Location of the heme-Fe atoms within the profile structure of a monolayer of cytochrome c bound to the surface of an ultrathin lipid multilayer film.

We have recently developed x-ray diffraction methods to derive the profile structure of ultrathin lipid multilayer films having one to five bilayers (e.g., Skita, V., W. Richardson, M. Filipkowski, A.F. Garito, and J.K. Blasie. 1987. J. Physique. 47:1849-1855). Furthermore, we have employed these techniques to determine the location of a monolayer of cytochrome c bound to the carboxyl group surface of various ultrathin lipid multilayer substrates via nonresonance x-ray diffraction (Pachence, J.M., and J.K. Blasie. 1987. Biophys. J. 52:735-747). Here an intense tunable source of x-rays (beam line X9-A at the National Synchrotron Light Source at the Brookhaven National Laboratory) was utilized to measure the resonance x-ray diffraction effect from the heme-Fe atoms within the cytochrome c molecular monolayer located on the carboxyl surface of a five monolayer arachidic acid film. Lamellar x-ray diffraction was recorded for energies above, below, and at the Fe K-absorption edge (E = 7,112 eV). An analysis of the resonance x-ray diffraction effect is presented, whereby the location of the heme-Fe atoms within the electron density profile of the cytochrome c/arachidic acid ultrathin multilayer film is indicated to +/- 3 A accuracy.     

Binding selectivity ofStreptococcus pyogenes and M-protein to epithelial cells differs from that of lipoteichoic acid

The ability of M-protein-positive (M⁺) and M-protein-negative (M^–) strains (including an M^– mutant lacking the structural gene for M-protein) ofStreptococcus pyogenes to attach to human pharyngeal, buccal, and tongue epithelial cells was compared. We observed that M⁺ strains ofS. pyogenes attached in significantly higher numbers to human pharyngeal epithelial cells than to human buccal or tongue cells. M^– strains did not exhibit high-level binding to any type of epithelial cell. Also, the adhesion of an M⁺ and an M^– strain ofS. pyogenes was low to all types of rat epithelial cells tested. The apparent differences in the surface components between human pharyngeal and buccal epithelial cells were confirmed by studies utilizing radiolabeled lectins.Ulex europaeus lectin with a specificity for fucosyl residues, andTriticum vulgaris lectin with a specificity for N-acetyl glucosamine and N-acetyl neuraminic acid residues, bound in higher amounts to human pharyngeal cells than to buccal cells. Pretreatment of pharyngeal epithelial cells with microgram quantities of highly purified type 6 M-protein or miligram quantities of lipoteichoic acid (LTA) derived fromS. pyogenes decreased the subsequent attachment of the organism. However, the binding specificities of³H-LTA were different from those of intact streptococci;³H-LTA bound comparably to human pharyngeal, buccal, and tongue epithelial cells, and it bound in higher quantities to rat epithelial cells. Also, although the adsorption ofS. pyogenes cells to pharyngeal cells was inhibited by the presence of fucose and galactose, these sugars had little effect on the binding of³H-LTA to epithelial cells. In contrast, the high adhesion of M⁺ strains but not M^– mutants to pharyngeal cells suggested that M-protein may play an important role. This possibility was supported by the observation that³H-labeled purified type 6 M-protein bound in higher concentrations to human pharyngeal epithelial cells than to human buccal cells. Furthermore, human pharyngeal epithelial cells were estimated to contain larger numbers of binding sites for M-protein than buccal cells, whereas the affinity of M-protein was similar to both cell types. These adsorption parameters are similar to those previously established for intact streptococcal cells.     

Mapping the immunodeterminants of the complete streptococcal M6 protein molecule. Identification of an immunodominant region

The immune response to the complete streptococcal M6 protein was examined by kinetic ELISA to determine the reactivity of rabbit and human sera to M6 peptides representing 82% of the native molecule. The results revealed that rabbits immunized with purified native M6 protein or whole streptococci responded by reacting early and predominantly to one of the three sequence repeat regions of the molecule, the B-repeat, antibodies which have been shown to be non-opsonic. Antibodies to peptides representing the hypervariable N-terminal and adjacent A-repeat regions appear when opsonic antibodies are detected in the serum. Antibodies to peptides located within the conserved C-terminal half of the molecule (proximal to the cell) were restricted even after several immunizations. An examination of human sera from individuals with no recent streptococcal infection (greater than 3 yr), revealed that those sera opsonic for M6 streptococci contained antibodies reactive predominantly to the N-terminal and A-repeat regions, supporting the view that opsonic antibodies are long lived. Nonopsonic human sera to M6 streptococci exhibited a low reactivity to all peptides. However, by Western blot analysis, all human sera tested contained antibodies to the conserved region of the molecule, whereas only sera opsonic for M6 streptococci reacted with the variable region. Evidence is presented supporting the view that antibodies to the conserved regions of the M molecule may be conformation dependent.     

