382 research outputs found
Immune adaptations that maintain homeostasis with the intestinal microbiota
No full textHumans harbour nearly 100 trillion intestinal bacteria that are essential for health. Millions of years of co-evolution have moulded this human-microorganism interaction into a symbiotic relationship in which gut bacteria make essential contributions to human nutrient metabolism and in return occupy a nutrient-rich environment. Although intestinal microorganisms carry out essential functions for their hosts, they pose a constant threat of invasion owing to their sheer numbers and the large intestinal surface area. In this Review, we discuss the unique adaptations of the intestinal immune system that maintain homeostatic interactions with a diverse resident microbiota
The Ligand and Function of the RegIII Family of Bactericidal C-Type Lectins
No full textBeginning at birth, the intestines of humans and other mammals are colonized with a diverse society of resident bacteria that play a crucial role in host nutrient metabolism. To maintain this commensal relationship, resident microbes must be prevented from crossing the intestinal epithelium into host tissues where they can cause inflammation and sepsis. The innate immune system plays a crucial role in preventing bacterial incursions across gut epithelial surfaces. Mucosal epithelial cells produce a variety of secreted antimicrobial proteins that help to prevent bacterial attachment and encroachment at epithelial surfaces. Among these, Paneth cells are specialized small intestinal epithelial cells that have been shown to produce and secrete antimicrobial proteins and peptides. To gain new insights into the adaptation of mucosal surfaces to microbial challenges, the Hooper lab has used DNA microarrays to screen for Paneth cell genes whose expression is modulated by intestinal microbes. This screen revealed that expression of two C-type lectins, RegIIIbeta and RegIIIgamma , is strongly induced following intestinal colonization with resident microbes. Two features suggested that members of the RegIII family may have microbicidal functions. First, they are C-type lectin family members. Other C-type lectins, including the mannose binding lectin, have well-characterized innate immune functions and play critical roles in microbial killing by recruiting complement. Second, I have shown that the murine RegIII lectins localize to intestinal crypt cells, including Paneth cell secretory granules, and that they bind to luminal bacteria harvested from intestinal conditions. Based on these observations, we hypothesized that this family of proteins may perform an innate immune function, specifically antimicrobial defense. The studies reported in this thesis characterize a family of C-type lectins. Specifically, we determined that these proteins interact with peptidoglycan by binding with high affinity to its glycan structure, representing a unique blend of peptidoglycan recognition and lectin function. Additionally, we have demonstrated that this binding results in the specific disruption of the Gram positive bacterial cell wall, where peptidoglycan is exposed, which is the first example of a family of directly bactericidal C-type lectins. We also present evidence for the regulation of these bactericidal proteins by colonization with an intestinal microflora. Therefore, the research presented in this thesis elucidates the function of three members of the RegIII family, in both mice and humans
A Novel Role for γδ Intraepithelial Lymphocytes in Antibacterial Defense of the Intestine
No full textThe mammalian intestine has coevolved with a highly complex population of enteric bacteria. For the most part, mammals and their intestinal microbiota maintain a mutually beneficial relationship. However, the symbiotic nature of this relationship depends on strict sequestration of intestinal microbes in the gut lumen, and damage to intestinal surfaces by chemical agents or microbial pathogens poses a serious threat of inflammation and sepsis. Therefore, the cells populating the intestinal epithelium have evolved strategies to maintain the integrity of the intestinal epithelium and to limit bacterial invasion. Gamma delta intraepithelial lymphocytes (gamma delta IEL) are unconventional T cells that intercalate under epithelial tight junctions of the intestine. While gamma delta IEL are numerically the most abundant T cell population in the body, their biology in intestinal tissues has remained obscure. The work in this thesis seeks to understand the role of gamma delta IEL in maintaining homeostasis with symbiotic intestinal microbes and in protecting against bacterial pathogens. My findings disclose that intestinal bacteria provide critical regulatory input to gamma delta IEL in the small and large intestine, and direct the production of proinflammatory and antibacterial factors in gamma delta IEL. Additionally, my in vivo studies disclose a novel role for delta gamma IEL in antibacterial defense of the intestine, revealing that gamma delta IEL protect the mucosal barrier in two general ways. First, gamma delta IEL protect against opportunistically invading commensals immediately after mucosal damage. Next, they also function to limit dissemination of invasive bacterial pathogens. My work suggests that a unique feature of gamma delta IEL relative to other intestinal immune cells is their early role in providing protection against invading bacteria immediately after challenge. Taken together, these findings disclose that gamma delta IEL participate in multifaceted antibacterial responses to promote beneficial host-microbial relationships in the intestine
Molecular Basis of Peptidoglycan Recognition by a Bactericidal Gut Lectin
No full textThe mammalian gut is densely populated by varied microbial species. This relationship is mutually beneficial as long as bacteria remain corralled in the gut lumen. The epithelium is protected by the secretion of antimicrobial proteins by specialized epithelial cells in the intestinal crypts. This molecular arsenal includes the RegIII family. RegIII proteins are novel in that they are C-type lectins that directly kill Gram-positive bacteria and thus play a vital role in antimicrobial protection of the mammalian gut. RegIII proteins bind their bacterial targets via interactions with cell wall peptidoglycan, but lack the canonical sequences that support calcium-dependent carbohydrate binding in other C-type lectins. Given these novel functions and the lack of structural clues, nothing was known about the molecular mechanisms by which RegIII family members recognize and bind to peptidoglycan. Furthermore, the question of how RegIII proteins specifically recognize target microbes in the presence of soluble peptidoglycan shed by bacteria in vivo still remained.
