Lora V. Hooper

Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells

The adult mouse intestine contains an intricate vascular network. The factors that control development of this network are poorly understood. Quantitative three-dimensional imaging studies revealed that a plexus of branched interconnected vessels developed in small intestinal villi during the period of postnatal development that coincides with assembly of a complex society of indigenous gut microorganisms (microbiota). To investigate the impact of this environmental transition on vascular development, we compared the capillary networks of germ-free mice with those of ex-germ-free animals colonized during or after completion of postnatal gut development. Adult germ-free mice had arrested capillary network formation. The developmental program can be restarted and completed within 10 days after colonization with a complete microbiota harvested from conventionally raised mice, or with Bacteroides thetaiotaomicron, a prominent inhabitant of the normal mouse/human gut. Paneth cells in the intestinal epithelium secrete antibacterial peptides that affect luminal microbial ecology. Comparisons of germ-free and B. thetaiotaomicron-colonized transgenic mice lacking Paneth cells established that microbial regulation of angiogenesis depends on this lineage. These findings reveal a previously unappreciated mechanism of postnatal animal development, where microbes colonizing a mucosal surface are assigned responsibility for regulating elaboration of the underlying microvasculature by signaling through a bacteria-sensing epithelial cell.

Angiogenins: a new class of microbicidal proteins involved in innate immunity

Although angiogenins have been implicated in tumor-associated angiogenesis, their normal physiologic function remains unclear. We show that a previously uncharacterized angiogenin, Ang4, is produced by mouse Paneth cells, is secreted into the gut lumen and has bactericidal activity against intestinal microbes. Ang4 expression is induced by Bacteroides thetaiotaomicron, a predominant member of the gut microflora, revealing a mechanism whereby intestinal commensal bacteria influence gut microbial ecology and shape innate immunity. Furthermore, mouse Ang1 and human angiogenin, circulating proteins induced during inflammation, exhibit microbicidal activity against systemic bacterial and fungal pathogens, suggesting that they contribute to systemic responses to infection. These results establish angiogenins as a family of endogenous antimicrobial proteins.

Host–microbial symbiosis in the mammalian intestine: exploring an internal ecosystem

The mammalian intestine contains a complex, dynamic, and spatially diversified society of nonpathogenic bacteria. Very little is known about the factors that help establish host-microbial symbiosis in this open ecosystem. By introducing single genetically manipulatable components of the microflora into germfree mice, simplified model systems have been created that will allow conversations between host and microbe to be heard and understood. Other paradigms of host-microbial symbiosis suggest that these interactions will involve an exchange of biochemical signals between host and symbionts as well as among the bacteria themselves. The integration of molecular microbiology, cell biology, and gnotobiology should provide new insights about how we adapt to a microbial world and reveal the roles played by our indigenous, nonpathogenic flora.

Oligosaccharides containing β1,4-linked N-acetylgalactosamine, a paradigm for protein-specific glycosylation

The carbohydrate moieties found on glycoproteins have long been recognized as having great potential to bear biologically important information. However, actual examples of systems in which oligosaccharides play defined physiological roles have remained limited. These oligosaccharides with known biologic functions typically have distinctive structural features and are generally confined to specific glycoproteins. Synthesis of structurally unique oligosaccharides on specific glycoproteins at defined times is essential if these structures are to fulfill their biologic purpose. Since cells produce many distinct oligosaccharides as newly synthesized glycoproteins pass through the endoplasmic reticulum and the Golgi, mechanisms are required to assure that the correct structures are added to the numerous glycoproteins being synthesized. Determining how synthesis of the vast array of oligosaccharides produced by each cell is regulated is essential for understanding the biologic importance of these complex structures. Asn-linked oligosaccharides arise by processing of a common precursor structure, which is transferred en bloc from dolichol to the nascent peptide chain in the endoplasmic reticulum (1). As a result Asn-linked oligosaccharides have a common core region and differ primarily in the number and location of their peripheral branches and terminal modifications. Since all newly synthesized glycoproteins pass through the same subcellular compartments and are exposed to the same transferases, structural differences in oligosaccharides on individual glycoproteins and/or at individual glycosylation sites must in some fashion reflect the influence of the protein moiety on one or more glycosyltransferases. This suggests that key glycosyltransferases recognize features encoded within the peptide as well as the oligosaccharide of the target glycoprotein. Among the three glycosyltransferases thus far demonstrated to display peptide as well as oligosaccharide recognition, UDP-glucose:glycoprotein glucosyltransferase, UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, and UDP-GalNAc:glycoprotein hormone b1,4-N-acetylgalactosaminyltransferase (b1,4-GalNAcT, reviewed in Ref. 2), one of the most extensively characterized is the b1,4-GalNAcT, which produces the terminal sequence GalNAcb1,4GlcNAcb1-R on glycoproteins that contain a specific peptide recognition determinant in addition to an appropriate oligosaccharide acceptor. The product of the b1,4-GalNAcTmay be further modified by the addition of sulfate, sialic acid, or fucose, thus producing a range of unique oligosaccharide structures defined by the presence of b1,4-linked GalNAc as illustrated in Fig. 1. Each of these structures has the potential to be recognized by a specific receptor or binding protein and thus mediate a distinct biological function. As will become apparent below, the b1,4-GalNAcT is a key component of a well characterized system, which includes unique oligosaccharide structures, highly specific glycosyltransferases, and oligosaccharide-specific receptors. This is therefore an excellent model system for understanding proteinspecific glycosylation.

