Alteration of the Cell Wall of Haemophilus influenzae Type b by Transformation with Cloned DNA: Association with Attenuated Virulence

Abstract

A virulent strain of Haemophilus influenzae type b was used to construct a lambda library of chromosomal DNA in Charon 4, amplified in Escherichia coli. From this library a recombinant (I-69) phage was isolated that contained a 10.2-kilobase-pair fragment of DNA eliciting H. influenzae transformants whose colonies had a distinctive opaque phenotype. Compared with their H. influenzae parent strains the opaque I-69 transformants had two defined cell wall alterations: one in the lipopolysaccharide (greater mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and one in the outer membrane proteins. The I-69 transformant of virulent type b strain Rd-/b+ had stable expression of type b capsule. In contrast to strain Rd-/b+, the Rd-/b+/I-69 transformant was serum sensitive in vitro and avirulent in vivo in rats. Thus the potential of H. influenzae type b organisms to cause invasive infection can be substantially attenuated by altering the expression of one or more genes that affect the cell wall composition.

Chemical structure of the lipopolysaccharide of Haemophilus influenzae strain I‐69 Rd−/b+

“…influenzae 1-69 Rd-/ b' is able to survive and multiply normally in vitro [4]. This shows that one (phosphorylated) dOclA in the lipopolysaccharide suffices for the survival of a gram-negative bacterium.…”

“…properties which are opposite to those found in the type-b-capsular, wildtype strains of H . injZuenzue [4]. This indicates that lipopolysaccharide is involved in modulating the virulence of H .…”

“…Furthermore, this strain differs from wild-type H . influenzae strains in that it is serum-sensitive and exhibits attenuated virulence in rats [4]. As will be shown, this lipopolysaccharide consists only of lipid A and one phosphorylated dOclA residue, and thus exhibits an exceptionally high degree of rough character.…”

“…Haemophilus injluenzae 1-69 Rd-/b+ [4] was cultivated to late logarithmic phase in trypticase soy broth (Difco, Detroit, USA) supplemented with yeast extract (30 g/l, Difco), hematin (10 mg/l, Sigma, St Louis, USA), and NAD (10 mg/l, Sigma). Cultivation was at constant pH 7.0 with aeration by atmospheric gas at 37°C in a fermenter (LKB, Sweden).…”

Characterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide of the deep rough mutant I‐69 Rd−/b+ of Haemophilus influenzae

“…Studies on the H. influenzae mutant strain I-69 Rd ¹ /b þ (hereafter referred to as I-69) revealed that it contained a truncated LPS composed of lipid A and a single phosphorylated Kdo residue (Zamze et al, 1987). I-69 was identified from a genomic library of chromosomal DNA in a lambda vector when it was observed that transformants using DNA from one of the lambda clones produced an opaque phenotype (Zwahlen et al, 1985). Such transformants were found to be exquisitely sensitive to complement-mediated killing by serum and were defective in either colonizing the nasopharynx or causing invasive infections of infant rats.…”