Andrew D. Cox

Colistin Resistance in Acinetobacter baumannii Is Mediated by Complete Loss of Lipopolysaccharide Production

ABSTRACT 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.

The position of phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae affects binding and sensitivity to C-reactive protein-mediated killing.

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.

Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae.

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.

Identification of a gene (lpt-3) required for the addition of phosphoethanolamine to the lipopolysaccharide inner core of Neisseria meningitidis and its role in mediating susceptibility to bactericidal killing and opsonophagocytosis.

We have identified a gene, lpt‐3, that is required for the addition of phosphoethanolamine to the 3‐position (PEtn‐3) on the β‐chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn‐3 substituent is characteristic of the LPS of a majority (≈ 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface‐accessible epitope. All strains of Nm that have PEtn‐3 possess the lpt‐3 gene. In some lpt‐3‐containing strains, the 3‐position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt‐3 resulted in loss of PEtn‐3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt‐3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a candidate immunogen for inclusion in an LPS‐based vaccine to protect against invasive meningococcal disease.

Neisserial Lipooligosaccharide Is a Target for Complement Component C4b INNER CORE PHOSPHOETHANOLAMINE RESIDUES DEFINE C4b LINKAGE SPECIFICITY

We identified Neisseria meningitidis lipooligosaccharide (LOS) as an acceptor for complement component C4b (C4b). Phosphoethanolamine (PEA) residues on the second heptose (HepII) residue in the LOS core structure formed amide linkages with C4b. PEA at the 6-position of HepII (6-PEA) was more efficient than 3-PEA in binding C4b. Strains bearing 6-PEA bound more C4b than strains with 3-PEA and were more susceptible to complement-mediated killing in serum bactericidal assays. Deleting 3-PEA from a strain that expressed both 3- and 6-PEA simultaneously on HepII did not decrease C4b binding. Glycose chain extension of the first heptose residue (HepI) influenced the nature of the C4b-LOS linkage. Predominantly ester C4b-LOS bonds were seen when lacto-N-neotetraose formed the terminus of the glycose chain extension of HepI with 3-PEA on HepII in the LOS core. Related LOS species with more truncated chain extensions from HepI bound C4b via amide linkages to 3-PEA on HepII. However, 6-PEA in the LOS core bound C4b even when the glycose chain from HepI bore lacto-N-neotetraose at the terminus. The C4A isoform exclusively formed amide linkages, whereas C4B bound meningococci preferentially via ester linkages. These data may serve to explain the preponderance of 3-PEA-bearing meningococci among clinical isolates, because 6-PEA enhances C4b binding that may facilitate clearance of 6-PEA-bearing strains resulting from enhanced serum killing by the classical pathway of complement.

Transmission dynamic modelling of the impact of human papillomavirus vaccination in the United Kingdom.

Many countries are considering vaccination against human papillomavirus (HPV). However, the long-term impact of vaccination is difficult to predict due to uncertainty about the prevalence of HPV infection, pattern of sexual partnerships, progression of cervical neoplasias, accuracy of screening as well as the duration of infectiousness and immunity. Dynamic models of human papillomavirus (HPV) transmission were developed to describe the infection spread and development of cervical neoplasia, cervical cancer (squamous cell and adenocarcinoma) and anogenital warts. Using different combinations of assumptions, 9900 scenarios were created. Each scenario was then fitted to epidemiological data and the best-fitting scenarios used to predict the impact of vaccination. Results suggest that vaccinating 12-year-old girls at 80% coverage will result in a 38-82% reduction in cervical cancer incidence and 44-100% reduction in anogenital warts incidence after 60 years of an ongoing vaccination programme if vaccine protection lasts 20 years on average. The marginal benefit of vaccinating boys depends on the degree of protection achieved by vaccinating girls.

The K1 Capsular Polysaccharide from Acinetobacter baumannii Is a Potential Therapeutic Target via Passive Immunization

ABSTRACT The 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.

Identification and Characterization of a Glycosyltransferase Involved in Acinetobacter baumannii Lipopolysaccharide Core Biosynthesis

ABSTRACT Although Acinetobacter baumannii has emerged as a significant cause of nosocomial infections worldwide, there have been few investigations describing the factors important for A. baumannii persistence and pathogenesis. This paper describes the first reported identification of a glycosyltransferase, LpsB, involved in lipopolysaccharide (LPS) biosynthesis in A. baumannii. Mutational, structural, and complementation analyses indicated that LpsB is a core oligosaccharide glycosyl transferase. Using a genetic approach, lpsB was compared with the lpsB homologues of several A. baumannii strains. These analyses indicated that LpsB is highly conserved among A. baumannii isolates. Furthermore, we developed a monoclonal antibody, monoclonal antibody 13C11, which reacts to an LPS core epitope expressed by approximately one-third of the A. baumannii clinical isolates evaluated to date. Previous studies describing the heterogeneity of A. baumannii LPS were limited primarily to structural analyses; therefore, studies evaluating the correlation between these surface glycolipids and pathogenesis were warranted. Our data from an evaluation of LpsB mutant 307::TN17, which expresses a deeply truncated LPS glycoform consisting of only two 3-deoxy-d-manno-octulosonic acid residues and lipid A, suggest that A. baumannii LPS is important for resistance to normal human serum and confers a competitive advantage for survival in vivo. These results have important implications for the role of LPS in A. baumannii infections.

A Heptosyltransferase Mutant of Pasteurella multocida Produces a Truncated Lipopolysaccharide Structure and Is Attenuated in Virulence

ABSTRACT Pasteurella multocida is the causative agent of fowl cholera in birds. In a previous study using signature-tagged mutagenesis, we identified a mutant, AL251, which was attenuated for virulence in mice and in the natural chicken host. Sequence analysis indicated that AL251 had an insertional inactivation of the gene waaQPM, encoding a putative heptosyl transferase, required for the addition of heptose to lipopolysaccharide (LPS) (M. Harper, J. D. Boyce, I. W. Wilkie, and B. Adler, Infect. Immun. 71:5440-5446, 2003). In the present study, using mass spectrometry and nuclear magnetic resonance, we have confirmed the identity of the enzyme encoded by waaQPM as a heptosyl transferase III and demonstrated that the predominant LPS glycoforms isolated from this mutant are severely truncated. Complementation experiments demonstrated that providing a functional waaQPM gene in trans can restore both the LPS to its full length and growth in mice to wild-type levels. Furthermore, we have shown that mutant AL251 is unable to cause fowl cholera in chickens and that the attenuation observed is not due to increased serum sensitivity.

Cytosolic detection of the bacterial metabolite HBP activates TIFA-dependent innate immunity

Detecting Gramnegative bacteria Invariant molecules specific to different classes of microbes, but not expressed by eukaryotic cells, alert the immune system to a potential invader. Gaudet et al. identified one such molecule expressed by a variety of Gram-negative bacteria: the monosaccharide heptose-1,7-bisphosphate (HBP) (see the Perspective by Brubaker and Monack). HBP is an intermediate in the synthesis of lipopolysaccharide, a major component of bacterial cell walls. Rather than alerting the immune system through traditional pathogen detection pathways, such as Toll-like receptors, HBP signals through the host protein TIFA (TRAF-interacting protein with forkhead-associated domain), which activates both innate and adaptive immune responses to control the infection. Science, this issue p. 1251; see also p. 1207 Eukaryotic cells use the host protein TIFA to sense the monosaccharide HBP, derived from Gram-negative bacteria. [Also see Perspective by Brubaker and Monack] 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 factor 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.