needs to coordinate the expression of a diverse number of cellular systems during the infection cycle (28
). In this study, we investigated the dynamic regulation of three of these systems, namely, flagella, the SPI1 T3SS, and type 1 fimbriae. We were able to demonstrate that these three systems are expressed sequentially during in vitro
growth. This hierarchy in gene expression could mirror the roles of these three systems during the infection cycle. According to this simplified model, Salmonella
first needs to swim to the sites of invasion in the distal small intestine. Logically then, the flagellar genes are expressed first. Upon reaching its target sites for invasion, Salmonella
stops synthesizing flagella as movement is no longer required and starts to synthesize the SPI1 T3SSs necessary for invasion. Once the bacterium enters the stationary phase, it stops synthesizing the SPI1 T3SSs and begins to express the type 1 fimbrial genes involved in intestinal colonization and persistence (1
). In vivo
, we imagine that this last step is necessary only in those bacteria that were unable to breach the intestinal epithelium.
As multiple studies have shown that extensive regulatory interactions exist between these three systems (5
), we hypothesized that regulatory cross talk may govern the hierarchy. However, we found that transcriptional hierarchy is controlled predominately by external factors, external in the sense that the hierarchy is not due to known interactions among these three systems. Cross talk, rather, appears to tune the hierarchy. In particular, cross talk between these three systems is critical for regulating gene expression during transition phases in the hierarchy. The one exception is the effect of FliZ on SPI1 gene expression dynamics.
Among the regulators underlying this cross talk, we found that FliZ is the most significant as it regulates both SPI1 and type 1 fimbrial gene expression, where the latter is a novel finding of this study. Moreover, FliZ's effect on SPI1 gene expression is profound, reducing the expression roughly 3-fold. While FimZ also regulates flagellar and SPI1 gene expression, the effects are minor and are really seen only when the regulator is constitutively expressed. Interestingly, unlike RtsB and FimZ, FliZ does not appear to directly regulate transcription in these three systems. Rather, FliZ appears to function through another transcription factor, be it FlhD4
), HilD (41
), or FimZ. While the underlying mechanism of action of FliZ is still unclear, our data imply that it is posttranslational in all three systems.
One outstanding question concerns the physiological role of this regulatory cross talk between the flagellar, SPI1, and type 1 fimbrial genes. In particular, cross talk has a relatively minor role in regulating the hierarchical expression of these three systems, at least under the conditions investigated in this study. As a comparison, the most well-characterized example of regulatory cross talk involves the hierarchical expression of carbohydrate transport and metabolic genes during growth on mixed substrates. In that case, transcriptional cross talk is used to enforce a strict hierarchy in carbohydrate utilization (17
). While we also cannot discount that other factors associated with the flagellar, SPI1, and type 1 fimbrial systems are involved in regulating the transcriptional hierarchy, we expect that the hierarchy is not due to cross talk but rather is regulated by external factors. Specifically, based on our current understanding, we believe that these three systems are regulated in response to the growth phase of the cell (Fig. ). Why then is cross talk employed?
If we consider the logic of this cross talk, then a simple pattern emerges (Fig. ). Specifically, cross talk appears to make the expression of these three systems mutually exclusive, though only to the degree to which they are expressed during the infection cycle. In this regard, it helps to reinforce the transcriptional hierarchy. For example, the expression of the flagellar genes represses the expression of the type 1 fimbrial genes and vice versa. This mutual repression is perfectly logical when considering that Salmonella cannot move and adhere/persist at the same time. Similarly, the expression of the flagellar genes enhances SPI1 gene expression, whereas the expression of the SPI1 gene represses flagellar gene expression. Regulation in this case would suggest that only actively motile cells try to invade. As a corollary, persisting cells do not invade, consistent with the fact that the expression of the type 1 fimbrial genes represses the expression of the SPI1 genes. Lastly, once the cells decide to invade, logically then motility is no longer required.
FIG. 8. Salmonella invasion program. (A) Diagram of transcriptional cross talk between the flagellar, SPI1, and type 1 fimbrial gene systems. (B) Inferred logic of transcriptional cross talk, where the decision to “move” results from flagellar (more ...)
One limitation of this model is that it does not account for all the other systems involved in the infection cycle. For example, Salmonella
has at least 13 distinct fimbrial systems (68
) whose expression might also be coordinated with the expression of flagellar, SPI1, and type 1 fimbrial genes. Salmonella
also possesses a nonfimbrial adhesin encoded in Salmonella
pathogenicity island 4 (SPI4) (28
). Previously, we demonstrated that the expression of the SPI4 adhesin is regulated by SprB, a SPI1-encoded regulator (73
). Aside from fimbriae and adhesins, Salmonella
also possesses a second T3SS encoded within Salmonella
pathogenicity island 2 (SPI2) (34
). The SPI2 T3SS is used to survive and replicate within host cells during systemic phases of infection. The SPI2 genes also play a role in inducing intestinal inflammation and are known to be regulated by HilD, a SPI1 regulator (7
). However, chemical and environmental cues are required to activate SPI2 gene expression, most notably low Mg2+
) and acidic pH (6
); HilD is not required for SPI2 gene expression during systemic infection (20
). How the cells coordinate the expression of SPI1 and SPI2 genes is still not well understood.
Clearly, this model of the coordinate expression of Salmonella virulence genes (Fig. ) is still incomplete, as it considers only a small subset of the systems involved in the infection cycle. In addition, it is based on just one mode of growth. Likely, cross talk is more significant when growth is irregular and the environment is variable, as opposed to our in vitro experiments where growth is uninterrupted and the environment is fixed. Further investigations are necessary to fully characterize the role of regulatory cross talk in coordinating gene expression during invasion. The significance of this study is that it is the first to systemically study the effect of regulatory cross talk on the expression dynamics of flagellar, SPI1, and type 1 fimbrial genes.