Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo , Which Can Be Used as a Target for Bactericidal Antibody

Abstract

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N-acetylneuramic acid (Neu5Ac) moiety to a terminal N-acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (>90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi.

Panel 8: Vaccines and immunology

“…Among surfaceexposed proteins identified as essential for NTHi survival, antibodies against Hel1 and Hel2 prevented bacteremia in a rat model [54]. A monoclonal antibody against ketodeoxyoctanoate (KDO) was bactericidal against NTHi in vitro and KDO decoration of lipooligosaccharide within NTHi biofilms from the chinchilla middle ear was shown ex vivo [55]. Immunization of chinchillas with outer membrane vesicles isolated from HMW1/ HMW2-and Hia-expressing NTHi prevented experimental OM [56].…”

“…While parenteral immunization routes (subcutaneous and intramuscular) are the mainstay approach to assess the immunogenicity and efficacy of most preclinical and clinical vaccine candidates [50][51][52][53][54][55][56]81], greater emphasis has more recently been placed on alternative immunization regimens for preventing otopathogen colonization or OM. Herein we provide an update on alternative approaches being explored for delivery of novel OM vaccines.…”

Analysis of Invasive Nontypeable Haemophilus influenzae Isolates Reveals Selection for the Expression State of Particular Phase-Variable Lipooligosaccharide Biosynthetic Genes

“…Addition of a ketodeoxyoctanoate (KDO) residue as the terminal sugar of LOS rather than N -acetylneuraminic acid (Neu5Ac) (Fig. 1) is present during NTHi biofilm formation in vivo (36), meaning this modification may cause chronic infection with NTHi. Previous studies examining the role of LOS phase variation in NTHi pathobiology during infection of human volunteers have investigated the ON/OFF status of LOS biosynthetic genes (19, 37) and have shown selection for particular ON/OFF states: lex2A and lic1A were shown to switch from OFF to ON during nasopharyngeal colonization (37).…”

Lipopolysaccharides: structure, function and bacterial identification

“…LOS vary with environment and culture conditions, their cores are often longer than usual cores and can extend up to 15 sugars. In non-typable H. influenzae (NTHi), a distal Kdo or Neu5Ac could be added to the distal part of the structure on Nacetyllactosamine (Apicella et al, 2018).…”