Specificity of marine microbial surface interactions.

The macromolecular surface components involved in intraspecific cell surface interactions of the green microalga Chlorella vulgaris and closely associated bacteria were investigated. The specific surface attachment between this alga and its associated bacteria is mediated by lectin-like macromolecules associated with the surfaces of these cells. The binding activity of these surface polymers was inhibited by specific simple sugars; this suggests the involvement of specific receptor-ligand binding sites on the interactive surfaces. Epifluorescent microscopic evaluation of bacteria-alga interactions in the presence and absence of the macromolecules that mediate these interactions showed that the glycoproteins active in these processes were specific to the microbial sources from which they were obtained. The demonstration and definition of the specificity of these interactions in mixed microbial populations may play an important role in our understanding of the dynamics of marine microbial populations in the sea.     

Carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1), apically expressed on human colonic M cells,are potential receptors for microbial adhesion

In the human gut mucosa, specialized M cells deliver intact foreign macromolecules and commensal bacteria from the lumen to organized mucosal lymphoid tissues triggering immune responses. M cells are also major sites of adhesion and invasion for enteric pathogens. The molecular features of M cell apical surfaces that promote microbial normal attachment are still largely unknown. We have demonstrated previously that in the human colonic epithelium, carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1) are integral components of the apical glycocalyx which participate in epithelial–microbial interactions. In this study, based on the reactivity of specific monoclonal antibodies and on immunoelectron microscopy, we show that M cells of human colonic solitary lymphoid follicles express CEA and CEACAM1 on the apical surface. Recently these highly glycosylated molecules have been characterized as protein receptors for different bacteria. This leads us to propose a role for CEA and CEACAM1 in the adherence of enteric bacteria to the apical membrane of colonic M cells. We also hypothesize that, unlike colonic enterocytes, M cells lack the defense mechanism that eliminates CEA and CEACAM1 upon microbial binding and which is based on vesiculation of microvillus plasma membrane.     

Diversification of CD1 Molecules Shapes Lipid Antigen Selectivity

Molecular studies of host–pathogen evolution have largely focused on the consequences of variation at protein–protein interaction surfaces. The potential for other microbe-associated macromolecules to promote arms race dynamics with host factors remains unclear. The cluster of differentiation 1 (CD1) family of vertebrate cell surface receptors plays a crucial role in adaptive immunity through binding and presentation of lipid antigens to T-cells. Although CD1 proteins present a variety of endogenous and microbial lipids to various T-cell types, they are less diverse within vertebrate populations than the related major histocompatibility complex (MHC) molecules. We discovered that CD1 genes exhibit a high level of divergence between simian primate species, altering predicted lipid-binding properties and T-cell receptor interactions. These findings suggest that lipid–protein conflicts have shaped CD1 genetic variation during primate evolution. Consistent with this hypothesis, multiple primate CD1 family proteins exhibit signatures of repeated positive selection at surfaces impacting antigen presentation, binding pocket morphology, and T-cell receptor accessibility. Using a molecular modeling approach, we observe that interspecies variation as well as single mutations at rapidly-evolving sites in CD1a drastically alter predicted lipid binding and structural features of the T-cell recognition surface. We further show that alterations in both endogenous and microbial lipid-binding affinities influence the ability of CD1a to undergo antigen swapping required for T-cell activation. Together these findings establish lipid–protein interactions as a critical force of host–pathogen conflict and inform potential strategies for lipid-based vaccine development.     

Influence of calcium and other cations on surface adhesion of bacteria and diatoms: A review

Association with a surface is an important aspect of survival for microorganisms in natural and manmade environments/Both bacteria and diatoms are involved in such associations. In many cases, this leads to surface fouling, which often results in surface deterioration and mechanical failure in industrial systems. We now know that microorganisms exploit many strategies to establish associations with surfaces. As in the case of other cellular processes, calcium ions seem to play an important role in adhesion of cells to surfaces. Calcium is involved in non-specific interactions such as neutralization of the electrical double layer between cell and substratum surface as well as specific adhesive interactions that cannot be replaced by other cations. The unique properties of calcium ions promote both specific and non-specific interactions with protein and polysaccha-ride adhesin molecules at the cell surface. As important, but less well understood, calcium ions also influence the way microbial cells interact with different substrata.     

