A thesis submitted

by
Riddhika Pandya
Graduate Student
M.Sc. Pharmacology & Toxicology

Guided By
Dr. Mrinal K. Bhattacharjee
 INDEX:

 GENRAL INTRODUCTION OF ACTINOBACILLUS

ACTINOMYCETEMCOMITANS

 AIM OF THE STUDY

 BACTERIAL STRAINS USED AND METHODS

 RESULTS

 DISCUSSION

 IMPORTANCE OF PROJECT

 FUTURE APPLICATIONS
 Actinobacillus Actinomycetemcomitans

• A.actinomycetemcomitans is gram negative facultative anaerobe , non-motile

cocobacillus and a member of pasteurellacea. Family. It is major causative
agent of Localized aggressive periodontitis.

• Apart from being a major periodontal pathogen, evidences of the presence of

their genomic DNA in lower respiratory tract, meninges of CNS and urinary
tract suggest that they are also non oral pathogens.

• A.a. is predisposing factor for several other major diseases like atherosclerosis
and related conditions of coronary artery disease, stroke, diabetes and
pregnancy complications, abscesses, meningitis, pneumonia, septicemia, UTI &
osteomyelitis.
Distribution:

 In united states, periodontitis Affects 14% of adults aged 45-54, 23% of

those aged 65-74. Mean prevalence was 0.53% among adolescents of all
racial origins , and A.a. was isolated in 97% of those cases .

 Adolescents of African-American descent were found to have a 15- fold

higher incidence of diseases than Caucasian Americans.

 Very few research that support genetic predisposition to Localized

aggressive periodontitis.
Phenotypes:
 Fresh clinical isolates of A.a. produce rough, star
positive colonies on plates.
 Cells adhere to each other and to solid surface.

 Smooth colonies grow as more

opaque, glistering, circular colonies with regular
borders .

 Natural conversion of rough to smooth has been

observed.
Oral and Extra-oral Pathology:

 A. a. s is one of the most powerful periodontal pathogen. It is naturally found in

dental plaque, periodontal pockets and gingival sulcus. It is also present in
periodontal pocket disease.

 Extra-oral pathologies includes, preterm low birth

weight, atherosclerosis, plaque buildup in the arteries, which creates greater
risk of stroke and heart attack.

 It also plays important role in accumulation of cholesterol in blood stream with

a support of macrophage derived foam cells. Thus, contribute role in formation
of atheroma.
Continue....

 0.6% of infective endocarditis are caused by A.a.

 A.a. is a member of clinically important HACEK group. ( Haemophilus

aphrophilus, A.a., cardiobacterium ominis, Eikenella corrodense, Kingella
Kinge) .

 This group of bacteria causes inflammation of heart valves .

 Studies show that infection by these bacteria may impact the effectiveness of
some medicines which are used to prevent heart attacks.
Mechanism of pathogenicity:
Virulence factors of A. a.

 One of the best studied virulence factor of A. a. is Leukotoxin ( a 14 kDa

secreted lipoprotein that belongs to RTX family.

 leukotoxin has been shown to kill polymorphonuclear leucocytes and

macrophages .

 Other ill defined virulence proteins of A. a. are

# Thioredoxins that inhibit lymphokine production.
# An unidentified supra antigen which cause T- cell apoptosis.
# Cytolethal distending toxin ( Cdt A, B, and C) causes arrest of cell
growth in G2 phase.
Natural Methods of gene transfer observed in A. a.

 Natural gene transfer or DNA translocation occur during several important

biological processes, such as
- infection by bacteriophages, conjugative DNA transfer of plasmids, T-
DNA transfer and natural genetic transformation.
General mechanism of Natural transformation

 Development of
competence

 Binding of DNA to the cell

surface

 Processing and Uptake of

DNA into cell

 Incorporation of DNA into

host genome via
homologous recombination.
 Continue…..

 Natural transformation of A. a. appears to be strain specific and is of too low

frequency to be useful in genetic studies.