In this dissertation, I have used NMR spectroscopy as an unbiased approach to study the molecular basis for peptidoglycan recognition by HIP/PAP, a human RegIII lectin. I have shown that HIP/PAP recognizes the peptidoglycan carbohydrate backbone, showing that ligand recognition by RegIII family members is unique compared to other peptidoglycan recognition proteins. This work also shows that HIP/PAP recognizes peptidoglycan in a calciumindependent manner via a conserved ‘EPN’ motif that is critical for bacterial killing. While EPN sequences govern calcium-dependent carbohydrate recognition in other C-type lectins, the unusual location and calcium-independent functionality of the HIP/PAP EPN motif suggest that this sequence is a versatile functional module that can support both calcium-dependent and calciumindependent carbohydrate binding. Further, these studies show that HIP/PAP binding affinity for carbohydrate ligands depends on carbohydrate chain length, supporting a binding model in which HIP/PAP molecules "bind and jump" along the extended polysaccharide chains of peptidoglycan, reducing dissociation rates and increasing binding affinity. I propose that dynamic recognition of highly multivalent carbohydrate epitopes in native peptidoglycan is an essential mechanism governing high affinity interactions between HIP/PAP and the bacterial cell wall
Interactions between the microbiota and the immune system
No full textThe large numbers of microorganisms that inhabit mammalian body surfaces have a highly coevolved relationship with the immune system. Although many of these microbes carry out functions that are critical for host physiology, they nevertheless pose the threat of breach with ensuing pathologies. The mammalian immune system plays an essential role in maintaining homeostasis with resident microbial communities, thus ensuring that the mutualistic nature of the host-microbial relationship is maintained. At the same time, resident bacteria profoundly shape mammalian immunity. Here, we review advances in our understanding of the interactions between resident microbes and the immune system and the implications of these findings for human health
You AhR What You Eat: Linking Diet and Immunity
No full textThe aryl hydrocarbon receptor (AhR) is responsible for the toxic effects of environmental pollutants such as dioxin, but little is known about its normal physiological functions. Li et al. (2011) now show that specific dietary compounds present in cruciferous vegetables act through the AhR to promote intestinal immune function, revealing AhR as a critical link between diet and immunity
Serum Amyloid A is a Retinol Binding Protein that Transports Retinol during Bacterial Infection
No full textRetinol plays a vital role in the immune response to infection, however it remains unclear which proteins mediate retinol transport during infection. Serum amyloid A (SAA) proteins are produced by the liver following acute systemic infection and are also induced by bacteria in the intestine. SAAs have been proposed to play a role in the inflammatory response to infection and injury, but their exact functions have not been well defined. In this dissertation, I present data that demonstrates the acute phase protein SAA is a novel retinol binding protein that transports retinol during infection. SAA proteins are induced by bacteria and additionally require retinol for their expression. I demonstrate that SAA’s requirement for retinol is not restricted to the small intestine, as mice on a vitamin A deficient diet have reduced SAA expression in the liver as well. Additionally, I demonstrate in fluorescence based binding assays that SAAs are capable of binding retinol at nanomolar affinities, which is comparable to a known retinol binding protein. I also found that SAA proteins associate with retinol in the serum following a bacterial challenge in wild-type mice. This phenotype was not observed in SAA1/2-/- mice following bacterial challenge. Furthermore, SAA1/2-/- mice have greater bacterial loads in their spleens and livers following bacterial infection. In parallel with my studies, Dr. Mehabaw Derebe, a post-doctoral researcher in the Hooper lab, recently solved the mSAA3 crystal structure, demonstrating the protein oligomerizes