Intact Gram-positive Bacteria Research Articles

Soluble bacterial constituents down-regulate secretion of IL-12 in response to intact Gram-positive bacteria

Intact Gram-positive bacteria induce production of large amounts of IL-12 from freshly isolated human monocytes. Here the bacterial structures and signalling pathways involved were studied and compared with those leading to IL-6 production, and to IL-12 production in response to LPS after IFN-γ pre-treatment. Intact bifidobacteria induced massive production of IL-12 (1 ng/ml) and IL-6 (>30 ng/ml) from human PBMC, whereas fragmented bifidobacteria induced IL-6, but no IL-12. IL-12 production induced by intact bifidobacteria was inhibited by pre-treatment with bifidobacterial sonicate, peptidoglycan, muramyl dipeptide, lipoteichoic acid, the soluble TLR2 agonist Pam 3Cys-SK 4, or anti-TLR2 antibodies. Blocking of phagocytosis by cytochalasin, inhibition of the JNK or NF-κB pathways or treatment with Wortmannin also reduced the IL-12 response to intact Gram-positive bacteria. LPS induced moderate levels of IL-12 (0.31 ng/ml), but only from IFN-γ pre-treated PBMC. This IL-12 production was enhanced by Wortmannin and unaffected by blocking the JNK pathway. Thus, intact Gram-positive bacteria trigger monocyte production of large amounts of IL-12 via a distinct pathway that is turned off by fragmented Gram-positive bacteria. This may be a physiological feedback, since such fragments may signal that further activation of the phagocyte via the IL-12/IFN-γ loop is unnecessary.

Monocytes can phagocytose Gram-negative bacteria by a CD14-dependent mechanism.

Phagocytosis of bacteria by monocytes and neutrophil granulocytes provides an important first line of defense against bacterial infections. Opsonization of bacteria with complement and phagocytosis by neutrophils is dependent on divalent cations and does not take place in blood that has been anticoagulated with EDTA. Monocytes, however, do carry out phagocytosis even in the presence of EDTA. We show here that this divalent cation-independent phagocytosis pathway requires the presence of the LPS receptor CD14 on the cell surface. This pathway is dependent on the availability of LPS binding protein, can be blocked by anti-CD14 Abs, by an excess of soluble CD14, by excess free LPS, or by an excess of unlabeled Gram-negative bacteria. In contrast, intact Gram-positive bacteria fail to inhibit this process. These experiments define a CD14-dependent phagocytosis pathway for Gram-negative bacteria that operates in monocytes in human whole blood. This pathway may be able to deal with bacterial pathogens that have developed resistance to complement-dependent opsonization and phagocytosis by neutrophils.

Mode of action of the staphylococcinlike peptide Pep 5: voltage-dependent depolarization of bacterial and artificial membranes.

The cationic staphylococcinlike peptide Pep 5 is shown to depolarize bacterial and planar lipid membranes in a voltage-dependent manner. An artificial valinomycin-induced potassium diffusion potential across the cytoplasmic membrane of Staphylococcus cohnii 22 was sufficient to promote Pep 5 action. Thus, evidence is provided that a membrane potential of sufficient magnitude is the only prerequisite for Pep 5 activity. The voltage dependence was elucidated by macroscopic conductance measurements with black lipid membranes. A threshold potential of about -90 to -100 mV, which was deduced from experiments with bacterial cells, could be confirmed. Single pores were resolved which often occur as short-lived bursts and fluctuate among different conductance levels. Pore diameters were calculated ranging from 0.1 to 1 nm. Succinylation of the lysine residues of Pep 5 resulted in prolonged pore lifetimes and maintenance of distinct conductance levels. However, the succinylated peptide required a higher threshold potential, approximately -150 mV, than the native peptide, which is probably the reason for the reduced activity of the modified peptide against intact gram-positive bacteria.