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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

Obg is a primarily cytoplasmic protein that predominantly fractionates together with the 50S ribosomal particles. a Samples of isolated subproteome fractions (as indicated; 15 μg) obtained from wild type FA1090 cultured under regular aerobic laboratory growth conditions were separated in 4-20 % gradient gels and probed with either polyclonal rabbit anti-ObgGC antisera or monoclonal mouse anti-MtrE antibodies. b FA1090 cell lysate was subjected to separation on a 10-47 % step sucrose gradient by ultracentrifugation at 174,000 × g for 4 h and monitored by UV absorbance at 254 nm. The positions of the 30S and 50S ribosomal subunits, the 70S monosomes, and polyribosomes are indicated. The level of ObgGC in the collected fractions was detected by immunoblotting with polyclonal rabbit anti-ObgGC antibodies. A representative polyribosome profile and immunoblot are shown
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Fig6: Obg is a primarily cytoplasmic protein that predominantly fractionates together with the 50S ribosomal particles. a Samples of isolated subproteome fractions (as indicated; 15 μg) obtained from wild type FA1090 cultured under regular aerobic laboratory growth conditions were separated in 4-20 % gradient gels and probed with either polyclonal rabbit anti-ObgGC antisera or monoclonal mouse anti-MtrE antibodies. b FA1090 cell lysate was subjected to separation on a 10-47 % step sucrose gradient by ultracentrifugation at 174,000 × g for 4 h and monitored by UV absorbance at 254 nm. The positions of the 30S and 50S ribosomal subunits, the 70S monosomes, and polyribosomes are indicated. The level of ObgGC in the collected fractions was detected by immunoblotting with polyclonal rabbit anti-ObgGC antibodies. A representative polyribosome profile and immunoblot are shown

Mentions: To examine the cellular localization of ObgGC, wild type FA1090 was cultured under standard laboratory conditions in GCBL and harvested at the mid-exponential phase of growth. The bacterial cells were lysed and the cell envelope proteins were separated from the cytosolic proteome by a sodium carbonate extraction procedure [58]. The crude cell envelopes were treated with sarkosyl to solubilize the inner membrane proteins, and the outer membrane was recovered by ultracentrifugation [59]. The same total amounts of purified subproteomes (15 μg) were separated by SDS-PAGE and probed with anti-ObgGC antisera (Fig. 6a). The outer membrane protein, MtrE, which contains an extended periplasmic tunnel [60], was used as the cell envelope protein marker. As expected, MtrE was absent in the cytosol and enriched in the sarkosyl-insoluble fraction, whereas the vast majority of ObgGC was present in the cytosolic protein fractions (Fig. 6a). A faint band of the same molecular weight was also detected in the cytoplasmic membrane. Many cytoplasmic proteins are repeatedly identified in different cell envelope proteomics studies and are often considered “contaminants” [61]. However, recent thorough sequential biochemical fractionations of E. coli, combined with mass spectrometry, demonstrated that many of these proteins, including Obg, form an actual peripheral inner membrane proteome linked via functional and/or structural oligomeric complexes [62].Fig. 6


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)

Obg is a primarily cytoplasmic protein that predominantly fractionates together with the 50S ribosomal particles. a Samples of isolated subproteome fractions (as indicated; 15 μg) obtained from wild type FA1090 cultured under regular aerobic laboratory growth conditions were separated in 4-20 % gradient gels and probed with either polyclonal rabbit anti-ObgGC antisera or monoclonal mouse anti-MtrE antibodies. b FA1090 cell lysate was subjected to separation on a 10-47 % step sucrose gradient by ultracentrifugation at 174,000 × g for 4 h and monitored by UV absorbance at 254 nm. The positions of the 30S and 50S ribosomal subunits, the 70S monosomes, and polyribosomes are indicated. The level of ObgGC in the collected fractions was detected by immunoblotting with polyclonal rabbit anti-ObgGC antibodies. A representative polyribosome profile and immunoblot are shown
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487204&req=5

Fig6: Obg is a primarily cytoplasmic protein that predominantly fractionates together with the 50S ribosomal particles. a Samples of isolated subproteome fractions (as indicated; 15 μg) obtained from wild type FA1090 cultured under regular aerobic laboratory growth conditions were separated in 4-20 % gradient gels and probed with either polyclonal rabbit anti-ObgGC antisera or monoclonal mouse anti-MtrE antibodies. b FA1090 cell lysate was subjected to separation on a 10-47 % step sucrose gradient by ultracentrifugation at 174,000 × g for 4 h and monitored by UV absorbance at 254 nm. The positions of the 30S and 50S ribosomal subunits, the 70S monosomes, and polyribosomes are indicated. The level of ObgGC in the collected fractions was detected by immunoblotting with polyclonal rabbit anti-ObgGC antibodies. A representative polyribosome profile and immunoblot are shown
Mentions: To examine the cellular localization of ObgGC, wild type FA1090 was cultured under standard laboratory conditions in GCBL and harvested at the mid-exponential phase of growth. The bacterial cells were lysed and the cell envelope proteins were separated from the cytosolic proteome by a sodium carbonate extraction procedure [58]. The crude cell envelopes were treated with sarkosyl to solubilize the inner membrane proteins, and the outer membrane was recovered by ultracentrifugation [59]. The same total amounts of purified subproteomes (15 μg) were separated by SDS-PAGE and probed with anti-ObgGC antisera (Fig. 6a). The outer membrane protein, MtrE, which contains an extended periplasmic tunnel [60], was used as the cell envelope protein marker. As expected, MtrE was absent in the cytosol and enriched in the sarkosyl-insoluble fraction, whereas the vast majority of ObgGC was present in the cytosolic protein fractions (Fig. 6a). A faint band of the same molecular weight was also detected in the cytoplasmic membrane. Many cytoplasmic proteins are repeatedly identified in different cell envelope proteomics studies and are often considered “contaminants” [61]. However, recent thorough sequential biochemical fractionations of E. coli, combined with mass spectrometry, demonstrated that many of these proteins, including Obg, form an actual peripheral inner membrane proteome linked via functional and/or structural oligomeric complexes [62].Fig. 6

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