Bactericidal Factor Produced by Haemophilus influenzae b: Partial Purification of the Factor and Transfer of Its Genetic Determinant

Abstract

When aerobically grown on complex media, Haemophilus influenzae b and unencapsulated variants, Rb strains, produced a bactericidal factor that was active against other Haemophilus species and certain genera of the Enterobacteriaceae. A total of 341 clinical isolates of Haemophilus were tested for susceptibility to the factor. Ninety-three percent of H. influenzae (nontypable), 75% of H. haemolyticus, 71% of H. parainfluenzae, and 22% of H. parahaemolyticus were susceptible. H. influenaze b strains were resistant producers of the bactericidal factor and H. influenzae f strains were susceptible nonproducers. Only one strain each of H. aegyptius and H. aphrophilus was isolated and each was susceptible and resistant, respectively. 143 clinical isolates of the Enterobacteriaceae were tested and of those 82% ofEscherichia coli, 85% ofSalmonella sp., and all Citrobacter sp., Shigella sp., and Yersinia sp. were sensitive to the bactericidal factor produced by H. influenzae b. Attempts to isolate the bactericidal activity from mechanically disrupted, solubilized, or osmotically shocked cells failed to release active bactericidal factor. However, we partially purified the bactericidal factor from the spent culture medium of aerobically grown H. influenzae b by a series of extractions. The ability to produce the bactericidal factor was transferable to nonproducer strains without also genetically transforming for type b encapsulation. The converse was also true in that type b capsules were produced by transformed H. influenzae Rd strains but no bactericidal factor was detected from these strains. Additionally, nitrosoguanidineinduced mutants ofH. influenzae b lost the ability to produce bactericidal factor without loss of their type-specific capsule, demonstrating that production of the bactericidal factor was genetically separable from production of the type capsule of H. influenzae b.We have reported that Haemophilus influenzae b produces a bactericidal factor that is active against Haemophilus species and certain members of the Enterobacteriaceae but not against gram-positive bacteria (13). Preliminary characterization of the bactericidal factor showed that it was a heat-stable protein of between 50,000 and 100,000 daltons in size, as determined by membrane filter retention. It was produced throughout the growth cycle ofH. influenzae when cultured aerobically. The bactericidal factor appeared to be different from "colicin-like" bacteriocins in that its production was not inducible by mitomycin C, it did not kill by "single-hit" kinetics and, as opposed to the strain specificity of bacteriocin production, we have observed that all H. influenzae b strains produced the factor. None of the clinical nontypable H. influenzae strains tested produced the bactericidal factor. It is interesting that H. influenzae Rb, i.e., type b strains that have lost the ability to produce a capsule, retained the capacity to produce the bactericidal factor. We have hypothesized that capsule production occurs independently of bactericidal factor ...

Mode of action of the Haemophilus bactericidal factor

“…Preliminary characterization suggested that the bacteriocin haemocin was a thermostable protein molecule of between 10,000 and 14,000 daltons based on sodium dodecyl sulfategel electrophoresis (11). It was not a "colicinlike" bacteriocin in that it was not inducible by ultraviolet radiation or treatment with mitomycin C, had a wide range of activity, and did not kill by "single-hit" kinetics.…”

Interaction of Haemocin with Escherichia coli

“…Haemophilus influenzae b and exhibits bactericidal activity against Haemophilus species and certain members of the Enterobacteriaceae family (9,10). We previously have shown that its mode of action involves the inhibition of deoxyribonucleic acid synthesis in both H. influenzae Rd strain C25 and Escherichia coli CR34 (6).…”

“…Haemocin is bactericidal for H. influenzae types a, c, d, e, and f, other Haemophilhs species, and certain members of the Enterobactericeae family (9,10 In a previous report we demonstrated that survival curves of strain C25 treated with haemocin were not consistent with a single-hit mechanism of killing because of the shoulder obtained in the initial portion of the curves (10). However, the survival curves of strain CR34 treated with haemocin appeared to be different from those of strain C25 under similar conditions.…”

Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae

“…Primer extension analysis revealed a putative transcriptional start site 34 bp upstream of the start codon, and the presence of a promoter immediately upstream of hmcI was confirmed by cloning the gene into a promoterless chloramphenicol acetyltransferase vector. To characterize the hmcI gene product, a His-HmcI fusion protein was constructed.Most type b-encapsulated strains of Haemophilus influenzae, including strains demonstrated to be genetically diverse by multilocus enzyme electrophoresis (22), produce the bacteriocin haemocin (HMC), which is toxic in vitro to virtually all non-type b strains of H. influenzae, both encapsulated and nonencapsulated (16,37,38). Data suggest that HMC exerts its toxicity by inhibiting DNA synthesis in susceptible strains (34); however, little is known about the genetic determinants conferring HMC production or immunity in H. influenzae.…”

“…Most type b-encapsulated strains of Haemophilus influenzae, including strains demonstrated to be genetically diverse by multilocus enzyme electrophoresis (22), produce the bacteriocin haemocin (HMC), which is toxic in vitro to virtually all non-type b strains of H. influenzae, both encapsulated and nonencapsulated (16,37,38). Data suggest that HMC exerts its toxicity by inhibiting DNA synthesis in susceptible strains (34); however, little is known about the genetic determinants conferring HMC production or immunity in H. influenzae.…”