Suppression of Alternative Lipooligosaccharide Glycosyltransferase Activity by UDP-Galactose Epimerase Enhances Murine Lung Infection and Evasion of Serum IgM.

Sandy M Wong,Mary Darby Jackson,Brian J Akerley

doi:10.3389/fcimb.2019.00160

Sandy M Wong, Mary Darby Jackson + Show 1 more

Open Access

https://doi.org/10.3389/fcimb.2019.00160

Copy DOI

Abstract

In pathogens that produce lipooligosaccharide (LOS), sugar residues within the surface-exposed LOS outer core mediate interactions with components of the host immune system, promoting bacterial infection. Many LOS structures are controlled by phase variation mediated by random slipped-strand base mispairing, which can reversibly switch gene expression on or off. Phase variation diversifies the LOS, however its adaptive role is not well-understood. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen that causes a range of illnesses in the upper and lower respiratory tract. In NTHi a phase variable galactosyltransferase encoded by lic2A initiates galactose chain extension of the LOS outer core. The donor substrate for Lic2A, UDP-galactose, is generated from UDP-glucose by UDP-galactose epimerase encoded by galE. Our previous fitness profiling of H. influenzae mutants in a murine lung model showed that the galE mutant had a severe survival defect, while the lic2A mutant's defect was modest, leading us to postulate that unidentified factors act as suppressors of potential defects in a lic2A mutant. Herein we conducted a genome-wide genetic interaction screen to identify genes epistatic on lic2A for survival in the murine lung. An unexpected finding was that galE mutants exhibited restored virulence properties in a lic2A mutant background. We identified an alternative antibody epitope generated by Lic2A in the galE mutant that increased sensitivity to classical complement mediated killing in human serum. Deletion of lic2A or restoration of UDP-galactose synthesis alleviated the galE mutant's virulence defects. These studies indicate that when deprived of its galactosyl substrate, Lic2A acquires an alternative activity leading to increased recognition of NTHi by IgM and decreased survival in the lung model. Biofilm formation was increased by deletion of galE and by increased availability of UDP-GlcNAc precursors that can compete with UDP-galactose production. NTHi's ability to reversibly inactivate lic2A by phase-variation may influence survival in niches of infection in which UDP-Galactose levels are limiting.

Highlights

Lipooligosaccharide (LOS), a short chain form of lipopolysaccharide (LPS) that lacks the long repetitive polysaccharide O-antigen of LPS, is the principal component of the outer-membrane of bacteria that produce it, mediating interactions with host immune defenses that determine the outcome of infection
In H. influenzae, LOS structure varies between strains, yet several features are conserved such as lipid A, an inner core usually composed of a single 3-deoxy-D-mannooctulosonic acid linked to three heptose residues, and an outer core containing a short heteropolymer of glucose and galactose residues in different configurations extending from the heptosyl residues (Moxon and Murphy, 2000)
The lic2A gene is subject to reversible inactivation by phase variation mediated by 5′CAAT-3′ repeats in its coding region (High et al, 1996), and this approach can reveal genes that may be required during natural infection in which lic2A can reversibly switch off by this mechanism

Summary

Introduction

Lipooligosaccharide (LOS), a short chain form of lipopolysaccharide (LPS) that lacks the long repetitive polysaccharide O-antigen of LPS, is the principal component of the outer-membrane of bacteria that produce it, mediating interactions with host immune defenses that determine the outcome of infection. Haemophilus influenzae, a Gram-negative bacterium that colonizes the human respiratory tract, causes a range of illnesses including otitis media, sinusitis, and exacerbation of chronic obstructive pulmonary disease (Klein, 1997; Murphy et al, 2004; Wiertsema et al, 2011; Ahearn et al, 2017). In H. influenzae, LOS structure varies between strains, yet several features are conserved such as lipid A, an inner core usually composed of a single 3-deoxy-D-mannooctulosonic acid linked to three heptose residues, and an outer core containing a short heteropolymer of glucose and galactose residues in different configurations extending from the heptosyl residues (Moxon and Murphy, 2000). Glucose and galactose extensions of the outer core often terminate in host-like structures including sialic acid, phosphorylcholine (PC), and N-acetylgalactosamine (Hood et al, 1999; Risberg et al, 1999) mediating numerous aspects of immune evasion. PC modification of LOS promotes persistence in the respiratory tract in an infant rat model of infection (Weiser et al, 1998), and mutants deficient in the N-acetylgalactosamine structure exhibit reduced lethality in a mouse model of bacteremia (Hood et al, 1996)

Methods

Results

Conclusion