Loss of DNA-membrane interactions and cessation of DNA synthesis in myeloperoxidase-treated Escherichia coli.

Abstract

Neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host defense functions. The mechanisms of action of most of these systems are incompletely understood. The present report indicates that microbicidal activity by a neutrophil-derived antimicrobial system, consisting of myeloperoxidase, enzymatically generated hydrogen peroxide, and chloride ion, is accompanied by prompt cessation of DNA synthesis in Escherichia coli, as determined by markedly reduced incorporation of [3H]thymidine into trichloracetic acid-precipitable material. Simultaneously, the myeloperoxidase system mediates a decline in the ability of E. coli membranes to bind hemimethylated DNA sequences containing the E. coli chromosomal origin of replication (oriC). Binding of oriC to the E. coli membrane is an essential element of orderly chromosomal DNA replication. Comparable early changes in DNA synthesis and DNAmembrane interactions were not observed with alternative oxidant or antibiotic-mediated microbicidal systems. It is proposed that oxidants generated by the myeloperoxidase system modify theE. coli membrane in such a fashion that oriC binding is markedly impaired. As a consequence chromosomal DNA replication is impaired and organisms can no longer replicate.Human neutrophils and monocytes employ a diverse array of antimicrobial effector systems to support their host-defense functions. Among these is one comprised of myeloperoxidase, hydrogen peroxide, and an oxidizable halide cofactor, usually chloride. The best characterized microbicide of the system is the hypohalous acid, HOCI, which gives rise to additional, derivative microbicidal oxidants, most notably chloramines (1,2). Characterization of the pathways by which loss of viability is effected by these oxidants remains an area of active investigation.Biochemical lesions induced by HOCI-in amounts just sufficient to cause loss of bacterial viability-are largely confined to the cell envelope. Membrane-associated functions that are inactivated in Escherichia coli include loss of FoF1 ATPase activity (3), succinate-dependent respiration (4, 5), nutrient transport (3, 6), and modification of proteins involved in remodeling the bacterial cell wall during growth and division (R.M.R. and H.R., unpublished observations). In contrast, cytosolic proteins with HOCI vulnerable sites, such as the sulfhydryl-containing enzyme aldolase, are oxidized in intact E. coli only by exposure to amounts of HOCI far in excess of those required to produce loss of viability (7,8). Thus, it appears that injuries to cell envelope structures precede those to cytosolic structures and that the surface injuries are sufficient to effect loss of viability.McKenna and Davies recently reported prompt cessation of DNA synthesis in E. coli incubated with low concentrations of HOCI (9). This inhibition was observed under conditions where protein synthesis was relatively spared and where membrane integrity was fully preserved. Among several proposed mechanisms for loss of DN...

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“…We have tested this idea, employing an otherwise isogenic pair of E. coli strains, one of which lacks GSH (16). The results affirm the importance of GSH as a defense against chlorine compounds (and, expectably, H 2 O 2 ).…”

“…Interestingly, no significant bacterial resistance to chlorine compounds (with the possible exception of Cryptosporidium parvum [13]) has been found, despite decades of water treatment with powerful chlorinebased oxidants. Previous studies have identified several potential bacterial targets of chlorine oxidants such as the electron transport chain (2), iron sulfur centers (15), the membraneassociated origin of DNA replication (16), adenine nucleotides (1-3), important metabolic enzymes such as succinate oxidase (17), and unsaturated fatty acids within membranes (26).…”

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