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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

Validation of polyclonal rabbit anti-ObgGC antisera. (a) The polyclonal rabbit anti-ObgGC antibodies were used to probe the whole-cell lysates derived from wild type FA1090 and isogenic Plac::obgGC as well as purified variants of ObgGC. The bacteria were harvested following 2 h of growth in GCBL with (+) and without (−) 100 μM IPTG, and the samples were matched by equivalent OD600 units. Purified recombinant proteins (40 ng) include wild type ObgGC with N-terminal 6 × His tag, (N-His-ObgGC) and N-His-ObgGC with T192AT193A substitutions. (b) Samples of whole-cell lysates derived from various Neisseria species, as indicated, were harvested from GCB and matched by equivalent OD600 units. All samples were separated in 4-20 % Mini-PROTEAN TGX precast gels, the proteins were transferred onto the nitrocellulose membrane and probed with polyclonal rabbit anti-ObgGC antisera raised against N-His-ObgGC
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Fig2: Validation of polyclonal rabbit anti-ObgGC antisera. (a) The polyclonal rabbit anti-ObgGC antibodies were used to probe the whole-cell lysates derived from wild type FA1090 and isogenic Plac::obgGC as well as purified variants of ObgGC. The bacteria were harvested following 2 h of growth in GCBL with (+) and without (−) 100 μM IPTG, and the samples were matched by equivalent OD600 units. Purified recombinant proteins (40 ng) include wild type ObgGC with N-terminal 6 × His tag, (N-His-ObgGC) and N-His-ObgGC with T192AT193A substitutions. (b) Samples of whole-cell lysates derived from various Neisseria species, as indicated, were harvested from GCB and matched by equivalent OD600 units. All samples were separated in 4-20 % Mini-PROTEAN TGX precast gels, the proteins were transferred onto the nitrocellulose membrane and probed with polyclonal rabbit anti-ObgGC antisera raised against N-His-ObgGC

Mentions: To begin the characterization of ObgGC, N- and C-terminally His-tagged versions of the wild type NGO1990, N-His-ObgGC and C-His-ObgGC (respectively), were overexpressed in E. coli BL21(DE3), and purified. The purified recombinant N-His-ObgGC was subsequently used to obtain polyclonal rabbit anti-ObgGC antisera. The antisera specifically recognized both the native and recombinant versions of ObgGC (Fig. 2a). The purified proteins migrated in SDS-PAGE more slowly than the native protein and accordingly with the deduced molecular mass of ObgGC (41.998 kDa) with the addition of the histidine epitope. Further, the antibodies cross-reacted with Obg homologs in the N. meningitidis serogroup B strains MC58 and NZ98/254 but failed to recognize Obg from N. weaveri and E. coli despite their 85 and 56 % identity to ObgGC, respectively (Fig. 2b and Table 1). It is possible that the anti-ObgGC antisera bind to the highly variable C-terminal domain of ObgGC.Fig. 2


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)

Validation of polyclonal rabbit anti-ObgGC antisera. (a) The polyclonal rabbit anti-ObgGC antibodies were used to probe the whole-cell lysates derived from wild type FA1090 and isogenic Plac::obgGC as well as purified variants of ObgGC. The bacteria were harvested following 2 h of growth in GCBL with (+) and without (−) 100 μM IPTG, and the samples were matched by equivalent OD600 units. Purified recombinant proteins (40 ng) include wild type ObgGC with N-terminal 6 × His tag, (N-His-ObgGC) and N-His-ObgGC with T192AT193A substitutions. (b) Samples of whole-cell lysates derived from various Neisseria species, as indicated, were harvested from GCB and matched by equivalent OD600 units. All samples were separated in 4-20 % Mini-PROTEAN TGX precast gels, the proteins were transferred onto the nitrocellulose membrane and probed with polyclonal rabbit anti-ObgGC antisera raised against N-His-ObgGC
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Validation of polyclonal rabbit anti-ObgGC antisera. (a) The polyclonal rabbit anti-ObgGC antibodies were used to probe the whole-cell lysates derived from wild type FA1090 and isogenic Plac::obgGC as well as purified variants of ObgGC. The bacteria were harvested following 2 h of growth in GCBL with (+) and without (−) 100 μM IPTG, and the samples were matched by equivalent OD600 units. Purified recombinant proteins (40 ng) include wild type ObgGC with N-terminal 6 × His tag, (N-His-ObgGC) and N-His-ObgGC with T192AT193A substitutions. (b) Samples of whole-cell lysates derived from various Neisseria species, as indicated, were harvested from GCB and matched by equivalent OD600 units. All samples were separated in 4-20 % Mini-PROTEAN TGX precast gels, the proteins were transferred onto the nitrocellulose membrane and probed with polyclonal rabbit anti-ObgGC antisera raised against N-His-ObgGC
Mentions: To begin the characterization of ObgGC, N- and C-terminally His-tagged versions of the wild type NGO1990, N-His-ObgGC and C-His-ObgGC (respectively), were overexpressed in E. coli BL21(DE3), and purified. The purified recombinant N-His-ObgGC was subsequently used to obtain polyclonal rabbit anti-ObgGC antisera. The antisera specifically recognized both the native and recombinant versions of ObgGC (Fig. 2a). The purified proteins migrated in SDS-PAGE more slowly than the native protein and accordingly with the deduced molecular mass of ObgGC (41.998 kDa) with the addition of the histidine epitope. Further, the antibodies cross-reacted with Obg homologs in the N. meningitidis serogroup B strains MC58 and NZ98/254 but failed to recognize Obg from N. weaveri and E. coli despite their 85 and 56 % identity to ObgGC, respectively (Fig. 2b and Table 1). It is possible that the anti-ObgGC antisera bind to the highly variable C-terminal domain of ObgGC.Fig. 2

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