Biology 322 Lecture Nov 15, 2010 Anouncements: see course web site Backtrack to a previous lecture: where do antibiotic resistance genes and alleles come from? Thinking about the nature of mutation & about mutation frequency Tuberculosis and Antibiotic Resistance 1
How do they confer resistance? Science 293: 1786 Sept. 7, 2001 2
Where do antibiotic-resistance alleles/genes come from? 3
Weapons of Microbial Drug Resistance Abound in Soil Flora http://fire.biol.wwu.edu/trent/trent/microbialresistance.pdf (+) = acquired from an extraspecies source What do we know about the transfer mechanisms? What types of genetic elements are involved? 4
A = adaptive implying a darwinian process What requirements for evolution by natural selection does the table on the previous page illustrate? 5
A recent article on antibiotic resistance is titled The bacteria fight back This is misleading: adaptive resistance happens because mutation happens and the antibiotic is simply selecting for preexisting mutations in the vast bacterial populations 6
rif s rif r (rif = the antibiotic rifamycin = rifampin) Target site alteration Rifampin (rifamycin) is a major drug used in the treatment of tuberculosis infections, and increasing rifampin resistance represents a worldwide clinical problem. Resistance to rifampin is caused by mutations in the rpob gene, encoding the beta-subunit of RNA polymerase. The wildtype Mycobacterium tuberculosis RNA polymerase is 1000X more sensitive to this antibiotic than the wildtype E. coli RNA polymerase Tough bug. Mycobacterium tuberculosis (red) likes nothing better than to be ingested by a macro- phage, its usual home. 7
Bacterial RNAP as an antibiotic target: Bacterial RNA polymerase (RNAP) is a proven target for broad-spectrum antibacterial therapy and for antituberculosis therapy RNAP is a suitable target for three reasons: (1) RNAP is an essential enzyme (permits efficacy) (2) bacterial RNAP-subunit sequences are highly conserved (permits broad-spectrum activity) (3) bacterial RNAP sequences and eukaryotic RNAP sequences are less highly conserved (permits therapeutic selectivity). The rifamycin antibacterial agents rifampin (also known as rifampicin) function by binding to and inhibiting bacterial RNAP The rifamycins are in clinical use in treatment of Gram-positive and Gram-negative bacterial infections, are first-line antituberculosis agents, and are among the few antituberculosis agents that can kill nonreplicating tuberculosis bacteria. For all major bacterial pathogens, including the tuberculosis pathogen, strains resistant to rifamycins have arisen Resistance to rifamycins involves substitution of residues within the rifamycin-binding site on bacterial RNAP, i.e., substitutions that directly decrease rifamycin binding 8
What we really need, Rich adds, is a good, cheap, point-of-service test. Wealthier countries have access to polymerase chain reaction tests that monitor variable TB organism genes, signaling within 24 hours whether the strain is resistant to the first-line drugs isoniazid and rifampin. 9
Follow-up on rifr mutants We will follow up on our rifr mutations by determining the location and nature of mutatgenic changes that can confer resistance to the antibiotic without severely compromising the ability of RNA polymerase to perform its cellular function Figure 1. The Rif-Resistant Regions of the RNAP b Subunit The bar on top schematically represents the E. coli b subunit primary sequence with amino acid numbering shown directly above. Gray boxes indicate evolutionarily conserved regions among all prokaryotic, chloroplast, archaebacterial, and eukaryotic sequences. indicate the four clusters where RifR mutations have been identified in E. coli. Mutations that confer RifR in E. coli and M. tuberculosis are indicated directly above (for E. coli) or below (for M. tuberculosis) as follows: D for deletions, V for insertions, and colored dots for amino acid substitutions (substitutions at each position are indicated in single amino acid code in columns above or below the positions). Color coding for the amino acid substitutions is as follows: yellow, residues that interact directly with the bound Rif green, residues that are too far away from the Rif for direct interaction purple, three positions that are substituted with high frequency (noted as a % immediately below the substitutions) in clinical isolates of RifR M. tuberculosis 10
How common should spontaneous mutation to antibiotic resistance be in a population not under selection? Before we address this question in bacterial, let s indulge in thinking about us. 11
