1 Title: A mutant Escherichia coli that attaches peptidoglycan to lipopolysaccharide and 2 displays cell wall on its surface 3 Authors: Marcin Grabowicz1, Dorothee Andres2, Matthew D. Lebar2, Goran Malojčić2, Daniel 4 Kahne2,3*, Thomas J. Silhavy1* 5 Affiliations: 6 1Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. 7 2Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, 8 Cambridge, MA 02138, USA. 9 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 45 10 Shattuck Street, Boston, MA 02115, USA. 11 *Correspondence should be addressed to T.J.S. ([email protected]) or D.K. 12 ([email protected]). 13 Competing interests: The authors declare no competing interests. 14 15 The lipopolysaccharide (LPS) forms the surface-exposed leaflet of the outer 16 membrane (OM) of Gram-negative bacteria, an organelle that shields the underlying 17 peptidoglycan (PG) cell wall. Both LPS and PG are essential cell envelope components that 18 are synthesized independently and assembled by dedicated transenvelope multiprotein 19 complexes. We have identified a point-mutation in the gene for O-antigen ligase (WaaL) in 20 Escherichia coli that causes LPS to be modified with PG subunits, intersecting these two 21 pathways. Synthesis of the PG-modified LPS (LPS*) requires ready access to the small PG 22 precursor pool but does not weaken cell wall integrity, challenging models of precursor 23 sequestration at PG assembly machinery. LPS* is efficiently transported to the cell surface 24 without impairing OM function. Because LPS* contains the canonical vancomycin binding 25 site, these surface- exposed molecules confer increased vancomycin-resistance by 26 functioning as molecular decoys that titrate the antibiotic away from its intracellular 27 target. This unexpected LPS glycosylation fuses two potent pathogen-associated molecular 28 patterns (PAMPs). 29 A peptidoglycan (PG) cell wall is an essential extracytoplasmic feature of most 30 bacteria (Singer et al., 1989); this essentiality has made its biogenesis a fruitful target for 31 antibiotics, including vancomycin and penicillin. The cell wall is directly exposed to the 32 extracellular milieu in Gram-positive bacteria, but is shielded in E. coli and other Gram-negative 33 species by a highly selective permeability barrier formed by the outer membrane (OM). The OM 34 prevents influx of antibiotics, such as vancomycin, restricting their access to intracellular targets 35 (Eggert et al., 2001; Ruiz et al., 2005). Lipopolysaccharide (LPS) forms the surface-exposed 36 outer leaflet of the OM and is key to the barrier function (Osborn et al., 1972; Kamio and 37 Nikaido, 1976; Nikaido, 2003). LPS is a glycolipid consisting of a ‘lipid A’ anchor within the 38 bilayer, and a set of covalently attached distal ‘core’ saccharides (Raetz and Whitfield, 2002). 39 LPS is made at the cytosolic leaflet of the inner membrane (IM) before being flipped to the 40 periplasmic leaflet (Zhou et al., 1998). A transenvelope complex of seven lipopolysaccharide 41 transport proteins (LptABCDEFG) delivers LPS from the IM to the OM (Ruiz et al., 2009; 42 Chng, Gronenberg, et al., 2010). A sub-complex of the β-barrel LptD and lipoprotein LptE 43 resides within the OM and accomplishes the final step of inserting LPS into the outer leaflet 44 (Chng, Ruiz, et al., 2010). 45 A recently described lptE mutation (lptE613) causes defective LPS transport and leads to 46 increased antibiotic sensitivity (Malojčić et al., 2014). To better understand the basis of the 47 lptE613 defect, we isolated spontaneous suppressors that restored antibiotic resistance. One such 48 vancomycin-resistant suppressor mapped to the waaL gene, the product of which is an IM 49 glycosyltransferase that attaches O-antigen (O-Ag) oligosaccharides to LPS (Ruan et al., 2012; 50 Han et al., 2012). Indeed, the suppressed strain is certainly no more vancomycin sensitive than is 51 the corresponding wild-type control (Figure 1a). However, this suppressor (waaL15 herein) was 52 not specific for lptE613 or even for LPS transport defects. The waaL15 mutation increases 53 vancomycin resistance in strains carrying bamB or bamE null mutations that disrupt the OM 54 barrier by causing defects in -barrel protein assembly (Figure 1b) (Ricci and Silhavy, 2012). 55 Moreover, waaL15 also increases vancomycin-resistance even in the wild-type strain (Figure 56 1a). The suppressor does not qualitatively improve the OM barrier, since it did not increase 57 resistance against other antibiotics (Figure 1b). So, waaL15 provides a vancomycin-specific 58 resistance mechanism across different strains. 