to form a tetramer. This tetramer unit contains a central pore-like cavity, lined with hydrophobic amino acid residues, which would allow a lipophilic ligand to bind. A single amino acid mutation within this hydrophobic core resulted in reduced mSAA3 retinol binding. This structural insight describes how SAA, as a small and mostly alpha-helical protein, can protect a lipophilic ligand from the aqueous environment. Altogether, these data demonstrated that SAAs are a family of microbe-induced retinol binding proteins, reveal a unique protein architecture involved in retinol binding, and provide insight into the acute response to infection
Unexpected Factors That Influence Coxsackievirus B3 Replication in Mouse Intestine
No full textCoxsackievirus is a human pathogen that frequently infect humans. Although many infections are asymptomatic, there can be severe outcomes, including heart inflammation and pancreas inflammation. Most studies with coxsackieviruses and other viruses use laboratory-adapted viral strains because of their efficient replication in cell culture. I used a cell culture-adapted strain of coxsackievirus B3 (CVB3), CVB3-Nancy, to examine viral replication and pathogenesis in orally inoculated mice. Using HeLa cell plaque assays with agar overlays, I noticed that some fecal viruses generated plaques >100 times as large as inoculum viruses. These large-plaque variants emerged following viral replication in several different tissues. I identified a single amino acid change, N63Y, in the VP3 capsid protein that was sufficient to confer the large-plaque phenotype. Wild-type CVB3 and N63Y mutant CVB3 had similar plaque sizes when agarose was used in the overlay instead of agar. I determined that sulfated glycans in agar inhibited plaque formation by wild-type CVB3 but not by N63Y mutant CVB3. Furthermore, N63Y mutant CVB3 bound heparin, a sulfated glycan, less efficiently than wild-type CVB3 did. While N63Y mutant CVB3 had a growth defect in cultured cells and reduced attachment, it had enhanced replication and pathogenesis in mice. Infection with N63Y mutant CVB3 induced more severe hepatic damage than infection with wild-type CVB3, likely because N63Y mutant CVB3 disseminates more efficiently to the liver. This work reinforce the idea that culture-adapted laboratory virus strains can have reduced fitness in vivo. N63Y mutant CVB3 may be useful as a platform to understand viral adaptation and pathogenesis in animal studies.
I also explored other factors that influence CVB3 infection in mouse intestine. First, a sex bias for severe sequelae from coxsackievirus infections has been observed in humans. We sought to examine if the phenomenon can be seen in mice and to further understand the mechanisms. Here we orally infected mice with CVB3 and confirmed that CVB3 replication in the intestine is sex-dependent. CVB3 replicated efficiently in male mice intestine, but not female mice. Overall these data suggest that sex and the immune response play an important role in CVB3 replication in the intestine and should be considered in light of the sex bias observed in human disease.
Previously, our lab has shown that intestinal microbiota promote replication and pathogenesis of several viruses, including poliovirus (PV), reovirus and CVB3. With that finding, we wanted to examine how microbiota enhance CVB3 infection. Bacteria in the colon produce millimolar quantities of butyrate and other short-chain fatty acids (SCFAs) through fermentation of dietary fiber. SCFAs are among the most abundant molecules in the distal gastrointestinal tract. To determine whether bacterial-derived SCFAs such as butyrate impact CVB3 replication in the intestine, I antibiotic treated mice and then supplied them with tributyrin, a form of butyrate that is absorbed in the distal gastrointestinal tract. I found that CVB3 replication and pathogenesis was restored in antibiotic-treated mice that received tributyrin. These results suggest that butyrate is sufficient to promote CVB3 replication. My preliminary data demonstrate that oral delivery of a histone deacetylase (HDAC) inhibitor, Vorinostat, is sufficient to restore CVB3 replication in antibiotic-treated mice, suggesting that the HDAC activity of butyrate may promote CVB3 infection.
