The Bacillus subtilis stressosome is a 1.8 MDa complex that is the focal point for activating the bacterium's general response to physical stress. In vitro studies demonstrated that the stressosome's core element can be formed from one or more of a family of paralogous proteins (RsbRA, -RB, -RC and -RD) onto which the system's activator protein (RsbT) and its principal inhibitor (RsbS) are bound. The RsbR components of the stressosome are envisioned to be the initial receptors of stress signalling with the stressosome structure itself serving as a device to integrate multiple stress signals for a coordinated response. In the current work, we examine several of the in vivo characteristics of the RsbR family members, including their expression and ability to form stressosomes to regulate s B . Translational fusions of lacZ to each rsbR paralogue revealed that rsbRA, -RB and -RC are expressed at similar levels, which remain relatively constant during growth, ethanol stress and entry into stationary phase. rsbRD, in contrast, is expressed at a level that is only slightly above background during growth, but is induced to 30 % of the rsbRA expression level following ethanol stress. Velocity sedimentation analyses of B. subtilis extracts from strains expressing single rsbR paralogues demonstrated that each incorporates RsbS into fast-sedimenting complexes. However, consistent with rsbRD's lower expression, the RsbRDdependent RsbS complexes were present at only 20 % of the level of the complexes seen in a wild-type strain. The lower stressosome level in the RsbRD strain is still able to hold RsbT's activity in check, implying that the RsbR/S component of stressosomes is normally in excess for the control of RsbT. Consistent with such a notion, reporter gene and Western blot assays demonstrate that although RsbT is synthesized at the same rate as RsbRA and RsbS, RsbT's ultimate level in growing B. subtilis is only 10 % that of RsbRA. Apparently, RsbT's inherent structure and/or its passage between the stressosome and its activation target compromises its persistence.
The septal association of Mycobacterium tuberculosis MtrB, the kinase partner of the MtrAB two-component signal transduction system, is necessary for the optimal expression of the MtrA regulon targets, including ripA, fbpB, and ftsI, which are involved in cell division and cell wall synthesis. Here, we show that MtrB, irrespective of its phosphorylation status, interacts with Wag31, whereas only phosphorylation-competent MtrB interacts with FtsI. We provide evidence that FtsI depletion compromises the MtrB septal assembly and MtrA regulon expression; likewise, the absence of MtrB compromises FtsI localization and, possibly, FtsI activity. We conclude from these results that FtsI and MtrB are codependent for their activities and that FtsI functions as a positive modulator of MtrB activation and MtrA regulon expression. In contrast to FtsI, Wag31 depletion does not affect MtrB septal assembly and MtrA regulon expression, whereas the loss of MtrB increased Wag31 localization and the levels of PknA/PknB (PknA/B) serine-threonine protein kinase-mediated Wag31 phosphorylation. Interestingly, we found that FtsI decreased levels of phosphorylated Wag31 (Wag31ϳP) and that MtrB interacted with PknA/B. Overall, our results indicate that MtrB interactions with FtsI, Wag31, and PknA/B are required for its optimal localization, MtrA regulon expression, and phosphorylation of Wag31. Our results emphasize a new role for MtrB in cell division and cell wall synthesis distinct from that regulating the MtrA phosphorylation activities.
Summary
Smoking increases susceptibility to becoming infected with and developing tuberculosis. Among the components of cigarette smoke, nicotine has been identified as the main immunomodulatory molecule; however, its effect on the innate immune system is unknown. In the present study, the effect of nicotine on molecules of the innate immune system was evaluated. Lung epithelial cells and macrophages were infected with Mycobacterium tuberculosis (Mtb) and/or treated with nicotine. The results show that nicotine alone decreases the expression of the Toll-like receptors (TLR)-2, TLR-4 and NOD-2 in all three cell types, as well as the production of the SP-D surfactant protein in type II pneumocytes. Moreover, it was observed that nicotine decreases the production of interleukin (IL)-6 and C-C chemokine ligand (CCL)5 during Mtb infection in epithelial cells (EpCs), whereas in macrophages derived from human monocytes (MDMs) there is a decrease in IL-8, IL-6, tumor necrosis factor (TNF)-α, IL-10, CCL2, C-X-C chemokine ligand (CXCL)9 and CXCL10 only during infection with Mtb. Although modulation of the expression of cytokines and chemokines appears to be partially mediated by the nicotinic acetylcholine receptor α7, blocking this receptor found no effect on the expression of receptors and SP-D. In summary, it was found that nicotine modulates the expression of innate immunity molecules necessary for the defense against tuberculosis.
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