Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.Acinetobacter baumannii is an emerging, opportunistic, Gram-negative bacterial pathogen (19). It is associated with a range of nosocomial infections, including bacteremia, pneumonia, meningitis, and urinary tract infections. Outbreaks, especially in intensive care unit settings, have been identified in numerous countries around the world (23). The treatment of these infections is hampered by the rapid rise in prevalence of A. baumannii strains that are resistant to almost all available antibiotics, including ␤-lactams, fluoroquinolones, tetracyclines, and aminoglycosides (23). In these multidrug-resistant (MDR) strains, colistin (also known as polymyxin E) is often the only remaining treatment (15), although colistin-resistant clinical isolates have already been reported (7,10,21). Intriguingly, some A. baumannii isolates have been shown to display heteroresistance to colistin, where an apparently colistin-susceptible strain (based upon the MIC) harbors a small proportion of colistin-resistant cells (9, 16). Under selective pressure both in vitro (33) and in vivo (10), heteroresistant A. baumannii strains can rapidly give rise to strains with high-level colistin resistance.Colistin is a cationic polypeptide antibiotic that is composed of a cyclic decapeptide linked by an ␣-amide linkage to a fatty acyl chain (15). Its structure differs from that of polymyxin B by only a single amino acid; the two antibiotics demonstrate comparable activities against a range of Gram-negative bacteria (6). Polymyxins are proposed to exert their antibacterial effect on Gram-negative bacteria via a two-step mechanism comprising initial binding to and permeabilization of the outer membrane, followed by destabilization of the cytoplasmic membrane (37). While the exact mechanism of bacterial killing is not clearly defined, a critical first step in the action of polymyxins is the electrostatic interaction between the positively charged peptide and the negatively charged ...

Host recognition of pathogen-associated molecular patterns (PAMPs) initiates an innate immune response that is critical for pathogen elimination and engagement of adaptive immunity. Here we show that mammalian cells can detect and respond to the bacterial-derived monosaccharide heptose-1,7-bisphosphate (HBP). A metabolic intermediate in lipopolysaccharide biosynthesis, HBP is highly conserved in Gram-negative bacteria, yet absent from eukaryotic cells. Detection of HBP within the host cytosol activated the nuclear facto κB pathway in vitro and induced innate and adaptive immune responses in vivo. Moreover, we used a genome-wide RNA interference screen to uncover an innate immune signaling axis, mediated by phosphorylation-dependent oligomerization of the TRAF-interacting protein with forkhead-associated domain (TIFA) that is triggered by HBP. Thus, HBP is a PAMP that activates TIFA-dependent immunity to Gram-negative bacteria.

The lic1 locus of Haemophilus influenzae controls the incorporation of environmental choline into lipopolysaccharide (LPS) as phosphorylcholine (ChoP) as well as the phase variation of this structure. ChoP is the target of an acute phase reactant in serum, C‐reactive protein (CRP), which mediates killing through the activation of complement when bound to the organism. Structural analysis of the oligosaccharide region of the H. influenzae LPS showed that ChoP is linked to different hexose residues on different chain extensions in strains Rd and Eagan. Differences in the molecular environment of ChoP affect the epitope defined by monoclonal antibody 12D9 and were associated with polymorphisms within LicD, a putative diphosphonucleoside choline transferase. Exchanging the licD genes between the two strains with ChoP on different chain extensions was sufficient to switch its position. Allelic variants with ChoP on a hexose on heptose III rather than heptose I were sensitive to CRP‐mediated serum bactericidal activity regardless of the genetic background. Differences in CRP‐mediated killing correlated with differences in the binding of CRP from human serum to whole bacteria. This suggests that, in addition to the mechanism involving phase variation, the structural rearrangements within the oligosaccharide contribute to evasion of innate and acquired immunity.

We have identified a gene for the addition of N‐acetylneuraminic acid (Neu5Ac) in an α‐2,3‐linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase‐variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N‐acetyl‐lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase‐variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non‐typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl‐α‐(2–3)‐lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an α‐2,3‐sialyltransferase activity, is the first reported phase‐variable sialyltransferase gene.

