1 Anthony Sung BioC 118Q Professor Doug Brutlag May 29 2002 Avoiding Selective Pressure Using Genomics to Design Anti Virulence Drugs Current antimicrobial drugs target genes that are essential for survival creating immense selection pressure for populations to develop resistance One way around this problem is for antibiotics to target virulence genes instead of essential genes Because many virulence genes do not affect survival anti virulence drugs would exert less selection pressure In the face of rising antimicrobial resistance genomics and bioinformatics offer new opportunities for understanding pathogenesis and identifying new drug targets Antimicrobial resistance is widespread rapidly rising and deadly A wide range of bacterial strains that were previously susceptible to antibiotics are now resistant these include 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 multiresistant Salmonella typhi which causes typhoid 1 Furthermore resistance can spread rapidly in 1987 less than 10 of Shigella dysenteriae isolates in Bangladesh were resistant to nalidixic acid in 1992 more than 90 of isolates were nalidixic acid resistant 2 Resistance has even risen against vancomycin the antibiotic of last resort increasing from 0 3 in 1989 to 7 9 in 1993 3 The threat posed by these drug resistant bacteria cannot be understated infectious diseases are one of the leading causes of death in the world accounting for 13 3 million deaths 25 of all deaths in 1998 and are related to 2 five 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 treat multiresistant forms of tuberculosis are over 100 times more expensive than the first line drugs 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 and huge numbers of bacteria increase the frequency of mutations within a population Once a 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 the incorporation of bacterial DNA from the environment and conjugation the exchange of genetic material between bacteria Furthermore genes coding for resistance are frequently found on transposons small units of DNA that are readily exchangeable Adding to the natural evolution of resistance current antibiotics create immense selection pressures that encourage the spread of resistance Because antibiotics kill nonresistant bacteria the remaining resistant bacteria face greatly reduced competition for nutrients and will rapidly proliferate resulting in a resistant population Furthermore antibiotics often kill innocent bystanders benign bacteria that would otherwise limit the spread of pathogens by competing for resources even worse the surviving bacteria often become reservoirs of resistance genes passing on these traits to foreign pathogens 7 Selection pressure favoring resistant bacteria might be reduced if antimicrobials targeted virulent genes instead of essential genes Pathogens want to survive the reason there is immense selection pressure on a population to evolve resistance is resistance is 3 necessary for survival If drugs inhibited virulent genes but did not kill the microbes this pressure would be reduced In fact pathogens find it to their advantage to mitigate their virulence provided they can do so without compromising their livelihood 8 Furthermore these harmless bacteria could actually help protect against other diseases because they would compete with and thus limit the proliferation of other pathogens 9 Anti virulence drugs would allow humans and microbes to coexist similar to the relationship between humans 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 compounds that killed microbes Although these methods successfully discovered many antibiotics they employed a trial and error methodology the goal of which was to discover compounds that killed the microbe in vitro rather than stopped virulence in vivo Today genomics and bioinformatics enable the rational drug design of antimicrobials targeted specifically at pathogenic genes By using techniques such as in vivo expression technology IVET differential fluorescence induction DFI and signature tagged mutagenesis STM scientists can discover which genes are activated by microbes during the process of infection these genes are often inactive and thus undetectable in vitro Genomics and bioinformatics can also be employed in the functional analysis of these gene sequences These technologies also allow scientists to use comparative genomics to identify the best drug targets Finally DNA microarrays can be used to construct expression profiles aiding target identification as well as allowing mechanism of action studies at all discovery phases 10 4 In IVET a vector that contains a promoterless resistance gene is randomly inserted into bacterial genomic DNA fragments The host is then infected incubating the modified bacteria in vivo The microbes are then recovered Because the vector is constructed so that the indicator gene is expressed only when the modified bacteria gene is activated 11 scientists can detect which genes are activated by screening for antibiotic resistance Because the incubation time can be varied IVET can be used to generate temporal information on gene expression DFI is used to identify infection specific processes First green fluorescence protein GFP is fused to different bacterial promoters GFP is used because it can usually be expressed in bacteria without disturbing its pathogenicity 12 When these modified microbes infect hosts transcription factors bind to promoters activating both virulence gene and GFP at
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