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The Neisseria gonorrhoeae Obg protein is an essential ribosome-associated GTPase and a potential drug target.

Zielke RA, Wierzbicki IH, Baarda BI, Sikora AE - BMC Microbiol. (2015)

Bottom Line: Serious complications associated with these infections are frequent and include pelvic inflammatory disease, ectopic pregnancy, and infertility.The cellular levels of Obg reach a maximum in the early logarithmic phase and remain constant throughout bacterial growth.Characterization of the GC Obg at the molecular and functional levels presented herein may facilitate the future targeting of this protein with small molecule inhibitors and the evaluation of identified lead compounds for bactericidal activity against GC and other drug-resistant bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, 433 Weniger Hall, 103 SW Memorial Pl, Corvallis, OR, 97330, USA. Ryszard.Zielke@oregonstate.edu.

ABSTRACT

Background: Neisseria gonorrhoeae (GC) is a Gram-negative pathogen that most commonly infects mucosal surfaces, causing sexually transmitted urethritis in men and endocervicitis in women. Serious complications associated with these infections are frequent and include pelvic inflammatory disease, ectopic pregnancy, and infertility. The incidence of gonorrhea cases remains high globally while antibiotic treatment options, the sole counter measures against gonorrhea, are declining due to the remarkable ability of GC to acquire resistance. Evaluating of potential drug targets is essential to provide opportunities for developing antimicrobials with new mechanisms of action. We propose the GC Obg protein, belonging to the Obg/CgtA GTPase subfamily, as a potential target for the development of therapeutic interventions against gonorrhea, and in this study perform its initial functional and biochemical characterization.

Results: We report that NGO1990 encodes Obg protein, which is an essential factor for GC viability, associates predominantly with the large 50S ribosomal subunit, and is stably expressed under conditions relevant to infection of the human host. The anti-Obg antisera cross-reacts with a panel of contemporary GC clinical isolates, demonstrating the ubiquitous nature of Obg. The cellular levels of Obg reach a maximum in the early logarithmic phase and remain constant throughout bacterial growth. The in vitro binding and hydrolysis of the fluorescent guanine nucleotide analogs mant-GTP and mant-GDP by recombinant wild type and T192AT193A mutated variants of Obg are also assessed.

Conclusions: Characterization of the GC Obg at the molecular and functional levels presented herein may facilitate the future targeting of this protein with small molecule inhibitors and the evaluation of identified lead compounds for bactericidal activity against GC and other drug-resistant bacteria.

No MeSH data available.


Related in: MedlinePlus

Biochemical properties of ObgGC. a GTPase cycle. Obg GTPases oscillate between active (ON, GTP-bound) and inactive (OFF, GDP-bound) states. b ObgGC binds mant-GTP and mant-GDP with guanine nucleotide-specific Mg2+ dependence. Binding of mant-GTP (red circles) and mant-GDP (blue squares) to N-His-ObgGC was assessed in the presence of varying concentrations of Mg2+. The averages with SEM from three independent experiments are shown. c Increase in Relative Fluorescence Units (RFU) of mant-GTP (red bars) and mant-GDP (blue bars) upon addition of different ObgGC variants: recombinant wild type ObgGC with N- and C-terminal 6 × His tag (N-His-ObgGC and C-His-ObgGC, respectively), and N-His-ObgGC with T192AT193A substitutions. The data shows averages with corresponding SEM of at least eight experiments performed on separate occasions. d Hydrolysis of mant-GTP by N-His-ObgGC (green) and C-His-ObgGC (red) was monitored by recording the decrease in fluorescence that is coupled to the conversion of mant-GTP-Obg to mant-GDP-Obg complexes. Data from at least four experiments were fitted to a single exponential decay equation. The fluorescence intensity of the mant-GTP in the absence of protein served as a control and is shown in black
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Fig3: Biochemical properties of ObgGC. a GTPase cycle. Obg GTPases oscillate between active (ON, GTP-bound) and inactive (OFF, GDP-bound) states. b ObgGC binds mant-GTP and mant-GDP with guanine nucleotide-specific Mg2+ dependence. Binding of mant-GTP (red circles) and mant-GDP (blue squares) to N-His-ObgGC was assessed in the presence of varying concentrations of Mg2+. The averages with SEM from three independent experiments are shown. c Increase in Relative Fluorescence Units (RFU) of mant-GTP (red bars) and mant-GDP (blue bars) upon addition of different ObgGC variants: recombinant wild type ObgGC with N- and C-terminal 6 × His tag (N-His-ObgGC and C-His-ObgGC, respectively), and N-His-ObgGC with T192AT193A substitutions. The data shows averages with corresponding SEM of at least eight experiments performed on separate occasions. d Hydrolysis of mant-GTP by N-His-ObgGC (green) and C-His-ObgGC (red) was monitored by recording the decrease in fluorescence that is coupled to the conversion of mant-GTP-Obg to mant-GDP-Obg complexes. Data from at least four experiments were fitted to a single exponential decay equation. The fluorescence intensity of the mant-GTP in the absence of protein served as a control and is shown in black

