The type III secretion system apparatus determines the intracellular niche of bacterial pathogens

Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon—Shigella IpaC, Salmonella SipC, or Chromobacterium CipC—determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established.Intracellular bacterial pathogens use a variety of elaborate means to survive within host cells. Postinvasion, some such as Legionella, Salmonella, and Chlamydia species modify bacteria-containing vacuoles to avoid death via phagosomal acidification or lysosomal fusion. Others, including Shigella, Listeria, Rickettsia, and Burkholderia species, rapidly escape from phagosomes into the cytosol of infected cells. Although escape from phagosomes by the classic intracytoplasmic Gram-positive bacterium Listeria monocytogenes is well understood (1), much less is known regarding how Gram-negative pathogens, including the model professional intracytoplasmic Shigella species, enter the cytosol.During the course of an infection, many Gram-negative pathogens, including Shigella, Salmonella, enteropathogenic Escherichia coli, and Yersinia species, use type III secretion systems (T3SSs) as injection devices to deliver multiple virulence proteins, referred to as effectors, directly into the cytosol of infected cells (2). T3SSs are composed of ∼20 proteins and sense host cell contact via a tip complex at the distal end of a needle filament, which then acts as a scaffold for the formation of a translocon pore in the host cell membrane. Although components of their type III secretion apparatus (T3SA) are relatively well conserved, each pathogen delivers a unique repertoire of effectors into host cells, likely accounting for the establishment of a variety of replicative niches. For example, Salmonella and Shigella secreted effectors promote the uptake of these bacteria into nonphagocytic cells, whereas those from Yersinia inhibit phagocytosis by macrophages.All four pathogenic Shigella species—Shigella flexneri, Shigella sonnei, Shigella boydii, and Shigella dysenteriae—deliver ∼30 effectors into host cells, the majority of which are encoded on a large virulence plasmid (VP) alongside the genes for all of the proteins needed to form a T3SA (3). These secreted proteins play major roles in Shigella pathogenesis, including host cell invasion and modulation of innate immune response. One effector, IpgD, promotes the efficiency of Shigella phagosomal escape, although it is not absolutely required for this process (4). Interestingly, IpaB and IpaC, components of the Shigella translocon, the portion of the T3SA that inserts into the host cell membrane, have been implicated to mediate phagosomal escape based on the behavior of recombinant proteins (5–7). The physiologic relevance of these findings has not yet been directly addressed, as strains that lack either of these two proteins are completely impaired in the delivery of Shigella effectors into host cells (8).Here, using a reductionist approach, we directly tested a role for the Shigella translocon apparatus in phagosomal escape. Using an innovative reengineering approach, we introduced a functional effectorless Shigella T3SA into a nonpathogenic laboratory strain of DH10B E. coli. Remarkably, upon entry into host epithelial cells, these bacteria efficiently escape from phagosomes. This demonstrates for the first time, to our knowledge, in the context of an infection, a direct role for the Shigella T3SA in mediating vacuole lysis. Despite structural conservation across T3SS families, we further observed that, in the absence of any type III effectors, the Ysc T3SA mediates little to no Yersinia phagosomal escape, suggesting that not all injectisomes have equivalent functions. Lastly, by exploring the functional interchangeability of translocon components of the Shigella, Salmonella, and Chromobacterium T3SA, we demonstrate that one translocon protein controls the extent to which these intracellular pathogens escape into the cytosol of infected cells, thus demonstrating a major role for the T3SA in determining the site of the replicative niche of intracellular bacteria.     