 Requirements for natural transformation:

# Presence of 9-bp uptake signal sequences in incoming DNA
# expression of tfox gene to activate genes of competence
# Induction of genes of competence

 Similar features were observed in another gram negative bacteriaum

H.influezae.
 Development of Competence and role of Tfox gene

• Induction of competence in A.a. requires the product of tfoX (sxy) gene

• Tfox protein itself does not bind to DNA but interacts with CRP(CAP)

• Turn on the genes of the competence regulon

• Thus expression of tfoX makes A.a. cell constitutively competent

 Significance of USS in natural transformation

 USSs are believed to be genomic identity tags

 A.a. seems to take up their own DNA preferentially
 This specificity arises from presence of USSs .
 Numerous copies of USSs found in genome of A.a.
 Cell surface proteins or transporters that bind the USS have not yet been
identified

 USS are often found in inverted-repeat pairs downstream from coding region.
 Need for natural transformation

 The lack of genetic tools

 Scarcity of standard and biological techniques which work well on A.a.

 Preference to take DNA containing sequences which are present frequently
in its own genome.
 Lack of treatments which permanently cure LAP by A.a.
 Aim of the study:

 The objective of this study is to demonstrate a novel mechanism of natural

transformation of A . a. with genomic and plasmid DNA present in micro
vesicles secreted by donor cells in growth medium.

 To demonstrate that A. a. can be naturally transformed by this method, both in

the presence or absence of Uptake signal sequences (USS) or Tfox gene.
Materials and Methods
Recipient strains of A. a.

Formal name of LIU names serotype Reference/Source

strains

 Strainsused: LIU 1235

DF2200Nal A David Figurski, Columbia
 Recepient strains of A. Actinomycetemcomitans
University

NJ1000Nal LIU 1195 B Dan Fine, UMDNJ

NJ 2700Nal LIU 1196 C Dan Fine, UMDNJ

IDH781Nal LIU 1231 D Dan Fine, UMDNJ

NJ9500Nal LIU 1201 E Dan Fine, UMDNJ

NJ9100Nal LIU 1193 F Dan Fine, UMDNJ

CU1000Nal LIU 1188 F David Figurski, Columbia

University
 Donor strains of A.a.
Name of strains Formal names Source/ Reference

LIU 1121 Y4Nal::katAIS903фKan Thomson et. al.1999

LIU 1212 Y4NalTn::katA(pJAK 17) pJAK 17 mobilized from E.coli into

LIU1121
LIU 1223 Y4nalTn::Kat(pMB 40) pMB40 mobilized from E.coli into Liu
1121

 All donor strains were catalase negative.

 pJAK 17 is a derivative of the broad host range plasmid RSF1010.
 pMB40 contains a two copies of USS cloned into pJAK13, a similar
derivative of RSF1010 .
 E.coli strain used:
LIU 4 (MV10Nal)
 Transformation with genomic donor DNA containing kanamycin resistance
marker present in vesicles in the growth medium of donor stains
 Set 1:

24
hours
culture tubes

Incubation for
different
length of
time
 Continue…..

 Set 2: recipient cells were resuspended in 2.5 ml of donor DNA instead of

100 µl.
 Transformation Assay : 2

 Transformation with plasmid DNA with and without USS present in growth
medium of donor strains LIU 1212 and LIU 1223 respectively.
 4 steps were as above.

Km(40) Cm(2) Km(40) Sp(20)

Transformation Assay : 3

Transformation of E.coli

LIU 4

Nal(20) Nal(20 Nal(20) Nal(20)

Km(50) Cm(50) Km(50) Sp(50)
Transformation assay 4

Transformation with heated growth medium

Transformation assay 5

Transformation with frozen supernatant

Catalase test

Individual colonies of transformant were picked with tooth pick without

touching the plate surface and dipped into eppendorf tube containing 1 ml
of 3% hydrogen peroxide
Results Transformation assay 1 and 2

Number of transformants

Fresh donor growth medium Frozen/thawed donor growth medium

Growth medium volume Growth medium volume

0.1 ml 2.5 ml 0.1 ml 2.5 ml

Recipient Serotype Incubation time Incubation time Incubation time Incubation time

2h 3h 5h 2h 3h 5h 2h 3h 5h 2h 3h 5h
Strains

1235 A 457 388 299 389 401 403 425 478 489 378 425 467

1195 B 1377 1490 1234 989 1189 1478 1340 1456 990 1290 1329 1365

1196 C 299 320 345 345 377 389 297 329 453 357 430 298

1231 D 378 344 390 289 385 489 408 369 478 376 402 389

1201 E 109 98 110 145 134 129 123 112 97 104 132 116

1193 F 119 128 113 145 90 138 97 156 123 154 145 134

1188 F 109 145 93 94 119 137 118 132 98 156 114 123
Gel electrophoresis:

PMB 78
positive
control

A.a.
transformant
Transformation assay 2

Transformants/ml Transformants/ml

(spectinomycin
Recipient strains Serotype (kanamycin
resistant)
resistant)
LIU 1235 A 154 + 34 47 + 16
LIU1195 B 206 + 29 123 + 45
LIU1196 C 173 + 21 78 + 13
LIU 1231 D Smooth 139 + 13 39 + 2

