EARLY AND DISCRETE CHANGES IN PERMEABILITY OF ESCHERICHIA COLI AND CERTAIN OTHER GRAM-NEGATIVE BACTERIA DURING KILLING BY GRANULOCYTES

Abstract

The effects of phagocytosis and killing on microbial structural integrity have not been well defined. Recently we have presented evidence suggesting that rapid killing of Escherichia coli in vitro by intact or disrupted rabbit polymorphonuclear leukocytes occurs without major structural disorganization (1, 2). This conclusion was based on the findings that: (a) degradation of structural constituents such as lipids and peptidoglycans was limited, (b) macromolecular synthesis, including the induction of/~-galactosidase continued after more than 95 % of the E.coli could no longer divide, (c) the killed E.coli population retained its ability to "step-up" macromolecular synthesis in response to enrichment of the medium with simple nutrients (3), and (d) by contrast, under identical conditions other microorganisms, such as gram-positive Micrococcus lysodeikticus and Bacillus megaterium, underwent more rapid and extensive destruction of envelope constituents and within minutes lost biosynthetic activity (i).This report concerns further studies on the effects of granulocytes on the envelope of E.coli. The results support our contention that killing of E.coli by granulocytes is not linked to general structural disruption. However, killing by granulocytes and purified fractions prepared from granulocytes is temporally closely related to an increase in permeability. Some of the properties of the activity in disrupted granulocytes that causes this permeability change in E.coli and certain other gram-negative microorganisms will be described.

Role of Charge and Hydrophobic Interactions in the Action of Bactericidal/Permeability-increasing Protein of Neutrophils on Gram-negative Bacteria

“…Generation of goat antibodies against purified rabbit BPI and preparation of IgG-rich fractions have been described (8 (3,12).…”

Partial characterization and purification of a rabbit granulocyte factor that increases permeability of Escherichia coli.

“…Sedimented granulocytes were resuspended in distilled water (3.0 X 108 granulocyte equivalents/ml) and ice-cold 0.4 N H2SO was added to a final concentration of 0.15 N H2SO4. The extraction, described more fully in a preceding paper (4) which contained at least as much biological activity per granulocyte equivalent as the homogenate with less than 5% of the homogenate protein. Normal rabbit alveolar macrophages (3.0 X 108 cells/ml) were extracted in the same manner.…”

“…Rapid killing of Escherichia coli by granulocytes occurs without gross microbial structural disorganization (1)(2)(3), but is associated with a discrete increase in permeability of the microbial envelope that is evident within minutes after the bacteria are exposed to granulocytes or bactericidal granulocyte fractions (4). Several investigators have proposed that the mode of action of bactericidal agents derived from granulocytes (5,6), or other sources (7,8), may include effects on permeability.…”

“…PI was measured by determining the susceptibility of E. coli to actinomycin D (Act D), an agent that normally does not cross E. coli's permeability barrier (11). Thus, unless indicated otherwise, PI was measured by determining the effect of a given fraction on bacterial [14C] leucine incorporation in the presence and absence of Act D as described in a preceding paper (4 Cycloheximide was added to all incubation mixtures in a final concentration of 0.5 mM to exclude incorporation of labeled amino acids into granulocyte protein. Incubations were carried out at 370C for 30 min.…”

“…E. coli (W) were grown in minimal medium buffered with triethanolamine at pH 7.75-7.9 and Serratia marcescens were grown in trypticase soy broth (Baltimore Biological Laboratories, Cockeysville, Md.) (4). The bacteria used were obtained from overnight cultures that were transferred to fresh medium and subcultured for approximately 2.5 h at 370C.…”