SIK2 orchestrates actin-dependent host response upon Salmonella infection

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Salmonella enterica is a gram-negative, facultative intracellular human pathogen, annually causing more than 100 million food- and waterborne infections worldwide. Salmonella Typhimurium causes severe gastroenteritis, which could turn into a systemic infection in children, immune-compromised, or elderly people (1, 2). Concurrently, multidrug resistant bacteria are globally emerging and threatening our health systems, calling for a better understanding of the underlying virulence mechanism and host response.Pathogenic bacteria have evolved the inherent ability to infect and to establish their niche within host cells. For colonizing nonphagocytic cells such as epithelial cells, Salmonella uses a trigger mechanism–based entry mode. Bacterial virulence factors are then injected via a Type III-secretion system (T3SS) into the host cell to induce cytoskeletal rearrangements leading to membrane ruffling and macropinocytosis-driven internalization into a sealed phagosome (3, 4). This specialized compartment is referred to as the Salmonella-containing vacuole (SCV) and serves as the intracellular replicative niche by hiding the bacteria from the humoral and cell-autonomous immune response (5). Salmonella invasion requires a cooperative action of several bacterial effector proteins hijacking multiple host targets. One of the main targets forcing Salmonella´s uptake is the actin cytoskeleton by subverting the host Rho GTPases system. Bacterial effector proteins such as SopE/SopE2 mimic host nucleotide exchange factors (GEFs) to stimulate Rac1 and CDC42 activity (6, 7). Once GTP-activated, Rho GTPases stimulate downstream pathways to drive actin filament (F-actin) assembly and rearrangement.The actin cytoskeleton network is regulated by actin-binding proteins (ABPs), which orchestrate assembly and disassembly of actin in higher networks (8). Monomeric, globular actin (G-actin) is nucleated into new actin filaments, or the existing F-actin is elongated, stabilized, or disassembled by ABPs. The major actin nucleation factor is the multimeric Arp2/3 complex, which generates branched actin filament networks. Formins generate long unbranched actin filaments and represent another actin nucleation family. Together with actin nucleation-promoting factors, small Rho GTPases control ABPs in a spatiotemporal manner. Actin polymerization and membrane ruffling are necessary for Salmonella invasion. Following Salmonella internalization, the SCV undergoes SPI-1–dependent biogenesis and is transported to a juxtanuclear position at 1 to 2 h postinfection (pi). At later time-points (4 to 6 h pi), SPI-2–dependent effector proteins are expressed to further mature the SCV, allowing bacterial proliferation. Pioneering work described that, at later stages of the infection (≥6 h pi), an actin meshwork around the SCV stabilizes and protects the vacuolar niche (9–13).Here, we report SIK2 as a Salmonella resistance factor and a regulator of the actin cytoskeleton. SIK2 belongs to the AMPK kinase family and was named after its homolog SIK1, found to be expressed upon high-salt diet-induced stress in rats (14, 15). SIK2 has been implicated into multiple biological roles including melanogenesis, cancer progression, and gluconeogenesis (16–18). SIK2 depletion results in a loss of SCV integrity and bacterial escape into the host cytosol, causing intracellular Salmonella hyperproliferation. Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. Hence, SIK2 may represent a cellular safeguard, which controls the actin cytoskeleton and SCV integrity, thereby serving as a prime regulator of Salmonella proliferation subsequent to cellular internalization.     

Type 1 interferon-dependent repression of NLRC4 and iPLA2 licenses down-regulation of Salmonella flagellin inside macrophages

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with Salmonella and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. Salmonella switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues that Salmonella perceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching of Salmonella to a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipids which in turn enhance expression of flagellin by Salmonella thereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression by Salmonella. These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection with Salmonella.

