Transcript Conjugative DNA transfer, antibiotic resistance and MDR bacteria

Conjugative DNA
transfer, antibiotic
resistance and MDR
bacteria
With thanks to Steve Matson
Who first created this lecture
Antibiotics – a medical miracle
The discovery of antibiotics
changed the medical landscape
http://www.nature.com/nature/journal/v406/n6797
Bacterial infection as cause of
death plummeted

Life expectancy increased by 8 years
between 1944 and 1972
Deaths in Scotland due to infectious disease per 100,0000
www.gro-scotland.gov.uk
Bacterial infection as cause of
death plummeted

Life expectancy increased by 8 years
between 1944 and 1972
Deaths in Scotland due to TB per 100,0000
www.gro-scotland.gov.uk
The antibiotic resistance
problem

Drug resistant bacteria are very wide
spread occurring throughout the world
The antibiotic resistance
problem

Drug resistance happens quickly

One study observed an increase from
0% to 28% drug resistant E. coli in less
than 5 years
The antibiotic resistance
problem

In 2005 there were more deaths in the US from
Methicillin resistant Staphylococcus aureus
than from AIDS
HIV 17.011 deaths
MRSA Staph aureus 19,000 deaths
Stats from CDC
The antibiotic resistance
problem

85% of the cases of MRSA Staph were acquired in
hospitals or other health care settings
HIV 17.011 deaths
MRSA Staph aureus 19,000 deaths
How antibiotics work
How do drug resistant bugs
arise?
evolution.berkeley.edu
How do drug resistant bugs
arise?
evolution.berkeley.edu
How do drug resistant bugs
arise?
evolution.berkeley.edu
How do drug resistant bugs
arise?
evolution.berkeley.edu
How did that 1st drug
resistant bug arise?

A simple error in DNA
replication that produced a
mutation




Occurs at low frequency
Mutation is on the
chromosome
Mutation affects either
ribosomal protein S12 or 16S
rRNA to produce streptomycin
resistance
Does not explain MDR bugs or
high rate of spread
How do we solve this puzzle?

We know that drug resistance spreads at
an alarming rate

Far too fast to be the result of single
mutations in the chromosome that arise
independently
How do we solve this puzzle?

We know that drug resistance spreads at
an alarming rate


Far too fast to be the result of single
mutations in the chromosome that arise
independently
We also know that bacteria become
resistant to more than a single drug

If this were the result of point mutations in
the chromosome the rate would be even
slower
The four waves of antibiotic resistance in Staph. aureus
Vancomycin resistant
There are many ways of
becoming drug resistant
Plasmids are a key to combining
them together in one bacterium
A plasmid is an extra-chromosomal DNA molecule
separate from the chromosomal DNA which is
capable of replicating independently of the
chromosomal DNA. In many cases, it is circular
and double-stranded. Plasmids usually occur
naturally in bacteria, but are sometimes found in
eukaryotic organisms
Two questions
To understand the rapid increase in multiple drug
resistant strains of bacteria there are two
questions we must answer.
1– how are plasmids rapidly
transferred in a bacterial
population?
 2 – how do plasmids encode
resistance to multiple drugs?

Bacterial conjugation




Driven by conjugative plasmids;
1st example called the fertility
factor or F
 found in some but not all E.
coli
 one of several different types
of conjugative plasmid
Mating only between cell with F
(F+) and cell without F (F–)
Transfer of information is oneway from donor to recipient
Cells must be in close cell-cell
contact for DNA transfer to occur
F Plasmid
William Hayes
• A 100 kbp plasmid (single copy) with ~ 100 genes
– Replicates inside host cell using host machinery for replication
– Partitions to daughter cells in a manner similar to chromosome
F Plasmid
• Contains genes encoding synthesis of pillin which is
assembled into pili that allow cell contact
• F+ cells have pili and F- cells lack pilli
• F+ inhibited from making contact with other F+ cells
F Plasmid
• F+ cells conjugate with F– cells
– F+ donates single-stranded copy of F to F– cell (rolling circle)
– F+ retains copy of plasmid, F- cell converted to F+ by
replication of ssDNA donated to the F- cell
– Allows F plasmid to rapidly spread through a bacterial
population
Bacterial Conjugation



Bacterial conjugation is
the primary mechanism
used to spread
antibiotic resistance
among bacterial
populations
There will be several
million infections
involving antibiotic
resistant bacteria this
year
This is now a very
significant health
problem
Pumping ssDNA
Pumping ssDNA
Tra I (H) = helicase
Tra Y (R)= nicks donor DNA at oriT
and remains covalently linked
during transfer
Tra D = links TraY to
Type 4 secretion machine
This machine can be a drug target
Look among existing drugs
for small molecules
that inhibit the Relaxase
1 nM
10 nM
Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12282-7
These inhibit DNA transfer!
Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12282-7
Plasmid transfer provides a
drug target
Plasmid transfer provides
other drug targets
Plasmids that replicate in similar ways (top, red and blue)
compete for resources, and the losing plasmid
is lost from the bacterial cell.
J. Am. Chem. Soc., 2004, 126 (47), pp 15402–15404
Plasmid transfer
provides a drug target
An aminoglycoside that binds the small RNA
causing plasmid incompatibility can mimic this natural process,
Causing elimination of a drug-resistance plasmid (bottom, green).
J. Am. Chem. Soc., 2004, 126 (47), pp 15402–15404
Transposable Genetic
Elements are also key to
antibiotic resistance

A variety of colorful names have been used to describe these
genetic elements







Controlling elements
Jumping genes
Roving genes
Mobile genetic elements
Transposons
Definition: Transposable genetic elements (transposons) are
DNA segments that can insert themselves at one or more sites
in a genome. They are ubiquitous among organisms and play
an important role in genome evolution.
Remarkably, almost 50% of our chromosomes consist of
transposable elements

We are still unsure of the normal genetic role, if any, of these
elements
Composite versus simple Tns


Composite Tns contain a variety of genes between two IS
elements
 Transposase is encoded by one of the elements
 Individual IS elements cannot move
Simple Tn contains short IRs at each end
 Encode their own transposase and other genes
Transposons carry drug
resistance genes onto
plasmids called R plasmids
The plasmid can then be
transferred to another
bacterium by conjugation
How does transposition occur?
Transposition is catalyzed by an enzyme, transposase, encoded by
the transposon
The ends of the transposon are critical for transposition
Our genome is filled with
transposons and their “fossils”


Human genome is typical in terms of abundance
and distribution of mobile elements
 How do we survive?
 Elements inserted into introns
 Vast majority of elements cannot move
There are instances of mutations caused by mobile
elements
R plasmids can become
increasingly complex
through natural selection
http://www.fbs.leeds.ac.uk/staff/profile.php?tag=ONeill_AJ
Integrases can move DNA
flanked by direct repeats
From plasmids to chromosome
and back
Research into this area is
key to combating TB and
other bacterial infections!
CDC