Cellular Location of Ribosomal Protein L7/L12 in Neisseria gonorrhoeae

Jeanne Quick

The University of Montana IBS-CORE Program

Mentor- Dr. Ralph Judd

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Introduction:

Neisseria gonorrhoeae infects up to one million people yearly in the US, causing urethritis and cervicitis, pelvic inflammatory disease, sterility, ectopic pregnancy, disseminated gonococcal infection and arthritis (2). Treatment costs billions of dollars each year. Further, gonococcal inflammation increases the risk of transmitting and acquiring other sexually transmitted disease agents such as HIV (6). Due to a lack of an animal model, we do not understand the physiological parameters of the gonococcus that influence disease. One area of undisputed importance is the synthesis of outer membrane proteins, molecules that both interact with the host (humans) and are vulnerable to attack by our immune system.

There is a strong need to understand the outer membrane protein synthetic mechanisms employed by N. gonorrhoeae, as well as all Gram negative bacteria. This obligate human pathogen is an excellent model organism to investigate how ribosomes synthesize outer membrane proteins. First, the Judd laboratory demonstrated that gonococci lack Braun's lipoprotein (8) which, in other Gram negative bacteria, anchors the outer membrane to the cell wall. Thus Bayer's junctions (4) can be observed without plasmolyzing the cell, facilitating studies on inner and outer membrane interaction. In addition, the Judd laboratory has developed techniques for isolating gonococcal cytoplasm, periplasm, inner-, mixed- and outer membranes.

In Escherichia coli, L7/L12 exists as a dimer when integrated into the ribosome (7). There are two distinct domains linked by a flexible "hinge". The N-terminal domain associates with ribosomal protein L10 and is responsible for the dimer interaction through a coiled-coil interaction of the alpha-helices. The globular C-terminal domain is involved in binding of elongation factors. As the nascent peptide chain is synthesized, the hinge region flexes to move the growing chain through the ribosome. There is speculation that L7/L12 operates in a different manner when synthesizing outer membrane or export molecules, which may explain the possible surface-exposure of this ribosomal protein.

There is evidence that ribosomal protein L7/L12 is exposed on the bacterial surface in bacteria. Work by Judd and Hill in the 1970s and 1980s showed that antiserum directed against 50S gonococcal ribosomes agglutinated whole gonococcal cells (unpublished observation), suggesting that a 50S ribosomal element was surface-exposed. In 1996, the Judd laboratory demonstrated that elongation factor Tu (EF-Tu) was the dominant molecule in the periplasm of N. gonorrhoeae (15). The L7/L12 protein is known to interact with EF-Tu, suggesting ribosomal activity near or at the bacterial outer membrane. The same year Oliveira and Splitter (13) showed that immunization with a recombinant L7/L12 protein of Brucella abortis elicited protective immunity, strongly supporting the hypothesis that the L7/L12 protein is available on the bacterial surface to interact with anti-L7/L12 antibody.

The work of Bayer (4) may explain the enigma of a ribosomal protein being surface-exposed. He showed that, at sites of inner and outer membrane fusion during biosynthesis of outer membrane proteins (i.e. Bayer's junctions), there was an interaction between ribosomes and the fused membranes. The ribosomes appeared to be associated with a "granular" appearing component in the fused membranes. The nature of this granular substance remains unknown, but it is possible the granularity is the result of the L7/L12 protein intercalating into the membrane. He also demonstrated that the junctions, which accounted for about 10% of the bacterial surface, migrated as proteins were installed in the outer membrane. Thus, the peptidoglycan was constantly being degraded and reformed as the Bayer's junctions traversed the bacterial surface. The L7/L12 protein may be involved in this process, explaining the apparent peptidoglycan association of the L7/L12 protein if the 14.5kDa peptidoglycan-associated protein identified in the 1980s proves to be the L7/L12 protein.