Adhesion and surface-aggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber

Adhesive interactions between Candida albicans and oral bacteria are generally thought to play a crucial role in the microbial colonization of denture acrylic, which may lead to denture stomatitis. This study investigated the influence of saliva on the adhesive interactions between C. albicans and Streptococcus sanguis or Actinomyces naeslundii on denture acrylic. First, bacteria were allowed to adhere to the acrylic surface from a flowing suspension, and subsequently yeasts were flowed over the acrylic surface. The organisms were assayed in the presence or absence of human whole saliva. All experiments were carried out in a parallel plate flow chamber and enumeration was done in situ with an image analysis system. In the absence of adhering bacteria, adhesion of C. albicans from buffer was more extensive than from saliva. However, in the presence of adhering bacteria, yeast adhesion from saliva was increased with respect to adhesion of yeasts from buffer, indicating that specific salivary components constitute a bridge between bacteria and yeasts. In all cases, yeast aggregates consisting of 3 to 5 yeast cells were observed adhering to the surface. A surface physico-chemical analysis of the microbial cell surfaces prior to and after bathing the microorganisms in saliva, suggests that this bridging is mediated by acid-base interactions since all strains show a major increase in electron-donating surface free energy parameters upon bathing in saliva, with no change in their zeta potentials. The surface physico-chemical analysis furthermore suggests that S. sanguis and A. naeslundii may use a different mechanism for adhesive interactions with C. albicans in saliva.     

Perspectives on microbial cell surface display in bioremediation

The display of heterologous proteins on the microbial cell surface by means of recombinant DNA biotechnologies has emerged as a novel approach for bioremediation of contaminated sites. Both bacteria and yeasts have been investigated for this purpose. Cell surface expression of specific proteins allows the engineered microorganisms to transport, bio-accumulate and/or detoxify heavy metals as well as to degrade xenobiotics. These otherwise would not be taken up and transformed by the microbial cell. This review focuses on the application of cell surface displays for the enhanced bio-accumulation of heavy metals by metal binding proteins. It also reviews the biodegradation of xenobiotics by enzymes/proteins expressed on microbial cell surfaces.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.     

Bacteriophage-host interactions leading to genome internalization

Bacteriophage infection is initiated by binding of the virion to a specific receptor located on the host surface. The genome is then released from the capsid and delivered to the host cytoplasm. Our knowledge of these early steps of infection has recently improved. The three-dimensional structure of numerous receptor binding proteins of tailed phages has been solved. Cryo-electron tomography has allowed characterization of the phage-host interactions in a cellular context and at nanometric resolution. The localization and motions of fluorescently labelled phages, receptors and viral DNA were monitored on individual bacteria. Altogether these approaches have revealed the intricacy of these early events and emphasize the link between infection and microbial architecture.     

A method of reversible biomolecular immobilization for the surface plasmon resonance quantitative analysis of interacting biological macromolecules

This article presents a new procedure for the immobilization of macromolecules on gold surfaces, with the purpose of studying macromolecular interactions by simple optical configurations rendering surface plasmon resonance. Gold surfaces were covered by a three-layer structure composed of poly-L-lysine irreversibly bound to gold, followed by a second layer of heparin and a third layer of polylysine. The three-layer structure of polylysine-heparin-polylysine remains irreversibly bound to gold, it prevents biomolecules from coming into direct contact with the metal surface, and it allows the irreversible binding of different proteins and polynucleotides. After binding of a macromolecule to the three-layer structure, the interaction with a second macromolecule can be studied, and then the complex formed by the two interacting macromolecules, together with the second heparin layer and the third polylysine layer, can be broken down just by treatment with an alkaline solution having a pH value above the pK value of the amino groups of polylysine. The first polylysine layer remains irreversibly bound to gold, ready to form a new three-layer structure and, therefore, to support a new macromolecular interaction on the same regenerated surface. Polynucleotide interactions, the proteolytic action of chymotrypsin, and the interaction between the component subunits of a heterotetrameric enzyme are described as examples of macromolecular interactions studied by using this system. The method may be especially suitable for developing of low-cost systems aimed to look for surface resonance signals, and it offers the advantage of allowing calculation of parameters related to the size and stoichiometry of the interacting macromolecules, in addition to the kinetic and equilibrium properties of the interaction.     

