Glycerol monolaurate (GML) is a naturally occurring surfactant that has potential use as an additive to tampons and wound dressings to reduce the incidence of certain bacterial toxin-mediated illnesses. In vitro studies were undertaken to evaluate the effect of GML on the growth of and toxin production by potentially pathogenic bacteria. GML inhibited the growth of clinical isolates of group A, B, F, and G streptococci at concentrations of 10 to 20 micrograms/ml. Exotoxin production, including that of pyrogenic exotoxins and hemolysins, was reduced by concentrations of GML that were below those inhibitory for growth as well as growth inhibitory. The growth of Staphylococcus aureus strains from patients with toxic shock syndrome and scalded skin syndrome was inhibited or delayed in the presence of 100 to 300 micrograms of GML per ml. Growth inhibition by GML could be overcome by the production of lipase. S. aureus elaboration of hemolysin, toxic shock syndrome toxin 1, and exfoliative toxin A was inhibited at GML concentrations below those necessary to inhibit growth. Results similar to those for S. aureus were obtained in tests of S. hominis. Escherichia coli growth and Salmonella minnesota growth were unaffected by GML, but an S. minnesota Re mutant was susceptible to growth-inhibitory activity. Endotoxin release into the medium from E. coli cells was also unaffected by GML, but the release or activity of E. coli hemolysin was increased by GML. Streptococcal pyrogenic endotoxin A production by an E. coli clone was not affectd by GML. These studies indicate that GML is effective in blocking or delaying the production of exotoxins by pathogenic gram-positive bacteria.

In this study, we demonstrate that the mechanism by which Staphylococcus aureus induces apoptosis in bovine mammary epithelial (MAC-T) cells involves caspases 8 and 3, two key components of a proteolytic cascade leading to apoptosis. In addition, internalized S. aureus induces expression of the inflammatory cytokines tumor necrosis factor alpha and interleukin-1␤ by MAC-T cells. These data suggest that the internalization of S. aureus could induce specific cellular responses in vivo that may ultimately impact the course of infection.Staphylococcus aureus is internalized by a variety of nonprofessional mammalian phagocytes (1,8,19). Work in our laboratories has shown that internalization requires a specific interaction between fibronectin binding protein and the host cell, presumably followed by signal transduction events that ultimately lead to rearrangement of the host cell cytoskeleton (4). We have also shown that internalization by bovine mammary epithelial cells induces apoptosis and that metabolically active intracellular S. aureus are needed for apoptosis to occur (1,20).Assessment of caspase activity. The induction of apoptosis, also called programmed cell death, often progresses through an ordered series of events involving a family of cysteine aspartyl proteases known as caspases. This family of proteins is divided into two major groups: initiator caspases, which are involved in triggering the cascade of events leading to cell death, and effector caspases, which, when activated by initiator caspases, catalyze the disassembly of cell structures (17). The caspase cascade can be initiated in response to the binding of an appropriate ligand to receptors on target cells or through self-aggregation of receptors in response to their increased density on the cell surface. To determine whether caspases are involved in apoptosis induction, two major caspase pathways were examined; the first is mediated through the most apical of the initiator caspases, caspase 8 (10), and the second is mediated through caspase 1. Either caspase 8 or 1 can activate caspase 3 and result in the cell disassembly associated with apoptosis.Bovine mammary epithelial cells, designated MAC-T (9), were infected with S. aureus (wild-type strain RN6390) essentially as described previously (1, 20). After either 3 or 6 h of infection, MAC-T cell culture supernatants were discarded, and the monolayers were washed three times with sterile phosphate-buffered saline (PBS, pH 7.2). The monolayers were harvested with sterile cell scrapers (Nalge Nunc International, Naperville, Ill.), and cells were collected by centrifugation at 450 ϫ g for 5 min at 4°C. Each cell pellet was washed once with sterile PBS and resuspended in 10 ml of sterile PBS. Cells were counted with a hemacytometer, pelleted again by centrifugation, and resuspended to a concentration of 10 8 cells/ml in cell lysis buffer (25 mM HEPES [pH 7.5], 5 mM MgCl 2 , 5 mM EDTA, 5 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, 10 g of leupeptin per ml; all reagents from Sigma, St. Lou...

