Bactericidal substance produced by <i>Haemophilus influenzae b</i>

Venezia, Richard A.; Robertson, Richard G.

doi:10.1139/m75-232

Cited by 23 publications

(26 citation statements)

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“…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.…”

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

Venezia

Matusiak

Robertson

1977

Antimicrob Agents Chemother

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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 ...

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“…Media. Brain heart infusion (BHI) agar and 3.7% BHI broth (Difco) were prepared and supplemented with hemin (Kodak) and nicotinamide adenine dinucleotide (NAD; Sigma) as previously described (sBHI) (13).…”

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

Venezia

Matusiak

Robertson

1977

Antimicrob Agents Chemother

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“…influenzae, both encapsulated and nonencapsulated (LiPuma et al, 1990;Venezia & Robertson, 1975;Venezia et al, 1977). The exclusive production of H M C by type b encapsulated H .…”

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Cloning of the haemocin locus of Haemophilus influenzae type b and assessment of the role of haemocin in virulence

et al. 1998

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The bacteriocin haemocin (HMC) is produced by most type b strains of Haemophilus influenzae, including strains determined to be genetically diverse, and is toxic to virtually all non-type b strains of H. influenzae, both encapsulated and non-encapsulated. Examination of the deduced amino acid sequences of several genes upstream of the previously identified HMC immunity gene (hmcl) revealed several features common to class II bacteriocins of certain Gram-positive bacteria. Mutagenesis of the open reading frame immediately upstream of hmcl resulted in a loss of the HMC production phenotype. When an HMC-producing strain of H. influenzae and the HMC-deficient isogenic mutant were compared for invasion in the infant-rat model, the HMC-producing strain was found to invade significantly earlier; however, a significantly higher number of rats infected with the isogenic mutant became bacteraemic as compared with those infected with the HMCproducing parent.

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“…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.…”

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“…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.…”

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Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae

et al. 1997

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The bacteriocin haemocin is produced by most type b strains of Haemophilus influenzae, including strains of diverse genetic lineage, and is toxic to virtually all nontypeable H. influenzae strains. An H. influenzae transformant bearing a plasmid with a 1.5-kbp chromosomal fragment capable of conferring haemocin immunity on a haemocin-susceptible H. influenzae mutant was selected by using partially purified haemocin. Deletional and site-directed mutagenesis localized the haemocin immunity gene to the 3 open reading frame (ORF) within this chromosomal fragment. Subcloning of this ORF demonstrated that it was sufficient to confer haemocin immunity on wild-type haemocin-susceptible H. influenzae strains as well as haemocin-susceptible strains of Escherichia coli. This ORF, designated hmcI, encodes a 105-amino-acid protein with an estimated molecular mass of 12.6 kDa. 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.Bacterial cells capable of bacteriocin production must be immune to the toxic effects of the bacteriocin protein. This immunity is usually mediated via the coproduction of a specific immunity protein. A number of bacteriocin immunity genes have been cloned and characterized. The majority of these are located within gene clusters that also encode bacteriocin structural, transporter, and accessory proteins (1,14,26). Although they generally encode relatively small peptides, little homology has been observed among characterized bacteriocin immunity genes. In this report, we describe the identification, characterization, and heterologous expression of hmcI, the gene responsible for conferring immunity against the H. influenzae bacteriocin HMC. MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. Bacterial strains and plasmids used are listed in Table 1. Escherichia coli strains were cultivated in LuriaBertani medium (LB) with appropriate antibiotics. Antibiotic concentrations (in micrograms per milliliter) used for plasmid selection in E. coli were as follows: carbenicillin, 40 to 50; chloramphenicol, 20 to 25; and ribostamycin, 40. H. influenzae strains were grown in brain heart infusion broth supplemented with 10 g of hemin and 10 g of ␤-NAD (Sigma Chemical Co., St. Lou...

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Bactericidal substance produced by Haemophilus influenzae b

Cited by 23 publications

References 9 publications

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

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

Cloning of the haemocin locus of Haemophilus influenzae type b and assessment of the role of haemocin in virulence

Cloning and characterization of the haemocin immunity gene of Haemophilus influenzae

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