Synthesis and Use of Mechanism-Based Protein-Profiling Probes for Retaining β-d-Glucosaminidases Facilitate Identification of Pseudomonas aeruginosa NagZ

The NagZ class of retaining exo-glucosaminidases play a critical role in peptidoglycan recycling in Gram-negative bacteria and the induction of resistance to beta-lactams. Here we describe the concise synthesis of 2-azidoacetyl-2-deoxy-5-fluoro-beta-d-glucopyranosyl fluoride as an activity-based proteomics probe for profiling these exo-glycosidases. This active-site directed reagent covalently inactivates this class of retaining N-acetylglucosaminidases with exquisite selectivity by stabilizing the glycosyl-enzyme intermediate. Inactivated Vibrio cholerae NagZ can be elaborated with biotin or a FLAG-peptide epitope using the Staudinger ligation or the Sharpless-Meldal click reaction and detected at nanogram levels. This ABPP enabled the profiling of the Pseudomonas aeruginosa proteome and identification at endogenous levels of a tagged protein with properties consistent with those of PA3005. Cloning of the gene encoding this hypothetical protein and biochemical characterization enabled unambiguous assignment of this hypothetical protein as a NagZ. The identification and cloning of this NagZ may facilitate the development of strategies to circumvent resistance to beta-lactams in this human pathogen. As well, this general strategy, involving such 5-fluoro inactivators, may prove to be of general use for profiling proteomes and identifying glycoside hydrolases of medical importance or having desirable properties for biotechnology.&nbsp

Insights from the redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains

H. pylori is a Gram-negative extracellular bacterium, first discovered by the Australian physicians Barry Marshall and Robin Warren in 1982, that colonises the human stomach mucosa. It is the leading cause of peptic ulcer and commonly infects humans worldwide with prevalence as high as 90% in some countries. H. pylori infection usually results in asymptomatic chronic gastritis, however 10-15% of cases develop duodenal or gastric ulcers and 1-3% develop stomach cancer. Infection is generally acquired during childhood and persists for life in the absence of antibiotic treatment. H. pylori has had a long period of co-evolution with humans, going back to human migration out of Africa. This prolonged relationship is likely to have shaped the overall host-pathogen interactions and repertoire of virulence strategies which H. pylori employs to establish robust colonisation, escape immune responses and persist in the gastric niche. In this regard, H. pylori lipopolysaccharide (LPS) is a key surface determinant in establishing colonisation and persistence via host mimicry and resistance to cationic antimicrobial peptides. Thus, elucidation of the H. pylori LPS structure and corresponding biosynthetic pathway represents an important step towards better understanding of H. pylori pathogenesis and the development of novel therapeutic interventions

Xer Recombinase and Genome Integrity in Helicobacter pylori, a Pathogen without Topoisomerase IV

In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis

Syntheses and biological investigations of kirkamide and oseltamivir hybrid derivatives

The C7N-aminocyclitol kirkamide was recently isolated from the plant obligate symbiont Candidatus Burkholderia kirkii and was hypothesized to be beneficial to the plant host due to its cytotoxic activity against insects and arthropods. To study its mechanism of action and inspired by its structural similarity with N-acetylglucosamine (GlcNAc) and oseltamivir, kirkamide-oseltamivir hybrid derivatives were synthesized and investigated for their biological activity. Interestingly, kirkamide analogues were reasonably potent against a known bacterial neuraminidase

Lipopolysaccharide Structural Differences between Western and Asian Helicobacter pylori Strains

Recent structural analysis of the lipopolysaccharide (LPS) isolated from Helicobacter pylori G27 wild-type and O-antigen ligase mutant resulted in the redefinition of the core-oligosaccharide and O-antigen domains. The short core-oligosaccharide (Glc–Gal–Hep-III–Hep-II–Hep-I–KDO) and its attached trisaccharide (Trio, GlcNAc–Fuc–Hep) appear to be highly conserved structures among H. pylori strains. The G27 LPS contains a linear glucan–heptan linker between the core-Trio and distal Lewis antigens. This linker domain was commonly identified in Western strains. In contrast, out of 12 partial LPS structures of Asian strains, none displayed the heptan moiety, despite the presence of Lewis antigens. This raises the question of how Lewis antigens are attached to the Trio, and whether the LPS structure of Asian strains contain another linker. Of note, a riban was identified as a linker in LPS of the mouse-adapted SS1 strain, suggesting that alternative linker structures can occur. In summary, additional full structural analyses of LPS in Asian strains are required to assess the presence or absence of an alternative linker in these strains. It will also be interesting to study the glucan-heptan linker moieties in pathogenesis as H. pylori infections in Asia are usually more symptomatic than the ones presented in the Western world

XerH-mediated recombination at mutant <i>difH</i> sequences.

<p>(A) The four A's in positions 19 to 22 of the <i>difH</i> sequence were changed to T's (empty stars), the G in position 18 was changed to C (black star) and the C in position 23 was changed to G (grey star). PCR tests were carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033310#pone-0033310-g001" target="_blank">Figure 1</a> on a clone harbouring WT <i>difH</i> repeats and two clones harbouring each mutant <i>difH</i> sequence (same sequence in each copy of <i>difH</i>) as indicated by the stars, <i>difH</i> M1, <i>difH</i> M2 and <i>difH</i> M3. No PCR product reflecting <i>difH</i> recombination was detected in <i>difH</i> M1, <i>difH</i> M2 mutants, whereas a weak band reflecting <i>difH</i> recombination was detected in the two clones with <i>difH</i> M3 sequences. Underlined nucleotides were found in a palindrome in all species studied. (B) <i>difH</i> recombination frequencies for WT <i>difH</i> and the <i>difH</i> M1, <i>difH</i> M2 and <i>difH</i> M3 mutant sequences carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033310#pone-0033310-g002" target="_blank">Figure 2B</a>. Experiments were performed in triplicate; horizontal bars indicate means and standard deviations.</p