The Contribution of Transcriptomic and Proteomic Analysis in Elucidating Stress Adaptation Responses of Listeria monocytogenes

Abstract

The foodborne transmission of Listeria monocytogenes requires physiological adaptation to various conditions, including the cold, osmotic, heat, acid, alkaline, and oxidative stresses, associated with food hygiene, processing, and preservation measures. We review the current knowledge on the molecular stress adaptation responses in L. monocytogenes cells as revealed through transcriptome, proteome, genetic, and physiological analysis. The adaptation of L. monocytogenes to stress exposure is achieved through global expression changes in a large number of cellular components. In addition, the cross-protection of L. monocytogenes exposed to different stress environments might be conferred through various cellular machineries that seem to be commonly activated by the different stresses. To assist in designing L. monocytogenes mitigation strategies for ready-to-eat food products, further experiments are warranted to specifically evaluate the effects of food composition, additives, preservatives, and processing technologies on the modulation of L. monocytogenes cellular components in response to specific stresses.

Introduction

L isteria monocytogenes is an important public health problem and food safety challenge. As a foodborne pathogen, this bacterium primarily targets immunosuppressed individuals, including the elderly, pregnant women, and newborns, leading to listeriosis, a disease that, although less frequent in occurrence (∼2.4 cases/million), is associated with relatively high (averaging 20%–30%) mortality rates (Mead et al., 1999). L. monocytogenes is associated with significant food safety control problems due to its wide distribution in nature and its capacity to survive and grow on the food products despite frequent exposure to harsh environmental conditions associated with food processing and preservation measures.

Upon encountering different environmental stress challenges along the food supply chain, it is presumed that L. monocytogenes cells depend on various stress-sensing mechanisms that detect stress-associated molecular hardships. On sensing such impending molecular stress problems, appropriate signal transduction processes are activated, subsequently leading to the mobilization of necessary stress protection measures through modifications in gene expression and protein function activities. Insights into gene expression changes mobilized during stress adaptation responses have been recently gained through transcriptome and proteome stress analysis in this bacterium. This article provides a review of cold, heat, osmotic, acid, alkaline, and oxidative stress responses in this bacterium.

Cold Stress Adaptation Responses of L. monocytogenes

Numerous molecular hardships confront L. monocytogenes cells exposed to cold stress such as increased membrane rigidity, reduced protein and enzyme activity, slow transport and nutrient uptake processes, stalled gene expression processes, and protein damage and alteration. Altered expression of numerous gene transcripts and proteins was observed in L. monocytogenes cells adapted to cold exposure (Chan et al., 2007; Cacace et al., 2010). Functional classification of genes and proteins identified in these two studies revealed that cold stress adaptation genetic responses of this bacterium promote (1) specific and general stress protection; (2) membrane fluidity and function; (3) resumption of gene expression events; (4) protein folding and degradation; (5) assimilation of carbon sources and cold protective nutrients; (6) oxidative stress protection; (7) energy production, and (8) specific amino acid and lipid biosynthesis pathways. A number of cold stress adaptation mechanisms that have been experimentally validated in this bacterium are listed in Table 1 and Figure 1A. Readers interested in more detail on these cold stress adaptation systems are directed to recent review articles (Tasara and Stephan, 2006; Chan and Wiedmann, 2009). The subsequent discussion therefore only focuses on the cold stress response systems recently described in this bacterium and not covered by previous reviews.

FIG. 1.

Graphical presentations depicting stress-affected cellular components and a selection of molecular stress adaptation responses documented in Listeria monocytogenes cells exposed to (A) cold stress, (B) heat stress, (C) osmotic stress, and (D) acid stress. Alterations are caused by stress exposure on cell envelope, nucleic acids (RNA and DNA), proteins, and metabolism. Such molecular alterations are presumably detected by different stress-sensing molecular systems involving cell envelope components, nucleic acids, and proteins leading to signal transduction events and subsequent activation of adaptive gene expression response regulators. Gene expression modulation leads to activation stress protection functions through increased protein synthesis and/or activity modulation..

Table 1.

