1. Genes Associated with Biofilm Formation in Mycobacterium smegmatis Molly D. McNab Oregon State University College of Veterinary Medicine Department of Biomedical Sciences Summer 2003 Mentor: Dr. Luiz Bermudez

2. What is Biofilm?  Biofilms are multicellular aggregates of bacteria and yeast that congregate on surfaces.  Biofilm may form on any surface exposed to biofilm-forming bacteria and some amount of water.  Biofilms are formed to protect the bacteria from host defenses, antibiotics, and from harsh environmental conditions.

3. Where are Biofilms Found? Bacterial biofilms are often a cause of infections associated with medical implants such as catheters and IV lines and other medical devices. Biofilms are found almost everywhere in nature, including rivers, lakes, soil, water pipes, and even inside the human body. A common type of bacterial biofilm-responsible for plaque.

4. Why Research Biofilms?  Due to the morphology of biofilms, bacteria capable of forming them are highly resistant to antibiotics, making treatment very difficult.  In the US alone, one million nosocomial (hospital acquired) infections each year are caused by bacterial biofilms, leading to longer hospitalization, surgery, and even death.

5. Biofilms and Infections:  Biofilms are responsible for Otitis Media, the most common acute ear infection.  Biofilms play a role in Bacterial Endocarditis (infection of the inner surface of the heart and its valves).  Biofilms form frequently in patients with Cystic Fibrosis (a chronic disorder resulting in increased susceptibility to serious lung infections).  Biofilms also play a role in Legionnaire's disease (an acute respiratory infection resulting from the aspiration of clumps of Legionnella biofilms detached from air and water heating/cooling and distribution systems).

6. How are Biofilms Formed? Biofilm formation relies on an exchange of chemical signals between cells in a process known as “quorum sensing.”

7. Quorum Sensing  Quorum sensing is required for natural biofilm formation.  When enough bacteria (a quorum) are present, diffusible signal molecules produced by the bacteria allow for communication with others in order to coordinate their behavior.

8. Mycobacteria: An Overview  There are >70 species of mycobacteria, many of which are human pathogens.  Of these, three are major pathogens: Mycobacterium tuberculosis Mycobacterium leprae Mycobacterium avium  Many species of mycobacteria are found in the environment where biofilm formation is demonstrated.

9. Incidence of Mycobacterial Infections  8-12 million new infections of M. tuberculosis are reported per year, particularly in developing countries.  2-3 million people die from TB each year  Antibiotic resistant strains of M. tuberculosis are very common and cause great public health concern.  In the USA, environmental mycobacteria are more common than M. tuberculosis in a clinical setting; this is due to their association with AIDS and the low incidence of TB in this country.  M. avium is an environmental mycobacteria that is capable of forming biofilm and often infects AIDS patients and those with chronic pulmonary diseases.

10. Mycobacterium smegmatis  Avirulent mycobacterium  Ability to form biofilm  Found in the environment  Shares many properties with other more virulent mycobacteria  Grows easily in a laboratory setting and is fairly receptive to genetic manipulation

11. My Research  Using Mycobacterium smegmatis as a model, I will investigate the genes that are important for biofilm formation in other more virulent mycobacteria.

12. Design of My Plasmid: pEMC 1 Kanamycin Resistance Mycobacterium Origin of Replication Promoter-less GFP M. avium Library

13. Step One:  In a 96-well plate (shown below) grow bacteria (5 colonies per well), transferred directly from an already prepared GFP promoter library, in 200µL 7H9 growth media with Kanamycin (50µg/mL). Incubate at 37°C for 3-4 days to increase bacterial concentration.

14. Step Two:  After 3-4 days, transfer 100µL from each well into a 96-well Polyvinyl Chloride (PVC) plate, which promotes biofilm formation.  Store the PVC plate at room temperature with slight agitation.  Read the intensity of GFP expression of each well on day one, to use as a control, and each following day up to day five.

15. Step Three:  Analyze the results of the GFP expression assay by comparing day five with the day one controls.  Individual wells whose GFP expression increases by at least two times are isolated from the original 96-well plates, then plated on 7H11 agar (with OADC and Km 50) and allowed sufficient time for growth.

