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Abstract

Fully characterized mycobacteriophages may be utilized as potential treatments for diseases caused by bacterial infections. Isolation and characterization of novel mycobacteriophages could lead to advances in the rising field of phage therapy. Using Mycobacterium smegmatis, a novel mycobacteriophage, was isolated and purified from tropical soil collected in Puerto Rico. The specimen was named Incognito. This serves as evidence that Puerto Rico’s tropical environment is adequate for the growth of phage populations.

1. Introduction

One of the most challenging problems facing scientists today is the constant resistance of bacterial infections to current antibiotics. Bacteria can grow resistant through several mechanism; some develop the ability to neutralize the antibiotic before it can do harm, others can rapidly pump the antibiotic out, and still others can change the antibiotic attack site so it cannot affect the function of the bacteria. Therefore, researchers have had to find new alternatives to fight the ever stronger resistance of bacterial infections. In recent years, bacteriophages have emerged as the ideal alternative and answer to the problem. Bacteriophages (phages) are bacterial viruses that infect, disrupt, and lyse bacterial cells resulting in

cell death. For example, mycobacteriophages are a specific group of viruses that only infect bacteria from the mycobacteria genus. Common bacteria from this genus include Mycobacterium smegmatis, which is harmless, and others like Mycobacterium tuberculosis and Mycobacterium leprae; both of which cause deadly bacterial infections. Most of these bacteria are constantly growing resistant to antibiotics, but in the future fully characterized mycobacteriophages may be utilized as potential treatments for these and other diseases.

By utilizing Mycobacterium smegmatis as a rapidly growing host (Endersena et al. 2013), it is possible to isolate and characterize a whole variety of mycobacteriophages found in the environment. In this manner, researchers are able to use genomics and proteomics to study the complete genetic information (genomes), and the structure and function of the phage’s proteins (proteome). These studies help scientists determine the unique genes that each phage may possess, giving clues and ultimately an indication into their possible antimicrobial properties.

The main objective of this project is to isolate, purify, and completely characterize a phage found in tropical soils of Puerto Rico. Phages are estimated to be the most abundant biological entities on Earth (Suttle 2005; Wommack & Colwell 2000).

Isolation and Purification of Novel Mycobacteriophage: Incognito Paola G. Caballero León1, Anthony Hernández Rivera2

1Department of Chemistry, RISE Program, University of Puerto Rico at Cayey 2Department of Biology, RISE Program, University of Puerto Rico at Cayey

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They are found in a variety of ecosystems; in other words they are easily found everywhere. Puerto Rico is characterized as being very diverse, in terms of climate and types of environments. The question is if this diverse tropical environment is sufficiently adequate for bacteriophages to thrive in. We hypothesize there is a whole variety of bacteriophages that can be found, successfully isolated, and purified in the tropical soils of Puerto Rico. By achieving this, we intend to observe if its respective genes and proteins hold any unique characteristics that may contribute in the further development of alternative means to fight bacterial infection.

2. Materials and Methods 2.1. Sample Collection and Preparation

A soil sample was collected and analyzed for the presence of mycobacteriophages. Using aseptic techniques, 10 mL of AD Supplement Smeg Master Mix were mixed with 1 mL of M. smegmatis bacteria and 0.5 g of the soil sample in a labeled tube. This enrichment was incubated at 37°C for 24 hours and then centrifuged for 15 minutes, at room temperature, and 3,000 rpm. Following this enrichment procedure, 1 mL of the supernatant was obtained, placed inside a microtube and centrifuged a second time at 10,000 rpm for 10 minutes, to obtain the phage filtrate. Without disturbing the pellet that formed, 500 μL of the supernatant were removed and placed inside a clean microtube for further processing.

