# Differential Proteomic Analysis of _Listeria monocytogenes_ during High-Pressure Processing
## 4 Discussion
### HPP Response-Associated Pathways  
Proteolysis and redox status both contribute to \(L\). _monocytogenes_ virulence, environmental stress adaption and the multifaceted interactions among the class three stress gene repressor (CtsR), the alternative general stress sigma factor (\(\sigma^{\text{B}}\)), and the positive regulatory factor A (PrfA) regulons. \(L\). _monocytogenes_ expresses several ATPases, including ClpC, ClpP and ClpE, associated with diverse cellular activities. ClpP is found in many prokaryotic cells and is often involved in stress response and virulence by functioning as a molecular chaperone [32]. An inconsistency of CtsR or Clp proteins in a cell may greatly damage the cell because Clp proteins eradicate misfolding proteins and regulate many critical cellular processes such as short-lived regulators. CtsR represses class III heat shock genes, including _clpP_, _clpE_, and the _clpC_ operon [33]. The high HPP-tolerant \(L\). _monocytogenes_ ScottA was established when overexpressing _clp_ genes with an inactive CtsR repressor by deleting a codon in the glycine-rich domain (_ctsRAGly_) [34]. ClpP and ClpC are both transcribed from \(\sigma^{\text{B}}\)-dependent promoters. This study revealed that HPP induced ClpP significantly and ClpX slightly, possibly facilitating protein folding and maturation under HPP stress (Table 1, Table S3). Except for the proteolytic core ClpP, the different cognate Clp-ATPases fine-tune the proteolytic response. ClpY (also called ATP-dependent protease ATPase subunit HsIU) and ClpQ (ATP-dependent protease ATPase subunit HsIV) were also slightly upregulated by 0.65 and 0.47 log\({}_{2}\) fold in H4/C, respectively. However, ClpB were slightly downregulated under the HPP 200 and 400 MPa treatments. ClpB, controlled by CtsR, induces thermal tolerance and virulence but not general stress tolerance in \(L\). _monocytogenes_[35]. Similar results for _Lactobacillus sanfranciscensis_ showed that only HPP but not other stress such as extreme temperatures, salt, acid or starvation could induce ClpP protein [36]. Since stress-induced ClpP promotes the intracellular survival of \(L\). _monocytogenes_ by modulating the presentation of protective antigens Listeriolysin O, ClpP may represent a potential vaccine target against \(L\). _monocytogenes_[37]. Taken together, we suggest that HPP induced the expression of the ubiquitous Clp protease system consisting of ClpP, ClpX, ClpY and ClpQ for better adaptation upon HPP stress. PrfA requires binding a signaling molecule, glutathione (GSH), to an entire active state. GSH is synthesized by the glutathione biosynthesis bifunctional protein (GshAB), and regulated by the GSH reductase and global transcriptional regulator (Spx), a thio-sensing and putative regulator of GSH biosynthesis [38]. The promoters of GSH reductase and Spxtranscription are \(\sigma^{\text{B}}\) dependent. GSH could be oxidized to GSSG, which can be reduced to GSH by GSH reductase. Furthermore, the downregulation of Spx resulting in lower PrfA regulon expression is controlled directly by \(\sigma^{\text{B}}\)[39]. A feedback regulation loop is operated by the Spx, ClpXP and CtsR regulon [32]. Spx is also a key target of the ClpXP protease complex in _Bacillus subtilis_. Spx contributes to CtsR regulon activity and directly mediates \(clpX\) expression. Hence, \(\sigma^{\text{B}}\) could affect PrfA activity through modulation of GSH homeostasis and Spx activity. The serine-protein kinase RsbW, an anti-sigma factor, inactivates its specific antagonist protein RsbV through phosphorylation. RsbW is released upon phosphorylation of RsbV and negatively regulates \(\sigma^{\text{B}}\) activity by blocking its ability to form an RNA polymerase holoenzyme [40]. Spx significantly increased by 1.02 and 1.73 fold in H2/C and H4/C, respectively (Table 1 and Table S3). GshAB and \(\sigma^{\text{A}}\) also increased, whereas RsbW decreased but not significantly (data not shown).