Signature-tagged mutagenesis and the hunt for virulence factors: Response.

Dr Perry has written an up-to-date article summarizing the signature-tagged mutagenesis (STM) approach for identifying virulence determinants. He has also provided a summary of the numerous virulence genes that have been reported using this approach in a variety of different pathogens. Every approach has its strengths and weaknesses and the same is true for STM. However, I feel that by placing undue emphasis on the potential limitations of STM, Dr Perry has inadvertently presented a misleading picture of the value of this method. There are three points I would like to emphasize.Firstly, despite any inherent limitations of STM, many new and interesting genes have been identified in every pathogen it has been applied to even though, to date, none of the screens has been comprehensive.Secondly, there seems to be the perception that if an approach is not predicted to identify all possible virulence determinants then it is of little value. In fact, no single approach could possibly identify every virulence determinant. Thus, to obtain a comprehensive picture of the virulence determinants of a pathogen it is important to use more than one approach – knowing the various strengths and weaknesses of each method allows one to choose complementary methods. The STM and in vivo expression technology (IVET) methods are quite complementary and we, and others, have applied both approaches to the same system; in every case there has been little or no overlap in the results, confirming the complementarity of the methods.Lastly, many of the potential limitations of STM have not proven to be serious problems in practice. For example, Dr Perry suggests that the need to use high infecting doses could mask subtle effects. In practice, this is not the case. When individual mutants isolated in our screen were tested for their in vivo competitive index (CI) it ranged from 0.36 (i.e. very little effect) to <0.000003 (Ref. 1xDarwin, A.J. and Miller, V.L. Mol. Microbiol. 1999; 32: 51–62Crossref | PubMedSee all ReferencesRef. 1). Another potential problem raised was that infection could be caused by a very small proportion of the input population and many false negatives will thus be obtained (i.e. virulent mutants will not establish an infection and, therefore, will appear to be avirulent). In fact, with careful experimental design and controls this is a very rare event. In our study, very few ‘mutants’ that passed through our two-stage STM screen proved to be fully virulent when tested individually against wildtype for an in vivo CI (Ref. 1xDarwin, A.J. and Miller, V.L. Mol. Microbiol. 1999; 32: 51–62Crossref | PubMedSee all ReferencesRef. 1). Additionally, it was suggested that extracellular virulence determinants might not be identified because they can be complemented by other mutants in the pool. I think it is far too early to determine if this is a serious limitation because no screen has been comprehensive. In fact, there is some evidence to the contrary, as we have identified an extracellular virulence factor1xDarwin, A.J. and Miller, V.L. Mol. Microbiol. 1999; 32: 51–62Crossref | PubMedSee all References1. It should also be kept in mind that many extracellular toxins do not have a direct effect on bacterial survival (e.g. cholera toxin) and thus would not be predicted to be identified in an STM screen.