An Overview of the Listeria monocytogenes Gene and Protein Systems Associated with Cold, Osmotic, Acid, Heat, Oxidative, and Alkaline Stress Adaptation Responses

		Phenotypically confirmed stress adaptation roles
Stress adaptation gene/protein system	Functional description	Cold stress	Osmotic stress	Acid stress	Heat stress	Oxidative stress	Alkaline stress	References
LisRK	Two component system	+	+	+	+			Cotter et al. (1999); Kallipolitis and Ingmer (2001); Sleator and Hill (2005)
KpdED	Two component system		+		+			Kallipolitis and Ingmer (2001)
Lmo 1172	Putative two component system protein	+						Chan et al. (2008)
Lmo 1060	Putative Two component system protein	+						Chan et al. (2008)
RsbV	Anti-antisigma factor protein.	+		+		+		Chaturongakul and Boor (2004)
RsbT	Serine kinase	+		+		+		Chaturongakul and Boor (2004)
SigB	Alternative sigma factor protein	+	+	+	+	+	+	Chan et al. (2007); Giotis et al. (2008a); Raengpradub et al. (2008)
SigC	Alternative sigma factor protein	+			+			Chan et al. (2008); Zhang et al. (2005)
SigH	Alternative sigma factor protein	+					+	Chan et al. (2008); Rea et al. (2004)
SigL/RpoN	Alternative sigma factor protein	+	+	+				Chan et al. (2008); Okada et al. (2006); Raimann et al. (2009)
MarR	MarR family transcription regulator						+	Rea et al. (2004)
Fur	Transcription regulator						+	Rea et al. (2004)
CtsR	Negative transcription regulator		+	+	+	+		Nair et al. (2000)
HrcA	Negative transcription regulator				+			Hu et al. (2007a)
MogR	Transcription repressor				+			van der Veen et al. (2009)
OppA	Oligopeptide permease	+						Borezee et al. (2000)
OpuC, Gbu	Osmolyte transporter	+	+					Angelidis and Smith (2003a, 2003b)
ProBA	Proline synthesis enzyme system		+					Sleator et al. (2001)
GAD	Glutamate decarboxylase system			+				Cotter et al. (2001)
Csps	Cold shock domain proteins	+	+			+		Loepfe et al. (2010); Schmid et al. (2009)
Hfq	RNA chaperone-	+	+					Christiansen et al. (2004)
Ctc	50S ribosomal protein L25		+					Gardan et al. (2003b)
HtrA	Serine protease		+	+	+	+		Wilson et al. (2006); Wonderling et al. (2004)
ClpB, ClpC, ClpE, ClpP	ATP dependent Clp chaperone and protease		+		+			Nair et al. (2000)
Fri	DNA binding protein of starved cells (Dps)	+			+	+		Dussurget et al. (2005)
Catalase	Catalase	+				+		Azizoglu and Kathariou (2010b)
SOD	Superoxide dismutase					+		Archambaud et al. (2006)
ADI	Arginine deiminase system			+				Ryan et al. (2009)
Lmo0038	Putative argmatine deiminase			+	+			Chen et al. (2009)
PgpH	Putative integral membrane and ppGpp hydrolase	+						Liu et al. (2006)
Lmo1078	Putative UDP glucose synthetase	+	+					Chassaing and Auvray (2007)
Bkd system	Branched chain a-keto acid dehydrogenase enzyme complex	+						Zhu et al. (2005)
F₀F₁-ATPase	Proton efflux membrane ATPase			+				Cotter et al. (2000)
LtrC	General stress response protein	+						Chan et al. (2007)
UvrR	DNA repair			+				Kim et al. (2006)

+ = Gene confirmed to have significant functional contributions under these stress conditions.

