesterol, sphingolipids, Rac1 and Cdc42 away from the plasma membrane into a large intracellular compartment. Equivalent results were obtained with the vesicular stomatitis virus. Therefore, COPI-facilitated maintenance of lipids may represent a novel, unifying mechanism essential for a wide range of pathogens, offering opportunities for designing new drugs.

International audience; Environmental enteric dysfunction (EED) is an inflammatory syndrome postulated to contribute to stunted child growth and to be associated with intestinal dysbiosis and nutrient malabsorption. However, the small intestinal contributions to EED remain poorly understood. This study aimed to assess changes in the proximal and distal intestinal microbiota in the context of stunting and EED and to test for a causal role of these bacterial isolates in the underlying pathophysiology. We performed a cross-sectional study in two African countries recruiting roughly 1,000 children aged 2 to 5 years and assessed the microbiota in the stomach, duodenum, and feces. Upper gastrointestinal samples were obtained from stunted children and stratified according to stunting severity. Fecal samples were collected. We then investigated the role of clinical isolates in EED pathophysiology using tissue culture and animal models. We find that small intestinal bacterial overgrowth (SIBO) is extremely common (>80%) in stunted children. SIBO is frequently characterized by an overgrowth of oral bacteria, leading to increased permeability and inflammation and to replacement of classical small intestinal strains. These duodenal bacterial isolates decrease lipid absorption in both cultured enterocytes and mice, providing a mechanism by which they may exacerbate EED and stunting. Further, we find a specific fecal signature associated with the EED markers fecal calprotectin and alpha-antitrypsin. Our study shows a causal implication of ectopic colonization of oral bacterial isolated from the small intestine in nutrient malabsorption and gut leakiness in vitro. These findings have important therapeutic implications for modulating the microbiota through microbiota-targeted interventions.

International audience; Environmental enteric dysfunction (EED) is an inflammatory syndrome postulated to contribute to stunted child growth and to be associated with intestinal dysbiosis and nutrient malabsorption. However, the small intestinal contributions to EED remain poorly understood. This study aimed to assess changes in the proximal and distal intestinal microbiota in the context of stunting and EED and to test for a causal role of these bacterial isolates in the underlying pathophysiology. We performed a cross-sectional study in two African countries recruiting roughly 1,000 children aged 2 to 5 years and assessed the microbiota in the stomach, duodenum, and feces. Upper gastrointestinal samples were obtained from stunted children and stratified according to stunting severity. Fecal samples were collected. We then investigated the role of clinical isolates in EED pathophysiology using tissue culture and animal models. We find that small intestinal bacterial overgrowth (SIBO) is extremely common (>80%) in stunted children. SIBO is frequently characterized by an overgrowth of oral bacteria, leading to increased permeability and inflammation and to replacement of classical small intestinal strains. These duodenal bacterial isolates decrease lipid absorption in both cultured enterocytes and mice, providing a mechanism by which they may exacerbate EED and stunting. Further, we find a specific fecal signature associated with the EED markers fecal calprotectin and alpha-antitrypsin. Our study shows a causal implication of ectopic colonization of oral bacterial isolated from the small intestine in nutrient malabsorption and gut leakiness in vitro. These findings have important therapeutic implications for modulating the microbiota through microbiota-targeted interventions.

Background This study aimed to compare the prevalence of intestinal parasite infestations (IPIs) in stunted children, compared to control children, in Ankasina and Andranomanalina Isotry (two disadvantaged neighborhoods of Antananarivo, Madagascar), to characterize associated risk factors and to compare IPI detection by real-time PCR and standard microscopy techniques. Methodology/Principal findings Fecal samples were collected from a total of 410 children (171 stunted and 239 control) aged 2–5 years. A single stool sample per subject was examined by simple merthiolate-iodine-formaldehyde (MIF), Kato-Katz smear and real-time PCR techniques. A total of 96.3% of the children were infested with at least one intestinal parasite. The most prevalent parasites were Giardia intestinalis (79.5%), Ascaris lumbricoides (68.3%) and Trichuris trichiura (68.0%). For all parasites studied, real-time PCR showed higher detection rates compared to microscopy (G. intestinalis [77.6% (n = 318) versus 20.9% (n = 86)], Entamoeba histolytica [15.8% (n = 65) versus 1.9% (n = 8)] and A. lumbricoides [64.1% (n = 263) versus 50.7% (n = 208)]). Among the different variables assessed in the study, age of 4 to 5 years (AOR = 4.61; 95% CI, (1.35–15.77)) and primary and secondary educational level of the mother (AOR = 12.59; 95% CI, (2.76–57.47); AOR = 9.17; 95% CI, (2.12–39.71), respectively) were significantly associated with IPIs. Children drinking untreated water was associated with infestation with G. intestinalis (AOR = 1.85; 95% CI, (1.1–3.09)) and E. histolytica (AOR = 1.9; 95% CI, (1.07–3.38)). E. histolytica was also associated with moderately stunted children (AOR = 0.37; 95% CI, 0.2–0.71). Similarly, children aged between 4 and 5 years (AOR = 3.2; 95% CI (2.04–5.01)) and living on noncemented soil types (AOR = 1.85; 95% CI, (1.18–2.09)) were associated with T. trichiura infestation. Conclusions/Significance The prevalence of IPIs is substantial in the studied areas in both stunted and control children, despite the large-scale drug administration of antiparasitic drugs in the country. This high prevalence of IPIs warrants further investigation. Improved health education, environmental sanitation and quality of water sources should be provided., Author summary In populations living in adverse conditions due to poverty, a wide variety of intestinal parasite infestations can be observed. These infestations are usually diagnosed by stool microscopy but can be easily overlooked if the procedures used are inaccurate or performed in a suboptimal way. In the present study, we investigated the prevalence of intestinal parasite infestations in stunted and control children aged from 2 to 5 years living in two disadvantaged neighborhoods of Antananarivo Madagascar. We also assessed risk factors for infestations and the diagnostic performance of microscopic techniques and real-time PCR for the detection of parasites. Almost all individuals were found to be infested with at least one parasitic species. Children aged between 4 and 5 years and mothers with low educational levels were found to be associated with infestation. Similarly, children drinking untreated water were associated with G. intestinalis and E. histolytica infestation. This latter species was also associated with moderately stunted children. Children between 4 and 5 years old and with no cemented soil type were associated with T. trichiura infestation. The high prevalence of intestinal parasite infestations among the study participants requires the improvement of health education, environmental sanitation and quality of water sources.