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Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide.

ABSTRACT: Previous studies have shown that changes in the expression of the Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) epitope reactive with monoclonal antibody (MAb) 5G8 can be correlated with alterations in the virulence of some Hib strains. To identify the locus involved in expression of this particular LOS epitope, a genomic library was constructed in the plasmid shuttle vector pGJB103 from Hib strain DL42, which constitutively expressed LOS reactive with MAb 5G8. This library was used to transform a second Hib strain (DL180) that normally does not express this LOS epitope, and a recombinant clone was identified that bound MAb 5G8. Subcloning of different regions of the Hib DL42 DNA insert in this recombinant plasmid determined that a 1.9-kb EcoRI fragment, designated lex-2, was responsible for transforming Hib strain DL180 to reactivity with MAb 5G8. Nucleotide sequence analysis revealed the presence of two contiguous open reading frames (ORFs) in lex-2, the first of which contained 18 tandem repeats of the nucleotide tetramer GCAA near its 5' end. Sequence analysis of PCR-derived products from MAb 5G8-reactive and -nonreactive Hib DL180 colonies established that 18 GCAA repeats were associated with expression of the LOS epitope that bound MAb 5G8. Mutational analysis determined that a functional ORF 2 was essential for expression of the MAb 5G8-reactive LOS epitope. The nucleotide tetramer GCAA repeat present in ORF 1 was also detected in at least two different chromosomal regions in all Hib strains tested. The availability of the cloned lex-2 locus should facilitate future analysis of the complex regulatory mechanisms involved in expression of LOS epitopes by this pathogen.

SUBMITTER: Jarosik GP

PROVIDER: S-EPMC303199 | biostudies-other | 1994 Nov

REPOSITORIES: biostudies-other

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Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide.

Jarosik G P GP Hansen E J EJ

Infection and immunity 19941101 11

Previous studies have shown that changes in the expression of the Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) epitope reactive with monoclonal antibody (MAb) 5G8 can be correlated with alterations in the virulence of some Hib strains. To identify the locus involved in expression of this particular LOS epitope, a genomic library was constructed in the plasmid shuttle vector pGJB103 from Hib strain DL42, which constitutively expressed LOS reactive with MAb 5G8. This library was u ...[more]

PMID: 7523298

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Project description:Haemophilus influenzae has an absolute aerobic growth requirement for either heme, or iron in the presence of protoporphyrin IX. Both iron and heme in the mammalian host are strictly limited in their availability to invading microorganisms. Many bacterial species overcome iron limitation in their environment by the synthesis and secretion of small iron binding molecules termed siderophores, which bind iron and deliver it into the bacterial cell via specific siderophore receptor proteins on the bacterial cell surface. There are currently no reports of siderophore production or utilization by H. influenzae.Comparative genomics revealed a putative four gene operon in the recently sequenced nontypeable H. influenzae strain R2846 that encodes predicted proteins exhibiting significant identity at the amino acid level to proteins involved in the utilization of the siderophore ferrichrome in other bacterial species. No siderophore biosynthesis genes were identified in the R2846 genome. Both comparative genomics and a PCR based analysis identified several additional H. influenzae strains possessing this operon. In growth curve assays strains containing the genes were able to utilize ferrichrome as an iron source. H. influenzae strains lacking the operon were unable to obtain iron from ferrichrome. An insertional mutation in one gene of the operon abrogated the ability of strains to utilize ferrichrome. In addition transcription of genes in the identified operon were repressible by high iron/heme levels in the growth media.We have identified an iron/heme-repressible siderophore utilization locus present in several nontypeable H. influenzae strains. The same strains do not possess genes encoding proteins associated with siderophore synthesis. The siderophore utilization locus may enable the utilization of siderophores produced by other microorganisms in the polymicrobial environmental niche of the human nasopharynx colonized by H. influenzae. This is the first report of siderophore utilization by H. influenzae.

Project description:Haemophilus somnus undergoes antigenic and structural phase variation in its lipooligosaccharide (LOS). A gene (lob-1) containing repetitive 5'-CAAT-3' sequences that may, in part, contribute to phase variation was cloned and sequenced (T. J. Inzana et al., Infect. Immun. 65:4675-4681, 1997). We have now identified another putative gene (lob-2A) immediately upstream from lob-1. Lob-2A contained homology to several LOS biosynthesis proteins of the family Pasteurellaceae and the LgtB and LgtE galactosyltransferases of Neisseria meningitidis and N. gonorrhoeae. Unlike lob-1, lob-2A contained 18 to 20 5'-GA-3' repeats 141 bp upstream of the termination codon as determined by PCR amplification of DNA from individual colonies. Twenty repeats were most common, but when 19 5'-GA-3' repeats were present a stop codon would occur 1 bp after the last 5'-GA-3' repeat. A 630-bp SalI-BsgI fragment within lob-2A was deleted, and a kanamycin resistance (Km(r)) gene was inserted into this site to create pCAATDeltalob2A. Following electroporation of pCAATDeltalob2A into H. somnus 738, several allelic exchange mutants were isolated. The LOS electrophoretic profile of one mutant, strain 738-lob2A1::Km, was altered, and the phase variation rate was reduced but phase variation was not eliminated. A variant with 19 5'-GA-3' repeats in lob-2A had an LOS profile similar to that of 738-lob2A1::Km, suggesting that lob-2A was turned off in this phase. Nanoelectrospray mass spectrometry (nES-MS) and nuclear magnetic resonance spectroscopy showed that 738-lob2A1::Km was deficient in the terminal betaGal(1-3)betaGlcNAc residue present in parent strain 738. Mutant 738-lob2A1::Km was significantly more sensitive to the bactericidal action of normal bovine serum and was less virulent in mice than was parent strain 738. When H. somnus 129Pt was electrotransformed with shuttle vector pLS88 containing lob-2A, its LOS electrophoretic profile was modified and additional N-acetylhexosamine residues were present, as determined by nES-MS analysis. These results indicated that lob-2A may be an N-acetylglucosamine transferase involved in LOS biosynthesis and phase variation and that LOS structure is important to H. somnus virulence.

Dataset Information

Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide.

Publications

Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide.

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