The innate immune system senses microbial pathogens through recognition of conserved entities collectively referred to as pathogen/microbe-associated molecular patterns (PAMPs/MAMPs). These entities interact with conserved pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), Nod-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), and C-type lectin receptors (CLRs) that are expressed by immune cells and other cell types. Activation of PRRs by PAMPs is dictated by the availability and expression levels of PAMPs at different stages of infection and results in host responses which are vital for inflammation and immunity against pathogens (1). However, some pathogens, including Salmonella spp., a facultative intracellular pathogen, have evolved the ability to use these host responses for their own replication and establishment of infection (2).Flagellin, the monomeric protein constituting bacterial flagella, is one of the key Salmonella effector molecules which binds and activates membrane-bound TLR-5 as well as the cytosolic sensor NLRC4 and plays a major role in generating inflammatory responses (3–5). In macrophages, flagellin as well as the rod protein PrgJ, which are inadvertently released into the host cytosol by the type III secretion system (T3SS), are detected by the NAIPs. In mice, seven NAIPs are present of which NAIP1 senses the T3SS needle protein, NAIP2 detects the T3SS inner rod protein, and NAIP5 and NAIP6 recognize flagellin (6–9). Humans however encode a single functional NAIP which has been recently shown to broadly detect multiple T3SS proteins and flagellin (10). Ligand binding to the NAIPs leads to recruitment and oligomerization of NLRC4 (11, 12). Activation of the NAIP-NLRC4 inflammasome by these effectors and activation of the NLRP3 inflammasome by an as yet unidentified aconitase-regulated Salmonella effector (13–15) results in caspase-1-dependent pyroptosis and production of active IL-1β which promotes clearance of the bacterium and protects the host against Salmonella (13, 16, 17). It is believed that as infection progresses, Salmonella circumvents this host-protective response by suppressing the expression of flagellin to lower than the resting levels usually expressed by bacteria in culture (18). Down-regulation of flagellin is essential for the bacterium to establish successful infection. Previous work has shown that a Salmonella Typhimurium strain modified to constitutively express flagellin (ST-FliC^ON) and therefore unable to naturally down-regulate flagellin expression is avirulent and cleared successfully from the host compared to its wild-type (WT) counterpart (17). Despite this central role of flagellin in Salmonella pathogenesis, the molecular mechanisms that regulate the physiological switch of Salmonella from a flagellin-high to a flagellin-low phenotype and aid in establishment of an intracellular niche within macrophages in vivo are incompletely understood.Upon entry into macrophages, Salmonella resides in a vacuole called the Salmonella-containing vacuole (SCV) where it shuts down expression of the Salmonella pathogenicity island 1 (SPI-1) and concomitantly switches on expression of Salmonella pathogenicity island 2 (SPI-2), which is activated by the PhoP/PhoQ two-component system (19) and encodes genes required for intracellular replication. Prior work has shown that shutdown of SPI-1 in growth media that mimic conditions associated with the SCV such as acidic pH and low Mg²⁺ is also accompanied by repression of flagellin (20, 21). This is because low pH and low Mg²⁺ activate the PhoP/PhoQ system (20, 22, 23) and activated PhoP is believed to suppress expression of flagellin (21). A noteworthy issue relating to these early studies is that effects on PhoP/PhoQ-regulated genes were examined only during in vitro culture of bacteria in growth medium and not in the context of S. Typhimurium residence within macrophages. Therefore, the physiological contribution of these mechanisms to flagellin repression of intracellular Salmonella remains debatable. For example, contrary studies have shown that the effect of low pH on flagellin protein expression is observed only at a very low pH (pH = 3) and not at pH 5 (20) which is close to the physiologically relevant pH of the SCV (24, 25). Likewise, neither variation of extracellular Mg²⁺ nor reduced Mg²⁺ in the SCV was found to play a role in PhoP activation by Salmonella inside macrophages (26). Consequently, the regulatory mechanisms conventionally thought to repress flagellin expression by Salmonella remain controversial and there is scarce evidence to suggest that these factors are responsible for down-regulation of flagellin by bacteria residing within macrophages. Moreover, the physiological mechanisms that regulate repression of flagellin in vivo are unknown.In this study we describe a host innate immune circuit that regulates expression of Salmonella flagellin during both the early/extracellular and the later/intracellular phases of macrophage infection with this pathogen. We find that during early infection of macrophages with S. Typhimurium, rapid NLRC4 inflammasome-dependent macrophage pyroptosis is necessary and sufficient for releasing a host lysophospholipid stimulus that promotes synthesis and release of flagellin from Salmonella. Unexpectedly, these host factors regulate not only the initial increase in flagellin production but also the later down-regulation of flagellin by Salmonella inside macrophages. This later effect is mediated by a natural type 1 IFN-dependent host negative feedback response that represses expression of NLRC4 and the lysophospholipid biosynthetic enzyme calcium-independent phospholipase A2 (iPLA2) within cells, causing a decline in intracellular lysophospholipids over time, which promotes eventual down-regulation of flagellin by intracellular bacteria. Our data identify host NLRC4 inflammasome activity as a temporal and biphasic regulator of expression of its own bacterial ligand, flagellin. We also describe a physiologically relevant type 1 IFN-mediated host mechanism that controls switching of Salmonella from a flagellin-high to a flagellin-low phenotype within macrophages in vivo. These findings have important implications for understanding the intricate evolutionary adaptations that shape host–pathogen cross-talk.     

