Elaine Tuomanen

Targeted disruption of the p50 subunit of NF-κB leads to multifocal defects in immune responses

NF-kappa B, a heterodimeric transcription factor composed of p50 and p65 subunits, can be activated in many cell types and is thought to regulate a wide variety of genes involved in immune function and development. Mice lacking the p50 subunit of NF-kappa B show no developmental abnormalities, but exhibit multifocal defects in immune responses involving B lymphocytes and nonspecific responses to infection. B cells do not proliferate in response to bacterial lipopolysaccharide and are defective in basal and specific antibody production. Mice lacking p50 are unable effectively to clear L. monocytogenes and are more susceptible to infection with S. pneumoniae, but are more resistant to infection with murine encephalomyocarditis virus. These data support the role of NF-kappa B as a vital transcription factor for both specific and nonspecific immune responses, but do not indicate a developmental role for the factor.

Recognition of a bacterial adhesin by an integrin: Macrophage CR3 (αMβ2, CD11b CD18 ) binds filamentous hemagglutinin of Bordetella pertussis

Abstract During the course of whooping cough, Bordetella pertussis interacts with alveolar macrophages and other leukocytes on the respiratory epithelium. We report here mechanisms by which these bacteria adhere to human macrophages in vitro. Whole bacteria adhere by means of two proteins, filamentous hemagglutinin (FHA) and pertussis toxin, either of which is sufficient to mediate adherence. FHA interacts with two classes of molecules on macrophages, galactose-containing glycoconjugates and the integrin CR3 ( α M β 2 , CD11bCD18). The interaction between CR3 and FHA involves recognition of the Arg-Gly-Asp (RGD) sequence at positions 1097–1099 in FHA. This study demonstrates that bacterial adherence can be based on the interaction of a bacterial adhesin RGD sequence with an integrin and that bacterial adhesins can have multiple binding sites characteristic of eukaryotic extracellular matrix proteins.

Neural Targeting of Mycobacterium leprae Mediated by the G Domain of the Laminin-α2 Chain

We report that the molecular basis of the neural tropism of Mycobacterium leprae is attributable to the specific binding of M. leprae to the laminin-alpha2 (LN-alpha2) chain on Schwann cell-axon units. Using recombinant fragments of LN-alpha2 (rLN-alpha2), the M. leprae-binding site was localized to the G domain. rLN-alpha2G mediated M. leprae binding to cell lines and to sciatic nerves of dystrophic dy/dy mice lacking LN-alpha2, but expressing laminin receptors. Anti-beta4 integrin antibody attenuated rLN-alpha2G-mediated M. leprae adherence, suggesting that M. leprae interacts with cells by binding to beta4 integrin via an LN-alpha2G bridge. Our results indicate a novel role for the G domain of LN-2 in infection and reveal a model in which a host-derived bridging molecule determines nerve tropism of a pathogen.

PAF Receptor Anchors Streptococcus Pneumoniae to Activated Human Endothelial Cells

Streptococcus pneumoniae can produce asymptomatic colonization or aggressive sepsis. We sought to differentiate the molecular mechanisms of these disparate courses. Cytokine or thrombin activation of human vascular endothelial cells and type II pneumocytes enhanced pneumococcal adherence relative to resting cells. Adherence and subsequent invasion was dramatically reduced by PAF receptor antagonists. Cells transfected with the PAF receptor gained the ability to support pneumococcal adherence. PAF or PAF receptor antagonists inhibited attachment and invasion. Adherence involved phosphorylcholine on the pneumococcal teichoic acid. Virulent pneumococci target the PAF receptor on activated human cells, a necessary step to facilitate subsequent invasion.

The role of complement in inflammation during experimental pneumococcal meningitis

The mechanism whereby an effective bactericidal inflammatory reaction develops in the subarachnoid space is not clearly defined. While normal cerebrospinal fluid is deficient in complement, immunoglobulin and leukocytes, these serum components appear in cerebrospinal fluid (CSF) during the course of bacterial meningitis. Using a rabbit model of pneumococcal meningitis we examined the role of the alternate complement pathway in three early events important to the defense of the subarachnoid space: leukocyte chemotaxis, phagocyte mediated bacterial killing, and clearance of bacterial components from the cerebrospinal fluid space. Rabbits treated with cobra venom factor to deplete complement were inoculated intracisternally with encapsulated (type II or XIX) pneumococci. Following complement depletion, there was a dramatic (at least 100-fold) decrease in the LD50 for these strains. Nevertheless, complement depletion did not affect the magnitude of CSF leucocytosis or the rate of clearance of bacterial particles from CSF. A short delay in the appearance of leukocytes in CSF was found in the absence of complement. The major effect of complement depletion, however, was to diminish the efficiency of leukocyte mediated killing of encapsulated bacteria in the CSF. Although the short delay in the onset of leukocytosis in the complement depleted animals is consistent with a chemotactic role of complement in the normal animal, the quantitatively normal leukocytosis in the complement depleted rabbits clearly indicates that important chemotaxins other than complement function in CSF. Inhibition of leukocytosis by indomethacin and diclofenac suggests that metabolite(s) of the arachidonic acid pathway may perform such a chemotactic role. A major role of complement in the defense of the subarachnoid space appears to be as an opsonin needed for the effective bactericidal activity of leukocytes. It is the lack of this function that best explains the greatly decreased LD50 value of encapsulated pneumococci in the complement depleted animal.