ABSTRACTA novel phenotype was recently identified in which specific strains ofEscherichia coliinhibit competingE. colistrains via a mechanism that was designated “proximity-dependent inhibition” (PDI). PDI-expressing (PDI+)E. coliis known to inhibit susceptible (PDI−)E. colistrains, including several enterohemorrhagic (EHEC) and enterotoxigenic (ETEC)E. colistrains. In this study, every strain from a genetically diverse panel ofE. coliO157:H7 (n= 25) and additional strains ofE. coliserovar O26 were susceptible to the PDI phenotype. LIVE/DEAD staining was consistent with inhibition by killing of susceptible cells. Comparative genome analysis identified the genetic component of PDI, which is composed of a plasmid-borne (Incl1) operon encoding a putative microcin and associated genes for transport, immunity, and microcin activation. Transfer of the plasmid to a PDI−strain resulted in transfer of the phenotype, and deletion of the genes within the operon resulted in loss of the inhibition phenotype. Deletion of chromosomally encodedtolCalso resulted in loss of the inhibitory phenotype, and this confirmed that the putative microcin is most likely secreted via a type I secretion pathway. Deletion of an unrelated plasmid gene did not affect the PDI phenotype. Quantitative reverse transcription (RT)-PCR demonstrated that microcin expression is correlated with logarithmic-phase growth. The ability to inhibit a diversity ofE. colistrains indicates that this microcin may influence gut community composition and could be useful for control of important enteric pathogens.

The goal of this study was to investigate the molecular interaction between superantigens and the T-cell receptor (TCR). Using a quantitative polymerase chain reaction (PCR) to assess T-cell proliferation profiles, we found that SEB, SEC1, SEC2 and SEC3 expanded human T cells bearing V beta 3, V beta 12, V beta 13.2, V beta 14, V beta 15, V beta 17 and V beta 20. SEC2 and SEC3 have the additional ability to expand T cells bearing V beta 13.1, and their expansion of V beta 3 was markedly reduced compared to SEB and SEC1. Based on the activity of SEC1 mutants containing single amino acid substitutions, we concluded that the differential abilities of these native toxins to stimulate V beta 3 and V beta 13.1 was determined by the residue in position 26, located in the base of the SEC alpha 3 cavity. The SEC1 mutant, in which Val in position 26 was substituted with the analogous SEC2/SEC3 residue (Tyr), generated a V beta expansion profile that was indistinguishable from those generated by SEC2 and SEC3. Using these findings, the co-ordinates of a recently reported murine TCR beta-chain crystal structure, and other documented information, we propose a compatible molecular model for the interaction of SEC3 with the T-cell receptor. In this model complex, the complementarity-determining regions (CDRs) 1 and 2 and the hypervariable loop 4 of the V beta element contact SEC3 predominantly through residues in the alpha 3 cavity of the toxin. CDR3 of the beta chain is not involved in any toxin contacts. The proposed model not only includes contacts identified in previous mutagenesis studies, but is also consistent with the ability of tyrosine and valine in position 26 to differentially affect the expansion of V beta s 3 and 13.1 by the SEC superantigens.

c The CD4؉ T-cell response is central for the control of Anaplasma marginale infection in cattle. However, the infection induces a functional exhaustion of antigen-specific CD4 ؉ T cells in cattle immunized with A. marginale outer membrane proteins or purified outer membranes (OMs), which presumably facilitates the persistence of this rickettsia. In the present study, we hypothesize that T-cell exhaustion following infection is induced by the upregulation of immunoinhibitory receptors on T cells, such as programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3). OM-specific T-cell responses and the kinetics of PD-1-positive (PD-1 ؉ ) LAG-3 ؉ exhausted T cells were monitored in A. marginale-challenged cattle previously immunized with OMs. Consistent with data from previous studies, OM-specific proliferation of peripheral blood mononuclear cells (PBMCs) and interferon gamma (IFN-␥) production were significantly suppressed in challenged animals by 5 weeks postinfection (wpi). In addition, bacteremia and anemia also peaked in these animals at 5 wpi. Flow cytometric analysis revealed that the percentage of PD-1 ؉ LAG-3 ؉ T cells in the CD4 ؉ , CD8 ؉ , and ␥␦ T-cell populations gradually increased and also peaked at 5 wpi. A large increase in the percentage of LAG-3 ؉ ␥␦ T cells was also observed. Importantly, in vitro, the combined blockade of the PD-1 and LAG-3 pathways partially restored OM-specific PBMC proliferation and IFN-␥ production at 5 wpi. Taken together, these results indicate that coexpression of PD-1 and LAG-3 on T cells contributes to the rapid exhaustion of A. marginale-specific T cells following infection and that these immunoinhibitory receptors regulate T-cell responses during bovine anaplasmosis.