1Anthony SungBioC 118QProfessor Doug BrutlagMay 29, 2002Avoiding Selective Pressure: Using Genomics to Design Anti-Virulence DrugsCurrent antimicrobial drugs target genes that are essential for survival, creatingimmense selection pressure for populations to develop resistance. One way around thisproblem is for antibiotics to target virulence genes instead of essential genes. Becausemany virulence genes do not affect survival, anti-virulence drugs would exert lessselection pressure. In the face of rising antimicrobial resistance, genomics andbioinformatics offer new opportunities for understanding pathogenesis and identifyingnew drug targets.Antimicrobial resistance is widespread, rapidly rising, and deadly. A wide rangeof bacterial strains that were previously susceptible to antibiotics are now resistant; theseinclude penicillin resistant Streptococcus pneumoniae, which causes pneumonia,penicillin and tetracycline resistant Neisseria gonorrhoeae, which causes gonorrhea,rifampicin and isoniazid resistant Myobacterium tuberculosis, which causes tuberculosis,multiresistant Shigella dysenteriae, which causes dysentery, and multiresistantSalmonella typhi, which causes typhoid.1 Furthermore, resistance can spread rapidly: in1987, less than 10% of Shigella dysenteriae isolates in Bangladesh were resistant tonalidixic acid; in 1992, more than 90% of isolates were nalidixic acid-resistant.2Resistance has even risen against vancomycin, the “antibiotic of last resort,” increasingfrom 0.3% in 1989 to 7.9% in 1993.3 The threat posed by these drug-resistant bacteriacannot be understated: infectious diseases are one of the leading causes of death in theworld, accounting for 13.3 million deaths (25% of all deaths) in 1998, and are related to2five of the ten leading causes of death in the U.S.4 Even if other drugs can be used,second- or third-line antimicrobials are often more expensive – the drugs needed to treatmultiresistant forms of tuberculosis are over 100 times more expensive than the first-linedrugs used to treat non-resistant forms.5 Resistant bacteria are also more difficult to treat,and patients may suffer longer while doctors try different antibiotics.Resistance is a natural biological phenomenon. Although mutations are rare,occurring in about 1 in 1,000,000 or 1 in 10,000,000 cells,6 the rapid reproduction andhuge numbers of bacteria increase the frequency of mutations within a population. Oncea resistance gene arises, it can be spread between bacteria, even across the species barrier,through transduction, the transfer of bacterial DNA by a virus; transformation, theincorporation of bacterial DNA from the environment; and conjugation, the exchange ofgenetic material between bacteria. Furthermore, genes coding for resistance arefrequently found on transposons, small units of DNA that are readily exchangeable.Adding to the natural evolution of resistance, current antibiotics create immenseselection pressures that encourage the spread of resistance. Because antibiotics kill non-resistant bacteria, the remaining resistant bacteria face greatly reduced competition fornutrients and will rapidly proliferate, resulting in a resistant population. Furthermore,antibiotics often kill “innocent bystanders,” benign bacteria that would otherwise limitthe spread of pathogens by competing for resources; even worse, the surviving bacteriaoften become reservoirs of resistance genes, passing on these traits to foreign pathogens.7Selection pressure favoring resistant bacteria might be reduced if antimicrobialstargeted virulent genes instead of essential genes. Pathogens want to survive; the reasonthere is immense selection pressure on a population to evolve resistance is resistance is3necessary for survival. If drugs inhibited virulent genes but did not kill the microbes, thispressure would be reduced. In fact, pathogens find it to their advantage to mitigate theirvirulence, provided they can do so without compromising their livelihood.8 Furthermore,these “harmless” bacteria could actually help protect against other diseases because theywould compete with, and thus limit the proliferation of, other pathogens.9 Anti-virulencedrugs would allow humans and microbes to coexist, similar to the relationship betweenhumans and the millions of harmless bacteria that live in their guts.Until recently, scientists lacked the technology to develop anti-virulence drugs.The antibiotics of the 1950s and 60s were discovered by whole-cell screening approaches– scientists either used combinatorial chemistry or looked directly for natural compoundsthat killed microbes. Although these methods successfully discovered many antibiotics,they employed a trial-and-error methodology, the goal of which was to discovercompounds that killed the microbe in vitro rather than stopped virulence in vivo.Today, genomics and bioinformatics enable the rational drug design ofantimicrobials targeted specifically at pathogenic genes. By using techniques such as invivo expression technology (IVET), differential fluorescence induction (DFI), andsignature-tagged mutagenesis (STM), scientists can discover which genes are activatedby microbes during the process of infection; these genes are often inactive, and thusundetectable, in vitro. Genomics and bioinformatics can also be employed in thefunctional analysis of these gene sequences. These technologies also allow scientists touse comparative genomics to identify the best drug targets. Finally, DNA microarrayscan be used to construct expression profiles, aiding target identification as well asallowing mechanism of action studies at all discovery phases.104In IVET, a vector that contains a promoterless resistance gene is randomlyinserted into bacterial genomic DNA fragments. The host is then infected, incubating themodified bacteria in vivo. The microbes are then recovered. Because the vector isconstructed so that the indicator gene is expressed only when the modified bacteria geneis activated,11 scientists can detect which genes are activated by screening for antibioticresistance. Because the incubation time can be varied, IVET can be used to generatetemporal information on gene expression.DFI is used to identify infection-specific processes. First, green fluorescenceprotein (GFP) is fused to different bacterial promoters; GFP is used because it can usuallybe expressed in bacteria without disturbing its pathogenicity.12 When these modifiedmicrobes infect
View Full Document
Unlocking...