A gene, NMB1638, with homology to the recently characterized gene encoding a phosphoethanolamine transferase, lpt-3, has been identified from the Neisseria meningitidis genome sequence and was found to be present in all meningococcal strains examined. Homology comparison with other database sequences would suggest that NMB1638 and lpt-3 represent genes coding for members of a family of proteins of related function identified in a wide range of gram-negative species of bacteria. When grown and isolated under appropriate conditions, N. meningitidis elaborated lipopolysaccharide (LPS) containing a lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues. In all meningococcal strains examined, each lipid A species contained the basal diphosphorylated species, wherein a phosphate group is attached to each glucosamine residue. Also elaborated within the population of LPS molecules are a variety of "phosphoforms" that contain either an additional phosphate residue, an additional phosphoethanolamine residue, additional phosphate and phosphoethanolamine residues, or an additional phosphate and two phosphoethanolamine residues in the lipid A. Mass spectroscopic analyses of LPS from three strains in which NMB1638 had been inactivated by a specific mutation indicated that there were no phosphoethanolamine residues included in the lipid A region of the LPS and that there was no further phosphorylation of lipid A beyond one additional phosphate species. We propose that NMB1638 encodes a phosphoethanolamine transferase specific for lipid A and propose naming the gene "lptA," for "LPS phosphoethenolamine transferase for lipid A."Lipopolysaccharide (LPS) is found in all gram-negative bacteria and is usually composed of three regions: lipid A, core oligosaccharide (OS), and a polysaccharide molecule often referred to as the "O-antigen." LPS from Neisseria meningitidis lacks the polysaccharide and is sometimes referred to as "lipooligosaccharide" (LOS) to reflect this. The core OS unit of N. meningitidis LPS comprises an inner core diheptose-N-acetylglucosamine backbone, wherein the two L-glycero-D-mannoheptose (Hep) residues can provide a point of attachment for outer core OS residues (8). Meningococcal LPS has been classified into 12 distinct LPS immunotypes (L1 to L12), originally defined by monoclonal antibody (MAb) reactivities (21) but further defined by structural analyses (4,5,7,9,14,16,26). The structural basis of the immunotyping scheme is primarily governed by the location of a phosphoethanolamine (PEtn) moiety on the distal heptose residue (HepII) at either the 3-or 6-position or absent, but is also dictated by the length and nature of OS extension from the proximal heptose residue (HepI) and the presence or absence of a glucose at HepII (17 [ Fig. 1]). The lipid A region of the LPS is responsible for much of the toxicity of the LPS molecule, and LPS is indeed sometimes referred to as "endotoxin." The lipid A region consists of a disaccharide of pyranosyl glucosamine r...

A genetic basis for the biosynthetic assembly of the globotetraose containing lipopolysaccharide (LPS) of Haemophilus influenzae strain RM118 (Rd) was determined by structural analysis of LPS derived from mutant strains. We have previously shown that the parent strain RM118 elaborates a population of LPS molecules made up of a series of related glycoforms differing in the degree of oligosaccharide chain extension from the distal heptose residue of a conserved phosphorylated inner-core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)-]-L-alpha-D-Hepp-(1-->5)-alpha-Kdo. The fully extended LPS glycoform expresses the globotetraose structure, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp. A fingerprinting strategy was employed to establish the structure of LPS from strains mutated in putative glycosyltransferase genes compared to the parent strain. This involved glycose and linkage analysis on intact LPS samples and analysis of O-deacylated LPS samples by electrospray ionization mass spectrometry and 1D (1)H-nuclear magnetic resonance spectroscopy. Four genes, lpsA, lic2A, lgtC, and lgtD, were required for sequential addition of the glycoses to the terminal inner-core heptose to give the globotetraose structure. lgtC and lgtD were shown to encode glycosyltransferases by enzymatic assays with synthetic acceptor molecules. This is the first genetic blueprint determined for H. influenzae LPS oligosaccharide biosynthesis, identifying genes involved in the addition of each glycose residue.

fThe emergence of extremely resistant and panresistant Gram-negative bacilli, such as Acinetobacter baumannii, requires consideration of nonantimicrobial therapeutic approaches. The goal of this report was to evaluate the K1 capsular polysaccharide from A. baumannii as a passive immunization target. Its structure was determined by a combination of mass spectrometric and nuclear magnetic resonance (NMR) techniques. Molecular mimics that might raise the concern for autoimmune disease were not identified. Immunization of CD1 mice demonstrated that the K1 capsule is immunogenic. The monoclonal antibody (MAb) 13D6, which is directed against the K1 capsule from A. baumannii, was used to determine the seroprevalence of the K1 capsule in a collection of 100 A. baumannii strains. Thirteen percent of the A. baumannii isolates from this collection were seroreactive to MAb 13D6. Opsonization of K1-positive strains, but not K1-negative strains, with MAb 13D6 significantly increased neutrophil-mediated bactericidal activity in vitro (P < 0.05). Lastly, treatment with MAb 13D6 3 and 24 h after bacterial challenge in a rat soft tissue infection model resulted in a significant decrease in the growth/survival of a K1-positive strain compared to that of a K1-negative strain or to treatment with a vehicle control (P < 0.0001). These data support the proof of principle that the K1 capsule is a potential therapeutic target via passive immunization. Other serotypes require assessment, and pragmatic challenges exist, such as the need to serotype infecting strains and utilize serotype-specific therapy. Nonetheless, this approach may become an important therapeutic option with increasing antimicrobial resistance and a diminishing number of active antimicrobials.

Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5’-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic acid (Neu5Ac), a sialic acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation (“serum-resistance”). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, legionaminic acid (Leg5Ac7Ac) and pseudaminic acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from CMP-Leg5Ac7Ac-treated mice were sensitive to human complement ex vivo, simulating in vitro findings. These data reveal critical roles for the Sia exocyclic side-chain in gonococcal serum-resistance. Such CMP-NulO analogs may provide a novel therapeutic strategy against the global threat of multidrug-resistant gonorrhea.