The possibility that the L7/L12 protein is present in Bayer's junctions will be explored by isolating "mixed membranes" from N. gonorrhoeae, a process that the Judd laboratory is well acquainted (5). Non-piliated, Opa^- N. gonorrhoeae MS11LOSa (a well characterized strain) will be used in the proposed studies. Techniques for gonococcal growth, phenotype selection, harvesting, etc., are extant in our laboratory (11, 12).

The hypothesis tested in this application was that ribosomal protein L7/L12 is a surface-exposed protein of Neisseria gonorrhoeae. The cellular location of L7/L12 was examined with Western blotting. The lysates of gonococcal whole cells, inner membrane, mixed membrane (i.e. Bayer's junctions), and outer membrane isolates, cytoplasmic and periplasmic isolates were probed with monoclonal antibodies (AMGC-1 and BSGC-2) specific for L7/L12. The Judd laboratory used this technique to demonstrate that gonococcal sialyltransferase was located in the outer membrane (5). Demonstration that L7/L12 is exposed on the bacterial surface would have a significant impact on our understanding of how outer membrane proteins are synthesized and exported and would justify efforts to secure funding to further investigate ribosomal function in Gram negative bacteria.

Materials and Methods:

Bacterial Strain, Growth and Harvesting:

Neisseria gonorrhoeae strain MS11LOSA were grown on clear typing media from freeze-backs available in the Judd laboratory. The bacteria were grown for 16hr on clear typing media at 37 ^oC in 5% CO₂ (11, 12). Transparent bacteria cells lacking opacity proteins and pili were streaked for isolation for several days prior to use in order to isolate for phenotype. Transparent (opa^-), non-piliated (pili^-)cells were harvested to an OD_600nm = 1.0 in Dulbecco's PBS (DPBS). Five milliliters of this suspension was inoculated into 100ml of GC broth in a 250ml side arm flask and incubated with shaking at 37^oC until the culture reached mid-log growth (about 5h) OD_600nm = 0.50. Optical density of the culture was taken every 30 minutes to follow the growth of the cells to mid log phase. Twenty milliliters of mid-log phase cells was then inoculated into another 100ml of GC broth in a 250ml side arm flask and incubated with shaking at 37^oC until the culture again reached mid-log growth. The optical densities from the growth of the cultures were plotted against time on a growth curve. Cells were harvested by centrifugation at 8000xg for 20minutes.

Sucrose Gradient Separation of Inner, Mixed and Outer Membrane Components:

Preparation of cells:

A modification of the procedure of Osborn and Munson (14) was used to isolate gonococcal membrane components. Pellets of whole cells were resuspended in 2.5ml (per 100ml of culture) of 200mM Tris-HCl, pH 8.0 and diluted with an equal volume of ice cold 1M sucrose, 200mM Tris-HCl, pH 8.0. The following pre-chilled reagents were added sequentially with vortex mixing: 10ml of 250mM EDTA; 40ml of N-acetylmuramidase lysozyme (5mg/ml in 3X dH₂O)(Sigma, St. Louis, MO); 5ml of ice cold 3xdH₂O added forcefully by pipet. The preparation was gently shaken overnight at 4^oC. Cells were then sonicated by two 20s bursts at 50% power using a Branson sonicator. Unbroken cells and cell debris were removed by centrifugation at 12000xg for 20m. The supernatant was decanted into fresh centrifuge tubes and was then ultracentrifuged at 240000xg for 2h at 4^oC in a Ti80 rotor (Beckman). Pellets were resuspended in 1ml 18% (w/w) sucrose and were then frozen.