Attachment factors of Bordetella pertussis: mimicry of eukaryotic cell recognition molecules.

Bacteria localize to specific target organs during infection by recognizing eukaryotic cell surface addresses. This address system exists to facilitate normal trafficking of host cells such as leukocytes. The mechanisms by which bacteria subvert host targeting codes provide a unique link between microbial pathogenesis and eukaryotic cell trafficking, and can point to a new array of antibodies and peptides of potential therapeutic value.

A sandwich adhesin on Streptococcus pneumoniae attaching to human oropharyngeal epithelial cells in vitro

Abstract Streptococcus pneumoniae attach to human pharyngeal epithelial cells through the specific interaction of bacterial surface adhesins with glycoconjugate receptors. The present study defines the adhesin as a molecule bridging between an anchoring site in the bacterial cell wall and the epithelial cell receptor. The nature of the adhesin was defined in three ways: First, the attachment of whole bacteria was reduced by trypsin, periodate and heat. Second, heat treatment of whole bacteria was shown to release material, which was able to reconstitute the adherence. The heat extract bound to epithelial cells, as shown by fluorescence labelling, and agglutinated latex beads covalently coupled with receptor oligosaccharide. Active material could be extracted by heat from both high and low adhering strains, but could reconstitute only attaching strains. Third, the bacterial component binding the adhesin was localized to protoplasts and cell wall fractions obtained by mechanical or deoxycholate induced lysis of pneumococci. Isolated pneumococcal surface components, which did not inhibit attachment, included peptidoglycan, C polysaccharide, Forssmann antigen, capsular polysaccharide and a phenol extract produced in analogy to streptococcal lipoteichoic acid. The procedure used to extract the adhesin was previously used to prepare the competence factor. The competence deficient mutant RA7 attached poorly compared to the competent R6 parent. The possible relatedness of attachment to competence is discussed.

Induction of meningeal inflammation by diverse bacterial cell walls.

Bacterial cell walls have been shown to be potent inflammatory agents. Cell wall components from various bacterial species can i) immunomodulate the activity of macrophages, T cells and B cells (1,2), ii) trigger the alternative pathway of complement activation (3-5) , iii) interact with polymorphonuclear leukocytes (6, 7), and iv) bind with humoral immune factors such as C-reactive protein (8). Such activities presumably contribute to inflammation provoked by cell walls in models of post4nfectious and immune diseases (9, 10). Recently, however, it has become increasingly clear that host reactions to cell walls can also contribute very significantly to the course of acute bacterial infections such as bacteremia (11-13) and meningitis (14-16).

The Pathophysiology of Pneumococcal Meningitis

The interactions between pneumococcal surface components and host defence systems that initiate pneumococcal meningitis have been studied in considerable molecular detail over the past decade. In this sense, the pneumococcus has served as a prototype for the unravelling of the genesis of inflammation caused by gram-positive bacteria. This review outlines the progression of these early events involving the cytokine cascade, the coagulation cascade, and leukocyte migration, and relates these processes to the production of blood-brain barrier permeability, the hallmark of injury in meningitis. This new understanding has radically altered the therapy of disease with the promise of greatly improved outcome.

Lectin domains in the toxin ofBordetella pertussis: selectin mimicry linked to microbial pathogenesis

The pathogenesis of many infectious diseases is critically determined by prokaryotic lectins which enable differential recognition and activation of targeted eukaryotic cells. Some bacterial adhesins mimic and co-opt eukaryotic cell-cell adhesion motifs. This is illustrated by the toxin ofBordetella pertussis. Pertussis toxin mediates intoxication of eukaryotic cells by elevation of cAMP and it serves as an adhesin binding the bacteria to ciliated cells and respiratory macrophages. These activities are mediated by the lectin-like properties of the binding oligomer of the toxin. A comparison of pertussis toxin and the selectins involved in leukocyte trafficking indicates that these prokaryotic and eukaryotic C-type lectins share some element of primary sequence similarity, three dimensional structure, and biological activities. Such mimicry suggests a link between eukaryotic cell-cell adhesion motifs and microbial pathogenesis.