Chemical defenses of seaweeds against microbial colonization

Any living or non-living surface immersed in seawaterrapidly acquires a bacterial biofilm. For living marineorganisms, biofilm formation can result in the death ofthe host, and thus there is strong evolutionary pressure formarine eukaryotes to evolve mechanisms which inhibit orcontrol the development of biofilms on their surfaces.Some marine eukaryotes are indeed successful incontrolling biofilms on their surfaces, and in manyinstances this control is achieved by the production ofinhibitory chemicals which act at or near the surface ofthe organism. In some cases these natural inhibitors aresimply toxic to bacteria. However, increasingly it appearsthat at least some of these compounds act by interferingspecifically with bacterial characteristics which effect theability of bacteria to colonize their hosts, such asattachment, surface spreading, or the production ofextracellular macromolecules. As an example, theAustralian seaweed Delisea pulchra appears tocontrol bacterial colonization by interfering with abacterial regulatory system (the acylated homoserinelactone system) that regulates several colonizationrelevant bacterial traits. Understanding how marineorganisms control specific bacterial colonization traitsshould provide us with insights into new technologies forthe control of biofilms on artificial surfaces.     

Human oral cavity as a model for the study of genome-genome interactions

The enormous diversity of culturable bacteria within the oral microbial community coupled with experimental accessibility renders the human oral cavity a valuable model to investigate genome-genome interactions. The complex interactions of oral bacteria result in the formation of biofilms on the surfaces of the oral cavity. One mechanism thought to be important in biofilm formation is the coaggregation of bacterial partners. In this paper, we examine the role of coaggregation in oral biofilms and develop protocols to elucidate the spatial organization of bacterial species retained within oral biofilms. To explore these issues, we have employed two experimental systems: the saliva-coated flowcell and the retrievable enamel chip. From flowcell studies, we have determined that coaggregation can greatly influence the ability of an oral bacterial species to grow and be retained within the developing biofilm. To examine the spatial architecture of oral biofilms, fluorescent in situ hybridization protocols were developed that successfully target specific members of the oral microbial community. Together, these approaches provide insight into the development of oral biofilms and expand our understanding of genome-genome interactions.     

Progression of Epiphytic Microflora in Wheat and Alfalfa Silages as Observed by Scanning Electron Microscopy

Wheat and alfalfa silages were examined by scanning electron microscopy and standard methods of microbial enumeration. Epiphytic microflora were present at levels of 10⁶ to 10⁸/g in the fresh-cut plants. This flora was initially observed microscopically primarily on the surfaces. After 4 days of fermentation, lactic acid bacteria were observed on the surface in high concentrations near open stomata and throughout the interior mesophyll air sac spaces. At 4 days, populations on interior surfaces were restricted to the exterior surfaces of the air sacs. After 8 days the mesophyllic cells showed marked deterioration, and bacteria were observed on their inner surfaces. At 32 days, the end of the fermentation, vascular bundles and epidermal cells remained intact whereas stomata and mesophyllic cells were collapsed and often contained microorganisms. It is concluded that the interior of the leaves offers substantial nutritional and environmental advantages to epiphytic flora and is an important if not major deterioration site in fermented products. Since little deterioration of exterior surfaces was observed, these sites may play a minor role in supplying nutrients for microbial growth.     

Scanning Electron Microscope Study of Bacteria Associated with the Rumen Epithelium of Sheep

Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery of bacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed.     

