High pressure processing (HPP) is commonly used in the food industry to inactivate foodborne pathogens and spoilage microorganisms. However, it has been reported that L. monocytogenes is able to recover after HPP treatment during long-term storage [18, 19, 36]. To study gene expression response during early recovery of L. monocytogenes and identify genes that are important for recovery, we performed RNA-seq of samples taken at different time points after HPP at two different pressure levels (200 MPa and 400 MPa). To account for strain-dependent variation in the HPP response, experiments were performed with two strains: RO15, a strain that was shown to be more resistant to HPP than others; and ScottA, a strain that is more sensitive to HPP .
L. monocytogenes can recover within one day after injury caused by HPP for 10 min at 450 MPa and 45 °C . Recovery of L. monocytogenes was also observed after six days of storage at 4 °C for injured bacteria following treatment with HPP at 550 MPa at 45 °C for 10 min . Our results show that viability was unaffected in the two strains tested after HPP treatment at 200 MPa and 8 °C for 8 (Fig. 1), but a marked reduction in viable counts of several log10 units was observed for both strains treated at 400 MPa. However, the reduction in viable counts was significantly higher in ScottA compared to RO15, which supports our previous observation that RO15 is more pressure tolerant .
The time-resolved RNA-seq data allowed us to perform gene-network analysis. To summarize the gene networks, we clustered genes assuming that genes within a cluster, and in linked clusters, are functionally related or interact during recovery from HPP [37, 38, 39]. HPP mainly affects expression of protein folding genes, PTS system genes, prophage genes, and cobalamin biosynthesis genes. We saw that in both strains, stress response genes, virulence genes, and ribosome hibernation promoting factor hpf gene were strongly linked to each other, indicating that during recovery from HPP, co-expression of these three factors was needed. It has also been reported that the general stress sigma factor B (σB) regulates hpf, prfA (encoding listeriolysin regulatory protein), and UspA1,2 (encoding universal stress proteins A1 and A2) . Environmental stress activates σB, which regulates more than 200 genes [40, 41]. In line with this, based on de novo motif discovery analysis, SigB transcription factor binding site-like motif was found in the upstream regions of the gene cluster (Cl6; Fig. 6, Table S14), which includes prfA and hpf in ScottA. This indicates that a general stress response was activated in L. monocytogenes by σB after HPP.
We have reported that a large portion (up to ~ 85%) of RNA-sequencing reads were mapped to Rli47 ncRNA, which was upregulated in both strains after pressure treatment. Similarly, previous studies have also reported that up to ~ 90% of all RNA-seq reads map to Rli47 ncRNA in L. monocytogenes [42, 43]. It has been shown that Rli47 plays a role in the response to acid stress  and oxidative stress . In line with these observations, our data suggests that Rli47 is also involved in HPP recovery based on high expression level after HPP treatment. It has also been shown that Rli47 is regulated by σB . This supports our observation of general stress response activation by σB after HPP. In addition, Rli53 expression was upregulated in RO15 but not in ScottA. Rli53 has been associated with antibiotic resistance . Our results indicate that Rli53 may also play a role in pressure resistance in RO15.
Cobalamin biosynthesis was the most significantly enriched GO term for upregulated genes in both strains. It has been shown that cobalamin plays a protective role against oxidative stress in bacteria . Cobalamin was also shown to be an essential cofactor for propanediol and ethanolamine utilization . Significant downregulation of cobalamin biosynthesis genes in L. monocytogenes has been reported in response to Rli47 deletion . Hence, upregulated cobalamin synthesis genes after HPP in this study can be related to increased levels of Rli47, which is regulated by σB. We therefore predicted that cobalamin biosynthesis genes were upregulated as part of the general stress response of HPP.
Stress conditions have been shown to induce prophages in L. monocytogenes [49, 50]. Upregulation of prophage genes in both strains after HPP indicates that pressure stress also induces prophages. In addition, co-regulation of different prophages within the same host has also been shown in L. monocytogenes . Similarly, our gene-network inference suggests that prophages were linked to each other within strains, coexpression of prophages genes were observed. Based on pan-genome analysis, we previously proposed that prophages and anti-CRISPR genes may play a role in pressure resistance in L. monocytogenes . In this study we observed that both anti-CRISPR genes (acrIIA1 (OCPFDLNE_02770, OCPFDLNE_02583) and acrIIA2 (OCPFDLNE_02582)) in RO15 were upregulated after HPP.
GO enrichment analysis of upregulated genes indicated that PTS systems were activated in both strains for most of the time points during the recovery phase. Upregulation of PTS genes has also been reported for other stress conditions in L. monocytogenes based on transcriptome analysis [51, 52]. Upregulated PTS systems were mostly galactitol-, fructose-, and mannose-specific PTS systems. These carbon sources play a role in cell-wall biosynthesis [53, 54]. Thus, upregulation of these sugar transporters may be an indication of increased uptake of these sugars for cell-wall biosynthesis and as a carbon source to allow recovery from injury caused by HPP.
CtsR is a negative regulator of heat-shock genes, mainly of the clp family of genes, and has been shown to be directly involved in barotolerance of L. monocytogenes [55, 56, 57, 58, 59]. Deletion of ctsR led to an increase in barotolerance of ScottA by 5 orders of magnitude . In addition, upregulation of PTS, heat-shock, and clp family genes has been reported for a ScottA ctsR mutant . Furthermore, ctsR is reportedly regulated by σB in Bacillus subtilis . Our results show upregulation of ctsR expression in both strains at some time points after treatment with 200 MPa but not with 400 MPa. Moreover, upregulation of genes was observed for heat-shock proteins of the clp family and chaperones in samples treated at both 200 MPa and 400 MPa in both strains (Fig. 3c, 4c). Especially clpE was one of the most significantly upregulated genes at several time points. It has been shown that heat-shock proteins are needed to deal with misfolded proteins, prevent cellular damage, and help cell recovery during pressure treatment . Our observation that genes for heat-shock proteins are upregulated indicates a similar role in protection and recovery of L. monocytogenes to/after HPP treatment.
