Transcript Biofilm Formation in Mycobacterium smegmatis

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
What is Biofilm?
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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.
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.
Why Research Biofilms?
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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.
Biofilms and Infections:
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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).
How are Biofilms Formed?
Biofilm formation relies on an exchange of
chemical signals between cells in a process
known as “quorum sensing.”
Quorum Sensing
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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.
Mycobacteria: An Overview
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There are >70 species of mycobacteria,
many of which are human pathogens.
Of these, three are major pathogens:
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Mycobacterium tuberculosis
Mycobacterium leprae
Mycobacterium avium
Many species of mycobacteria are found
in the environment where biofilm
formation is demonstrated.
Incidence of Mycobacterial Infections
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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.
Mycobacterium smegmatis
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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
My Research
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Using Mycobacterium smegmatis as a
model, I will investigate the genes that
are important for biofilm formation in
other more virulent mycobacteria.
Design of My Plasmid:
Promoter-less GFP
M. avium Library
pEMC 1
Kanamycin Resistance
Mycobacterium Origin of Replication
Step One:
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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.
Step Two:
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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.
Step Three:
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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.
Sample GFP Expression Assay Results
Day One (Control)
Day Five
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
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
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
Step Four:
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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)
Step Five:
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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.
Step Six:
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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.
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)
Discussion:
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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
Model of Mycobacterial Cell Wall
Lipoarabinomannon (LAM)
cell wall
Glycopeptidolipid (GPL)
Trehalose
Mycolic acid
cytoplasmic
membrane
Peptidoglycan
cytoplasma
* GPL is associated with biofilm formation in mycobacteria
Discussion, continued:
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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.
Discussion, continued:
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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.
Conclusions:
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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.
Acknowledgements
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HHMI
Luiz Bermudez
Yoshitaka Yamazaki
OSU College of Veterinary Medicine