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

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.

Inactivation of the sfgtr4 Gene of Shigella flexneri Induces Biofilm Formation and Affects Bacterial Pathogenicity

“…Here, we showed that production and secretion of IcsA were not affected in the SfpgdA and Sfgtr4 mutants ( Figure 3 A), suggesting the existence of additional mechanisms involved in biofilm formation by Shigella . More recent study reported that the biofilm formation by Gram-negative Haemophilus influenzae was increased by deletion of galE gene and the remarkable availability of UDP-GlcNAc precursors [ 38 ]. The same study reported that the suppression of alternative lipooligosaccharides glycosyltransferase activity by UDP-Galactose epimerase enhanced murine lung infection and evasion of serum IgM [ 38 ].…”

“…One might consider that the ability of WT Shigella to modify its surface, by targeting either PG and/or LPS, is to subvert bacterial detection by the host immune system. Neutrophil-phagocytosed Shigella are killed rapidly by different mechanisms including proteolytic enzymes, antimicrobial proteins, and reactive oxygen species [ 38 ]. Thus, further investigations are needed to unravel mechanisms underpinning Shigella ’s circumvention and/or counteraction of the host’s immune responses.…”

Underlying Glycans Determine the Ability of Sialylated Lipooligosaccharide To Protect Nontypeable Haemophilus influenzae from Serum IgM and Complement

“…Specifically, we investigated antibody-dependent complement activity and identified structures on the surface of H. influenzae that are targets for binding and subsequent killing by serum antibodies and complement. Antibody and the classical pathway of complement are required for the bactericidal effect of normal human serum on NTHi (21,26,27). The majority of naturally occurring antibody directing the complement-mediated killing of NTHi is specific to the LOS, and anti-LOS IgM titers correlate with increased serum bactericidal activity (25).…”

“…While IgG antibodies against NTHi are present in most individuals, the majority of individuals lack abundant IgM against the LOS of wild-type (WT) NTHi (25). Nevertheless, bactericidal natural or naturally acquired serum IgM against NTHi mutants with truncated LOS structures has been detected (21,26), and these antibodies may recognize NTHi expressing LOS structures modified by phase variation or in response to environmental signals encountered during infection.…”

Learning from –omics strategies applied to uncover Haemophilus influenzae host-pathogen interactions: Current status and perspectives

“…Microevolution in experimental settings [60] , [61] B. Genome-wide genetic screening B1. HITS* based in vivo genetic screenings [12] , [67] , [68] B2. Tn-seq based in vitro genetic screenings [69] , [70] , [71] , [72] , [73] C. Genome-wide gene expression profiling C1.…”