Sucrose Gradient:

A isopycnic sucrose step gradient was layered from bottom to top in 37ml polypropolene tubes by adding the following w/w sucrose solutions (all contained 1mM EDTA and 200mM dithiothreitol(DTT)): 6ml of 60% sucrose overlaid with 5ml each of 55%, 50%, 45%, 35%, 25% and 20% sucrose. One ml of the membrane fraction was layered on top of the gradient and centrifuged in an SW28 rotor (Beckman) at 80000xg for 48h at 4^oC. In the second and all following runs the tubes were layered with: 6ml of 60% sucrose overlaid with 6ml each of 50%, 45%, 35%, 25% and 20% sucrose. One ml of the membrane fraction was layered on top of the gradient and centrifuged in an SW28 rotor (Beckman) at 80000xg for 72h at 4^oC. Following centrifugation 1ml fractions were collected from the top of the gradient using a peristaltic pump. Fractions were analyzed by refractometry and absorbance at 280_nm. The data from the spectophotometer printout and the refractive indexes were plotted. Three peaks within absorbing fractions in the density range of ~1.05 - ~1.26 were pooled, dialyzed, lyophilized and solubilized at 10mg/ml in 2X SDS solubilizing solution with 2ME and boiled for 10 minutes for use in SDS-PAGE and Western blotting experiments.

Periplasmic extraction:

Periplasmic proteins were released from freshly harvested bacteria as described by Ames(1). Bacteria were swabbed from culture plates, suspended in 1.5 ml of ice cold DPBS to an OD_600nm of 0.68 (~5 x 10⁹ cells/ml), and washed 3X in DPBS. The DPBS was gently but thoroughly removed from the final washed pellet, 20 ml of chloroform (Baker) added, and the mixture vortexed. After a 15 min, 22^oC, 100 ml of ice cold 0.01 M Tris-HCl, pH 8.0 was added, the preparation vortexed, and pelleted by centrifugation at 10,000 x g for 5 min. The Tris-HCl containing the released periplasmic material was carefully aspirated and placed in a second microfuge tube.

Several tubes were run at once. The Tris-extract from a couple of the tubes was pooled and frozen. 100ml of 2X solubilizing solution was added to another tube, boiled for 10 minutes, and used for SDS-PAGE, and Western Blotting.

Cytoplasmic extraction:

Cytoplasmic proteins were released from freshly harvested bacteria as described by Ames(1). Bacteria were swabbed from culture plates, suspended in 1.5 ml of ice cold DPBS to an OD_600nm of 0.68 (~5 x 10⁹ cells/ml), and washed 3X in DPBS. In 100ml of dPBS cells were then sonicated on ice 4-5 times with 20s bursts at 50% power using a Branson sonicator. The preparation was then spun to separate out the cells and debris in a microfuge at 4^oC. The supernatant containing the cytoplasmic components was then aspirated and put into a fresh tube in ice, followed by ultracentrifugation in the airfuge at 100,000xg for one hour. Supernatant was collected and the aliquots were frozen.

Several tubes were run at once. The supernatant (cytoplasm) from a couple of the tubes was pooled and frozen. A portion of the supernatant (cytoplasm) was solublized in an equal volume of 2X solubilizing solution, boiled for 10 minutes, and used for SDS-PAGE and Western Blotting.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting:

The components of the membrane fractions, the periplasm isolation and the cytoplasm isolations were separated by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) following procedures outlined by Judd (9). Gels were fixed in 25% isopropyl alcohol, 7% glacial acetic acid in deionized water (fixer/destain) for 16 hours, stained in 0.25% Coomassie Brilliant Blue R (0.25g/100ml fixer/destain) for one hour and destained in fixer/destain until the background was almost clear, then placed in dH₂O until the background was completely clear.

Western blots were performed on the samples following procedures by Batteiger, et.al(3) and Judd(10). Proteins were transferred to a Millipor Immobilon polyvinylidene difluoride (PVDF) membrane by immunoblotting in 20mM sodium phosphate buffer, pH of 8. Blots were run overnight, 16 hours, at 0.6 Amperes using a BIO-RAD Trans-Blot Cell. All blots were blocked for one hour in 0.05% Tween-20 in Delbecos PBS (dPBST). Follow this initial blocking blots were probed with an anti-L7/L12 antibody in dPBST at 4^oC for 16 hours (overnight). The antibodies were available in the Judd laboratory and were used at the following dilutions: AMGC1 at 1:150 and BSCG2 at 1:100 in dPBST. Blots were then washed three times in dPBST, five minutes per wash. Blots were then probed with a secondary antibody, anti-mouse IgG at a1:500 dilution in dPBST for one hour at room temperature. Blots were again washed three times in dPBST, five minutes per wash, and were developed in a 4-cholor-1-napthol, hydrogen peroxide solution.