Sequence homology of group A streptococcal Pep M5 protein with other coiled-coil proteins

Group A streptococcal Pep M5 protein, an antiphagocytic determinant of the bacteria, is an alpha-helical coiled-coil molecule, and exhibits significant sequence homology with tropomyosin and myosin, but to a lesser degree with other coiled-coil proteins. Moreover, Pep M5 is more homologous to myosin than to tropomyosin, and the homologies are more numerous between the C-terminal domain of the Pep M5 protein and the S2 fragment of myosin. The C-terminal domain of the Pep M5 protein exhibits extensive sequence identity with the C-terminal region of Pep M6 molecule, another M protein serotype. Thus, regions within two M protein serotypes are homologous to the S2 region of the myosin molecule. These observations are consistent with the immunological findings of other investigators and thus may explain some of the previously reported immunological cross-reactions between antigens of the group A streptococcus and mammalian heart tissue.     

Isolation of Clostridium perfringens Type B in an Individual at First Clinical Presentation of Multiple Sclerosis Provides Clues for Environmental Triggers of the Disease

We have isolated Clostridium perfringens type B, an epsilon toxin-secreting bacillus, from a young woman at clinical presentation of Multiple Sclerosis (MS) with actively enhancing lesions on brain MRI. This finding represents the first time that C. perfringens type B has been detected in a human. Epsilon toxin’s tropism for the blood-brain barrier (BBB) and binding to oligodendrocytes/myelin makes it a provocative candidate for nascent lesion formation in MS. We examined a well-characterized population of MS patients and healthy controls for carriage of C. perfringens toxinotypes in the gastrointestinal tract. The human commensal Clostridium perfringens type A was present in approximately 50% of healthy human controls compared to only 23% in MS patients. We examined sera and CSF obtained from two tissue banks and found that immunoreactivity to ETX is 10 times more prevalent in people with MS than in healthy controls, indicating prior exposure to ETX in the MS population. C. perfringens epsilon toxin fits mechanistically with nascent MS lesion formation since these lesions are characterized by BBB permeability and oligodendrocyte cell death in the absence of an adaptive immune infiltrate.     

Identification of chemosensory sensilla activating antennular grooming behavior in the Caribbean spiny lobster,Panulirus argus

Crustaceans such as crabs and lobsters clean or 'groom' their olfactory organ, the antennule, by wiping it through a pair of mouthpart appendages, the third maxillipeds. In the lobster, only a few chemicals found in prey extracts, especially glutamate, elicit grooming. Chemosensory input driving grooming is likely to be mediated via sensilla located on antennules and third maxillipeds. Chemosensory antennular sensilla are innervated by neurons with central projections either to the glomerular olfactory lobe (aesthetasc sensilla) or to non-glomerular antennular neuropils (nonaesthetasc sensilla). By selectively ablating the chemosensory sensilla on the antennules and the third maxillipeds we have determined that the aesthetascs are necessary and sufficient to drive grooming behavior. Chemosensory activation of antennular grooming behavior likely follows a 'labeled-line' model in that aesthetasc neurons tuned to glutamate provide adequate input via the olfactory lobe to motor centers in the brain controlling antennular movements.     

Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium

The Frank-Starling relationship of the heart has, as its molecular basis, an increase in the activation of myofibrils by calcium as the sarcomere length increases. It has been suggested that this phenomenon may be due to myofilaments moving closer together at longer lengths, thereby enhancing the probability of favorable acto-myosin interaction, resulting in increased calcium sensitivity. Accordingly, we have developed an apparatus so as to obtain accurate measurements of myocardial interfilament spacing (by synchrotron X-ray diffraction) as a function of sarcomere length (by video microscopy) over the working range of the heart, using skinned as well as intact rat trabeculas as model systems. In both these systems, lattice spacing decreased significantly as sarcomere length was increased. Furthermore, lattice spacing in the intact muscle was significantly smaller than that in the skinned muscle at all sarcomere lengths studied. These observations are consistent with the hypothesis that lattice spacing underlies length-dependent activation in the myocardium.     