Salmonella enterica serovar Typhimurium adhesion and cytotoxicity during epithelial cell stress is reduced by Lactobacillus rhamnosus GG

Background

Physiological stressors may alter susceptibility of the host intestinal epithelium to infection by enteric pathogens. In the current study, cytotoxic effect, adhesion and invasion of Salmonella enterica serovar Typhimurium (S. Typhimurium) to Caco-2 cells exposed to thermal stress (41°C, 1 h) was investigated. Probiotic bacteria have been shown to reduce interaction of pathogens with the epithelium under non-stress conditions and may have a significant effect on epithelial viability during infection; however, probiotic effect on pathogen interaction with epithelial cells under physiological stress is not known. Therefore, we investigated the influence of Lactobacillus rhamnosus GG and Lactobacillus gasseri on Salmonella adhesion and Salmonella-induced cytotoxicity of Caco-2 cells subjected to thermal stress.

Results

Thermal stress increased the cytotoxic effect of both S. Typhimurium (P = 0.0001) and nonpathogenic E. coli K12 (P = 0.004) to Caco-2 cells, and resulted in greater susceptibility of cell monolayers to S. Typhimurium adhesion (P = 0.001). Thermal stress had no significant impact on inflammatory cytokines released by Caco-2 cells, although exposure to S. Typhimurium resulted in greater than 80% increase in production of IL-6 and IL-8. Blocking S. Typhimurium with anti-ShdA antibody prior to exposure of Salmonella decreased adhesion (P = 0.01) to non-stressed and thermal-stressed Caco-2 cells. Pre-exposure of Caco-2 cells to L. rhamnosus GG significantly reduced Salmonella-induced cytotoxicity (P = 0.001) and Salmonella adhesion (P = 0.001) to Caco-2 cells during thermal stress, while L. gasseri had no effect.

Conclusion

Results suggest that thermal stress increases susceptibility of intestinal epithelial Caco-2 cells to Salmonella adhesion, and increases the cytotoxic effect of Salmonella during infection. Use of L. rhamnosus GG as a probiotic may reduce the severity of infection during epithelial cell stress. Mechanisms by which thermal stress increases susceptibility to S. Typhimurium colonization and by which L. rhamnosus GG limits the severity of infection remain to be elucidated.     

Actin polymerization as a key innate immune effector mechanism to control Salmonella infection

Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1β recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1β production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.A critical step in disease pathogenesis for many clinically important bacteria is their ability to infect and survive within host cells such as macrophages. Salmonella enterica, a pathogen that resides and replicates within macrophages, causes a range of life-threatening diseases in humans and animals, and accounts for 28 million cases of enteric fever worldwide each year (1). S. enterica infects phagocytes by a process that requires cytoskeletal reorganization (2). This bacterium resides in a Salmonella-containing vacuole (SCV) within host macrophages, and this intracellular lifestyle enables them to avoid extracellular antimicrobial killing, evade adaptive immune responses, and potentially to spread to new sites to seed new infectious foci within host tissue, which eventually develop into granulomas (3). Survival and growth of S. enterica within phagocytes is critical for virulence (4) and host restriction of the intracellular bacterial load is, therefore, paramount in surviving salmonellosis. Salmonella delivers microbial effector proteins into the host cell via the type III secretion systems (T3SS), mediated by the Salmonella pathogenicity island-1 and -2 (SPI-1 and SPI-2), to subvert cellular functions and facilitate intracellular survival (5).Microbes are recognized by macrophages through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), which initiate innate immune responses, including cytokine production and pathogen killing (6). NLRs drive the formation of inflammasomes—macromolecular protein complexes—comprising one or more NLRs, usually an adaptor protein (ASC) and the effector protein caspase-1, which then cleaves prointerleukin-1β (IL-1β) and pro–IL-18 into biologically active cytokines, and initiates macrophage cell death by pyroptosis (7). NLRC4, in concert with NAIPs 1, 2, 5, and 6, is a key PRR that forms an inflammasome complex upon sensing flagellin and/or the inner rod or needle proteins (PrgJ and PrgI, respectively) of the SPI-1 T3SS of S. enterica serovar Typhimurium (S. Typhimurium) (8–11). Activation of the NLRC4 inflammasome by Salmonella infection results in IL-1β and IL-18 production driven by an ASC-dependent pathway and macrophage pyroptosis driven by an ASC-independent pathway (12, 13). A second, noncanonical, NLR signaling pathway has been described, which requires caspase-11 to initiate delayed cell death and NLRP3 inflammasome activation (14–16). Effective clearance of Salmonella infection in host cells may therefore require a coordinated effort between different inflammasome signaling pathways.We, and others, have shown that NLRC4 is important in regulating bacterial burden of S. Typhimurium in vivo (17–19). A recent study revealed that Salmonella-infected epithelial cells are extruded from the intestinal epithelium in a process that requires NLRC4 (20). The molecular mechanism behind how NLRC4 restricts bacterial burden in macrophages infected with Salmonella is still unknown. Here, we identify an actin-dependent mechanism that controls NLRC4-mediated regulation of bacterial replication in macrophages infected with S. Typhimurium. Activation of NLRC4 in infected macrophages mediates the production of reactive oxygen species (ROS) to inhibit bacterial replication and limits additional bacterial uptake by inducing mechanical stiffening the cell via actin polymerization. Overall, we describe a previously unidentified effector mechanism, governed by actin and the NLRC4 inflammasome, to control Salmonella infection in macrophages.     

Inflammatory bactericidal lectin RegIIIβ: Friend or foe for the host?

In the inflamed gut, the bactericidal lectin RegIIIβ is massively produced by intestinal mucosa. RegIIIβ binds peptidoglycan and lipid A respectively, and thus can kill certain Gram-positive and Gram-negative bacteria, including the gut commensal microbiota and enteropathogenic bacteria. Considering the expression pattern and bactericidal activity, RegIIIβ is believed to be a host defense factor for protecting against the infection with enteropathogenic bacteria. However, it was poorly understood how RegIIIβ recognizes the target bacteria and kill them, and how RegIIIβ plays role(s) in infectious diarrhea. Therefore, our recent study has focused on RegIIIβ-target recognition, killing of Gram-negative bacteria, and host protective functions of RegIIIβ for infectious diarrhea inflicted by Salmonella Typhimurium. Here, we discuss novel insights into the protective role of RegIIIβ in infectious diarrhea, and propose avenues towards novel therapeutic interventions for Salmonella diarrhea.     

Importance of antibody and complement for oxidative burst and killing of invasive nontyphoidal Salmonella by blood cells in Africans

Bacteremia caused by nontyphoidal strains of Salmonella is endemic among African children. Case-fatality rates are high and antibiotic resistance increasing, but no vaccine is currently available. T cells are important for clearance of Salmonella infection within macrophages, but in Africa, invasive Salmonella disease usually manifests in the blood and affects children between 4 months and 2 y of age, when anti-Salmonella antibody is absent. We have previously found a role for complement-fixing bactericidal antibody in protecting these children. Here we show that opsonic activity of antibody and complement is required for oxidative burst and killing of Salmonella by blood cells in Africans. Induction of neutrophil oxidative burst correlated with anti-Salmonella IgG and IgM titers and C3 deposition on bacteria and was significantly lower in African children younger than 2 y compared with older children. Preopsonizing Salmonella with immune serum overcame this deficit, indicating a requirement for antibody and/or complement. Using different opsonization procedures, both antibody and complement were found to be necessary for optimal oxidative burst, phagocytosis and killing of nontyphoidal Salmonella by peripheral blood cells in Africans. Although most strains of African nontyphoidal Salmonella can be killed with antibody and complement alone, phagocytes in the presence of specific antibody and complement can kill strains resistant to killing by immune serum. These findings increase the likelihood that an antibody-inducing vaccine will protect against invasive nontyphoidal Salmonella disease in African children.     

