Salmonella invasion is controlled through the secondary structure of the hilD transcript.

Virulence functions of bacterial pathogens are often energetically costly and thus are subjected to intricate regulatory mechanisms. In Salmonella, invasion of the intestinal epithelium, an essential early step in virulence, requires the production of a multi-protein type III secretion apparatus. The pathogen mitigates the overall cost of invasion by inducing it in only a fraction of its population. This constitutes a successful virulence strategy as invasion by a small number is sufficient to promote the proliferation of the non-invading majority. Such a system suggests the existence of a sensitive triggering mechanism that permits only a minority of Salmonella to reach a threshold of invasion-gene induction. We show here that the secondary structure of the invasion regulator hilD message provides such a trigger. The 5' end of the hilD mRNA is predicted to contain two mutually exclusive stem-loop structures, the first of which (SL1) overlaps the ribosome-binding site and the ORF start codon. Changes that reduce its stability enhance invasion gene expression, while those that increase stability reduce invasion. Conversely, disrupting the second stem-loop (SL2) represses invasion genes. Although SL2 is the energetically more favorable, repression through SL1 is enhanced by binding of the global regulator CsrA. This system thus alters the levels of hilD mRNA and is so sensitive that changing a single base pair within SL1, predicted to augment its stability, eliminates expression of invasion genes and significantly reduces Salmonella virulence in mice. This system thus provides a possible means to rapidly and finely tune an essential virulence function. Author summary Pathogenic bacteria tightly regulate the expression of their virulence functions to balance survival and proliferation within an animal host against the fitness costs that these functions engender. Salmonella has evolved an energetically favorable means to invade the intestinal epithelium, required for its virulence, with only a small proportion of its population expressing the needed genes, while the remainder reaps the benefits. This work shows that the threshold of invasion induction is finely controlled through the message secondary structure of the activator hilD in conjunction with the invasion repressor CsrA. This sensitive system may thus allow Salmonella rapidly to adjust the dynamics of its invading population in response to signals within the animal.