Results:

The gonococcal strain MS11LOSA was successfully grown and the phenotype opa-/pilli- was isolated and used in the subcellular separations. Growth curves bringing the bacteria to their mid-log phase and second mid-log phase were also successful, see figures 1 and 2 below.

Figure1. Optical density of the culture was read at 30 minute

increments to follow the growth of the bacteria, end point of

first mid log phase, OD=0.52.

Figure 2. Optical density of the culture was read at 30 minute

increments to follow the growth of the bacteria, end point of

second mid log phase, OD=0.54.

Inner membranes, mixed membranes, outer membranes, cytoplasm, and periplasm were isolated from gonococcal stain MS11LOSA. Inner-, mixed-, and outer-membrane fractions were separated by isopycnic sucrose gradient centrifugation. Sucrose gradients were collected from top to bottom in 1 ml fractions. Optical density of all fractions was read with a spectophotometer at 280nm, and sucrose density was determined through a measure of refractive index. The absorbance data (from the spectophotometer) of the membrane isolation fractions were plotted in combination with the refractive index of every fifth fraction. The plotted absorbance points produced three visible peeks (see figure 3), showing isolation of the membranes. The inner membrane peak can be found at the density of 1.1g/ml, the outer membrane peak at 1.2g/ml and the mixed membrane peak between the other two.

Figure 3. Absorbance and sucrose density data of fractions from sucrose gradient separation of gonococcal membranes. Peak 1, density of 1.1= inner membranes, peak 2, density of 1.15 = mixed membranes, peak 3, density of 1.2 = outer membranes.

The fractions within these peaks were pooled to make three samples. These samples were dialyzed, lyophilized, and solubulized. Solubilized samples were standardized and run in SDS-PAGE gels and Western blots. Comparing SDS-PAGE gels from these pooled membrane fractions showed the membrane isolations to be successful. Additional SDS-PAGE gels and Western blots probed with AMGC-1 and BSCG-2 were performed on these samples, the periplasm and cytoplasm isolations. The results from one of these sets are shown in figure 4. Western blots performed on the membrane fractions probed with BSCG-2 or AMGC-1 showed no significant bands and therefore no presence of L7/L12 in any of the membrane fractions. Western blots on the cytoplasmic and periplasmic isolations probed with BSCG-2 showed a very significant amount of L7/L12 present in the cytoplasm and also a large amount in the periplasm. Western blots on the cytoplasm and periplasm probed with anti-MTRC showed no bands, signifying a lack of this surface exposed protein.

Figure 4. SDS-Page and Western blot of whole cell and subcellular fractions of Neisseria gonorrhoeae strain MS11LOSA. Lysates were separated in a 15% acrylamide gel and stained with Coomassie brilliant blue (CBB) or transferred to a PVDF membrane and probed with anti-L7/L12 BSGC2 monoclonal antibody (anti-L7/L12 BSGC2 Mab). MS11LOSA – whole cell lysate; cytoplasm – isolated cytoplasm; periplasm – isolated periplasm; inner membranes – sucrose gradient isolated inner membranes (see Figure 3); mixed membranes - sucrose gradient isolated mixed membranes (see Figure 3); outer membranes - sucrose gradient isolated outer membranes (see Figure 3); PSMW – prestained molecular mass markers, expressed in kilodaltons (k) were the BioRad prestained marker kit; MW – molecular mass markers, expressed in k were the BioRad low molecular mass kit.