The bacterial two-component systems (TCS) are part of signal transduction pathways with an important role in cold stress sensing. Deletions of lisR and two putative TCS (lmo1172 and lmo1060) genes were recently shown to impair cold adaptation in this bacterium (Chan et al., 2008). In addition, putative TCS genes lhkA, yycJ, and yycF have also been found to be transcriptionally activated in response to cold stress in L. monocytogenes, but their cold adaptation functional significance remains to be further investigated (Liu et al., 2002; Chan et al., 2007). The cold shock domain family proteins (Csps) and RNA helicases found in this bacterium are also important in cold growth functions. Csps are crucial in resolution of stabilized RNA secondary structures, which confront cold stress-exposed bacterial cells, leading to stalled transcription and translation events. Schmid et al. (2009) examined a series of csp-deleted L. monocytogenes mutants and found that mutants lacking cspA (cspL) and cspD genes were significantly impaired during cold growth. RNA helicases are also critical in RNA secondary structure resolution and thus vital to the resumption of transcription and translation events that get stalled under cold stress. The DEAD box RNA helicases are ATP-dependent RNA helicase proteins characterized by the presence of the D-E-A-D (Asp-Glu-Ala-Asp) amino acid sequence in the conserved helicase motif II. Three putative DEAD box RNA helicases (lmo0866, lmo1722, and lmo1450) were found to be part of the cold stress response transcriptome determined in L. monocytogenes (Chan et al., 2007). Meanwhile, a transposon mutant disrupted in the lmo0866 gene homolog of L. monocytogenes displaying significantly impaired cold growth was recently described further supporting the role of this putative RNA helicase protein in cold stress adaptation processes of this bacterium (Azizoglu and Kathariou, 2010a).

Heat Stress Adaptation Responses of L. monocytogenes

Proteins exposed to elevated heat stress are denatured, leading to aggregate formation and loss of function, including enzyme activity. Organisms adapting to heat stress must mobilize cellular mechanisms to restore membranes and nucleic acids functions, remove heat degraded proteins, and produce new proteins to restore metabolic functions lost through thermal stress destruction of proteins. Genome-wide transcriptome analysis has revealed that heat stress responses in L. monocytogenes involve the recruitment of various genes with roles in heat shock and SOS responses, as well as cell division and cell wall synthesis (Hu et al., 2007a, 2007b; van der Veen et al., 2007). This heat shock response is associated with increased production of a specific set of proteins, the heat shock proteins (Hsps), which includes highly conserved chaperones and ATP-dependent proteases, involved in damaged protein refolding and degradation. L. monocytogenes contains two heat shock stress response protein groups: the class I and III proteins that are negatively controlled through HrcA and CstR transcription repression mechanisms, and the σ^B controlled proteins, which include the class II stress response proteins, as well as class IV proteins with roles in general stress adaptation functions (Hu et al., 2007a, 2007b). The HrcA repressor regulon includes chaperonin proteins GroES, GroEL, and DnaK. The activation of these proteins at elevated temperature provides cellular protection by assisting in protein folding and assembling (Hu et al., 2007a). Further, the DnaK operon also includes the DnaJ and GrpE proteins, which are proposed to be important for stimulating the ATPase activity of DnaK (Liberek et al., 1991). CstR repressor-controlled ATP-dependent proteases are ClpP, ClpE, and ClpC, which degrade damaged or misfolded proteins, and ClpB, which reactivates the protein aggregates (Hu et al., 2007b). A ctsR-deleted mutant is more tolerant to heat stress consistent with CtsR in transcriptional repression of class III stress response gene expression (Hu et al., 2007a). Table 1 and Figure 1B include various protein systems so far shown to contribute to heat stress adaptation responses of L. monocytogenes. These include genes of two component signal transduction systems (lisR and kdpE) as well as various negative (hrcA, ctsR, and mogR) and positive (σ^B and σ^C) transcriptional regulators (Kallipolitis and Ingmer, 2001; Zhang et al., 2005; Hu et al., 2007a, 2007b; van der Veen et al., 2009).

Proteome analysis of heat stress responses in L. monocytogenes cells revealed increased production of GroEL and DnaK, which is consistent with the role of these highly conserved Hsps, in removal of altered proteins in L. monocytogenes cells exposed to heat stress (Sokolovic et al., 1990; Morange et al., 1993). Another heat stress response proteome study identified the general stress response protein Ferritin (also referred as ferritin like proteins, Flp) to be the predominant heat stress-induced protein in this bacterium (Hebraud and Guzzo, 2000). The fri gene transcripts were found to be heat stress inducible, whereas fri-gene-deleted L. monocytogenes cells had increased heat stress sensitivity (Hebraud and Guzzo, 2000; Dussurget et al., 2005). Agoston et al. (2009) compared proteomes associated with mild and prolonged heat treatments on L. monocytogenes and discovered that a large number of metabolic proteins are suppressed by heat stress exposure. In addition, they also found that the Hsp DnaN, which is a beta subunit of DNA polymerase III, was highly induced in response to different heat shock treatments. Induction of DnaN as observed in this study thus suggests that L. monocytogenes also counteracts heat stress challenges by modulation of DNA replication processes apart from the elevated activity of protein folding and degradation mechanisms. Heat stress exposure also induces recruitment of SOS response and DNA repair genes, as well as PrfA-controlled virulence genes, while repressing the cell division and cell wall synthesis genes in line with suppressed cell division and cellular elongation observed under heat stress (van der Veen et al., 2007).

