Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

et al. (2012) Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A. PLoS Pathog 8(10): e1002978. doi:10.1371/journal.ppat.1002978 Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A Mar Reine s 0 Enrique Llobet 0 Ka the M. Dahlstro m 0 Camino Pe rez-Gutie rrez 0 Catalina M. Llompart 0 Nuria Torrecabota 0 Tiina A. Salminen 0 Jose A. Bengoechea 0 Ralph R. Isberg, Tufts University School of Medicine, United States of America 0 1 Laboratory Microbial Pathogenesis, Fundacio d'Investigaci o Sanita`ria de les Illes Balears (FISIB), Recinto Hospital Joan March , Bunyola, Spain, 2 Consejo Superior de Investigaciones Cient ficas (CSIC), Madrid, Spain, 3 Structural Bioinformatics Laboratory , Department of Biosciences, A bo Akademi University , Turku , Finland Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21uC, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37uC, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 39-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37uC. Western blot analyses indicate that LpxR exhibits latency at 21uC, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo2-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21uC. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS. - Funding: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Fellowship support to C.M.L. from Govern Illes Balears is gratefully acknowledged. M.R. is the recipient of a JAE PreDOC fellowship (JAEPre_07_00250). This work has been funded by the Sigrid Juselius Foundation, and the Tor, Joe and Pentti Borgs Foundation to T.A.S. and by a grant from Consejo Superior de Investigaciones Cientficas (Spain) (Intramural Program 200820I174) to J.A.B. Competing Interests: The authors have declared that no competing interests exist. Lipopolysaccharide (LPS) is one of the major surface components of Gram-negative bacteria. The molecular structure of LPS is rather unique: an amphiphile with a hydrophobic region, the socalled lipid A, adjacent to a densely negatively charged polysaccharide. In Escherichia coli K-12, the lipid A is a b(19-6)-linked disaccharide of glucosamine phosphorylated at the 1 and 49 positions with positions 2, 3, 29, and 39acylated with R-3hydroxymyristoyl groups, the so-called lipid IVA. The 29and 39R-3-hydroxymyristoyl groups are further acylated with laureate (C12) and myristate (C14), respectively, by the action of the socalled late acyltransferases LpxL (HtrB) and LpxM (MsbB), respectively [1]. When E. coli is grown at 12uC, LpxP, the coldtemperature-specific late acyltransferase, acts instead of LpxL adding palmitoleate (C16:1) [1]. Although the enzymes required to synthesize the lipid A are conserved throughout all Gram-negative bacteria there is heterogeneity on lipid A structure among Gramnegative bacteria compared to the E. coli K-12. This is due to differences in the type and length of fatty acids, in the presence of decorations such as aminoarabinose or phosphoethanolamine and even in the removal of groups such as phosphates or fatty acids from lipid A [2]. LPS plays a crucial role during recognition of microbial infection by the host immune system. In fact, the lipid A moiety is a ligand of the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 complex [3]. The stimulation of this receptor complex triggers the activation of signalling cascades resulting in the induction of antimicrobial genes and release of cytokines, thereby initiating inflammatory and immune defence responses. Perusal of the literature demonstrates that changes in the number of acyl chains and in the phosphorylation status of the headgroup greatly affect the biological activity of lipid A. It is not surprising that some pathogens modulate their lipid A structure to alter their detection by the host; being these regulated changes important virulence traits (for a review see [4]). Furthermore, given the importance of the LPS structure to the homeostasis of the outer membrane, it is possible that the aforementioned changes may also affect the physiology of the outer membrane as was recently demonstrated for Salmonella [5]. Lipopolysaccharide (LPS) is one of the major surface components of Gram-negative bacteria. The LPS contains a molecular pattern recognized by the innate immune system. Not surprisingly, the modification of the LPS pattern is a virulence strategy of several pathogens to evade the innate immune system. Yersinia enterocolitica causes food-borne infections in animals and humans (yersiniosis). Temperature regulates most, if not all, virulence factors of yersiniae including the structure of the LPS lipid A. At 21uC, lipid A is mainly hexa-acylated and may be modified with aminoarabinose or palmitate. In contrast, at 37uC, Y. enterocolitica expresses a unique tetraacylated lipid A. In this work, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure expressed by the pathogen at 37uC, the host temperature. Our findings also revealed that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by a Yersinia protein translocated into the cytosol of macrophages and the reduced activation of the LPS receptor complex due to the expression of a LpxRdependent deacylated LPS. The g (...truncated)