Identifying Active Phage Lysins through Functional Viral Metagenomics

Recent metagenomic sequencing studies of uncultured viral populations have provided novel insights into the ecology of environmental bacteriophage. At the same time, viral metagenomes could also represent a potential source of recombinant proteins with biotechnological value. In order to identify such proteins, a novel two-step screening technique was devised for cloning phage lytic enzymes from uncultured viral DNA. This plasmid-based approach first involves a primary screen in which transformed Escherichia coli clones that demonstrate colony lysis following exposure to inducing agent are identified. This effect, which can be due to the expression of membrane-permeabilizing phage holins, is discerned by the development a hemolytic effect in surrounding blood agar. In a secondary step, the clones identified in the primary screen are overlaid with autoclaved Gram-negative bacteria (specifically Pseudomonas aeruginosa) to assay directly for recombinant expression of lytic enzymes, which are often encoded proximally to holins in phage genomes. As proof-of-principle, the method was applied to a viral metagenomic library constructed from mixed animal feces, and 26 actively expressed lytic enzymes were cloned. These proteins include both Gram-positive-like and Gram-negative-like enzymes, as well as several atypical lysins whose predicted structures are less common among known phage. Overall, this study represents one of the first functional screens of a viral metagenomic population, and it provides a general approach for characterizing lysins from uncultured phage.The field of metagenomics has expanded rapidly in recent years, providing access to environmental microorganisms that would remain unapproachable by standard, culture-based methods. The foundation of metagenomics lies in the direct extraction of DNA/RNA from environmental samples (e.g., soil, water, or feces) without prior isolation of individual microbial species (reviewed in references 18 and 32). It has been estimated that only a small proportion of naturally occurring microbes—approximately 1% of soil bacteria, for instance—are culturable under standard laboratory conditions (31). In this light, metagenomics has become an increasingly common tool for studying diverse ecosystems, from around the globe to within the human body.Overall, metagenomics research can be divided into two general categories: sequence-based and functional. In the former, environmental DNA is sequenced in mass and compared with genetic databases to address broad questions of ecology, taxonomy, and diversity. Some of the most extensive metagenomic studies to date have been sequence based in nature, benefiting from the development of high-throughput sequencing technologies. Notable examples include a 76-megabase study of an acid mine biofilm (33), a 1-gigabase analysis of the Sargasso Sea (35), and a 6.3-gigabase sampling of global oceanic samples (25). In functional metagenomics, by contrast, environmental genes are recombinantly expressed within a host organism, which is monitored for the acquisition of a desired phenotype. Rather than providing insight into entire ecosystems, functional studies aim to identify individual molecules with biomedical or industrial value. Targeted compounds may be either proteins (usually enzymes) encoded directly by environmental genes or small molecules synthesized by several enzymes of a gene cluster. Numerous classes of molecules have been identified to date, with particular interest in the areas of biosynthesis, biomass degradation, and antibiotic discovery (reviewed in references 2, 34, and 36).While bacteria provide the majority of DNA to most metagenomic pools, recent studies have begun focusing on subsets of total environmental populations. A prominent example is viral metagenomics, in which viral particles (predominately bacteriophage) are purified from cellular material prior to DNA extraction (reviewed in references 10 and 12). Although the yield of DNA from environmental phage isolates is generally low, PCR amplification techniques have been developed to overcome this issue (4, 26). Viral metagenomic analyses have been conducted on a growing number of samples, including ones purified from soil (15), seawater (4, 39), and human feces (3). These studies have revealed a remarkable abundance of novel sequences, supporting the notion that phage represent the largest source of untapped genetic diversity on the planet (19). Despite this wealth of information, viral metagenomic studies to date have remained predominantly sequence based in nature. In this regard, functional screens of viral metagenomes could provide a large source of recombinant molecules.Recently one class of phage-encoded protein has received particular attention from the biotechnology field: phage lytic enzymes (also referred to as endolysins or lysins) (reviewed in references 16 and 17). These peptidoglycan hydrolases are expressed late in the infective cycle of double-stranded DNA phage, and—along with a membrane-permeabilizing protein known as a holin—they are responsible for disrupting the bacterial cell envelope and freeing progeny viral particles. Despite this conserved biological function, phage lysins (especially Gram-positive ones) are a