Achondroplasia is a completely penetrant, autosomal dominant disorder characterized by disproportionate short stature -- the arms and legs are short compared with the head and trunk. More than 80% of the people who have achondroplasia are born to parents of normal stature and represent a new germline mutation Michael Dunn is best known for his recurring role as the villain Dr. Miguelito Loveless on The Wild Wild West (1965-68). 12
Achondroplasia and the mutagenic male An estimate of the mutation rate in FGFR is based upon the accumulated data of newborn studies in four cities: In a total of 242,257 births, seven infants had achondroplasia From these numbers, the rate of mutation of the normal to the achondroplasia allele is calculated to be 1.4 X 10-5 mutations in the achondroplasia gene per gamete = 1 mutation per 69,216 copies of the gene ** Who is at fault? ** It has been established that fathers are the source of all achondroplasia mutations The probability of having an affected offspring increases explonentially with the father s age 50% of children with achondroplasia are born to father older than 35 years old. 13
Sex, Errors and the Genome by Mark Ridley Natural History (6/2001) At conception, human embryos average about 200 copying errors and about 50% of the embryos have a botched number of chromosomes. WHO IS TO BLAME? 14
When a thirty year old man breeds with a 30 year old woman: his DNA (in his sperm cells) has been copied 430 times against her 33 cell division (in egg cells). with thirteen times as many errata in his DNA, about 185 of the 200 copying mistakes in each human conception may come from the sperm. however, a woman s eggs are more likely to carry serious errors in chromosome numbers, and these errors increase with maternal age. The germline mutation rate in human males, especially older males, is generally much higher than in females, presumably because in males there are many more germ-cell divisions Why is number of cell divisions correlated with mutation rate? 15
Every time a human cell divides it has to replicate 6 X 10 9 base pairs of DNA Every time an E. coli divides it has to replicate 5 X 10 6 base pairs of DNA Every time a base pair is copied, there is a very small but finite probability that an error will be introduced at that site Furthermore, if the DNA molecule has sustained damage that remains unrepaired, then mistakes will occur during DNA replication of that site by so-called error-prone bypass polymerases What sort of damage occurs routinely to DNA? 16
The Mutagenesis lab is a great illustration of the principle that the power of bacterial genetics is the potential for studying rare events We have looked at rare spontaneous [and EMS-induced] mutations: Selection for rif s rif r (rif = the antibiotic rifamycin) Screen for Forward mutation lac+ lac - (lac = lactose) Selection for Reverse mutation lac- lac + How to explain the differences in mutation rates? 17
MUTATION JARGON GENE MUTATION = POINT MUTATION (scales of mutation is small and is localized to a specific region, a single nucleotide or a few adjacent base pairs) at the DNA level: single base pair substitutions: transitions & transversions single (or a few) base pair addition or deletion: indels gene mutation by transposon insertion at the level of gene expression: promoter mutations splicing mutations regulatory mutations at the protein level: nonsense missense [neutral] silent frameshift at the level of gene function: loss-of-function gain-of-function [neutral CHROMOSOME MUTATION involves segments of chromosomes or whole chromosomes or whole genomes alterations in chromosome structure and number deletion, duplications, translocations and inversions CNVs: copy number variations 18
Decoding mutation jargon The (achondroplasia) mutations just discussed are single base substitutions. The most striking is achondroplasia, in which 153 of 154 analysed cases are due to a glycine to arginine substitution at codon 1,138. The mutations are in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3). Of the 153 mutations, 150 were guanine to adenine transitions and three were guanine to cytosine transversions of the same nucleotide. This means that all the cases of achondroplasia are due to changes in one nucleotide a nucleotide with the highest known mutation rate (about 10-5 per generation). There are mutations at other sites in this gene, but the phenotypes are different. The achondroplasia mutations are all at a CpG nucleotide pair, known to be a mutation hot-spot. What do you know about CpG islands? 19
MORE JARGON: gain and loss-of-function 20