Glucose metabolism: Focus on gut microbiota,the endocannabinoid system and beyond

The gut microbiota is now considered as a key factor in the regulation of numerous metabolic pathways. Growing evidence suggests that cross-talk between gut bacteria and host is achieved through specific metabolites (such as short-chain fatty acids) and molecular patterns of microbial membranes (lipopolysaccharides) that activate host cell receptors (such as toll-like receptors and G-protein-coupled receptors). The endocannabinoid (eCB) system is an important target in the context of obesity, type 2 diabetes (T2D) and inflammation. It has been demonstrated that eCB system activity is involved in the control of glucose and energy metabolism, and can be tuned up or down by specific gut microbes (for example, Akkermansia muciniphila). Numerous studies have also shown that the composition of the gut microbiota differs between obese and/or T2D individuals and those who are lean and non-diabetic. Although some shared taxa are often cited, there is still no clear consensus on the precise microbial composition that triggers metabolic disorders, and causality between specific microbes and the development of such diseases is yet to be proven in humans. Nevertheless, gastric bypass is most likely the most efficient procedure for reducing body weight and treating T2D. Interestingly, several reports have shown that the gut microbiota is profoundly affected by the procedure. It has been suggested that the consistent postoperative increase in certain bacterial groups such as Proteobacteria, Bacteroidetes and Verrucomicrobia (A. muciniphila) may explain its beneficial impact in gnotobiotic mice. Taken together, these data suggest that specific gut microbes modulate important host biological systems that contribute to the control of energy homoeostasis, glucose metabolism and inflammation in obesity and T2D.     

Synergy between bacterial infection and genetic predisposition in intestinal dysplasia

Accumulating evidence suggests that hyperproliferating intestinal stem cells (SCs) and progenitors drive cancer initiation, maintenance, and metastasis. In addition, chronic inflammation and infection have been increasingly recognized for their roles in cancer. Nevertheless, the mechanisms by which bacterial infections can initiate SC-mediated tumorigenesis remain elusive. Using a Drosophila model of gut pathogenesis, we show that intestinal infection with Pseudomonas aeruginosa, a human opportunistic bacterial pathogen, activates the c-Jun N-terminal kinase (JNK) pathway, a hallmark of the host stress response. This, in turn, causes apoptosis of enterocytes, the largest class of differentiated intestinal cells, and promotes a dramatic proliferation of SCs and progenitors that serves as a homeostatic compensatory mechanism to replenish the apoptotic enterocytes. However, we find that this homeostatic mechanism can lead to massive over-proliferation of intestinal cells when infection occurs in animals with a latent oncogenic form of the Ras1 oncogene. The affected intestines develop excess layers of cells with altered apicobasal polarity reminiscent of dysplasia, suggesting that infection can directly synergize with the genetic background in predisposed individuals to initiate SC-mediated tumorigenesis. Our results provide a framework for the study of intestinal bacterial infections and their effects on undifferentiated and mature enteric epithelial cells in the initial stages of intestinal cancer. Assessment of progenitor cell responses to pathogenic intestinal bacteria could provide a measure of predisposition for apoptotic enterocyte-assisted intestinal dysplasias in humans.     

Outer membrane vesicles blebbing contributes to B. vulgatus mpk-mediated immune response silencing

The Gram negative intestinal symbiont Bacteroides vulgatus mpk is able to prevent from induction of colonic inflammation in Rag1^?/? mice and promotes immune balance in Il2^?/? mice. These inflammation-silencing effects are associated with B. vulgatus mpk-mediated induction of semi-mature dendritic cells, especially in the colonic lamina propria (cLP). However the beneficial interaction of bacteria with host immune cells is limited due to the existence of a large mucus layer covering the intestinal epithelium. How can intestinal bacteria overcome this physical barrier and contact the host immune system?