tremendously diverse group of proteins whose enzymatic specificity includes various bonds within the peptidoglycan macromolecule. They include glycosyl hydrolases that target the polysaccharide backbone (muramidases/lysozymes and glucosaminidases), alanine amidases that target the initial l-alanine of the pentapeptide stem, and endopeptidases that target subsequent peptide bonds in the stem or cross bridge. While lysins of Gram-negative phage generally consist of an enzymatic domain alone, Gram-positive lysins are modular and combine an N-terminal lytic domain with a C-terminal binding domain that can recognize various epitopes within the target cell envelope.Although researchers have known of lysins for decades, interest has increased markedly in recent years after it was proposed that they could act as novel anti-infective agents against Gram-positive pathogens, whose peptidoglycan is directly accessible from the extracellular space (8, 23, 28). A growing number of in vitro and in vivo studies have confirmed the ability of recombinantly expressed lysins to kill such organisms, and their appeal lies in both the potency and the specificity they demonstrate toward individual Gram-positive species. This enzybiotic value of phage lysins goes alongside additional proposed applications in the areas of food (11), agricultural (20), veterinary (7), and industrial science (21, 40).Considering this potential, lytic enzymes represent an intriguing functional target for viral metagenomic screens. At the same time, identifying lysins in this manner would present several distinct challenges. Aside from general concerns common to all functional screens (e.g., protein expression and solubility), metagenomic lysin identification would face the following particular issues. (i) Clonal toxicity: recombinant lysin expression is typically well tolerated by host bacteria, since the enzymes are sequestered in the cytoplasm away from the peptidoglycan layer. Holins, on the other hand, interact nonspecifically with plasma membranes and are generally toxic to an Escherichia coli host, inducing bacteriolysis from within (9). Since holins are short (∼100 residues) and are often encoded adjacent to lysins, they can lead to selective toxicity of many of the clones one hopes to identify. In a metagenomic screen, where numerous lysins are present within a single library, this effect could lead to a significant loss of positive hits. (ii) Target bacterial species: in standard phage genomic screens, lysin-encoding clones are selected by their ability to kill the host bacterium of the encoding phage, which generally demonstrates the highest sensitivity (27). In a metagenomic screen, however, numerous host species of unknown origin could be present within a sample, confounding this choice of screening agent.To address these issues, we have devised a novel functional strategy for the general cloning of lytic enzymes from uncultured phage DNA. It utilizes a plasmid-based E. coli expression system and consists of a two-step process. Following induction by arabinose, clones are first screened for holin-mediated lysis by a hemolytic effect they create in the surrounding blood agar. These initial hits are then restreaked as patches and overlaid with Gram-negative cells whose outer membranes have been permeabilized by autoclaving, serving as a general source of peptidoglycan. The clones are observed for surrounding Gram-negative clearing zones to assay directly for the recombinant production of lytic enzymes encoded adjacent to the holins. As proof-of-principle, we applied our methodology to a viral metagenomic library constructed from mixed animal feces, identifying 26 actively expressed lysins of diverse molecular architectures. The first of its kind, this study presents a general model for lysin identification through viral metagenomics, highlighting the potential of this field for cloning of proteins of biotechnological or academic value.     

Mutagenesis of a bacteriophage lytic enzyme PlyGBS significantly increases its antibacterial activity against group B streptococci

Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. Intrapartum antibiotic prophylaxis (IAP) is the current prevention strategy given to pregnant women with confirmed vaginal GBS colonization. Due to antibiotic resistance identified in GBS, we previously developed another strategy using a bacteriophage lytic enzyme, PlyGBS, to reduce vaginal GBS colonization. In this study, various DNA mutagenesis methods were explored to produce PlyGBS mutants with increased lytic activity against GBS. Several hyperactive mutants were identified that contain only the endopeptidase domain found in the N-terminal region of PlyGBS and represent only about one-third of the wild-type PlyGBS in length. Significantly, these mutants not only have 18–28-fold increases in specific activities compared to PlyGBS, but they also have a similar activity spectrum against several streptococcal species. One of the hyperactive mutants, PlyGBS90-1, reduced the GBS colonization from >5 logs of growth per mouse to <50 colony-forming units (cfu) 4 h post treatment (∼4-log reduction) using a single dose in a mouse vaginal model. A reduction in GBS colonization before delivery should significantly reduce neonatal GBS infection providing a safe alternative to IAP.