Salmonella enterica serovar Typhi is a human-restricted Gram-negative bacterial pathogen responsible for causing an estimated 27 million cases of typhoid fever annually, leading to 217,000 deaths, and current vaccines do not offer full protection. The O-antigen side chain of the lipopolysaccharide is an immunodominant antigen, can define host-pathogen interactions, and is under consideration as a vaccine target for some Gram-negative species. The composition of the O-antigen can be modified by the activity of glycosyltransferase (gtr) operons acquired by horizontal gene transfer. Here we investigate the role of two gtr operons that we identified in the S Typhi genome. Strains were engineered to express specific gtr operons. Full chemical analysis of the O-antigens of these strains identified gtr-dependent glucosylation and acetylation. The glucosylated form of the O-antigen mediated enhanced survival in human serum and decreased complement binding. A single nucleotide deviation from an epigenetic phase variation signature sequence rendered the expression of this glucosylating gtr operon uniform in the population. In contrast, the expression of the acetylating gtrC gene is controlled by epigenetic phase variation. Acetylation did not affect serum survival, but phase variation can be an immune evasion mechanism, and thus, this modification may contribute to persistence in a host. In murine immunization studies, both O-antigen modifications were generally immunodominant. Our results emphasize that natural O-antigen modifications should be taken into consideration when assessing responses to vaccines, especially O-antigen-based vaccines, and that the Salmonellagtr repertoire may confound the protective efficacy of broad-ranging Salmonella lipopolysaccharide conjugate vaccines.

Lipopolysaccharide, Phase variation, O-antigen, Salmonella enterica, serum resistance

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
3	%Ac	?	23.1
	aLRhap	104.0	70.7	75.6	81.0	70.9	20.3


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
3	%Ac	-	2.14-2.16
	aLRhap	5.10	4.15	5.02	3.77	3.96	1.34


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
3	%Ac		23.1/2.14-2.16
	aLRhap	104.0/5.10	70.7/4.15	75.6/5.02	81.0/3.77	70.9/3.96	20.3/1.34

The spectrum also has 1 signal at unknown position (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](C)O[C@@H](*)[C@H](O)[C@@H]1OC(C)=O

188.179 g/mol (C₈H₁₂R₂O₅, R = next and previous repeats, not counted in mol weight)