Osmotic Stress Responses of L. monocytogenes

The high level of sugars and sodium salts typically used as food preservers threaten bacterial cells with dehydration, which leads to increased intracellular solute concentrations and disruption of various biological functions. Proteome analysis has been used to study osmotolerance responses in L. monocytogenes. Eleven osmotic stress acclimation proteins in this bacterium were identified, which include the GbuA, AppA, Ctc, DnaK, HtrA, and OpuC proteins (Duche et al., 2002a, 2002b; Abram et al., 2008). Specific osmotic stress adaptation protein systems currently known in this bacterium are listed in Table 1 and Figure 1C.

The accumulation of osmolytes constitutes one of the main osmotic stress adaptation strategies in L. monocytogenes. The BetL, Gbu, and OpuC transport systems, involved in glycine betaine and carnitine uptake, are important during growth of this bacterium under osmotic stress (Fraser et al., 2003). The membrane-based and osmotically inducible K⁺ uptake system, the Kdp-ATPase system, has been associated with osmotic stress protection roles in L. monocytogenes cells. The absence of the KdpE sensor and its regulator KdpD is associated with impaired L. monocytogenes growth under NaCl osmotic stress (Kallipolitis and Ingmer, 2001). Proline is also another osmolyte shown to be exploited for osmoprotection in L. monocytogenes. The disruption of the proBA locus encoding the ProA and ProB proteins was found to increase osmotic stress sensitivity of L. monocytogenes cells, indicating that proline biosynthesis genes may also be crucial in osmotic stress adaptation of this bacterium (Sleator et al., 2001).

Single-gene deletions targeting clpC, clpP, and htrA genes are all also associated with defective cellular growth under NaCl osmotic stress tolerance in this bacterium (Wonderling et al., 2004; van der Veen et al., 2007). These observations thus suggest that these protein chaperones are also vital in resolution of protein damages and restoration of cellular functions that get impaired by osmotic stress. An lmo1078-gene-deleted L. monocytogenes EGD-e strain was described, which is also defective in growth under NaCl osmotic stress (Chassaing and Auvray, 2007). Lmo1078 is a putative UDP-glucose phosphorylase. This protein has been proposed to facilitate cell wall and membrane lipid composition maintenance, and its functions seem important during osmotic and cold stress adaptation of L. monocytogenes cells.

Acid Stress Adaptation Responses of L. monocytogenes

The acid stress challenges faced by L. monocytogenes cells include organic acids used as food preservatives and decontaminants as well as inorganic acids encountered within the gastrointestinal tracts of human hosts. The antibacterial mechanisms against organic acid stress are generally presumed to depend on their dissociation in the bacterial cell cytoplasm, leading to acidification as well as proton and anion influx. Proton influx inhibits the cellular ATP synthesis and catabolite transport capacities, whereas the acidification and ionic influx into the cytoplasm disrupts metabolic functions as well as induce damage to proteins, nucleic acids, and cell membranes. Table 1 and Figure 1D list various acid stress response protein systems confirmed in this bacterium.

Bowman et al. (2010) recently examined transcriptome responses to organic acid stress in L. monocytogenes. They found that organic acid salt stress exposure (21 mM sodium diacetate at pH 5.0) was associated with a broad range of gene expression changes, including an increased activation of σ^B, PrfA, HrcA, and CtsR targeted regulons, as well as oxidative stress defenses, DNA repair, intermediary metabolism, cell-wall modification, and cofactor and fatty acid biosynthetic genes. A proteome analysis of L. monocytogenes ScottA cells exposed to food preservation organic acid salts also revealed an increased production of oxidoreductases and lipoproteins, and reduced expression of DNA-binding proteins, alpha amylase, and SecA (Mbandi et al., 2007). The repression of DNA-binding proteins (involved in transcriptional regulation), alpha amylase (involved in metabolizing complex carbon sources in readily available energy sources), and SecA (involved in translocation of proteins) may therefore be interpreted as downregulation of cell metabolic activity caused by organic acid stress exposure of L. monocytogenes cells.