2.2. Isolation of Phage

A small agar plate for the culture of M. smegmatis bacteria was used for the streak protocol. A sterilized wooden stick was inserted inside the filtrate and was then used to streak about one-third of the agar plate. A new stick was used to streak the adjacent area of the first streak, overlapping the original streaked area once. The third quadrant was streaked in the same manner. After the streak protocol, 2 mL of LB Top Agar for M. smegmatis were obtained, mixed with 0.25 mL of M. smegmatis bacteria and dispensed over the streaked agar plate. This plate was then incubated at 37°C for 24 hours and then examined for the presence of plaques.

2.3. Purification of Phage

One plaque was aseptically obtained from the plate with a micropipette tip, added to a microtube that contained 25 μL of phage buffer, and vortexed to ensure maximum contact of the phage with the buffer. This isolated phage sample was then used to perform the streak protocol on a new M. smegmatis agar plate and obtain the first purification of the phage. Single plaque purification was repeated three times to obtain a purified population of the phage.

From the third purification, one plaque was aseptically obtained and mixed with 10 mL of AD Supplement Smeg Master Mix and 1 mL of M. smegmatis bacteria. This second enrichment was then filtered and a Medium Titer Phage Lysate (MTPL) was obtained, which is a concentrated liquid sample of the phage. A spot test of

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eight 10 μL serial dilutions of the titer lysate was performed onto a prepared agar plate with the LB top agar and M. smegmatis bacteria already added and solidified.

2.4. Characterization of Phage 2.4.1. Proteomics

Polyacrylamide gel electrophoresis was carried out in order to isolate, separate, and visualize the mycobacteriophage capsid proteins. Initially, 20 μL of the MTPL were obtained, centrifuged and mixed with 25 μL of Beta-mercaptoethanol (BME). Subsequently, the sample was boiled for 2 minutes and then cooled down for an additional 2 minutes, in order to completely denaturalize the protein. Afterwards, 17 μL of this sample were loaded to one of the wells in the gel. The electrophoresis was carried out at 200 volts for 30 minutes. The gel was washed with water for another 30 minutes.

2.4.2. Electron Microscopy

Electron Microscopy grids were placed, shiny side up, on the very edge of a double sided tape. Afterwards, 10 μL of the phage sample were added to the grid. Once the sample had settled and attached onto the grid for at least 2 minutes, the excess fluid was wicked off with filter paper. This same procedure was repeated two more times, the first with 10 μL of water, and the second with 10 μL of 1% uranyl acetate to stain the phage sample.

3. Results

3.1. Soil Samples

A total of 11 soil samples were collected from all over the island of Puerto Rico in search of a phage. The enrichment process and streak protocol were performed with each soil sample until phage plaques were obtained. The environmental data for each soil sample is reported in Table 1.

3.2. Isolation of Phage

Soil sample #9 was taken from underneath a plantain tree at a depth of approximately 10 cm, in a rural area of Hatillo, PR. The coordinates of the location were 18.473341, -66.785332 (Fig 1).

Table 1. Eleven soil samples were collected and analyzed for any phage presence.

Fig. 1 Localization of soil sample #9

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The soil sample was saturated. After completing the streak protocol, the filtrate of this sample proved to contain a mycobacteriophage due to the presence of four turbid plaques (Fig 2).

3.3. Purification of Phage

The isolation of the phage was followed by the single plaque purification process. The purifications were repeated three times to obtain a pure population of the phage (Fig. 3). Due to the turbid nature of the plaques, edited pictures of this process are provided, because they facilitate the identification of the plaques (Fig. 4).

The spot test served as an empirical test to analyze the concentration of phage in the MTPL (Fig. 5). We observed complete lysis in dilutions 1 through 4. Three isolated turbid plaques were present in dilution 5. Complete infection of M. smegmatis will be carried out with these five dilutions in order to obtain a web pattern and subsequently, the high titer phage lysate (HTPL). With this data, the concentration of phage was calculated to be 3 x 107 Plaque Forming Units (PFU) per milliliter (PFU/mL).

