The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

capsular polysaccharide, Campylobacter jejuni, capsule modification genes

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O[C@H](CO)[C@@H]2O[C@@H](O[C@@H]3[C@@H](C(=O)NC(CO)CO)O[C@H](*)[C@H](O)[C@H]3O[C@@H]3O[C@@H]([C@@H](CO)OC)[C@H](OP(N)(=O)OC)[C@@H](OC)[C@@H]3O)[C@H](NC(C)=O)[C@H]2OP(N)(=O)OC)O[C@H](CO)[C@H]1O

990.793 g/mol (C₃₃H₆₀R₂N₄O₂₆P₂, R = next and previous repeats, not counted in mol weight)

The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

capsular polysaccharide, Campylobacter jejuni, capsule modification genes

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6	C7
4,5	bDRibf	106.3	?	?	?	?
4,2	Ac
4	bDGalfN	106.1	?	75.4	?	?	?
3,3	Me	60.8
3,6	Me	60.8
3	aXDLglcHepp	98.3	?	?	?	?	?	?
6	xXEtN
	aDGlcpA	99.0	?	?	?	?	?


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6	H7
4,5	bDRibf	5.36	?	?	?	?
4,2	Ac
4	bDGalfN	4.97	?	4.25	?	?	?
3,3	Me	3.63
3,6	Me	3.56
3	aXDLglcHepp	5.58	?	?	?	?	?	?
6	xXEtN
	aDGlcpA	5.14	?	?	?	?	-


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6	C7/H7
4,5	bDRibf	106.3/5.36	?/?	?/?	?/?	?/?
4,2	Ac
4	bDGalfN	106.1/4.97	?/?	75.4/4.25	?/?	?/?	?/?
3,3	Me	60.8/3.63
3,6	Me	60.8/3.56
3	aXDLglcHepp	98.3/5.58	?/?	?/?	?/?	?/?	?/?	?/?
6	xXEtN
	aDGlcpA	99.0/5.14	?/?	?/?	?/?	?/?	

The spectrum also has 19 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O[C@H](CO)[C@@H]2O[C@@H](O[C@@H]3[C@@H](C(=O)NCCO)O[C@H](*)[C@H](O)[C@H]3O[C@@H]3O[C@@H]([C@@H](CO)OC)[C@H](O)[C@@H](OC)[C@@H]3O)[C@H](NC(C)=O)[C@H]2O)O[C@H](CO)[C@H]1O

774.723 g/mol (C₃₀H₅₀R₂N₂O₂₁, R = next and previous repeats, not counted in mol weight)

The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

capsular polysaccharide, Campylobacter jejuni, capsule modification genes

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,5	bDRibf	106.0	?	?	?	?
4,2	Ac
4,3	Subst	55.0
4	bDGalfN	106.9	?	81.0	?	?	?
	aDGlcpA	99.0	?	?	?	?	?
6	x?GroN
6	xXEtN


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,5	bDRibf	5.38	?	?	?	?
4,2	Ac
4,3	Subst	3.77
4	bDGalfN	5.03	?	4.76	?	?	?
	aDGlcpA	5.22	?	?	?	?	-
6	x?GroN
6	xXEtN


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,5	bDRibf	106.0/5.38	?/?	?/?	?/?	?/?
4,2	Ac
4,3	Subst	55.0/3.77
4	bDGalfN	106.9/5.03	?/?	81.0/4.76	?/?	?/?	?/?
	aDGlcpA	99.0/5.22	?/?	?/?	?/?	?/?	
6	x?GroN
6	xXEtN

The spectrum also has 13 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O[C@H](CO)[C@@H]2O[C@@H](O[C@@H]3[C@@H](C(=O)NC(CO)CO)O[C@H](*)[C@H](O)[C@H]3O)[C@H](NC(C)=O)[C@H]2OP(N)(=O)OC)O[C@H](CO)[C@H]1O

677.55 g/mol (C₂₃H₄₀R₂N₃O₁₈P, R = next and previous repeats, not counted in mol weight)

The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.

capsular polysaccharide, Campylobacter jejuni, capsule modification genes

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,5	bDRibf	105.9	?	?	?	?
4,2	Ac
4	bDGalfN	106.7	?	76.8	?	?	?
	aDGlcpA	99.0	?	?	?	?	?
6	x?GroN
6	xXEtN


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,5	bDRibf	5.38	?	?	?	?
4,2	Ac
4	bDGalfN	4.98	?	4.17	?	?	?
	aDGlcpA	5.22	?	?	?	?	-
6	x?GroN
6	xXEtN


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,5	bDRibf	105.9/5.38	?/?	?/?	?/?	?/?
4,2	Ac
4	bDGalfN	106.7/4.98	?/?	76.8/4.17	?/?	?/?	?/?
	aDGlcpA	99.0/5.22	?/?	?/?	?/?	?/?	
6	x?GroN
6	xXEtN

The spectrum also has 13 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@H]1[C@H](O[C@H](CO)[C@@H]2O[C@@H](O[C@@H]3[C@@H](C(=O)NC(CO)CO)O[C@H](*)[C@H](O)[C@H]3O)[C@H](NC(C)=O)[C@H]2O)O[C@H](CO)[C@H]1O

584.528 g/mol (C₂₂H₃₆R₂N₂O₁₆, R = next and previous repeats, not counted in mol weight)