One mechanism is the production of outer membrane vesicles (OMVs) via ubiquitous blebbing of the outer membrane. These proteoliposomes have the ability to traverse the mucus layer. Hence, OMVs play an important role in immunomodulation and the maintenance of a balanced gut microbiota. Here we demonstrate that the stimulation of bone marrow derived dendritic cells (BMDCs) with isolated OMVs originated from B. vulgatus mpk leads to the induction of a tolerant semi-mature phenotype. Thereby, microbe- associated molecular patterns (MAMPs) delivered by OMVs are crucial for the interaction and the resulting maturation of immune cells. Additional to the binding to host TLR4, a yet unknown ligand to TLR2 is indispensable for the conversion of immature BMDCs into a semi-mature state. Thus, crossing the epithelial mucus layer and directly contact host cells, OMV mediate cross-tolerance via the transport of various Toll-like receptor antigens. These features make OMVs to a key attribute of B. vulgatus mpk for a vigorous acellular prevention and treatment of systemic diseases.     

Activation of the abundant nuclear factor poly(ADP-ribose) polymerase-1 by Helicobacter pylori

Modification of eukaryotic proteins is a powerful strategy used by pathogenic bacteria to modulate host cells during infection. Previously, we demonstrated that Helicobacter pylori modify an unidentified protein within mammalian cell lysates in a manner consistent with the action of a bacterial ADP-ribosylating toxin. Here, we identified the modified eukaryotic factor as the abundant nuclear factor poly(ADP-ribose) polymerase-1 (PARP-1), which is important in the pathologies of several disease states typically associated with chronic H. pylori infection. However, rather than being ADP-ribosylated by an H. pylori toxin, the intrinsic poly(ADP-ribosyl) polymerase activity of PARP-1 is activated by a heat- and protease-sensitive H. pylori factor, resulting in automodification of PARP-1 with polymers of poly(ADP-ribose) (PAR). Moreover, during infection of gastric epithelial cells, H. pylori induce intracellular PAR-production by a PARP-1-dependent mechanism. Activation of PARP-1 by a pathogenic bacterium represents a previously unrecognized strategy for modulating host cell signaling during infection.     

A surprising sweetener from enteropathogenic Escherichia coli

Infections with enteropathogenic Escherichia coli (EPEC) are remarkably devoid of gut inflammation and necrotic damage compared to infections caused by invasive pathogens such as Salmonella and Shigella. Recently, we observed that EPEC blocks cell death using the type III secretion system (T3SS) effector NleB. NleB mediated post-translational modification of death domain containing adaptor proteins by the covalent attachment of N-acetylglucosamine (GlcNAc) to a conserved arginine in the death domain.  N-linked glycosylation of arginine has not previously been reported in mammalian cell biology and the precise biochemistry of this modification is not yet defined. Although the addition of a single GlcNAc to arginine is a seemingly slight alteration, the impact of NleB is considerable as arginine in this location is critical for death domain interactions and death receptor induced apoptosis. Hence, by blocking cell death, NleB promotes enterocyte survival and thereby prolongs EPEC attachment to the gut epithelium.     

Increased expression of HIP/PAP and regenerating gene III in human inflammatory bowel disease and a murine bacterial reconstitution model

Although microorganisms play a role in gut inflammation, it remains uncertain which epithelial genes are expressed in response to luminal flora and whether these molecules are also involved in pathologic mucosal inflammation. Germ-free mice were orally challenged with a bacterial suspension prepared from conventionally housed mice (bacterial reconstitution). Thereafter, the differential gene expression in gut epithelial cells was identified by differential display. The expression of the identified genes was also examined in dextran sulfate sodium (DSS)-induced colitis and human inflammatory bowel disease (IBD) epithelial cells. Regenerating gene III (Reg III) was strongly induced in gut epithelial cells following bacterial reconstitution, as well as in the colitis initiated by DSS. The mRNA expression of hepatocarcinoma-intestine-pancreas/pancreatic associated protein (HIP/PAP), a human counterpart of Reg III, was enhanced in colonic epithelial cells of patients with IBD. Reg III mRNA expression was localized in the epithelial cells including goblet cells and columnar cells in mice; on the other hand, HIP/PAP-expressing cells were correlated with Paneth cell metaplasia in human colon. Epithelial expression of Reg III or HIP/PAP was induced under mucosal inflammation initiated by exposure to commensal bacteria or DSS as well as inflamed IBD colon.     

