The Structure of Maltooctaose-Bound Escherichia coli Branching Enzyme Suggests a Mechanism for Donor Chain Specificity.

Glycogen is the primary storage polysaccharide in bacteria and animals. It is a glucose polymer linked by α-1,4 glucose linkages and branched via α-1,6-linkages, with the latter reaction catalyzed by branching enzymes. Both the length and dispensation of these branches are critical in defining the structure, density, and relative bioavailability of the storage polysaccharide. Key to this is the specificity of branching enzymes because they define branch length. Herein, we report the crystal structure of the maltooctaose-bound branching enzyme from the enterobacteria E. coli. The structure identifies three new malto-oligosaccharide binding sites and confirms oligosaccharide binding in seven others, bringing the total number of oligosaccharide binding sites to twelve. In addition, the structure shows distinctly different binding in previously identified site I, with a substantially longer glucan chain ordered in the binding site. Using the donor oligosaccharide chain-bound Cyanothece branching enzyme structure as a guide, binding site I was identified as the likely binding surface for the extended donor chains that the E. coli branching enzyme is known to transfer. Furthermore, the structure suggests that analogous loops in branching enzymes from a diversity of organisms are responsible for branch chain length specificity. Together, these results suggest a possible mechanism for transfer chain specificity involving some of these surface binding sites.

Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins.

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

Enterococcal bacteremia in mice is prevented by oral administration of probiotic Bacillus spores.

Whether and how probiotics promote human health is a controversial issue. Their claimed benefit for counteracting gastrointestinal infection is linked predominantly to reducing pathogen abundance within the intestinal microbiota. Less understood mechanistically is the reported value that probiotics could have in reducing systemic infections. Enterococcus faecalis is an opportunistic pathogen that causes systemic infection after translocation through the intestinal epithelium, particularly in hospitalized and immune-depleted patients receiving antibiotic therapy. In this study, we used an E. faecalis mouse infection model with wild-type and isogenic mutant strains deficient in genes of the E. faecalis Fsr (fecal streptococci regulator) quorum-sensing system. We show that E. faecalis translocation from the mouse gut into the blood is mediated by the Fsr quorum-sensing system through production of the protease GelE, which compromises intestinal epithelium integrity. Furthermore, we demonstrate that orally administered probiotic Bacillus subtilis spores blocked E. faecalis translocation from the gut to the bloodstream and subsequent systemic infection in mice by inhibiting Fsr activity. These findings demonstrate that a key aspect of Enterococcus pathogenesis is controlled by quorum sensing, which can be targeted with probiotic Bacillus spores.

Alternative approaches to treat bacterial infections: targeting quorum-sensing.

Introduction: The emergence of multi- and pan-drug-resistant bacteria represents a global crisis that calls for the development of alternative anti-infective strategies. These comprise anti-virulence approaches, which target pathogenicity without exerting a bacteriostatic or bactericidal effect and are claimed to reduce the development of resistance. Because in many pathogens, quorum-sensing (QS) systems control the expression of virulence factors, interference with QS, or quorum-quenching, is often proposed as a strategy with a broad anti-virulence effect.Areas covered: We discuss the role and regulatory targets of QS control in selected Gram-positive and Gram-negative bacteria, focusing on those with clinical importance and QS control of virulence. We present the components of QS systems that form possible targets for the development of anti-virulence drugs and discuss recent research on quorum-quenching approaches to control bacterial infection.Expert opinion: While there has been extensive research on QS systems and quorum-quenching approaches, there is a paucity of in-vivo research using adequate animal models to substantiate applicability. In-vivo research on QS blockers needs to be intensified and optimized to use clinically relevant setups, in order to underscore that such drugs can be used effectively to overcome problems associated with the treatment of severe infections by antibiotic-resistant pathogens.

Chlamydia trachomatis Lipopolysaccharide Evades the Canonical and Noncanonical Inflammatory Pathways To Subvert Innate Immunity.

