Signature Tagged Mutagenesis of Haemophilus influenzae

Herbert, Mark A. (2001). Signature Tagged Mutagenesis of Haemophilus influenzae. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f994

Abstract

Signature Tagged Mutagenesis (STM) is a validated means of creating and labelling transposon generated bacterial mutants that can be screened in vivo for loss of virulence. The pathogenic H. influenzae strain Rd-b+ was mutagenized with Tn1545-Ʌ3 and a variety of stable single gene disruption mutants were created. Tu1545-Ʌ3 was adapted to contain signature tags, and transposons that contained pre-selected tags, that amplified and hybridized efficiently, were introduced into Rd-b+ by conjugation.

A biologically relevant animal model, the five-day old infant rat model, was adapted for STM and two thousand two hundred H. influenzae mutants were screened for those that could not survive in vivo. One hundred and twenty six mutants were identified, of which 39 were almost completely unable to survive and 87 had reduced survival. Transposon insertion sites were amplified by ARB-PCR. One of the disrupted genes, IS1016, controls amplification of the type b capsulation locus and is a known virulence determinant; hence this hit validates the technique. The mutant did not express capsule implying that the capsulation locus may be under transcriptional control by IS 1016. Other mutants that were completely absent in output pools included those with hits in the genes encoding malate oxidoreductase and L-lactate permease. The implication is that lactate may be a major carbon source for H. influenzae in vivo and may be a signal for the phenotypic shift that occurs with in vivo growth and survival. A better appreciation of the dual role of some house keeping genes in virulence was gained through a collaborative study of the in vivo survival of central metabolic pathway mutants created by site-directed mutagenesis of H. influenzae (J. Reidl, Wurzburg).

STM has advanced the understanding of the pathogenesis and physiology of H. influenzae, and this knowledge may ultimately lead to the development of new vaccines and antibiotics.