It has been shown that antibiotic resistant L. monocytogenes strains are more resistant to 400 MPa pressure treatment compared to antibiotic-susceptible strains . Our previous pan-genome study  also showed that barotolerant strains have slightly different amino acid sequences for norB encoding a protein involved in resistance against quinolones. Interestingly, different strains showed variations in their expression of norB. Significant upregulation of norB was observed in barotolerant RO15 at several time points after 400 MPa treatment, including early time points. However, norB was only upregulated at the 24 h time point after 400 MPa treatment in barosensitive ScottA. This supports the observation that differences in antibiotic resistance genes might provide a different barotolerance level within L. monocytogenes strains.
Ribosome damage can lead to cell death after HPP . Ribosome hibernation, that is dimerisation of 70S ribosomes leading to translationally inactive 100S particles, has been reported to occur as L. monocytogenes adapts to different stress conditions . Ribosome hibernation involves the gene product of hpf (ribosome hibernation promoting factor) and upregulation of hpf was seen in both strains. Downregulation of hpf was observed at the 48 h time point after 200 MPa treatment only in RO15. In addition, in RO15, the GO term “translation” was also mainly enriched for upregulated genes at the late time points in RO15 at both 200 and 400 MPa (Fig. 2). However, no enrichment of GO term “translation” was observed for upregulated genes in ScottA at 400 MPa (Fig. 2). Collectively, this indicates that L. monocytogenes keeps the translation inactive by inducing Hpf-mediated ribosome hibernation for a certain time after HPP. Moreover, there are differences between the strains in how long this hibernation lasts. The barotolerant strain RO15 seems to reactivate translation faster than the sensitive strain ScottA.
Based on morphological and physiological characterization, the cellular wall or membrane are targets to improve efficacy of HPP to inactivate L. monocytogenes . We observed that peptidoglycan-synthesis genes such as, murG, murC, murD, and pbp2A were upregulated in both strains after HPP. Upregulation of peptidoglycan-synthesis genes with simultaneous downregulation of cell-division genes indicates that an active cell-wall repair occurs in both strains after HPP. pbp2A encodes a penicillin-binding protein that was shown to contribute to β-lactam resistance and cell morphology in L. monocytogenes . To further investigate the role of this gene in response to HPP, a mutant carrying a deletion of the corresponding gene (lmo2229) was generated in L. monocytogenes strain EGDe and tested for resistance against HPP. The results show that the lmo2229 mutant was significantly more sensitive to HPP than the parental wildtype strain (Fig. 8). This shows that pbp2A is an important factor in L. monocytogenes for recovery after HPP.
Peptidoglycan of bacteria consists of a backbone of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) units interconnected with peptide side-chains . It has been shown that bacteria are able to recycle N-Acetylglucosamine from peptidoglycan using the proteins encoded by nagA and nagB [53, 65]. The genomes of L. monocytogenes RO15 and ScottA each contain two copies of nagA and nagB, respectively, and one of the copies of each nagA and nagB are organized in an operon. Expression of these nagAB copies were upregulated in both strains after HPP at 200 and 400 MPa. The second copy of nagA was not differentially expressed in either strains. Interestingly, the second copy of nagB gene (OCPFDLNE_02454) was only upregulated in RO15. This difference might partly explain the barotolerance difference between strains. Increasing protein levels of the NagB are associated with increased growth rate in E. coli . Thus, more efficient biosynthesis of cell-wall peptidoglycans due to higher NagB levels may contribute to the higher barotolerance of RO15.
HPP creates a mechanical force that may result in deformation of the membrane. Mechanosensitive channels were shown to respond to membrane stress and help bacteria to cope with this stress . We were intrigued by the observation that the mscL gene encoding a MS channel protein of large conductance was upregulated after 400 MPa pressure treatment in both strains. In addition, ykuT (encoding small MS channel protein) was upregulated at the 200 MPa 48 h time-point in RO15. However, the obtained lmo1013 mutant showed no significant difference in susceptibility/resistance to HPP indicating that the small MS channel protein was not directly involved in pressure resistance or only has a minor effect.
Gene expression profiling under pressure treatment in Listeria was studied previously by Bowman et al.  using L. monocytogenes strain S2542. Notably, a significant negative correlation was observed for log2 FC results between the study by Bowman et al.  and our study. Our RNA-seq results were validated using ddPCR (Fig. 7) and they are consistent for two different strains under different pressure levels and several time points. We speculate that discordance between the results could potentially arise from different growth conditions or different methods for measuring gene-expression levels.
Overall, these findings may lead to new approaches to improve HPP efficacy. For example, we observed that the mannose phosphotransferase system (Man-PTS) was upregulated after HPP treatment. Man-PTS is the receptor for class IIa bacteriocins, such as pediocin or garvicin [68, 69, 70]. Thus, increased expression of these receptors may provide an opportunity to pre-treat food with IIa bacteriocins, which may increase susceptibility to HPP. However, dltD upregulation in RO15 may lead to incorporation of more alanine residues , which increases the positive charge and consequently reduces affinity to cationic antimicrobials and bacteriocins. Interestingly, dltD was downregulated in ScottA indicating pre-treatment might be more effective for barosensitive strains. In addition, among peptidoglycan biosynthesis genes, deletion of pbp2A causes significant susceptibility to HPP. Hence new approaches could be sought by using peptidoglycan cross-linking.