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. 2017 Apr;6(2):e00432.
doi: 10.1002/mbo3.432. Epub 2017 Jan 7.

Functional and structural studies on the Neisseria gonorrhoeae GmhA, the first enzyme in the glycero-manno-heptose biosynthesis pathways, demonstrate a critical role in lipooligosaccharide synthesis and gonococcal viability

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Functional and structural studies on the Neisseria gonorrhoeae GmhA, the first enzyme in the glycero-manno-heptose biosynthesis pathways, demonstrate a critical role in lipooligosaccharide synthesis and gonococcal viability

Igor H Wierzbicki et al. Microbiologyopen. 2017 Apr.

Abstract

Sedoheptulose-7-phosphate isomerase, GmhA, is the first enzyme in the biosynthesis of nucleotide-activated-glycero-manno-heptoses and an attractive, yet underexploited, target for development of broad-spectrum antibiotics. We demonstrated that GmhA homologs in Neisseria gonorrhoeae and N. meningitidis (hereafter called GmhAGC and GmhANM , respectively) were interchangeable proteins essential for lipooligosaccharide (LOS) synthesis, and their depletion had adverse effects on neisserial viability. In contrast, the Escherichia coli ortholog failed to complement GmhAGC depletion. Furthermore, we showed that GmhAGC is a cytoplasmic enzyme with induced expression at mid-logarithmic phase, upon iron deprivation and anaerobiosis, and conserved in contemporary gonococcal clinical isolates including the 2016 WHO reference strains. The untagged GmhAGC crystallized as a tetramer in the closed conformation with four zinc ions in the active site, supporting that this is most likely the catalytically active conformation of the enzyme. Finally, site-directed mutagenesis studies showed that the active site residues E65 and H183 were important for LOS synthesis but not for GmhAGC function in bacterial viability. Our studies bring insights into the importance and mechanism of action of GmhA and may ultimately facilitate targeting the enzyme with small molecule inhibitors.

Keywords: Neisseria gonorrhoeae; crystal structure; drug target; sedoheptulose-7-phosphate isomerase GmhA.