From legumes to leukocytes: biological roles for sulfated carbohydrates.

Carbohydrates attached to proteins and lipids characteristically display complex and heterogeneous structures. However, it is becoming increasingly clear that carbohydrates with definite biological functions also exhibit unique structural features. A number of glycoproteins and glycolipids have been shown to bear oligosaccharides containing sulfate. Often, addition of a sulfate moiety turns a relatively common structural motif into a unique carbohydrate with the potential to be recognized by a specific receptor or lectin. This is clearly the case in three systems in which sulfated oligosaccharides have been shown to play a well‐defined biological role: 1) control of the circulatory half‐life of luteinizing hormone, 2) symbiotic interactions between leguminous plants and nitrogen‐fixing bacteria, and 3) homing of lymphocytes to lymph nodes. The rapidly growing list of glycoproteins and glycolipids identified as bearing sulfated oligosaccharides suggests that sulfated carbohydrates play important biological roles in numerous other systems as well.—Hooper, L. V., Manzells, S. M., Baenziger, J. U. From legumes to leukocytes: biological roles for sulfated carbohydrates. FASEB J. 10, 1137‐1146 (1996)

Analyzing the molecular foundations of commensalism in the mouse intestine.

We maintain complex societies of nonpathogenic microbes on our mucosal surfaces. Although the stability of this flora is important for human health, very little is known about how its constituents communicate with us to forge stable and mutually advantageous relationships. The vast majority of these indigenous microbes reside in the intestine. Recent studies of a gut commensal, Bacteroides thetaiotaomicron, has revealed a novel signaling pathway that allows the microbe and host to actively collaborate to produce a nutrient foundation that can be used by this bacterium. This pathway illustrates the type of dynamic molecular interactions that help define commensal relationships.

29 Combining gnotobiotic mouse models with functional genomics to define the impact of the microflora on host physiology

Publisher Summary This chapter describes the combining gnotobiotic mouse models with functional genomics to define the impact of the microflora on host physiology. Determining the effects of commensals on host biology has been challenging. Because host-microbial relationships are generally characterized by dynamic and reciprocal interactions among bacteria, host cells, and components of mucosal immune systems, cultured cells may not accurately portray in vivo responses to commensals. However, the fusion of several recently developed technologies with other more established methods has made it possible to use in vivo models to define the effects of specific components of the microflora on host gene expression, in quantitative terms, with a high degree of cellular resolution. First, gnotobiotic techniques and involving the use of germ-free mice, allow the impact of colonization by a single bacterial species to be monitored in a genetically defined host. Second, high-density Deoxyribo-nucleic acid (DNA) microarrays provide a means for conducting comprehensive and relatively unbiased assessments of host responses to microbes. Third, laser capture microdissection (LCM) can be used to recover specified cell populations from complex tissues prior to and following colonization of gnotobiotic mice. Fourth, quantitative real-time RT-PCR allows quantitative measurements of colonization-associated changes in the levels of specific mRNAs within microdissected cells.

Clinical Microbiology in the Year 2025

This article offers a mini-preview of whats to come in the field of clinical microbiology, and its the first such undertaking that any of the authors has ever attempted without tongue firmly planted in cheek. Obviously, the scenario portrayed herein is an exercise in pure fantasy, based loosely on the evolutionary pace of clinical microbiology witnessed over the past 25 years. Unlike readers of Sports Illustrateds yearly predictions of champions and losers, however, the reader will have to wait longer than a single season to prove us right or wrong (23 years to be exact). Had the task been the opposite—i.e., to reminisce about the state of clinical microbiology a quarter century ago—we would have discussed the introduction of the Analytab Products bacterial identification system or the first-generation AutoMicrobic system (AMS, Vitek Systems, Inc.) originally designed for use in the U.S. space program. We could generate a smirk by recollecting that the role of the clinical microbiologist in the mid-1970s was to identify all microbial life forms recovered from clinical specimens and then to provide susceptibility test results for each by disk diffusion testing. It was the clinician who would then sort through the myriad of results and decide which organism(s) deserved a therapeutic response. We could prompt a grimace or two by recalling that blood cultures were monitored visually for evidence of bacterial growth once or twice per day and that all anaerobes were identified to species level no matter how much time or how many biochemical reactions were required to do the trick. Interestingly, the two aspects of clinical microbiology that havent changed much since the mid-1970s are the identification of fungal diseases and the identification of parasitic diseases. n nAs a discipline and profession, clinical microbiology has “come a long way baby” (Virginia Slims, circa 1970), but we have just begun the molecular diagnostics learning curve, and its hard to predict just how far we can take this new tool from a technical, practical, and economic standpoint. Because of the recent explosion of technology, many clinical microbiologists have openly speculated that ours is a dying profession—one that will ultimately be consumed by the growing molecular diagnostic beast. With that in mind, heres a mythical take on the future of the clinical microbiology laboratory. One additional note: this might represent the only publication in the history of the Journal of Clinical Microbiology that contains no references. Why is this? The references that would have been cited havent been written yet!

Response from Jeffrey I. Gordon et al.: Commensal bacteria make a difference

The importance of the gut microbiota has been recognized since the days of Pasteur. What makes today different from yesterday, and tomorrow so exciting, is that we now have the tools to identify the molecular mechanisms that regulate assembly of the microbiota and determine how its components affect postnatal mammalian development and adult physiology.