Genes Associated with Biofilm Formation in Mycobacterium smegmatis

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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

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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.

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Where are Biofilms Found?
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.
Bacterial biofilms are often a cause of
infections associated with medical implants such
as catheters and IV lines and other medical
devices.
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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.

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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).

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

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Mycobacteria An Overview

• There are gt70 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.

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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.

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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

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My Research

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

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Design of My Plasmid
Promoter-less GFP
M. avium Library
pEMC 1
Kanamycin Resistance
Mycobacterium Origin of Replication
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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 37C for 3-4 days to increase
  bacterial concentration.

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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.

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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.

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Sample GFP Expression Assay Results
117 127 115 122 113 141 123 115 103 131
53 63 56 61 65 68 65 66 67 75
102 101 63 67 74 94 84 82 83 105
74 58 66 67 69 68 62 65 66 117
88 68 116 69 80 116 72 73 68 75
56 55 56 59 56 58 60 60 63 76
61 65 79 58 86 103 60 90 122 139
73 84 63 66 67 63 62 82 67 71
Day One (Control)
141 133 116 142 121 141 154 152 141 143
64 69 51 65 77 65 59 59 62 68
119 114 69 94 88 117 158 89 133 130
101 68 71 91 92 76 66 57 61 123
101 98 157 96 93 122 95 79 80 87
59 49 50 52 51 55 53 53 58 84
66 60 119 51 109 124 57 90 144 149
88 107 62 66 80 60 58 89 67 98
Day Five
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Analysis of Results
1.44 1.55 1.21 1.05 1.01 1.16 1.07 1.00 1.25 1.32 1.37 1.09
1.17 1.60 1.21 1.10 0.91 1.07 1.18 0.96 0.91 0.89 0.93 0.91
1.35 1.66 1.17 1.13 1.10 1.40 1.19 1.24 2.00 1.09 1.60 1.24
2.05 1.52 1.36 1.17 1.08 1.36 1.33 1.12 1.06 0.88 0.92 1.05
1.56 1.21 1.15 1.44 1.35 1.39 1.16 1.05 1.32 1.08 1.18 1.16
1.27 1.07 1.05 0.89 0.89 0.88 0.91 0.95 0.88 0.88 0.92 1.11
1.72 1.31 1.08 0.92 1.51 0.88 1.27 1.20 0.95 1.00 1.18 1.07
1.67 1.74 1.21 1.27 0.98 1.00 1.19 0.95 0.94 1.09 1.00 1.38
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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 37C for 3-4 days.
• Steps one and two are repeated with the new plate
  (1 colony per well)

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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.

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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.

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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)

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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
• 1Recht, 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

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Model of Mycobacterial Cell Wall
Lipoarabinomannon (LAM)
Glycopeptidolipid (GPL)
Trehalose
cell wall
Mycolic acid
cytoplasmic membrane
Peptidoglycan
cytoplasma
GPL is associated with biofilm formation in
mycobacteria
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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.
  2762 pp. 1523-1530. 2001.

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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.

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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.

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Acknowledgements

• HHMI
• Luiz Bermudez
• Yoshitaka Yamazaki
• OSU College of Veterinary Medicine