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Conjugative DNA transfer, antibiotic resistance and MDR bacteria
With thanks to Steve MatsonWho first created this lecture
Antibiotics – a medical miracle
The discovery of antibiotics
changed the medical landscape
http://www.nature.com/nature/journal/v406/n6797
Life expectancy increased by 8 years between 1944 and 1972
Bacterial infection as cause of death plummeted
www.gro-scotland.gov.uk
Deaths in Scotland due to infectious disease per 100,0000
Life expectancy increased by 8 years between 1944 and 1972
Bacterial infection as cause of death plummeted
www.gro-scotland.gov.ukDeaths in Scotland due to TB per 100,0000
The antibiotic resistance problem
Drug resistant bacteria are very wide spread occurring throughout the world
The antibiotic resistance problem
Drug resistance happens quicklyOne 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
MRSA Staph aureus 19,000 deaths
HIV 17.011 deaths
Stats from CDC
The antibiotic resistance problem
85% of the cases of MRSA Staph were acquired in hospitals or other health care settings
MRSA Staph aureus 19,000 deaths
HIV 17.011 deaths
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 rateFar 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 rateFar 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 drugIf this were the result of point mutations in
the chromosome the rate would be even slower
Plasmids are a key to combiningthem 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
1– how are plasmids rapidly transferred in a bacterial population?
2 – how do plasmids encode resistance to multiple drugs?
To understand the rapid increase in multiple drug resistant strains of bacteria there are two questions we must answer.
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 one-way
from donor to recipient Cells must be in close cell-cell
contact for DNA transfer to occur
F Plasmid
• 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
William Hayes
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
Tra I (H) = helicase
Tra Y (R)= nicks donor DNA at oriTand remains covalently linked during transfer
Tra D = links TraY to Type 4 secretion machine
Look among existing drugsfor small moleculesthat inhibit the Relaxase
1 nM
10 nM
Proc Natl Acad Sci U S A. 2007 Jul 24;104(30):12282-7
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
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