Identification of three oligo‐/polysaccharide‐specific ligases in <i>Yersinia enterocolitica</i>

Pinta, Elise; Li, Zhilin; Batzilla, Julia; Pajunen, Maria I.; Kasanen, Tiina; Rabsztyn, Kamila; Rakin, Alexander; Skurnik, Mikael

doi:10.1111/j.1365-2958.2011.07918.x

Cited by 17 publications

(23 citation statements)

References 60 publications

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“…LPS from the mutant strains and the WT counterparts was isolated using the modified protocol of Hitchcock & Brown (1983) as described previously (Pinta et al, 2012). In brief, 0.5 ml overnight bacterial culture grown at 22uC was pelleted in a microcentrifuge (13 000 g; 3 min).…”

Section: Rfah Of Y Enterocolitica O:3mentioning

confidence: 99%

“…Mouse mAb 898 specific for ECA recognizing ManpNAcA (Meier-Dieter et al, 1989), as well as mAbs TomA6 and 2B5, specific for Y. enterocolitica O:3 LPS O-antigen and OC, respectively, were used to analyse LPS (Al-Hendy et al, 1991a;Pekkola-Heino et al, 1987;Pinta et al, 2012). Peroxidase-conjugated anti-mouse antibodies (P0447; Dako Cytomation) were used as secondary antibodies in immunostaining.…”

Section: Rfah Of Y Enterocolitica O:3mentioning

confidence: 99%

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Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Leskinen

Varjosalo

et al. 2015

Microbiology

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The antiterminator RfaH is required for the expression of LPS, capsule, haemolysin, exotoxin, haemin uptake receptor and F pilus. As these structures are critical for bacterial virulence, loss of RfaH usually leads to attenuation. Here, we inactivated the rfaH gene of Yersinia enterocolitica O:3 to study its role in this enteropathogen. RNA sequencing of the WT and DrfaH strain transcriptomes revealed that RfaH acted as a highly specific regulator that enhanced the transcription of the operons involved in biosynthesis of LPS O-antigen and outer core (OC), but did not affect the expression of enterobacterial common antigen. Interestingly, the transcriptome of the DrfaH strain was very similar to that of an O-antigen-negative mutant. This indicated that some of the changes seen in the DrfaH strain, such as the genes involved in outer membrane homeostasis or in the stress-response-associated Cpx pathway, were actually due to indirect responses via the loss of O-antigen. The decreased amount of LPS on the DrfaH strain cell surface resulted in an attenuated stress response, and lower resistance to compounds such as SDS and polymyxin B. However, the DrfaH strain was significantly more resistant to complement-mediated killing by normal human serum. Taken together, our results revealed a novel role of RfaH acting as a highly specific regulator of O-antigen and OC of LPS in Y. enterocolitica O:3. It may be speculated that RfaH might have an in vivo role in controlling tissue-specific expression of bacterial surface oligo/polysaccharides.

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Section: Rfah Of Y Enterocolitica O:3mentioning

confidence: 99%

Section: Rfah Of Y Enterocolitica O:3mentioning

confidence: 99%

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Leskinen

Varjosalo

et al. 2015

Microbiology

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“…1). The LA-IC structure is very similar to that of other Yersinia; however, the IC of individual Y. enterocolitica O : 3 LPS moieties can be substituted by either OC or OPS but not by both and we suggested that this is due to the fact that both OC and OPS would be linked to the same Hep II residue of IC (Pinta et al, 2012). The OC is a hexasaccharide and the OPS is a homopolymer (Fig.…”

Section: Introductionmentioning

confidence: 85%

“…WaaL OS , OC-specific ligase; WaaL PS , OPS-specific ligase; ECA-specific ligase is unknown (Pinta et al, 2012). YeO3-trs24 mutant grown at 22 uC, the yield of LPS was slightly lower than from 37 uC and was 2.2 % and 2.7 %, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Mouse monoclonal antibody (mAb) 898 specific for ECA (Meier-Dieter et al, 1989), recognizing ManpNAcA was a kind gift of Professor D. Bitter-Suermann (Institute of Medical Microbiology, Medical School of Hannover, Germany). MAbs TomA6 and 2B5 which are specific for Y. enterocolitica O : 3 OPS and OC, respectively, have been described previously (al-Hendy et al, 1991a;Pekkola-Heino et al, 1987;Pinta et al, 2012). Horse-radish peroxidase (HRP) or alkaline phosphatase (AP) -conjugated secondary swine antirabbit or goat anti-mouse antibody (Dako A/S) was used as a secondary antibody in immunoblotting analysis.…”

Section: Methodsmentioning

confidence: 99%

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Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O : 3

et al. 2013

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Yersinia enterocolitica serotype O : 3 produces two types of lipopolysaccharide (LPS) molecules to its surface. In both types the lipid A (LA) structure is substituted by inner core (IC) octasaccharide to which either outer core (OC) hexasaccharide or homopolymeric O-polysaccharide (OPS) is linked. In addition, enterobacterial common antigen (ECA) can be covalently linked to LPS, however, via an unknown linkage. To elucidate the relationship between ECA and LPS in Y. enterocolitica O : 3 and the effect of temperature on their expression, LPS was isolated from bacteria grown at 22 6C and 37 6C by consequent hot phenol/water and phenol-chloroform-light petroleum extractions to obtain LPS preparations free of ECA linked to glycerophospholipid. In immunoblotting, monoclonal antibodies TomA6 and 898, specific for OPS and ECA, respectively, reacted both with ladder-like bands and with a slower-migrating smear suggesting that the ECA and OPS epitopes coexist on the same molecules. These results were supported by immunoblotting with a monovalent Y. enterocolitica O : 3 ECAspecific rabbit antiserum. Also, two or three 898-positive (and monovalent-positive) TomA6-negative bands migrated at the level of the LA-IC band in LPS samples from certain OC mutants, most likely representing LA-IC molecules carrying 1-3 ECA repeat units but no OPS. These bands were also present in Y. enterocolitica O : 9 OC mutants; however, coexistence of ECA and OPS in the same molecules could not be detected. Finally, the LA-IC-ECA bands were missing from LPS of bacteria grown at 37 6C and also the general reduction in wild-type bacteria of ECA-specific monovalentreactive material at 37 6C suggested that temperature regulates the expression of ECA. Indeed, RNAsequencing analysis showed significant downregulation of the ECA biosynthetic gene cluster at 37 6C.

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Yersinia Surface Structures and Bacteriophages

Skurnik

2012

Advances in Yersinia Research

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Identification of three oligo‐/polysaccharide‐specific ligases in Yersinia enterocolitica

Cited by 17 publications

References 60 publications

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

Enterobacterial common antigen and O-specific polysaccharide coexist in the lipopolysaccharide of Yersinia enterocolitica serotype O : 3

Yersinia Surface Structures and Bacteriophages

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