Rough 176 + 19.5

LIU 1201 E 189 + 18 98 + 19
LIU1193 F 142 + 24 56 + 9
LIU 1188 F 154 + 9 67 + 4
Gel electrophoresis

pMB40
positive
control

A.a.
transformants
Transformation assay 3
Recipient strains Serotype Transformants/ml Transformants/ml
(kanamycin (chloramphenicol
resistant) resistant )
LIU 1235 A 201 + 14 45 + 5
LIU1195 B 270 + 37 17 + 9
LIU1196 C 145 + 12 34 + 7
LIU 1231 D Smooth 34 + 4 11 + 3

Rough 103 + 6
LIU 1201 E 245 + 45 82 + 21
LIU1193 F 305 + 19 78 + 15
LIU 1188 F 197 + 34 46 + 13
Gel electrophoresis

PJAK 17
positive
control

A.a.
transformants
Transformation assay 4

With LIU 1212 spent medium

Recipient strain Transformants/ml Transformants/ml

(kanamycin resistant) (chloramphenicol resistant )

LIU 4 1077 + 45 1014 + 65

With LIU 1223 spent medium

Recipient strain Transformants/ml Transformants/ml

(kanamycin resistant) (spectinomycin resistant)

LIU 4 1538 + 78 962 + 92

Gel electrophoresis of genomic DNA transformants

pMB 79
positive
control

E.coli
genomic
DNA
transformants
Gel electrophoresis of plasmid DNA transformants with USS

pMB 40
positive
control

E.coli plasmid
DNA
transformants
Gel electrophoresis of plasmid DNA transformants without USS

pJAK 17
positive
control

E.coli plasmid DNA

transformants
Transformation assay 5
Discussion

 Preliminary studies show that mechanism of natural transformation in A. a. is

dependent on expression of tfox gene and presence of USS and development
of competence.

 A novel method of natural transformation is independent of both TfoX and

USS.
 The possible explanation for this behavior is that bacteria may undergo
process of adhesion and fusion with outer membrane of recipient strain of A.
a. whereby the DNA can be delivered inside the recipient cytoplasm.

 The donor DNA then undergoes a homologous recombination with the

resident genomic DNA of the recipient cell resulting in allelic exchange.
Continue….

 A. a. can be transformed with genomic as well as plasmid DNA with or

without USS present in the growth medium of donor cells.
 Hypothetical mechanism for this transformation is that the DNA containing
donor micro vesicles of A.a. in medium had interaction with recipient
bacteria through fusion or adhesion mechanisms.
 It was seen that all the six serotypes of A. a. were naturally transformable.
 A better method than the TfoX dependent method which does not work for F
serotype A. a.
 Transformation of E. coli with DNA from donor A. a. cells suggest the
possibility of interspecies transfer of genetic material by this method.
 This method is a much easier method than the conventional method of using
isolated pure DNA.
 As high transformation frequency was achieved using growth medium as a
source of DNA.
Continue….

 Heating or freezing of growth medium does not affect transformation efficacy

suggesting that the DNA secreted into the growth medium is protected in
micro vesicle made up of some structure that is resistant to heating and
freezing.
 DNA present in growth medium of A. a. can also be used to transform other
species of bacteria.
 The bacterium takes in DNA at a rate that is independent of the total amount
of DNA present in the growth medium. (Why???)
 Importance of project

 Provide focus on presence of DNA in membrane vesicle released by A.a. in

growth medium during their growth.

 Shows the possibility of involvement of DNA in pathogenesis of LAP by

A.a.
Future applications

 Provide good genetic tool to study A.a. genome over the present standard
molecular and biological techniques.

 Synthetic proteoliposomes have been developed to deliver drugs to tumors and

specific tissues, although off-target effects are a continuous problem. Making use
this adherence and entry mechanism that target native outer membrane vesicle to
host cells could improve the specific targeting of engineered therapeutic
liposomes for LAP.