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Figures

Figure 1
Figure 1
GmhAGC is pivotal for N. gonorrhoeae growth and lipooligosaccharide (LOS) synthesis. (a) The N. gonorrhoeae FA1090 gmh AGC conditional knockout strain, ∆gmh AGC/P lac ::gmh AGC, was streaked out from frozen glycerol stock on solid media supplemented with 20 μmol/L isopropyl β‐D‐1‐thiogalactopyranoside (IPTG). After 18 hr incubation at 37°C in the presence of 5% atmospheric CO 2, the colonies were passaged onto plates either with (+) or without IPTG (−). (b) Whole cell lysates of FA1090 wild‐type and isogenic ∆gmh AGC/P lac ::gmh AGC harvested from plates with (+) or without 20 μmol/L IPTG (−) were either probed with polyclonal rabbit antisera or subjected to LOS extraction using proteinase K followed by silver staining. (c–e) The FA1090 ∆gmh AGC/P lac ::gmh AGC cells were collected from solid media supplemented with 20 μmol/L IPTG, suspended to OD 600 of 0.1, washed twice, divided, and cultured either in the presence or absence of IPTG for 3 hr. At time point designated as 0 hr, corresponding cultures were back‐diluted to the same density (OD 600 of 0.1) into fresh media with or without IPTG and incubated for additional 6 hr. Samples of bacterial cultures were collected every hour for GmhAGC and Ng‐MIP immunoblotting analysis, LOS, and whole cell protein profiles (c), monitoring of bacterial proliferation by measurement of density of the cultures at OD 600, (d) and spotting serially diluted bacteria on solid media with IPTG for CFU scoring (e). Whole cell lysates were matched by the same OD 600 units. As loading control, samples separated by SDSPAGE were either probed with anti‐Ng‐MIP antibodies or stained with Coomassie brilliant blue G‐250. The migration of molecular mass marker (kDa) is indicated on the left. All experiments were performed in three biological replicates. Means and SEM are presented on graphs; *< .05
Figure 2
Figure 2
Assessment of GmhAGC subcellular localization and expression patterns. (a) N. gonorrhoeae wild‐type FA1090 cells were harvested during mid‐exponential phase and subjected to proteome extraction to separate cytoplasmic/periplasmic proteins (C), cell envelopes (CE), naturally released membrane vesicles (MVs), and soluble proteins in culture supernatants (SS). Individual fractions were loaded based on the total amount of protein (μg) and probed with antisera as indicated on the left. (b–c) Wild‐type N. gonorrhoeae FA1090 was cultured in liquid media and at indicated time points, OD 600 measurements were taken (b) and samples were withdrawn and processed for immunoblotting analysis of GmhAGC and Ng‐MIP (c). (d) Quantities of GmhAGC and lipooligosaccharide (LOS) in wild‐type FA1090 during in vitro conditions relevant to different infection sites [standard growth under aerobic conditions (SGC), iron deprivation (‐Iron), presence of normal human serum (+NHS), and anaerobiosis (‐O2)], were assessed by probing the whole cell lysates with anti‐GmhAGC antibodies and silver staining of proteinase K‐extracted LOS, respectively. Immunoblotting experiments with antisera against Ng‐MIP, TbpB, and AniA were used as markers for ubiquitous expression, iron‐limiting, and anaerobic conditions, respectively. (e) Effect of overexpression of GmhAGC on LOS amounts was examined by harvesting wild‐type FA1090 and isogenic ∆gmh AGC/P lac ::gmh AGC grown on solid media with different isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) concentrations (0, 20, or 1,000 μmol/L). Whole cell lysates were either probed with anti‐GmhAGC antisera or treated with proteinase K and LOS was visualized by silver staining. Experiments were performed in biological triplicates and representative immunoblots and silver‐stained gels are shown. Mean values and corresponding SEM are presented on the graph. Migration of a molecular mass marker (kDa) is indicated on the left
Figure 3
Figure 3
GmhAGC expression in a panel of N. gonorrhoeae isolates. Whole cell lysates of E. coli, wild‐type FA1090, ∆gmh AGC/P lac ::gmh AGC grown either with (+) or without (−) isopropyl β‐D‐1‐thiogalactopyranoside (IPTG), and 36 additional strains of N. gonorrhoeae were resolved on a 10–20% Tris‐Glycine gel and probed with anti‐GmhAGC antibodies. Immunoblotting with anti‐Zwf antisera was used as a loading control. Migration of a molecular mass marker (kDa) is indicated on the left
Figure 4
Figure 4
Hindering GmhAGC isomerase activity does not influence N. gonorrhoeae growth. (a) Wild‐type FA1090 and isogenic conditional mutants carrying either native gmh AGC (∆gmh AGC/P lac ::gmh AGC) or mutated variants of GmhAGC (∆gmh AGC/P lac ::gmh AGCE65A or ∆gmh AGC/P lac ::gmh AGCH183A) were collected from solid media with (+) and without (−) isopropyl β‐D‐1‐thiogalactopyranoside (IPTG). Expression of individual GmhAGC variants and lipooligosaccharide (LOS) patterns were examined in whole cell extracts by immunoblotting and silver staining, respectively. Samples were matched by equivalent OD 600 units. Migration of a molecular mass marker (kDa) is indicated on the left. (b) Wild‐type FA1090 and conditional mutants ∆gmh AGC/P lac ::gmh AGC, ∆gmh AGC/P lac ::gmh AGCE65A, and ∆gmh AGC/P lac ::gmh AGCH183A were collected from solid media supplemented with 20 μmol/L IPTG, suspended in liquid media to OD 600 of 0.1, cultured for 3 hr, back‐diluted to equal OD 600 of 0.2, serially diluted, and spotted on solid media in the presence (+) and absence (−) of IPTG. CFUs were scored. The data show averages of CFUs with corresponding SEM of at least three separate experiments; *< .05
Figure 5
Figure 5
Homologs of GmhA from N. gonorrhoeae and N. meningitidis function interchangeably. (a) N. gonorrhoeae FA1090 wild‐type and isogenic conditional gmhA mutants bearing either endogenous (∆gmh AGC/P lac ::gmh AGC) or N. meningitidis‐derived gmh ANM (∆gmh AGC/P lac ::gmh ANM), as well as N. meningitidis MC58 wild‐type and conditional gmhA mutants carrying gmhA alleles (∆gmh ANM/P lac ::gmh ANM or ∆gmh ANM/P lac ::gmh AGC) were harvested from GCB after 22 hr of incubation either with (+) or without (−) isopropyl β‐D‐1‐thiogalactopyranoside (IPTG). Whole cell lysates were probed with anti‐GmhAGC antibodies or treated with proteinase K and lipooligosaccharide (LOS) was visualized by silver staining. Migration of a molecular mass marker (kDa) is indicated on the left. (b) Wild‐type and mutant strains of N. gonorrhoeae and N. meningitidis, as described above, were collected from GCB supplemented with 20 μmol/L IPTG and 15 μmol/L IPTG, respectively. Bacteria were suspended in gonococcal base liquid (GCBL) to OD 600 of 0.1, cultured for 3 hr, back‐diluted to equal OD 600 of 0.2, serially diluted, and plated on GCB with (+) and without (−) IPTG. CFUs were counted following 22 hr of incubation. Experiments were performed in three independent replicates. Mean values and SEM are presented; *< .05
Figure 6
Figure 6
The structure of N. gonorrhoeae GmhAGC. (a) The ribbon representation of GmhAGC tetramer with monomers colored in green, yellow, cyan, and magenta. Zn2+ ions are shown as gray spheres. (b) The structure of GmhAGC monomer is colored in rainbow colors from blue (N‐terminus) to red (C‐terminus). The α‐helices are labeled according to Seetharaman et al., 2006. (c) A close‐up view of the Zn2+‐binding site of GmhAGC. The coordinating side chains are shown in stick representation. The σA‐weighted 2FOFC electron density map countered at 1.0 σ is displayed as blue mesh

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