Mentions: GTPases cycle between being turned “on” in the GTP-bound state and turned “off” in the GDP-bound state (Fig. 3a). In each state, G proteins undergo conformational changes and downstream effectors sense the GTP-bound protein complexes. Switch-off involves the exchange of GTP for GDP or hydrolysis of the γ-phosphate of GTP. The fluorescent N-methyl-3’-O-anthranoyl (mant) guanine nucleotide analogs, mant-GTP and mant-GDP, have been widely utilized for examining the nucleotide binding and GTP hydrolysis of various G-proteins including Obg homologs. The highly environmentally sensitive fluorescence of the mant group enables detection of nucleotide-protein interaction [22, 24, 26, 38, 40–43]. The binding of GTP to Obg requires the presence of physiological Mg2+ concentrations in C. crescentus, E. coli, and V. harveyi, whereas Obg-GDP complexes form over a wide range of Mg2+ concentrations [22, 24, 26]. The total intracellular Mg2+ content is about 100 mM in E. coli and B. subtilis and includes bound and free Mg2+, with the latter ranging from 1–5 mM [44–46].Fig. 3


The Neisseria gonorrhoeae Obg protein is an essential ribosome-associated GTPase and a potential drug target.

Zielke RA, Wierzbicki IH, Baarda BI, Sikora AE - BMC Microbiol. (2015)

Biochemical properties of ObgGC. a GTPase cycle. Obg GTPases oscillate between active (ON, GTP-bound) and inactive (OFF, GDP-bound) states. b ObgGC binds mant-GTP and mant-GDP with guanine nucleotide-specific Mg2+ dependence. Binding of mant-GTP (red circles) and mant-GDP (blue squares) to N-His-ObgGC was assessed in the presence of varying concentrations of Mg2+. The averages with SEM from three independent experiments are shown. c Increase in Relative Fluorescence Units (RFU) of mant-GTP (red bars) and mant-GDP (blue bars) upon addition of different ObgGC variants: recombinant wild type ObgGC with N- and C-terminal 6 × His tag (N-His-ObgGC and C-His-ObgGC, respectively), and N-His-ObgGC with T192AT193A substitutions. The data shows averages with corresponding SEM of at least eight experiments performed on separate occasions. d Hydrolysis of mant-GTP by N-His-ObgGC (green) and C-His-ObgGC (red) was monitored by recording the decrease in fluorescence that is coupled to the conversion of mant-GTP-Obg to mant-GDP-Obg complexes. Data from at least four experiments were fitted to a single exponential decay equation. The fluorescence intensity of the mant-GTP in the absence of protein served as a control and is shown in black
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Related In: Results  -  Collection

