This exploratory study was conducted to provide insight on environmental factors that influence the expression of the Ysa TTS system and to gain some perspective on how the Ysa TTS system affects interactions between Y. enterocolitica
biovar 1B and a susceptible host. To accomplish these initial goals, an experimental approach was used that yielded an extended collection of Y. enterocolitica
strains that harbored transcriptional fusions between Ysa TTS-related genes and the reporter operon lacZYA
. A systematic survey of environmental parameters that influence the expression of these genes revealed important information, some of which is new and some of which confirms or extends observations from other studies. First, expression of a variety of genes distributed throughout the YSA PI was examined, and all of the genes displayed similar regulatory patterns regardless of their predicted functions. The common pattern of expression may reflect that many of the genes within the YSA PI form operons or individual transcriptional units that are coregulated. All of the genes examined were induced during the early logarithmic phase of growth and respond to temperature, pH, and NaCl, including the regulatory genes ysrS
. The way that these environmental cues are sensed by the cell and mechanistically lead to changes in gene expression remains to be defined. At this point we can only speculate on the exact environmental cues sensed by the YsrS-YsrR and RcsC-YojN-RcsB signal transduction systems. Changes in temperature, pH, and salt concentrations might be specifically monitored. Alternatively, these chemical and physical conditions have a profound effect on cell membrane structure or integrity; the membrane itself may serve as a common site for signal integration. There is some evidence that this is the case for the activation of the RcsC-YojN-RcsB system in E. coli
and S. enterica
). Passage from the terrestrial environment through the gastrointestinal tract exposes the bacterium to a variety of conditions that assault the cell membrane, including changes in pH, temperature, ionic strength, membrane-perturbing antimicrobial peptides, and bile salts.
Genetic analysis demonstrated that ysrS
is required for the expression of many YSA PI genes and that rcsB
is important for full expression of the same genes. This suggests that both the YsrR-YsrS and RcsC-YojN-RcsB phosphorelay systems control the expression of the Ysa TTS system. Interestingly, inactivation of ysrS
also affected the transcription of rcsB
, but inactivation of rcsB
had only a small effect on ysrS
. The dominant effect of the ysrS
mutation is consistent with YsrR-YsrS acting upstream of RcsC-YojN-RcsB in controlling the expression of the Ysa TTS system. We propose that the YsrR-YsrS phosphorelay system directly or indirectly affects genes within the YSA PI and of the RcsC-YojN-RcsB phosphorelay system. Changes in the levels of the RcsC-YojN-RcsB system and its activity in turn exert additional modulatory effects on YSA PI genes. A complete regulatory model must also include the cyclic AMP-CRP regulatory system, which was previously shown to be required for the expression of the Ysa TTS system (32
). Testing a regulatory model will provide a foundation for understanding the molecular mechanisms used by Y. enterocolitica
to integrate environmental cues that affect the expression of the Ysa TTS system.
Expression of YSA-related genes was best induced when strains were cultured in a relatively-high-ionic-strength, nutrient-rich growth medium. Curiously, we observed that Ysa TTS-related genes were best induced at 26°C, but not at 37°C. This is consistent with previous studies that documented that Ysp secretion by the Ysa TTS system in vitro is promoted at low temperatures. This finding may seem to strike a blow against the possibility that the Ysa TTS system has a role in the colonization of a mammalian host by Y. enterocolitica
. However, this is not the only occasion where a putative virulence factor of a pathogenic species of Yersinia
was observed to be best expressed in vitro at a temperature that is lower than that of a warm-blooded animal. Other genes, such as inv
, the urease gene cluster, and the O-antigen gene cluster, display similar levels of temperature-dependent regulation (10
). Only inv
have been further shown to require specialized conditions that allow for induction at higher temperatures (25
). Yet, all of these genes affect pathogenic outcomes of a Y. enterocolitica
infection in experimentally infected mice. Furthermore, the O-antigen gene cluster notwithstanding, these genes promote colonization or progression of disease in gastrointestinal system-associated tissues. This set of results therefore helped to guide the second part of this exploratory study, which focused on evaluating whether the Ysa TTS system affected bacterial-host interactions and, in particular, played a role during early stages of infection when bacteria are predominantly found in gastrointestinal tissues.
The first set of experiments that were conducted to confirm and extend the previously reported observation that Ysa TTS-defective mutants displayed a modest 10-fold reduction in the ability to cause the mortality of orally infected BALB/c mice (17
). The collection of mutants tested here included strains that had insertion mutations in regulatory genes (rcsB
) and a structural gene (ysaU
) that map to the YSA PI or a distant location in the chromosome. All of the mutants tested displayed abilities to cause mortality that were similar to that of the isogenic wild-type strain of Y. enterocolitica
. It is not clear why we were unable to reproduce the previously reported result. Nonetheless, the two studies are consistent in that it appears that the Ysa TTS system is not required by Y. enterocolitica
biovar 1B to cause a progressive systemic infection in BALB/c mice that leads to mortality. As a follow-up analysis, the same mutants were tested for the ability to colonize tissues that commonly have large numbers of bacteria during the systemic phase of infection, such as Peyer's patches, mesenteric lymph nodes, liver, and spleen. None of the mutants displayed differences in their potentials to multiply in these tissues. This is consistent with the disease mortality analysis indicating that the Ysa TTS system does not influence systemic phases of infection in mice.
This led us to address whether the Ysa TTS system has a role during the gastrointestinal phase of infection. We were also encouraged to refocus the analysis in this way since the regulatory experiments revealed that Ysa TTS-related genes respond to many of the same environmental conditions as other genes involved in this stage of infection. Mice were infected with Y. enterocolitica
or a ysaU
mutant, after which colonization of the terminal ileum, cecum, and Peyer's patches was evaluated 24 h postinfection. This time point was selected because it ensured that the infection had not disseminated to deep tissues but exceeded the normal retention time for ingested material (18
). This investigation revealed that the Ysa TTS system affects colonization of the intestinal terminal ileum. While this analysis was informative, it also required a large number of animals, so to further test the validity of this result, we utilized in vivo competition assays. This experimental approach measures the ability of a mutant, when mixed with wild-type bacteria, to competitively colonize the host. This type of analysis required fewer animals per experiment and provided the opportunity to investigate many different Ysa TTS mutants for defects in the competitive colonization of host tissues. Importantly, this alternative experimental approach also provided evidence indicating that the Ysa TTS system contributes to colonization of the terminal ileum.
To provide additional perspective on the magnitude of the colonization defect of Ysa TTS mutants, we measured the competitive colonizations of an isogenic set of mutants consisting of strains whose Ysa TTS pathway, Ysc TTS pathway, or both secretion pathways were blocked. It turned out that blockage of individual TTS systems reduced the ability of Y. enterocolitica to competitively colonize the terminal ileum, cecum, and Peyer's patches. Blockage of both TTS systems exacerbated the effect in each case. The data are consistent with previous observations that the Ysc TTS system has an important role in the colonization of Peyer's patches even at an early time point of infection. Importantly, this study revealed the Ysa TTS system influences Y. enterocolitica colonization of the gastrointestinal tract and provides a foundation for future studies. It will be interesting to understand which type of host cells is targeted by the Ysa and Ysc TTS system effector proteins. Understanding this facet of bacterial-host interactions should shed some light on why Y. enterocolitica biovar 1B maintains two distinct contact-dependent TTS systems.