Discussion:

The focus of this project was identifying the cellular location of gonococcal L7/L12, a ribosomal protein. Techniques for separating inner and outer membranes and finding a mixed membrane fraction were an important part of this research. It was hypothesized that L7/L12 was a surface exposed protein of Neisseria gonorrhoeae, results do not show support of this. The membrane components, inner- mixed- and outer-membranes, were successfully separated as can be seen in the SDS-PAGE gel in figure 4. Bands showing the presence of ribosomal protein L7/L12 in any of the sucrose separated membrane fractions were not seen in the Western blots probed with AMGC-1 or BSGC-2. The lack of bands in these blots implies that this protein is not found in the membrane, however the protein could be present in one or more of the membrane components and could have been stripped away by the techniques used to physically separate them. To test this possibility, immunoelectronmicroscopy studies will be performed on MS11LOSA strain Neisseria gonorrhoeae. The bacteria will probed with the same antibodies used in this experiment to look for the location of ribosomal protein L7/L12.

The Western blots showed L7/L12 to be in the cytoplasm isolate, this was expected because whole ribosomes are located in the cytoplasm. A band showing the presence of ribosomal protein L7/L12 also appeared periplasm isolate. This finding was not expected and is being researched further to determine if the antibody detected was only L7/L12 or if entire ribosomes are located in the periplasm. This will be determined by running Western blots on the same isolates and probing them with an antibody directed at whole ribosomes.

A recent paper by Spence and Clark(16), presented evidence that gonococcal protein L7/L12 is membrane associated and surface exposed in gonococci. They describe the function of this protein in infection as a bridge between the host (humans) and the bacteria. The accuracy of these findings is questionable when compared with the results from the present experiment showing ribosomal protein L7/L12 not to be in the membrane components. Neisseria gonorrhoeae does not have Braun's lipoprotein (8) as other bacteria and do to this it does not contain its components as well as other bacteria with the lipoprotein. The L7/L12 protein found by Spence and Clark on the bacterial surface, could have leaked out from the periplasm. The immunoelectronmicroscopy studies discussed above will aid in understanding the actual location of this protein.

Acknowledgements

This work was funded by an IBS-CORE Undergraduate Research Fellowship to Jeanne Quick through a grant from the Howard Hughes Medical Institute to The University of Montana.

Special thanks to Dr. Ralph Judd for being so generous with his time and lab space. Thanks also to Deb Nycz, Dr. Scott Manning, and Leoned Gines for technical assistance and valuable discussions.

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Executive Summary

An experiment was conducted to determine the location of a ribosomal protein, L7/L12, in the bacteria Neisseria gonorrhoeae. This bacteria is associated with the sexually transmitted disease gonorrhea. If this protein is found to be on the outside surface of the bacteria it may serve as a target for a diagnostic test, or a vaccine. Methods used to explore this possibility are as follows: A specific strain of the bacterium was grown and specific properties were selected for. The bacterial cells were burst open by number of different means. The components of the bacterial membrane were separated by a “sucrose gradient,” which separates particles out by their buoyant densities. The layers of the gradient containing the membrane fractions were run in acrylamide gels, which separate proteins by their size. Smaller proteins travel faster and therefore further. The different proteins in each sample produce a unique and descriptive pattern of bands. These proteins are then transferred to a blot, a paper like membrane, which can be treated with antibodies. The antibodies will detect the presence of a specific protein on the blot by appearing as a band when developed. The antibodies used in this experiment were directed against the L7/L12 protein. No bands appeared in any of the membrane samples. Bands were seen only in the cytoplasm and periplasm of the bacteria cell. These findings suggest that the L7/L12 protein is not exposed on the surface of the gonorrhea bacteria. More research is currently being conducted on the periplasm to determine the nature of the protein located there. The process of physically separating the membranes could have stripped the protein off. Additional studies are underway using electronmicroscopy to detect the presence of the L7/L12 protein within the entire bacterial cell. The protein may be on the surface of an intact bacterium.

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