No abstract

Campylobacter jejuni polysaccharide capsules (CPS) are characterized by the presence of nonstoichiometric O-methyl phosphoramidate (MeOPN) modifications. The lack of stoichiometry is due to phase variation at homopolymeric tracts within the MeOPN transferase genes. C. jejuni strain 81-176 contains two MeOPN transferase genes and has been shown previously to contain MeOPN modifications at the 2 and 6 positions of the galactose (Gal) moiety in the CPS. We demonstrate here that one of the two MeOPN transferases, encoded by CJJ81176_1435, is bifunctional and is responsible for the addition of MeOPN to both the 2 and the 6 positions of Gal. A new MeOPN at the 4 position of Gal was observed in a mutant lacking the CJJ81176_1435 transferase and this was encoded by the CJJ81176_1420 transferase. During routine growth of 81-176, the CJJ81176_1420 transferase was predominantly in an off configuration, while the CJJ81176_1435 transferase was primarily on. However, exposure to normal human serum selected for cells expressing the CJJ81176_1420 transferase. MeOPN modifications appear to block binding of naturally occurring antibodies to the 81-176 CPS. The absence of MeOPN-4-Gal resulted in enhanced sensitivity to serum killing, whereas the loss of MeOPN-2-Gal and MeOPN-6-Gal resulted in enhanced resistance to serum killing, perhaps by allowing more MeOPN to be put onto the 4 position of Gal.IMPORTANCE Campylobacter jejuni undergoes phase variation in genes encoding surface antigens, leading to the concept that a strain of this organism consists of multiple genotypes that are selected for fitness in various environments. Methyl phosphoramidate modifications on the capsule of C. jejuni block access of preexisting antibodies in normal human sera to the polysaccharide chain, thus preventing activation of the classical arm of the complement cascade. We show that the capsule of strain 81-176 contains more sites of MeOPN modifications than previously recognized and that one site, on the 4 position of galactose, is more critical to complement resistance than the others. Exposure to normal human serum selects for variants in the population expressing this MeOPN modification.

Structural alterations in proteins have a significant impact on their function and body physiology. Glycation via nonenzymatic forms of cross-linking leads to proteins’ conformational changes, the macromolecule being recognized as a stable fibrillary structure, oligomerization, and becoming advanced glycation end products (AGEs). Protein that undergoes glycation-related modifications, namely, β-sheet enriched structural changes, are recognized as amyloid. In the current study, we characterized a single protein modified in vitro under physiological conditions to represent a protein glycation model. The glycation altered the helical conformation of serum albumin (SA) and promoted the formation of a β-sheet enriched with amyloid fibrils detected at multidimensional levels. The nanoscale resolution by spectroscopy in the presence of thioflavin-T (ThT) and 8-anilinonaphthalene-1-sulfonic acid (8-ANS) showed binding of the fibrils formed in the presence of glucose (GLU) and the carbonyl metabolites methylglyoxal (MGO) and glycolaldehyde (GAD). In the presence of MGO and GAD, the SA becomes insoluble aggregates, demonstrated by TEM microscopy and dynamic light scattering (DLS). The protein oligomerization was visualized when separated via SDS gel electrophoresis and mass photometry (MP) assays. Following the glycation, eventually, the material polymerized and became stiffer. The level of stiffness was analyzed by a rheometer that revealed a quick alteration under MGO and GAD. This is the first study to combine multiple spectroscopy assays, imaging, and rheology measurements of SA and to demonstrate a resolution on a nanoscale structural toward better resolution of the conformational changes of glycated SA, oligomerization, and protein aggregations under physiological conditions.

Enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni (CJ), and Shigella sp. are major causes of bacterial diarrhea worldwide, but there are no licensed vaccines against any of these pathogens. Most current approaches to ETEC vaccines are based on recombinant proteins that are involved in virulence, particularly adhesins. In contrast, approaches to Shigella and CJ vaccines have included conjugate vaccines in which Shigella lipopolysaccharides (LPS) or CJ capsule polysaccharides are chemically conjugated to proteins. We have explored the feasibility of developing a multi-pathogen vaccine by using ETEC proteins as conjugating partners for CJ and Shigella polysaccharides. We synthesized three vaccines in which two CJ polysaccharides were conjugated to two recombinant ETEC adhesins based on CFA/I (CfaEB) and CS6 (CssBA), and LPS from Shigella flexneri was also conjugated to CfaEB. The vaccines were immunogenic in mice as monovalent, bivalent and trivalent formulations. Importantly, functional antibodies capable of inducing hemaglutination inhibition (HAI) of a CFA/I expressing ETEC strain were induced in all vaccines containing CfaEB. These data suggest that conjugate vaccines could be a platform for a multi-pathogen, multi-serotype vaccine against the three major causes of diarrheal disease worldwide.

The Campylobacter jejuni capsule type HS1 complex is one of the most common serotypes identified worldwide, and consists of strains typing as HS1, HS1/44, HS44 and HS1/8. The capsule structure of the HS1 type strain was shown previously to be composed of teichoic-acid like glycerol-galactosyl phosphate repeats [4-)-α-D-Galp-(1–2)-Gro-(1-P-] with non-stoichiometric fructose branches at the C2 and C3 of Gal and non-stoichiometric methyl phosphoramidate (MeOPN) modifications on the C3 of the fructose. Here, we demonstrate that the capsule of an HS1/44 strain is identical to that of the type strain of HS1, and the capsule of HS1/8 is also identical to HS1, except for an additional site of MeOPN modification at C6 of Gal. The DNA sequence of the capsule locus of an HS44 strain included an insertion of 10 genes, and the strain expressed two capsules, one identical to the HS1 type strain, but with no fructose branches, and another composed of heptoses and MeOPN. We also characterize a HS1 capsule biosynthesis gene, HS1.08, as a fructose transferase responsible for the attachment of the β-D-fructofuranoses residues at C2 and C3 of the Gal unit. In summary, the common component of all members of the HS1 complex is the teichoic-acid like backbone that is likely responsible for the observed sero-cross reactivity.