SdrC induces staphylococcal biofilm formation through a homophilic interaction

The molecular pathogenesis of many Staphylococcus aureus infections involves growth of bacteria as biofilm. In addition to polysaccharide intercellular adhesin (PIA) and extracellular DNA, surface proteins appear to mediate the transition of bacteria from planktonic growth to sessile lifestyle as well as biofilm growth, and can enable these processes even in the absence of PIA expression. However, the molecular mechanisms by which surface proteins contribute to biofilm formation are incompletely understood. Here we demonstrate that self‐association of the serine‐aspartate repeat protein SdrC promotes both bacterial adherence to surfaces and biofilm formation. However, this homophilic interaction is not required for the attachment of bacteria to abiotic surfaces. We identified the subdomain that mediates SdrC dimerization and subsequent cell‐cell interactions. In addition, we determined that two adjacently located amino acid sequences within this subdomain are required for the SdrC homophilic interaction. Comparative amino acid sequence analysis indicated that these binding sites are conserved. In summary, our study identifies SdrC as a novel molecular determinant in staphylococcal biofilm formation and describes the mechanism responsible for intercellular interactions. Furthermore, these findings contribute to a growing body of evidence suggesting that homophilic interactions between surface proteins present on neighbouring bacteria induce biofilm growth.     

Binding of Streptavidin to Bacteria or Fungi and Its Applications in Detecting These Microbes

We have investigated the characteristics and utilities of streptavidin-binding to gram-negative and gram-positive bacteria and Candida spp. The pre-treatment of these microbes with chemical reagents such as CHCl₃, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte-smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi-digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte-smears).     

Phylogenetic diversity of bacteria associated with ascidians in Eel Pond (Woods Hole, Massachusetts, USA)

Recent studies have revealed that many marine invertebrates are closely associated with diverse microorganisms, frequently resulting in the production of compounds of biomedical interest. Thus far, ascidians have not been widely examined for the presence of bacterial associations, although the production of secondary metabolites is well documented. In the present study, we examined the gonad of Molgula manhattensis and the tunic surfaces of Botryllus schlosseri, Didemnum sp., and Botrylloides violaceus for the presence of associated bacteria. These ascidians are common inhabitants of the coastal ocean of Cape Cod, Massachusetts. We used denaturing gradient gel electrophoresis (DGGE) as well as cloning and sequencing of 16S rDNA to analyze the microbial communities. There is a strong evidence that spiroplasma-like bacteria colonize the gonad of the solitary ascidian M. manhattensis. The bacteria might be vertically transmitted and may be involved in the production of secondary metabolites that deter predators of the ascidians. The bacterial communities found on the tunic surfaces of the colonial ascidians were found to be more diverse. However, in all cases the bacterial communities were predominated by alpha-proteobacteria. Alpha-proteobacteria related to the obtained sequences have been identified as symbionts in a variety of hosts, suggesting a specific role for these bacteria. However, based on our data it is difficult to differentiate between persistently and only transiently associated bacteria. Overall, this study demonstrates that ascidian species are associated with diverse bacterial populations. Future studies will aim to elucidate the precise relationships between bacteria and ascidians and to identify bioactive compounds that might be produced as a result of these relationships.     

Diffusion through a Double-Sided Plate: Development of a Method to Study Alga-Bacterium Interactions

Bacteria and algae isolated from a wastewater oxidation pond were inoculated onto opposing surfaces of double-layer agar plates (Lutri plates) to determine the usefulness of such plates for studying microbial interactions. The altered growth characteristics of various algae depending on the species of bacteria on the adjacent medium surface indicated that there was diffusion of extracellular products through the agar, suggesting that this simple assay can be used for screening potential interactions of actively growing organisms.     

Specific capture of mammalian cells by cell surface receptor binding to ligand immobilized on gold thin films

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic fibroblast growth factor (bFGF) binding receptor was targeted on baby hamster kidney (BHK-21) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.