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Fig3: Biochemical properties of ObgGC. a GTPase cycle. Obg GTPases oscillate between active (ON, GTP-bound) and inactive (OFF, GDP-bound) states. b ObgGC binds mant-GTP and mant-GDP with guanine nucleotide-specific Mg2+ dependence. Binding of mant-GTP (red circles) and mant-GDP (blue squares) to N-His-ObgGC was assessed in the presence of varying concentrations of Mg2+. The averages with SEM from three independent experiments are shown. c Increase in Relative Fluorescence Units (RFU) of mant-GTP (red bars) and mant-GDP (blue bars) upon addition of different ObgGC variants: recombinant wild type ObgGC with N- and C-terminal 6 × His tag (N-His-ObgGC and C-His-ObgGC, respectively), and N-His-ObgGC with T192AT193A substitutions. The data shows averages with corresponding SEM of at least eight experiments performed on separate occasions. d Hydrolysis of mant-GTP by N-His-ObgGC (green) and C-His-ObgGC (red) was monitored by recording the decrease in fluorescence that is coupled to the conversion of mant-GTP-Obg to mant-GDP-Obg complexes. Data from at least four experiments were fitted to a single exponential decay equation. The fluorescence intensity of the mant-GTP in the absence of protein served as a control and is shown in black
Mentions: GTPases cycle between being turned “on” in the GTP-bound state and turned “off” in the GDP-bound state (Fig. 3a). In each state, G proteins undergo conformational changes and downstream effectors sense the GTP-bound protein complexes. Switch-off involves the exchange of GTP for GDP or hydrolysis of the γ-phosphate of GTP. The fluorescent N-methyl-3’-O-anthranoyl (mant) guanine nucleotide analogs, mant-GTP and mant-GDP, have been widely utilized for examining the nucleotide binding and GTP hydrolysis of various G-proteins including Obg homologs. The highly environmentally sensitive fluorescence of the mant group enables detection of nucleotide-protein interaction [22, 24, 26, 38, 40–43]. The binding of GTP to Obg requires the presence of physiological Mg2+ concentrations in C. crescentus, E. coli, and V. harveyi, whereas Obg-GDP complexes form over a wide range of Mg2+ concentrations [22, 24, 26]. The total intracellular Mg2+ content is about 100 mM in E. coli and B. subtilis and includes bound and free Mg2+, with the latter ranging from 1–5 mM [44–46].Fig. 3

Bottom Line: Serious complications associated with these infections are frequent and include pelvic inflammatory disease, ectopic pregnancy, and infertility.The cellular levels of Obg reach a maximum in the early logarithmic phase and remain constant throughout bacterial growth.Characterization of the GC Obg at the molecular and functional levels presented herein may facilitate the future targeting of this protein with small molecule inhibitors and the evaluation of identified lead compounds for bactericidal activity against GC and other drug-resistant bacteria.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, 433 Weniger Hall, 103 SW Memorial Pl, Corvallis, OR, 97330, USA. Ryszard.Zielke@oregonstate.edu.

ABSTRACT

Background: Neisseria gonorrhoeae (GC) is a Gram-negative pathogen that most commonly infects mucosal surfaces, causing sexually transmitted urethritis in men and endocervicitis in women. Serious complications associated with these infections are frequent and include pelvic inflammatory disease, ectopic pregnancy, and infertility. The incidence of gonorrhea cases remains high globally while antibiotic treatment options, the sole counter measures against gonorrhea, are declining due to the remarkable ability of GC to acquire resistance. Evaluating of potential drug targets is essential to provide opportunities for developing antimicrobials with new mechanisms of action. We propose the GC Obg protein, belonging to the Obg/CgtA GTPase subfamily, as a potential target for the development of therapeutic interventions against gonorrhea, and in this study perform its initial functional and biochemical characterization.

Results: We report that NGO1990 encodes Obg protein, which is an essential factor for GC viability, associates predominantly with the large 50S ribosomal subunit, and is stably expressed under conditions relevant to infection of the human host. The anti-Obg antisera cross-reacts with a panel of contemporary GC clinical isolates, demonstrating the ubiquitous nature of Obg. The cellular levels of Obg reach a maximum in the early logarithmic phase and remain constant throughout bacterial growth. The in vitro binding and hydrolysis of the fluorescent guanine nucleotide analogs mant-GTP and mant-GDP by recombinant wild type and T192AT193A mutated variants of Obg are also assessed.

Conclusions: Characterization of the GC Obg at the molecular and functional levels presented herein may facilitate the future targeting of this protein with small molecule inhibitors and the evaluation of identified lead compounds for bactericidal activity against GC and other drug-resistant bacteria.

No MeSH data available.


Related in: MedlinePlus