Role of SalK/SalR in Virulence 
Given the multiple roles of TCSTS in bacteria, we evaluated the effect of salKR deletion on the general biological characteristics of SS2 prior to in vivo work.
First, the ability of the mutant strain to retain spectinomycin resistance was assessed.
The spectinomycin resistance phenotype was stable in DeltasalKR during in vitro culture (data not shown).
Next, the growth kinetics curves of DeltasalKR were compared with that of WT, and no obvious distinctions were observed.
When streaked on THB plates with 5% sheep blood, DeltasalKR and WT colonies displayed a similar haemolytic phenotype.
Finally, cell morphology and ultrastructure of these two strains were examined under light microscope and transmission electron microscope respectively, however, no significant differences were found (Fig. 4).
An experimental infection model in piglets was designed to assess the role of SalK/SalR in virulence.
As shown in Figure 5, all six SPF-piglets inoculated intravenously with WT developed most of the typical disease symptoms, including high fever, limping, swollen joints, shivering, CNS failure and respiratory failure within 24 hrs.
Five of them died on day 2 post-infection, and the last one on day 3.
In contrast, six infected piglets with an avirulent strain, 05HAS68 (as negative control), survived nearly without any obvious symptoms until the end of the experiment (14 days after infection).
Wherein the six piglets challenged with the DeltasalKR mutant were all alive and symptom-free during the entire course monitored.
This was similar to those observed in the negative control group, indicating that DeltasalKR totally loses its lethality.
The experiments of functional complementation with the complemented strain CDeltasalKR showed clearly that the reintroduction of salKR into DeltasalKR restored its high pathogenicity.
Five out of six piglets succumbed to the lethal infections of CDeltasalKR and died, with median survival times of 2 to 3 days.
The last one recovered from serious clinical symptoms and survived at the experiments end.
All these results strongly suggest that SalK/SalR plays an important role in the pathogenesis of Chinese isolates of highly invasive SS2.
To better evaluate the virulence attenuation of DeltasalKR, colonization experiments were carried out.
As shown in Table 1, wild-type bacteria were found in all of the SS2 specific tissues of WT-infected piglets, including heart, lung, liver, kidney, spleen, tonsil, brain, blood, joints and so on.
In contrast, in the mutant-inoculated group, no mutant bacteria could be recovered from any of the examined tissue specimens.
Obviously, lack of SalK/SalR badly impaired the capability of 05ZYH33 to colonize the various tissues of piglets.
In the WT/DeltasalKR coinfection group, besides the detection of wild-type bacteria in all the specimens, DeltasalKR mutant bacteria could also be isolated from some susceptible tissues (such as heart, kidney, tonsil and brain) at a lower degree than the wild-type strain.
We anticipated that the DeltasalKR mutant is still able to colonize these organs, is just due to the WT-mediated impairment of host defense against SS2 infection in these tissues.
