Showing papers by "B. Brett Finlay published in 2018"

Stunted childhood growth is associated with decompartmentalization of the gastrointestinal tract and overgrowth of oropharyngeal taxa.

Abstract: 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.

Identifying the etiology and pathophysiology underlying stunting and environmental enteropathy: study protocol of the AFRIBIOTA project

Abstract: Globally one out of four children under 5 years is affected by linear growth delay (stunting). This syndrome has severe long-term sequelae including increased risk of illness and mortality and delayed psychomotor development. Stunting is a syndrome that is linked to poor nutrition and repeated infections. To date, the treatment of stunted children is challenging as the underlying etiology and pathophysiological mechanisms remain elusive. We hypothesize that pediatric environmental enteropathy (PEE), a chronic inflammation of the small intestine, plays a major role in the pathophysiology of stunting, failure of nutritional interventions and diminished response to oral vaccines, potentially via changes in the composition of the pro- and eukaryotic intestinal communities. The main objective of AFRIBIOTA is to describe the intestinal dysbiosis observed in the context of stunting and to link it to PEE. Secondary objectives include the identification of the broader socio-economic environment and biological and environmental risk factors for stunting and PEE as well as the testing of a set of easy-to-use candidate biomarkers for PEE. We also assess host outcomes including mucosal and systemic immunity and psychomotor development. This article describes the rationale and study protocol of the AFRIBIOTA project. AFRIBIOTA is a case-control study for stunting recruiting children in Bangui, Central African Republic and in Antananarivo, Madagascar. In each country, 460 children aged 2–5 years with no overt signs of gastrointestinal disease are recruited (260 with no growth delay, 100 moderately stunted and 100 severely stunted). We compare the intestinal microbiota composition (gastric and small intestinal aspirates; feces), the mucosal and systemic immune status and the psychomotor development of children with stunting and/or PEE compared to non-stunted controls. We also perform anthropological and epidemiological investigations of the children’s broader living conditions and assess risk factors using a standardized questionnaire. To date, the pathophysiology and risk factors of stunting and PEE have been insufficiently investigated. AFRIBIOTA will add new insights into the pathophysiology underlying stunting and PEE and in doing so will enable implementation of new biomarkers and design of evidence-based treatment strategies for these two syndromes.

Global Profiling of Proteolysis from the Mitochondrial Amino Terminome during Early Intrinsic Apoptosis Prior to Caspase-3 Activation

Abstract: The human genome encodes ∼20 mitochondrial proteases, yet we know little of how they sculpt the mitochondrial proteome, particularly during important mitochondrial events such as the initiation of apoptosis. To characterize global mitochondrial proteolysis we refined our technique, terminal amine isotopic labeling of substrates, for mitochondrial SILAC (MS-TAILS) to identify proteolysis across mitochondria and parent cells in parallel. Our MS-TAILS analyses identified 45% of the mitochondrial proteome and identified protein amino (N)-termini from 26% of mitochondrial proteins, the highest reported coverage of the human mitochondrial N-terminome. MS-TAILS revealed 97 previously unknown proteolytic sites. MS-TAILS also identified mitochondrial targeting sequence (MTS) removal by proteolysis during protein import, confirming 101 MTS sites and identifying 135 new MTS sites, revealing a wobbly requirement for the MTS cleavage motif. To examine the relatively unknown initial cleavage events occurring before the well-studied activation of caspase-3 in intrinsic apoptosis, we quantitatively compared N-terminomes of mitochondria and their parent cells before and after initiation of apoptosis at very early time points. By identifying altered levels of >400 N-termini, MS-TAILS analyses implicated specific mitochondrial pathways including protein import, fission, and iron homeostasis in apoptosis initiation. Notably, both staurosporine and Bax activator molecule-7 triggered in common 7 mitochondrial and 85 cellular cleavage events that are potentially part of an essential core of apoptosis-initiating events. All mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD009054.

A Nonpyroptotic IFN-γ-Triggered Cell Death Mechanism in Nonphagocytic Cells Promotes Salmonella Clearance In Vivo.

Abstract: The cytokine IFN-γ has well-established antibacterial properties against the bacterium Salmonella enterica in phagocytes, but less is known about the effects of IFN-γ on Salmonella-infected nonphagocytic cells, such as intestinal epithelial cells (IECs) and fibroblasts. In this article, we show that exposing human and murine IECs and fibroblasts to IFN-γ following infection with Salmonella triggers a novel form of cell death that is neither pyroptosis nor any of the major known forms of programmed cell death. Cell death required IFN-γ-signaling via STAT1-IRF1-mediated induction of guanylate binding proteins and the presence of live Salmonella in the cytosol. In vivo, ablating IFN-γ signaling selectively in murine IECs led to higher bacterial burden in colon contents and increased inflammation in the intestine of infected mice. Together, these results demonstrate that IFN-γ signaling triggers release of Salmonella from the Salmonella-containing vacuole into the cytosol of infected nonphagocytic cells, resulting in a form of nonpyroptotic cell death that prevents bacterial spread in the gut.

Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection

Abstract: Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.

The Third Transmembrane Domain of EscR Is Critical for Function of the Enteropathogenic Escherichia coli Type III Secretion System.

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.

Characterization of the two conformations adopted by the T3SS inner-membrane protein PrgK.

Abstract: The pathogenic bacterium Salmonella enterica serovar typhimurium utilizes two type III secretion systems (T3SS) to inject effector proteins into target cells upon infection. The T3SS secretion apparatus (the injectisome) is a large macromolecular assembly composed of over twenty proteins, many in highly oligomeric states. A sub-structure of the injectisome, termed the basal body, spans both membranes and the periplasmic space of the bacterium. It is primarily composed of three integral membranes proteins, InvG, PrgH, and PrgK, that form ring structures through which components are secreted. In particular, PrgK possesses a periplasmic region consisting of two globular domains joined by a linker polypeptide. We showed previously that in isolation, this region adopts two distinct conformations, of with only one is observed in the assembled basal body complex. Here, using NMR spectroscopy, we further characterize these two conformations. In particular, we demonstrate that the interaction of the linker region with the first globular domain, as found in the intact basal body, is dependent upon the cis conformation of the Leu77-Pro78 peptide. Furthermore, this interaction is pH-dependent due to coupling with hydrogen bond formation between Tyr75 and His42 in its neutral Nδ1 H tautomeric form. This pH-dependent interaction may play a role in the regulation of the secretion apparatus disassembly in the context of bacterial infection.