Chlamydia trachomatis is the most common bacterial cause of sexually transmitted infections. C. trachomatis sexually transmitted infections are commonly asymptomatic, implying a pathogenic strategy for the evasion of innate inflammatory immune responses, a paradox as the C. trachomatis outer membrane contains lipopolysaccharide (LPS), a known potent agonist of inflammatory innate immunity. Here, we studied the ability of chlamydial LPS to activate the proinflammatory canonical and noncanonical inflammasome pathways in mouse bone marrow-derived macrophages (BMDM). We show, in comparison to Escherichiacoli LPS, that C. trachomatis LPS-treated BMDM produce significantly less IL-6, TNF, and type I interferon mRNA, indicating that downstream signaling through the canonical TLR4 myddosome and triffosome pathways was blocked. We confirmed this in C. trachomatis LPS-treated BMDM by showing the lack of NF-κB and IRF3 phosphorylation, respectively. Interestingly, C. trachomatis LPS bound CD14 and promoted its endocytosis; however; it did not promote efficient TLR4/MD-2 dimerization or endocytosis, known requirements for myddosome and triffosome signaling pathways. We further found that transfection of BMDM with C. trachomatis LPS did not cause pyroptotic cell ballooning, cytotoxicity, or IL-1ß secretion, all characteristic features of noncanonical inflammasome activation. Western blotting confirmed that cytosolic C. trachomatis LPS failed to signal through caspase-11, as shown by the lack of gasdermin D, caspase-1, or IL-1ß proteolytic cleavage. We propose that chlamydiae evolved a unique LPS structure as a pathogenic strategy to avoid canonical and noncanonical innate immune signaling and conclude that this strategy might explain the high incidence of asymptomatic infections.IMPORTANCEChlamydia trachomatis is the most common bacterial cause of sexually transmitted infections (STI). C. trachomatis STI are commonly asymptomatic, implying a pathogenic strategy for the evasion of innate inflammatory immune responses, a paradox as the C. trachomatis outer membrane contains lipopolysaccharide (LPS), a known potent agonist of inflammatory innate immunity. Here, we found that C. trachomatis LPS is not capable of engaging the canonical TLR4/MD-2 or noncanonical caspase-11 inflammatory pathways. The inability of C. trachomatis LPS to trigger innate immunity inflammatory pathways is related to its unique fatty acid structure. Evolutionary modification of the LPS structure likely evolved as a pathogenic strategy to silence innate host defense mechanisms. The findings might explain the high incidence of asymptomatic chlamydial genital infection.

Pathogen elimination by probiotic Bacillus via signalling interference.

Probiotic nutrition is frequently claimed to improve human health. In particular, live probiotic bacteria obtained with food are thought to reduce intestinal colonization by pathogens, and thus to reduce susceptibility to infection. However, the mechanisms that underlie these effects remain poorly understood. Here we report that the consumption of probiotic Bacillus bacteria comprehensively abolished colonization by the dangerous pathogen Staphylococcus aureus in a rural Thai population. We show that a widespread class of Bacillus lipopeptides, the fengycins, eliminates S. aureus by inhibiting S. aureus quorum sensing-a process through which bacteria respond to their population density by altering gene regulation. Our study presents a detailed molecular mechanism that underlines the importance of probiotic nutrition in reducing infectious disease. We also provide evidence that supports the biological significance of probiotic bacterial interference in humans, and show that such interference can be achieved by blocking a pathogen's signalling system. Furthermore, our findings suggest a probiotic-based method for S. aureus decolonization and new ways to fight S. aureus infections.

Crystal Structures of Escherichia coli Branching Enzyme in Complex with Linear Oligosaccharides.

Branching enzyme is responsible for all branching of glycogen and starch. It is an unusual member of the α-amylase family because it has both α-1,4-amylase activity and α-1,6-transferase activity [Drummond, G. S., et al. (1972) Eur. J. Biochem. 26, 168-176]. It also does not react with shorter glucans, though it will bind much longer substrates and substrate mimics [Binderup, K., et al. (2002) Arch. Biochem. Biophys. 397, 279-285]. In an effort to better understand how branching enzyme interacts with its polymeric substrate, we have determined the structure of Δ112 Escherichia coli branching enzyme bound to maltoheptaose and maltohexaose. Together, these structures define six distinct oligosaccharide binding sites on the surface of E. coli branching enzyme. Most of these binding sites surround the edge of the ß-barrel domain and are quite far from the active site. Surprisingly, there is no evidence of oligosaccharide binding in the active site of the enzyme. The closest bound oligosaccharide resides almost 18 Å from the active site. Mutations to conserved residues in binding sites I and VI had a debilitating effect on the activity of the enzyme.