| Salmonella enterica serovar Typhi (S. typhi ) uses type IVB pili, encoded adjacent to the viaB locus required for Vi antigen synthesis, to facilitate both eukaryotic cell attachment and bacterial self-association. Due to the exceptional host restriction of S. typhi, there is no animal model for serovar Typhi systemic infection of humans. In attempt to seek evidence that might point to an important role for the type IVB pili in the pathogenesis of enteric fever, we have investigated two other Salmonella serovars, Dublin and Paratyphi, which are not as pathogenic for humans as is serovar Typhi. First, the pilS mutant of serovar Dublin was shown to invade cultured human intestinal INT407 cells to an extent only 20% that of the wild-type parent. The wild-type serovar Dublin strain, but not the pilS mutant, self-associated. The data suggested that the serovar Dublin Type IVB pil operon might increase the human-invasiveness of serovar Dublin, compared to pil-free strains. Next, we have discovered that the serovar Paratyphi C shufflon is essentially inactive because each of the Rci 19-bp substrates has acquired a single basepair insertion. A PilV protein is thus synthesized whenever the pil operon is active, and bacterial self-association therefore does not occur in serovar Paratyphi C. Taken together, the data suggested that serovar Typhi bacterial self-association using Type IVB pili and regulated by PilV protein might, in fact, be important in the pathogenesis of epidemic enteric fever. We thus proposed that a novel means of control of protein expression was operative in the shufflons of serovars Typhi and Dublin. Specifically, the rate of Rci-catalyzed inversion of DNA encoding the C-terminal portions of the PilV proteins controls PilV protein synthesis. Here, it was initially shown that DNA inversion per se is required for inhibition of gene expression from invertible DNA. Next, it was shown that inversion frequencies of xylE-encoding DNA, bracketed by Rci substrate sequences, are affected by changes in the 19 bp consensus sequences which are essential components of Rci substrate DNA. The affinity of Rci for these sequences affects inversion frequencies, so that a greater affinity is predictive of faster inversion and therefore less synthesis of product encoded by invertible DNA. In vivo, the frequency of Rci-mediated inversion is influenced by the extent of DNA supercoiling, with increasing levels of expression of invertible genes as novobiocin inhibits DNA supercoiling and thus Rci action. This inhibition of DNA supercoiling results in increased synthesis of PilV proteins as Rci activity decreases, and, in turn, bacterial self-association decreases. In conclusion, shufflon inversion activity and PilV expression are regulated by the extent of DNA supercoiling, which responds to environmental cues. This potentially represents a novel means of control of bacteria aggregation and dispersal, which play an important role in the development of bacterial infections. |
