2D proteome analysis initiates new Insights on the SalmonellaTyphimurium LuxS protein

Abstract

BackgroundQuorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2.ResultsDifferential proteome analysis of wildtype S. Typhimurium versus a luxS mutant revealed relatively few changes beyond the known effect on phase 2 flagellin. However, two highly differentially expressed protein spots with similar molecular weight but differing isoelectric point, were identified as LuxS whereas the S. Typhimurium genome contains only one luxS gene. This observation was further explored and we show that the S. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-βla and LuxS-PhoA fusion proteins, we demonstrate that S. Typhimurium LuxS can substitute the cognate signal peptide sequences of β-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in S. Typhimurium. This was further confirmed by fractionation of S. Typhimurium protein extracts, followed by Western blot analysis.Conclusion2D-DIGE analysis of a luxS mutant vs. wildtype Salmonella Typhimurium did not reveal new insights into the role of AI-2/LuxS in Salmonella as only a small amount of proteins were differentially expressed. However, subsequent in depth analysis of the LuxS protein itself revealed two interesting features: posttranslational modification and potential translocation across the cytoplasmic membrane. As the S. Typhimurium LuxS protein does not contain obvious signal motifs, it is speculated that LuxS is a new member of so called moonlighting proteins. These observations might have consequences in future studies on AI-2 quorum signaling in S. Typhimurium.

A Chemical Biology Approach to Interrogate Quorum-Sensing Regulated Behaviors at the Molecular and Cellular Level

“…(Soni, et al, 2008) In a different report, only 4 proteins were differentially expressed, 1 of which was LuxS itself, and 2 others were unidentified proteins. (Kint, et al, 2009) The differences between the current study and the two previous proteomics-based studies of S. typhimurium are readily interpreted. For one, employing Δ luxS mutants in the previous studies compared to the use of a QS inhibitor in our study is a distinct difference.…”

The superoxide dismutase SodA is targeted to the periplasm in a SecA‐dependent manner by a novel mechanism

“…sphaeroides and P. denitrificans, and may be widespread in Gram-negative bacteria, but has previously not been investigated systematically or mechanistically. Other proteins lacking a signal peptide, such as LuxS in Salmonella (Kint et al, 2009) and an aberrant PhoA in Synechococcus (Ray et al, 1991), have also been detected in the periplasm, and may be targeted in a similar way. As Mn/Fe-SODs are required for successful infection by many pathogens (Hale, 1991;McGee and Mobley, 1999;Purdy et al, 1999;Verneuil et al, 2006;Kang et al, 2007) and indeed can be a major antigen (McMillan et al, 2004), this finding forces a re-evaluation of the role of SodA in pathogenesis as well as expanding on the known mechanisms of targeting proteins to the periplasm.…”

Pathogen espionage: multiple bacterial adrenergic sensors eavesdrop on host communication systems

“…LuxS exhibits AI‐2‐independent activity in S . Typhimurium by modulating flagellar phase variation to favour expression of the more immunogenic phase‐1 flagellin (Karavolos et al ., ; Kint et al ., ). Formation of biofilms by S .…”

“…In S. Typhimurium, AI-2 affects the expression of the Lsr system involved in its own uptake Xavier and Bassler, 2003;De Keersmaecker et al, 2005). LuxS exhibits AI-2-independent activity in S. Typhimurium by modulating flagellar phase variation to favour expression of the more immunogenic phase-1 flagellin (Karavolos et al, 2008a;Kint et al, 2009). Formation of biofilms by S. Typhimurium is dependent on the small RNA micA whose expression is affected by luxS via a yet unknown mechanism (Kint et al, 2010).…”