Structure-Function Relationships of the Neisserial EptA Enzyme Responsible for Phosphoethanolamine Decoration of Lipid A: Rationale for Drug Targeting

Bacteria cause disease by two general mechanisms: the action of their toxins on host cells and induction of a pro-inflammatory response that can lead to a deleterious cytokine/chemokine response (e.g., the so-called cytokine storm) often seen in bacteremia/septicemia. These major mechanisms may overlap due to the action of surface structures that can have direct and indirect actions on phagocytic or non-phagocytic cells. In this respect, the lipid A (endotoxin) component of lipopolysaccharide (LPS) possessed by Gram-negative bacteria has been the subject of literally thousands of studies over the past century that clearly identified it as a key virulence factor in endotoxic shock. In addition to its capacity to modulate inflammatory responses, endotoxin can also modulate bacterial susceptibility to host antimicrobials, such as the host defense peptides termed cationic antimicrobial peptides. This review concentrates on the phosphoethanolamine (PEA) decoration of lipid A in the pathogenic species of the genus Neisseria [N. gonorrhoeae and N. meningitidis]. PEA decoration of lipid A is mediated by the enzyme EptA (formerly termed LptA) and promotes resistance to innate defense systems, induces the pro-inflammatory response and can influence the in vivo fitness of bacteria during infection. These important biological properties have driven efforts dealing with the biochemistry and structural biology of EptA that will facilitate the development of potential inhibitors that block PEA addition to lipid A

B Part of It protocol: a cluster randomised controlled trial to assess the impact of 4CMenB vaccine on pharyngeal carriage of Neisseria meningitidis in adolescents.

INTRODUCTION: South Australia (SA) has the highest notification rate of invasive meningococcal disease in Australia with the majority of cases due to serogroup B. Neisseria meningitidis is carried in the pharynx, with adolescents having the highest rates of carriage. A vaccine designed to offer protection against serogroup B (4CMenB) is licensed in Australia. The SA MenB vaccine carriage study aims to assess the impact of 4CMenB on carriage of N. meningitidis in adolescents. METHODS AND ANALYSIS: This is a parallel cluster randomised controlled trial enrolling year 10, 11 and 12 school students (approximately 16-18 years of age) throughout SA, in metropolitan and rural/remote areas. Schools are randomised to intervention (4CMenB vaccination at baseline) or control (4CMenB vaccination at study completion) with randomisation stratified by school size and socioeconomic status, as measured by the Index of Community Socio-Educational Advantage (Australian Curriculum). Oropharyngeal swabs will be taken from all students at visit 1, and 12 months later from year 11 and 12 students. Students unvaccinated in 2017 will receive vaccine at the 12-month follow-up. Carriage prevalence of N. meningitidis will be determined by PCR at baseline and 12 months following 4CMenB vaccination and compared with carriage prevalence at 12 months in unvaccinated students. A questionnaire will be completed at baseline and 12 months to assess risk factors associated with carriage. The primary outcome of carriage prevalence of disease causing N. meningitidis at 12 months will be compared between groups using logistic regression, with generalised estimating equations used to account for clustering at the school level. The difference in carriage prevalence between groups will be expressed as an OR with 95% CI. ETHICS AND DISSEMINATION: The study was approved by the Women's and Children's Health Network Human Research Ethics Committee (WCHN HREC). The protocol, informed consent forms, recruitment materials, social media and all participant materials have been reviewed and approved by the WCHN HREC and updated on ClinicalTrials.gov. Results will be published in international peer-reviewed journals and presented at national and international conferences. The study findings will be provided in public forums and to study participants and participating schools. TRIAL REGISTRATION NUMBER: ACTRN12617000079347. NCT03089086; Pre-results

Clonal expansion of new penicillin-resistant clade of neisseria meningitidis serogroup w clonal complex 11, Australia

In Western Australia, Neisseria meningitidis serogroup W clonal complex 11 became the predominant cause of invasive meningococcal disease in 2016. We used core-genome analysis to show emergence of a penicillin-resistant clade that had the penA_253 allele. This new penicillin-resistant clade might affect treatment regimens for this disease

Genomic epidemiology and population structure of Neisseria gonorrhoeae from remote highly endemic Western Australian populations

Background: Neisseria gonorrhoeae causes gonorrhoea, the second most commonly notified sexually transmitted infection in Australia. One of the highest notification rates of gonorrhoea is found in the remote regions of Western Australia (WA). Unlike isolates from the major Australian population centres, the remote community isolates have low rates of antimicrobial resistance (AMR). Population structure and whole-genome comparison of 59 isolates from the Western Australian N. gonorrhoeae collection were used to investigate relatedness of isolates cultured in the metropolitan and remote areas. Core genome phylogeny, multilocus sequencing typing (MLST), N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) in addition to hierarchical clustering of sequences were used to characterize the isolates. Results: Population structure analysis of the 59 isolates together with 72 isolates from an international collection, revealed six population groups suggesting that N. gonorrhoeae is a weakly clonal species. Two distinct population groups, Aus1 and Aus2, represented 63% of WA isolates and were mostly composed of the remote community isolates that carried no chromosomal AMR genotypes. In contrast, the Western Australian metropolitan isolates were frequently multi-drug resistant and belonged to population groups found in the international database, suggesting international transmission of the isolates. Conclusions: Our study suggests that the population structure of N. gonorrhoeae is distinct between the communities in remote and metropolitan WA. Given the high rate of AMR in metropolitan regions, ongoing surveillance is essential to ensure the enduring efficacy of the empiric gonorrhoea treatment in remote WA

The structure of the bacterial oxidoreductase enzyme DsbA in complex with a peptide reveals a basis for substrate specificity in the catalytic cycle of DsbA enzymes

Oxidative protein folding in Gram-negative bacteria results in the formation of disulfide bonds between pairs of cysteine residues. This is a multistep process in which the dithiol-disulfide oxidoreductase enzyme, DsbA, plays a central role. The structure of DsbA comprises an all helical domain of unknown function and a thioredoxin domain, where active site cysteines shuttle between an oxidized, substrate-bound, reduced form and a DsbB-bound form, where DsbB is a membrane protein that reoxidizes DsbA. Most DsbA enzymes interact with a wide variety of reduced substrates and show little specificity. However, a number of DsbA enzymes have now been identified that have narrow substrate repertoires and appear to interact specifically with a smaller number of substrates. The transient nature of the DsbA-substrate complex has hampered our understanding of the factors that govern the interaction of DsbA enzymes with their substrates. Here we report the crystal structure of a complex between Escherichia coli DsbA and a peptide with a sequence derived from a substrate. The binding site identified in the DsbA-peptide complex was distinct from that observed for DsbB in the DsbA-DsbB complex. The structure revealed details of the DsbA-peptide interaction and suggested a mechanism by which DsbA can simultaneously show broad specificity for substrates yet exhibit specificity for DsbB. This mode of binding was supported by solution nuclear magnetic resonance data as well as functional data, which demonstrated that the substrate specificity of DsbA could be modified via changes at the binding interface identified in the structure of the comple

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

The Gram-negative bacterial cell envelope consists of an inner membrane (IM) that surrounds the cytoplasm, and an asymmetrical outer-membrane (OM) that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS), phospholipids, outer membrane proteins (OMPs) and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the correct biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation and isomerisation pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, these conserved pathways have been modified to suit the lifestyle of each organism