The advent of molecular methods made necesary to revise the
Koch´s postulates, formulated in 1890, as general guidelines that should be followed to identify pathogens causing diseases. As a result,
Stanley Falkow established the
molecular version of Koch's postulates to guide the identification of
microbial genes encoding virulence factors. Falkow established five experimental criteria that a gene must fulfill to be considered a virulence factor. A criterium almost never addressed by scientists is "
The gene, which causes virulence, must be expressed during infection." It has been always considered enough to test whether specific inactivation of the gene is associated to a measurable loss of virulence. Actually, the golden standard is to demonstrate
in vivo (using suitable animal/plant models) that allelic replacement of the mutated gene leads to restoration of virulence.
However, wouldn´t it be interesting to know exactly when and where the virulence gene is expressed? Certainly this may help to understand the
in vivo role of the virulence factor: is it required only for the initial colonization of the tissues? Is it necesary to fight phagocytic cells?, is the expression of this factor coordinated with those of other factors?,...Moreover, are there virulenece factors expressed only
in vivo and therefore absolutely dispensable
in vitro? These questions led scientists to develop experimental approaches to enlighten virulence gene expression. Conceptually, the methods were originally conceived upon the premise (now considered fact) that most virulence genes are transcriptionally induced
at one or more times during infection