Modulation of immunity and inflammatory gene expression in the gut,in inflammatory diseases of the gut and in the liver by probiotics

The potential for the positive manipulation of the gut microbiome through the introduction of beneficial microbes, as also known as probiotics, is currently an active area of investigation. The FAO/WHO define probiotics as live microorganisms that confer a health benefit to the host when administered in adequate amounts. However, dead bacteria and bacterial molecular components may also exhibit probiotic properties. The results of clinical studies have demonstrated the clinical potential of probiotics in many pathologies, such as allergic diseases, diarrhea, inflammatory bowel disease and viral infection. Several mechanisms have been proposed to explain the beneficial effects of probiotics, most of which involve gene expression regulation in specific tissues, particularly the intestine and liver. Therefore, the modulation of gene expression mediated by probiotics is an important issue that warrants further investigation. In the present paper, we performed a systematic review of the probiotic-mediated modulation of gene expression that is associated with the immune system and inflammation. Between January 1990 to February 2014, Pub Med was searched for articles that were published in English using the Me SH terms “probiotics " and " gene expression " combined with “intestines", "liver", "enterocytes", "antigen-presenting cells", "dendritic cells", "immune system", and "inflammation". Two hundred and five original articles matching these criteria were initially selected, although only those articles that included specific gene expression results(77) were later considered for this review and separated into three major topics: the regulation of immunity and inflammatory gene expression in the gut, in inflammatory diseases of the gut and in the liver. Particular strains of Bifidobacteria, Lactobacilli, Escherichia coli, Propionibacterium, Bacillus and Saccharomyces influence the gene expression of mucins, Toll-like receptors, caspases, nuclear factor-κB, and interleukins and lead mainly to an anti-inflammatory response in cultured enterocytes. In addition, the interaction of commensal bacteria and probiotics with the surface of antigenpresenting cells in vitro results in the downregulation of pro-inflammatory genes that are linked to inflammatory signaling pathways, whereas other anti-inflammatory genes are upregulated. The effects of probiotics have been extensively investigated in animal models ranging from fish to mice, rats and piglets. These bacteria induce a tolerogenic and hyporesponsive immune response in which many genes that are related to the immune system, in particular those genes expressing anti-inflammatory cytokines, are upregulated. By contrast, information related to gene expression in human intestinal cells mediated by the action of probiotics is scarce. There is a need for further clinical studies that evaluate the mechanism of action of probiotics both in healthy humans and in patients with chronic diseases. These types of clinical studies are necessary for addressing the influence of these microorganisms in gene expression for different pathways, particularly thosethat are associated with the immune response,and to better understand the role that probiotics might have in the prevention and treatment of disease.     

Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity

Obesity and type 2 diabetes are characterized by altered gut microbiota, inflammation, and gut barrier disruption. Microbial composition and the mechanisms of interaction with the host that affect gut barrier function during obesity and type 2 diabetes have not been elucidated. We recently isolated Akkermansia muciniphila, which is a mucin-degrading bacterium that resides in the mucus layer. The presence of this bacterium inversely correlates with body weight in rodents and humans. However, the precise physiological roles played by this bacterium during obesity and metabolic disorders are unknown. This study demonstrated that the abundance of A. muciniphila decreased in obese and type 2 diabetic mice. We also observed that prebiotic feeding normalized A. muciniphila abundance, which correlated with an improved metabolic profile. In addition, we demonstrated that A. muciniphila treatment reversed high-fat diet-induced metabolic disorders, including fat-mass gain, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance. A. muciniphila administration increased the intestinal levels of endocannabinoids that control inflammation, the gut barrier, and gut peptide secretion. Finally, we demonstrated that all these effects required viable A. muciniphila because treatment with heat-killed cells did not improve the metabolic profile or the mucus layer thickness. In summary, this study provides substantial insight into the intricate mechanisms of bacterial (i.e., A. muciniphila) regulation of the cross-talk between the host and gut microbiota. These results also provide a rationale for the development of a treatment that uses this human mucus colonizer for the prevention or treatment of obesity and its associated metabolic disorders.