Fig. 4 Enhanced and edited pictures of the purification process

Fig. 3 Single plaque purification process and results

Fig. 5 Spot test- Empirical test

Fig. 2 Positive phage results- four turbid plaques

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3.4. Characterization of Phage 3.4.1. Proteomics

Using polyacrylamide gel electrophoresis (SDS-PAGE), the capsid protein content of the mycobacteriophage was isolated and separated. Our phage sample was loaded onto well #3 of the polyacrylamide gel (Fig. 6). The other wells contain phage samples that belong to other researchers.

4. Discussion

Our phage was found among a series of 11 soil samples collected from various areas in the island of Puerto Rico. It can be classified as a mycobacteriophage, because it infected M. smegmatis, a member of the Mycobacteria genus. This novel mycobacteriophage was named Incognito due to its elusive nature. Identifying its plaques for the first time was difficult because of its turbidity. There were weeks when the phage seemed to disappear,

causing doubts of its presence. By successfully isolating Incognito, we have proven that soils in Puerto Rico are adequate for the growth of phage populations. Our main objective was to purify and completely characterize Incognito to see if its genes and proteins hold any unique characteristics.

Temperate phages have the ability to carry out either the lysogenic or the lytic cycle of reproduction and they usually form turbid and cloudy plaques (Science Education Alliance, 2012). The observed plaques fit this description; which led us to conclude that Incognito is most likely a temperate phage. For this reason, edited pictures of the purification were provided.

The spot test that was carried out served as an empirical test to determine the concentration of the phage. Apart from the circles of complete lysis in grids 1 through 4, there were clear lytic plaques all over the plate. This is evidence of bacterial contamination. Nonetheless, the contamination can be ignored, because the objective was to simply observe in which dilutions Incognito was present. Since three turbid plaques were observed in dilution of 10-5, we used that number as the PFU (plaque forming units) and divided it by 0.01 mL, which was the volume added of the dilution. The concentration of our phage in the MTPL was 3 x 107 PFU/mL.

The capsid proteins of our phage were isolated and separated by using polyacrylamide gel electrophoresis. After image analysis, we can observe significant

Fig. 6 Polyacrylamide gel electrophoresis Well #3 corresponds to our phage’s capsid proteins: Incognito

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similarities between the capsid protein content of Incognito (well #3) and that of our respective peers (wells #2, #6, and #7). Further characterization will be carried out. By doing this, we strive to contribute to the global scientific community in the ever more important development of phage therapy as the future alternative for treating all kinds of pathogenic bacteria.

Acknowledgements

We would like to thank the RISE and Howard Hughes Programs for this great experience in which we gained invaluable research and laboratory experience. Special thanks to our RISE lab technician Giovanni Cruz and Teaching Assistants (TAs) Joseph Perez and Gustavo Martínez. We would also like to give special acknowledgements to Dr. Michael Rubin and Dr. Edwin Vazquez for their guidance and constant support.

Literature Cited

Endersena L, Coffeya A, Neveb H, MaAuliffec O, Rossc RP, O’Mahonya JM. 2013. Isolation and characterisation of six novel mycobacteriophages and investigation of their antimicrobial potential in milk. International Dairy Journal. 28(1): 8-14.

Suttle CA. 2005 Viruses in the sea. Nature. 437: 356-361.

Wommack KE, Colwell RR. 2000. Virioplankton: viruses in acquatic

ecosystems. Microbiology and Molecular Biology Reviews, 64: 69–114.

References

Center of Disease Control and Prevention. 2013 Get Smart: Know When Antibiotics Work. Available from: http://www.cdc.gov/getsmart/antibiotic-use/antibiotic-resistance-faqs.html#define-antibiotic-resistance (Accessed April 30, 2014)

Rubin M, Vázquez E. 2012 Mycobacteriophages Proteomics: From Genotype to Phenotype (There and Back Again) pp 1-20

Science Education Alliance, Howard Hughes Medical Institute. SEA- PHAGES Resource Guide.