16. Sample GFP Expression Assay Results 117127115122113141123115103131 53635661656865666775 10210163677494848283105 745866676968626566117 8868116698011672736875 56555659565860 6376 61657958861036090122139 73846366676362826771 141133116142121141154152141143 64695165776559 6268 11911469948811715889133130 1016871919276665761123 10198157969312295798087 59495052515553 5884 6660119511091245790144149 881076266806058896798 Day One (Control) Day Five

17. Analysis of Results 1.441.551.211.051.011.161.071.001.251.321.371.09 1.171.601.211.100.911.071.180.960.910.890.930.91 1.351.661.171.131.101.401.191.24 2.00 1.091.601.24 2.05 1.521.361.171.081.361.331.121.060.880.921.05 1.561.211.151.441.351.391.161.051.321.081.181.16 1.271.071.050.89 0.880.910.950.88 0.921.11 1.721.311.080.921.510.881.271.200.951.001.181.07 1.671.741.211.270.981.001.190.950.941.091.001.38

18. Step Four:  32 individual colonies from each well are picked up and transferred to a 96-well plate with the 7H9 + Km50 + 10% OADC growth medium described in step one.  This is then incubated at 37°C for 3-4 days.  Steps one and two are repeated with the new plate (1 colony per well)

19. Step Five:  Analyze the results of the GFP expression assay by comparing day five with the day one controls.  Individual wells whose GFP expression is increased by at least two times are isolated again and prepared for plasmid extraction.

20. Step Six:  The plasmids from the wells with the most green intensity are extracted, transformed into E. coli, extracted again, and then sent for sequencing.  The sequences are then matched up with the genomes M. avium and M. tuberculosis to find the specific gene or protein and its function.

21. Results: Genes found to play a role in biofilm formation:  Glycosyltransferase (CDC 1551)  GuaB2 (H37Rv) IMPDH (CDC 1551)  Rv0538, Rv0539 (H37Rv)  Rv 3412, Rv 3413c (H37Rv)  Rv 3526 (H37Rv)  Rv 0359, Rv0358 (H37Rv)

22. Discussion:  The absence of glycopeptidolipids (GPLs) in the outermost layer of the cell wall abolishes the ability of M. smegmatis and M. avium to form biofilm on PVC. 1  Glycosyltransferase (CDC 1551) catalyzes the addition of Rha (3-O-Me-rhamnose) to 6-d-Tal (6-deoxytalose) and is essential for the expression of mature GPLs. 2 1 Recht, J. et al. Glycopeptidolipid Acetlylation Affects Sliding Motility and Biofilm formation in Mycobacterium Smegmatis. J. Bacteriology. Oct 2001. p. 5718-5724. 2 Torsten M. et al. Identification and Recombinant Expression of a Mycobacterium avium Rhamnosyltransferase Gene (rtfA) Involved in Glycopeptidolipid Biosynthesis. J. Bacteriology. Nov 1998 p. 5567-5573

23. cytoplasma Peptidoglycan Mycolic acid Glycopeptidolipid (GPL) cell wall cytoplasmic membrane * GPL is associated with biofilm formation in mycobacteria Trehalose Lipoarabinomannon (LAM) Model of Mycobacterial Cell Wall

24. Discussion, continued:  GuaB2 (H37Rv), which is the same as IMPDH (CDC1551), is an inosine 5’ monophosphate dehydrogenase. This protein catalyzes the first reaction unique to GMP biosynthesis, which in turn synthesizes GDP and is necessary for GPL expression.  IMPDH also has a key role in the growth of many cell types. Escobar-Henriques, M. et al. Transcriptional Regulation of the Yeast GMP Synthesis Pathway by Its End Products. J of Biological Chemistry. 276:2 pp. 1523-1530. 2001.

25. Discussion, continued:  Rv0539 (H37Rv) is a probable glycosyltransferase and, as mentioned before, is important for the expression of mature GPLs.  Rv 3413c (H37Rv) is a probable Alanine and Proline rich protein, function unknown  Rv 3526 (H37Rv) is a possible oxidoreductase and is probably involved in cellular metabolism.  Rv 0359 (H37Rv) is a probable conserved integral membrane protein  Rv0358, Rv 3412, and Rv0538 (H37Rv) are conserved hypothetical proteins, functions are not yet known.

26. Conclusions:  Several genes isolated through research seem to play a role in glycopeptidolipid (GPL) biosynthesis or expression.  The relationship of GPLs to successful biofilm formation in M. Avium and M. Smegmatis on PVC is affirmed.

27. Acknowledgements  HHMI  Luiz Bermudez  Yoshitaka Yamazaki  OSU College of Veterinary Medicine