Research Article: Contribution of the Cpx envelope stress system to metabolism and virulence regulation in Salmonella enterica serovar Typhimurium

Date Published: February 4, 2019

Publisher: Public Library of Science

Author(s): Sivaraman Subramaniam, Volker S. Müller, Nina A. Hering, Hans Mollenkopf, Daniel Becker, Ann Kathrin Heroven, Petra Dersch, Anne Pohlmann, Karsten Tedin, Steffen Porwollik, Michael McClelland, Thomas F. Meyer, Sabine Hunke, Axel Cloeckaert.


The Cpx-envelope stress system regulates the expression of virulence factors in many Gram-negative pathogens. In Salmonella enterica serovar Typhimurium deletion of the sensor kinase CpxA but not of the response regulator CpxR results in the down regulation of the key regulator for invasion, HilA encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we provide evidence that cpxA deletion interferes with dephosphorylation of CpxR resulting in increased levels of active CpxR and consequently in misregulation of target genes. 14 potential operons were identified to be under direct control of CpxR. These include the virulence determinants ecotin, the omptin PgtE, and the SPI-2 regulator SsrB. The Tat-system and the PocR regulator that together promote anaerobic respiration of tetrathionate on 1,2-propanediol are also under direct CpxR control. Notably, 1,2-propanediol represses hilA expression. Thus, our work demonstrates for the first time the involvement of the Cpx system in a complex network mediating metabolism and virulence function.

Partial Text

An important group of bacterial regulatory sensing systems are the two-component systems, each of which enable bacteria to sense and respond to a specific subset of environmental changes and stress factors [1–3]. Two-component systems recognize environmental changes via a membrane-anchored sensor kinase that mediates the response through phosphorylation and dephosphorylation of its cognate response regulator [1]. The phosphorylated response regulator modulates the expression of target genes [3]. The Cpx-envelope stress system is a two-component system ubiquitous among Gram-negative pathogens [4, 5]. It is composed of the sensor kinase CpxA, the response regulator CpxR and the auxiliary periplasmic protein CpxP that inhibits CpxA presumably through a direct dynamic interaction [6, 7]. The Cpx-system corresponds to signals that induce envelope stress such as elevated pH, increased osmolarity, indole, adrenalin, surface contact and accumulation of adhesin subunits [5, 8–12]. Activation of the Cpx-system results in CpxA autophosphorylation and subsequently the phosphoryl group transferred to CpxR [6, 13]. Interestingly, all these signals typically emerge during early stages of infection in the gut and, accordingly, the Cpx-system could be linked to the virulence of enteropathogenic Escherichia, Salmonella, Shigella, Vibrio and Yersinia species [4, 5, 14–16]. A deletion of the Salmonella Cpx-system showed significantly reduced abilities to colonize tissue and inner organs in pigs [17, 18].

In the last decades the Cpx-envelope stress system of Gram-negative bacteria has been extensively studied and assigned to be crucially involved during the invasion of host cells [4, 5, 14, 83]. Previous studies revealed for the food-born pathogen S. Typhimurium that deletion of the sensor kinase CpxA represses expression of the SPI-1 T3SS regulator HilA and attenuates virulence but surprisingly, deletion of the response regulator CpxR had neither effect on hilA expression nor on virulence when the bacteria were grown under mild acid conditions [20]. Here, we confirmed this effect for S. Typhimurium SL1344 grown in SPI-1 inducing medium. Interestingly, we observed in addition that a S. Typhimurium strain lacking CpxA and CpxR showed no decrease in hilA expression. Notably, CpxA represents a bifunctional sensor kinase that is able not only to phosphorylate its signaling partner CpxR but also to hold the balance between active and inactive CpxR by dephosphorylation [6, 51]. In previous studies it has been reported that CpxR can be phosphorylated by additional phosphate donors as the acetyl-CoA pathway, independent of CpxA [49]. In this case, CpxR would remain in an activated phosphorylated state in the absence of CpxA and therefore CpxR would be able to inhibit hilA expression. Interestingly, similar effects were reported for the QseCB quorum sensing system involved in virulence regulation of uropathogenic E. coli (UPEC) [50]. Deletion of the sensor kinase QseC but not of the response regulator QseB significantly attenuated intracellular bacterial community formation and virulence [50]. Moreover, a qseBC deletion mutant behaved like wild-type strain contradicting the hypothesis that QseC may function through different response regulators but suggesting that dephosphorylation of QseB is required for virulence gene expression [50]. In agreement with this we suggest that the phosphatase activity of CpxA is important for hilA expression by inactivating the inhibitory effect of phosphorylated CpxR.




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