59 The domesticated E. coli K-12 does not produce the normal substrate (O-Ag) of WaaL 60 (Liu and Reeves, 1994) and a waaL deletion does not suppress vancomycin sensitivity, 61 indicating that waaL15 is a gain of function mutation. Thus, the WaaL15 mutant O-Ag ligase, 62 which contains an F332S substitution, must have an altered activity. Silver-staining of isolated 63 LPS confirmed that WaaL15 modifies LPS with additional sugars to produce an additional 64 glycoform (LPS*), detected as a higher molecular-weight band that is absent in waaL+ (Figure 65 2a). WaaL can use two minor saccharide substrates to modify LPS in E. coli K-12: 66 enterobacterial common-antigen (ECA) and colanic acid (CA). ECA-modified LPS is a minor 67 constituent of the OM (Schmidt et al., 1976; Meredith et al., 2007). Production of CA is 68 regulated by the Rcs phospho-relay stress response system, and CA-modified LPS (called ‘M- 69 LPS’) is only detectable during severe envelope stress (Meredith et al., 2007). Perhaps waaL15 70 had improved the utility of one, or both, of these substrates. However, LPS silver-staining 71 revealed that LPS* remained detectable when we inactivated biosynthesis of ECA (rff), CA 72 (cpsG), or both these polysaccharides (rff cpsG) (Figure 2a). Moreover, if we increase the 73 amounts of a competing substrate by introducing the rcsC137 mutation to activate expression of 74 the genes for CA biosynthesis (Gottesman et al., 1985), we actually observed lowered LPS* 75 abundance at the expense of increased M-LPS (Figure 2b). Notably, the decrease in LPS* 76 correlated with a significant reduction in vancomycin-resistance, providing evidence that LPS* 77 molecules directly mediate the resistance (Figure 2c). Similarly, if O-antigen biosynthesis is 78 restored by introducing a wild-type wbbL gene, we observe lowered LPS* at the expense of 79 wild-type LPS and vancomycin resistance is reduced. We conclude that WaaL15 is able to use a 80 new substrate and thereby generate a previously uncharacterized LPS glycoform that provides a 81 specific mechanism for vancomycin resistance. 82 All native WaaL substrates contain carbohydrates linked to a common undecaprenyl 83 (Und) lipid carrier. PG biosynthesis involves a disaccharide pentapeptide (DPP) linked to the 84 same Und carrier, a molecule called lipid II (Figure 3a). To directly determine if lipid II is a 85 substrate for WaaL15, we treated isolated LPS* with the muralytic enzyme mutanolysin (Figure 86 3a and 3b). Digestion of purified LPS*, but not LPS, liberated near-stoichiometric quantities of 87 fragments that were identified by mass spectrometry as DPP or derivatives with a tetrapeptide 88 stem (Figure 3c). Importantly, there is no evidence for cross-linked products suggesting that lipid 89 II was the source of the LPS* glycosylation (Lebar et al., 2013). 90 There are several carboxypeptidases in the periplasm that remove the terminal D-Alanine 91 (D-Ala) from DPP to produce the tetrapeptide derivative. Indeed, E. coli PG contains negligible 92 amounts of pentapeptide stems (Figure 3—figure supplement 1). Figure 3c shows that about 93 50% of the LPS* is sequestered before it can be attacked by one of these carboxypeptidases. It 94 seemed likely that sequestration happens because the molecule is transported from the periplasm 95 to the cell surface. 96 Peptide stems from adjacent peptidoglycan strands in the cell wall are cross-linked via 97 transpeptidation between the penultimate D-Ala on one stem and a meso-diaminopimelic acid 98 (m-DAP) residue on a nearby stem (Vollmer et al., 2008). Extensive cross-linking produces a 99 rigid macromolecular meshwork that is vital to cell wall function. Vancomycin binds and 100 sequesters the terminal D-Ala-D-Ala residues of a pentapeptide stem in order to inhibit 101 transpeptidation (Perkins, 1969). Since LPS* was the product of DPP ligation onto LPS, then 102 this modified glycoform should contain vancomycin binding sites. We assessed the ability of 103 purified LPS* to bind vancomycin in vitro. LPS* was immobilized on a carboxymethylated 104 dextran (CM3) chip and we used surface plasmon resonance to monitor interactions with 105 differing concentrations of vancomycin. We were able to measure specific binding of 106 vancomycin to LPS* and to obtain a Kd = 0.48 ± 0.08 µM (Figure 4a and Figure 4—figure 107 supplement 1), which is comparable to a reported Kd for vancomycin-lipid II interactions in 108 vesicles (Al-Kaddah et al., 2010). Clearly, LPS* molecules include high affinity binding sites for 109 vancomycin. 110 The ability of LPS* to directly bind vancomycin suggested a possible resistance 111 mechanism, namely that vancomycin is titrated outside the cell. To test this hypothesis, we 112 performed live cell microscopy using a fluorescent vancomycin-BODIPY. We used a wild-type 113 strain