The cell envelope of gram-negative bacteria consists of an inner (IM) and an outer membrane (OM) separated by an aqueous compartment, the periplasm, which contains the peptidoglycan layer. The OM is an asymmetric bilayer, with phospholipids in the inner leaflet and lipopolysaccharides (LPS) facing outward (29, 32). The OM is an effective permeability barrier that protects the cells from toxic compounds, such as antibiotics and detergents, thus allowing bacteria to inhabit several different and often hostile environments. LPS is responsible of most of the permeability properties of the OM and consists of the lipid A moiety (a glucosamine-based phospholipid) linked to the short core oligosaccharide and the distal O-antigen polysaccharide chain. The core oligosaccharide can be further divided into an inner core, composed of 3-deoxy-Dmanno-octulosanate (KDO) and heptose, and an outer core, which has a somewhat variable structure. LPS is essential in most gram-negative bacteria, with the notable exception of Neisseria meningitidis (39).The biogenesis of the OM implies that the individual components are transported from the site of synthesis to their final destination outside the IM by crossing both hydrophilic and hydrophobic compartments. The machinery and the energy source that drive this process are not yet understood.The lipid A-core moiety and the O-antigen repeat units are synthesized at the cytoplasmic face of the IM and are separately exported via two independent transport systems, namely, the O-antigen transporter Wzx (13, 17) and the ATP binding cassette (ABC) transporter MsbA that flips the lipid A-core moiety from the inner leaflet to the outer leaflet of the IM (12,28,45). O-antigen repeat units are then polymerized in the periplasm by the Wzy polymerase and ligated to the lipid A-core moiety by the WaaL ligase (reference 29 and references therein). Escherichia coli K-12 LPS is missing the O antigen, as an IS5 insertion disrupts its synthesis (18). Very recently, a modified LPS in which repeating units of colanic acid, a cell surface polysaccharide synthesized by enteric bacteria in the presence of envelope-damaging stresses (42), are ligated to the core oligosaccharide in a WaaL-dependent manner has been described (21).How LPS reaches the OM is less well understood. A protein complex in the OM of E. coli composed of LptD (formerly Imp), an essential ␤-barrel OM protein (6), and LptE (formerly RlpB), an essential OM lipoprotein, has recently been implicated in LPS assembly (43). Depletion of either protein results in similar OM biogenesis defects, including increased LPS levels, abnormal membrane structures, and activation of the OM enzyme PagP (43). These findings indicate that the LptD/LptE complex is responsible for LPS assembly at the outer surface of the OM (43). LptD has also been shown to be

Methicillin-resistantStaphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans 1,2 . Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients 3 . Previous vaccine development programs have not been successful 4 . A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc) 5,6 . It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS 7 , with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences.Novel prevention and treatment strategies against major antibioticresistant pathogens such as MRSA are urgently needed but are not within reach because some of the most critical virulence strategies of these pathogens are not understood 8 . The pathogenic potential of prominent healthcare-associated (HA)-MRSA and recently emerged livestock-associated (LA)-MRSA strains is thought to rely on particularly effective immune evasion strategies, whereas communityassociated (CA)-MRSA strains often produce more aggressive toxins 1,2 . Most humans have high overall levels of antibodies against S. aureus as a consequence of preceding infections, but antibody titres differ strongly for specific antigens and are often not protective in immunocompromised patients, for reasons that are not clear 3 . A large percentage of human antibodies against S. aureus is directed against WTA 5,9,10 , which is largely invariant. However, some S. aureus lineages produce altered WTA, which modulates, for instance, phage susceptibility 7,11 .To investigate whether ...

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.

In enterobacteria, the CsgD protein activates production of two extracellular structures: thin aggregative fimbriae (curli) and cellulose. While curli fibres promote biofilm formation and cell aggregation, the evidence for a direct role of cellulose as an additional determinant for biofilm formation is not as straightforward. The MG1655 laboratory strain of Escherichia coli only produces limited amounts of curli and cellulose; however, ectopic csgD expression results in strong stimulation of curli and cellulose production. We show that, in a csgD-overexpressing derivative of MG1655, cellulose production negatively affects curli-mediated surface adhesion and cell aggregation, thus acting as a negative determinant for biofilm formation. Consistent with this observation, deletion of the bcsA gene, necessary for cellulose production, resulted in a significant increase in curli-dependent adhesion. We found that cellulose production increased tolerance to desiccation, suggesting that the function of cellulose might be related to resistance to environmental stresses rather than to biofilm formation. Production of the curli/cellulose network in enterobacteria typically takes place at low growth temperature (,32 6C), but not at 37 6C. We show that CsgD overexpression can overcome temperature-dependent control of the curliencoding csgBA operon, but not of the cellulose-related adrA gene, suggesting very tight temperature control of cellulose production in E. coli MG1655.

Peptidoglycan (PGN) is a unique and essential structural part of the bacterial cell wall. PGNs from two contrasting Gram-negative plant pathogenic bacteria elicited components characteristic of the innate immune system in Arabidopsis thaliana, such as transcription of the defense gene PR1, oxidative burst, medium alkalinization, and formation of callose. Highly purified muropeptides from PGNs were more effective elicitors of early defense responses than native PGN. Therefore, PGN and its constituents represent a Microbe-Associated Molecular Pattern (MAMP) in plant-bacterial interactions. PGN and muropeptides from aggressive Xanthomonas campestris pv. campestris were significantly more active than those from Agrobacterium tumefaciens, which must maintain host cell viability during infection. The structure of muropeptide components and the distinctive differences are described. Differing defense-eliciting abilities appear to depend on subtle structural differences in either carbohydrate or peptide groups.