Phase-Variable Glycosylation in Nontypeable Haemophilus influenzae

Abstract

Non-typeable Haemophilus influenzae (NTHi) is a leading cause of respiratory tract infections worldwide and continues to be a global health burden. Adhesion and colonisation of host cells are crucial steps in bacterial pathogenesis, and in many strains of NTHi interaction with the host is mediated by the high molecular weight adhesins HMW1A and HMW2A. These adhesins are N-glycoproteins which are modified by cytoplasmic glycosyltransferases HMW1C and HMW2C. Phase variation in the number of short sequence repeats in the promoters of hmw1A and hmw2A directly affects their expression. Here, we report the presence of similar variable repeat elements in the promoters of hmw1C and hmw2C in diverse NTHi isolates. In an ex vivo assay, we systematically altered substrate and glycosyltransferase expression and showed that both of these factors affected the site-specific efficiency of glycosylation on HMW-A. Glycosylation occupancy was incomplete at many sites, variable between sites, and generally lower close to the C-terminus of HMW-A. We investigated the causes of this variability. As HMW-C glycosylates HMW-A in the cytoplasm, we tested how secretion affected glycosylation on HMW-A and showed that retaining HMW-A in the cytoplasm indeed increased glycosylation occupancy across the full length of the protein. Sitedirected mutagenesis showed that HMW-C had no inherent preference for glycosylating asparagines in NxS or NxT sequons. This work provides key insights into factors contributing to the heterogenous modifications of NTHi HMW-A adhesins, expands knowledge of NTHi population diversity and pathogenic capability, and is relevant to vaccine design for NTHi and related pathogens..

Semi-processive hyperglycosylation of adhesin by bacterial proteinN-glycosyltransferases

“…Our results suggest that glycoprotein production systems based on NGT expression in E. coli may suffer from the low UDP-Glc levels (typically, 1-2 mM), 56 as that may lead to premature product inhibition. In agreement with other reports, 31 we found that the glycosylation of HMW1ct is highly dependent on the levels of NGTs. This may limit the usefulness of future vaccines against glycosylated HMW adhesins, as bacteria may express different NGT levels under changing conditions, resulting in differently glycosylated adhesins.…”

“…This effect has been observed before in an ex vivo expression system of HiNGT and HMW1A (full-length H. influenzae adhesin), where the increasing expression of HMW1A resulted in a reduction of site-specific glycan occupancy. 31 We performed overnight glycosylation reactions in which the ratio ApNGT:HMW1ct was kept constant at 1:100, and the ratio of HiNGT:HMW1ct at 1:10, while the concentration of HMW1ct was varied from 5 μM to 100 μM ( Figure S9), and the UDP-Glc concentration was fixed at 1 mM. Indeed, upon increasing the concentration of HMW1ct in the ApNGT-catalyzed reaction, the final distribution of glycoforms reduced from 7-Glc to 9-Glc (5 μM HMW1ct) to 1-Glc to 5-Glc (100 μM HMW1ct).…”

Systematic Analysis of REBASE Identifies Numerous Type I Restriction-Modification Systems with Duplicated, Distinct hsdS Specificity Genes That Can Switch System Specificity by Recombination

“…SSR tracts located in the open reading frame of a gene can result in the gene being in frame and expressed (ON) or, due to a frameshift downstream of the SSR tract, out of frame and not expressed (OFF). SSR tracts also occur in the promoter of a number of genes ( 9 – 11 ), and variation in length of these SSRs can lead to ON-OFF switching of gene expression or result in a gradient of high- to low-level expression dependent on the length of the SSR tract ( 12 ). Several bacterial pathogens also contain well-characterized cytoplasmic N 6 -adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems, that exhibit phase-variable expression.…”

Streptococcus suis Encodes Multiple Allelic Variants of a Phase-Variable Type III DNA Methyltransferase, ModS, That Control Distinct Phasevarions

“…Phase variation is the rapid and reversible switching of gene expression, and it plays an important role in the pathogenesis of many organisms, where it is usually associated with expression of surface factors such as capsules (5) and adhesins (6,7) and with lipooligosaccharide biosynthesis (8). Randomized gene expression as a result of phase variation can complicate vaccine development, as it results in an unstable antigenic repertoire, and is potentially a major cause of vaccine escape.…”