Taken all together, I identified several unexpected factors that may influence CVB3 replication in mouse intestine although much remains open for exploration
Characterization of Host and Microbiota Derived Signals that Regulate the Locus of Enterocyte Effacement of EHEC
No full textHumans are populated by an extensive community of microorganisms, primarily in organs such as the skin, mucosal membranes in the mouth, reproductive organs, and the gut. This complex community, termed the microbiota, is in part comprised of bacteria, many of which have intimate associations with their hosts to promote physiological homeostasis. These organisms, commonly termed commensal bacteria, have a rich and long history with their human hosts and accomplish important functions such as providing the host with nutrients, developing the immune system, and preventing colonization by pathogenic organisms in a process known as colonization resistance. These functions are especially apparent within the gastrointestinal (GI) tract, which contains the richest and most densely populated community of microbes in the body. Healthy gut function relies on the proper structure and balance of this microbial community. Disruption of the community, termed dysbiosis, has been associated with a plethora of diseases such as increased susceptibility to GI infections, neurological disorders, intestinal inflammation, and cancer progression. Dysbiosis is most commonly caused by pharmacological interventions with antibiotics or infection with a GI pathogen.
The microbiota is regarded as a barrier against intestinal pathogens, partly due to intense competition for a limited supply of nutrients and space. This suggests that GI pathogens have evolved mechanisms to overcome colonization resistance and outcompete the resident microbiota for resources within the GI tract. Microbiota and host-derived metabolites have a significant impact on the abilities of GI pathogens to successfully establish intestinal infection and the subsequent development of disease. However, the precise mechanism by which microbiota or host metabolites affect the pathogenesis of GI pathogens is not well understood. Many of these nutrients, whether host-, diet-, or microbiota-derived, serve as chemical cues for incoming pathogens. These signals are used by pathogens to gauge resource availability, microbiota composition, host physiology, and location within the intestines to properly deploy virulence strategies that allow for colonization. Microbiota-derived small molecules include toxins, antimicrobials, oligopeptides, hormones, and products of microbial metabolism of host-derived and dietary molecules. Pathogens can directly sense many of these host- and microbiota-derived small molecules, which in turn can regulate their virulence mechanisms. Taken together, developing therapeutics that target the signaling pathways that control virulence-associated functions in pathogens represent an attractive alternative or secondary strategy to tackle bacterial infections.
In a previous study, our group conducted a candidate-based screen of 372 independent mutants to look for novel regulators of the T3SS [1]. The candidates of this screen consisted primarily of transcription factors, two-component regulatory systems, anti-terminators and anti-toxins. This work generated a great number of hits that potentially regulate the T3SS of EHEC. Our work sought to characterize novel signaling pathways that directly affect the virulence of enterohemorrhagic Escherichia coli (EHEC) through characterization of some of the hits of said screen in particular the transcriptional regulators ExuR and FadR. Understanding of these signaling pathway could lead us to develop novel strategies to drive down the virulence of enteric pathogens and improve colonization resistance as an alternative approach to control bacterial infections.
Here, we found that EHEC senses and utilizes galacturonic-acid (GalA) as a nutrient during infection and moonlights as a signal to downregulate the expression of virulence associated genes. Furthermore, we demonstrated that a pectin-rich diet, which is a source of GalA, increased mice tolerance towards a Citrobacter rodentium infection, a surrogate mice model for EHEC infection. AE pathogens like EHEC and C. rodentium thrive in an inflamed environment. During the onset phase of inflammation, the host-derived polyunsaturated omega-6 long-chain fatty acid (LCFA), arachidonic acid (AA) becomes elevated to produce endogenous lipid signaling molecules like prostaglandins and leukotrienes that act as inducers of inflammation. EHEC can sense long-chain fatty acids through the FadR response regulator. We found that AA is processed by EHEC using canonical LCFA signaling pathways involving the FadL LCFA transporter, the FadD acyl-CoA synthase and the FadR transcriptional regulator.
In conclusion, we characterized the signaling pathways that mediate the sensing of galacturonic-acid and arachidonic-acid. We demonstrated that a diet high in pectin can effectively be used to control an infection by C. rodentium by effectively modulating the levels of GalA and affecting virulence in an ExuR dependent manner. We also showed that EHEC is capable of sensing a host produced long chain fatty acid like arachidonic acid to regulate its virulence. These studies highlight the complexity that underlies regulation of the locus of enterocyte effacement and perhaps will serve as a starting point for the development of new strategies to control enteric infections
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