Other acid stress response proteomes induced mainly by exposure to hydrochloric acid have been determined in L. monocytogenes (Wemekamp-Kamphuis et al., 2004; Phan-Thanh and Jansch, 2006). The identified acid stress proteins indicate that proteins involved in respiration (enzyme dehydrogenases and reductases), osmolyte transport, protein folding and repair, general stress resistance, flagella synthesis, and metabolism are all recruited as part of acid stress responses in L. monocytogenes cells. Cellular proton permeability involves altering the composition of the membrane lipid bilayer. Along these lines, increased straight chain fatty acid concentrations and decreased levels of branched chain fatty acids were observed in cell membranes of L. monocytogenes cells exposed to acid stress (Giotis et al., 2007). Accelerated electron transfer through enhanced oxidation–reduction potential might be another mechanism employed by bacteria that are exposed to acid stress to dispel protons. The induction of dehydrogenases (GuaB, PduQ, and Lmo0560), reductases (YcgT), and respiratory enzymes associated with acid stress responses in L. monocytogenes cells may therefore also indicate acid stress adaptive mechanisms, which involve active cellular proton efflux (Phan-Thanh and Jansch, 2006). Along the same lines, the F₀F₁ ATPase also plays an important role during L. monocytogenes acid stress adaptation, where it is thought to contribute to proton efflux events (Cotter et al., 2000).

The glutamate decarboxylase (GAD) acid resistance system serves important acid stress adaptation roles in this bacterium (Cotter et al., 2001; Wemekamp-Kamphuis et al., 2004). The L. monocytogenes arginine deiminase (ADI) system includes enzymes encoded by arcA, arcB, and arcC, which are involved conversion of arginine to ornithine, as well as the antiporter arcD, which transfers ornithine outside the cell in exchange for arginine (Ryan et al., 2009). An argR and arcA deletion mutant showed impaired survival under mild (pH 3.5) and lethal (pH 4.8) acid stress conditions, respectively. The lmo0038, encoding a putative peptidylarginase deiminase family gene, has also been suggested to have acid stress adaptation functions in this bacterium (Chen et al., 2009). The TCS LisRK has been linked to acid stress response modulation probably through its role in sensing of acid stress-related environmental stimuli (Cotter et al., 1999; Sleator and Hill, 2005). In addition, L. monocytogenes cells lacking HtrA are acid stress sensitive consistent with the requirement of HtrA protease functions in removal of acid stress-damaged proteins (Wonderling et al., 2004).

L. monocytogenes Response to Alkaline Stress

Environmental L. monocytogenes strains inhabiting food-processing environments may frequently encounter sublethal alkaline stress associated with detergents and sanitizing agents applied as food hygiene measures. The adaptive strategies deployed in microorganisms during alkaline stress adaptation include metabolic changes to increase acid generation, as well as the induction of transporters and enzymes involved in proton retention, and cell surface modifications that promote cytoplasmic proton retention. Transcriptome analysis has revealed that as much as 390 gene transcripts in L. monocytogenes cells are differentially expressed in response to alkaline stress exposure. Genes mobilized for alkaline stress adaptation participate in general stress responses, solute transport, and various metabolic pathways (Giotis et al., 2010). In another study the proteome analysis of L. monocytogenes cells exposed to alkaline stress revealed the synthesis or over expression of a limited number of proteins as compared to the repressed expression of proportionally large number of proteins (Giotis et al., 2008b). Meanwhile, the stress protective chaperones DnaK and GroEL were also induced by alkaline stress exposure. By screening of a library of Tn917- lac insertional mutants in L. monocytogenes LO28, Gardan et al. (2003a) identified 12 mutant strains that were sensitive to alkaline stress, and these mutants were disrupted in genes encoding putative transporter proteins. At present there is, however, still less known about specific alkaline stress adaptation protein systems in this bacterium.

Oxidative Stress Adaptation Responses

L. monocytogenes cells must combat oxidative stress encountered in external environments and during host infection. In food environments, oxidative stress might develop due to atmospheric modification as well as by chemical reagents applied as detergents and disinfectants. Reactive oxygen species (ROS) are also produced as byproducts of metabolism and can accumulate due to respiratory chain impairment or metabolic alterations encountered in other stress situations (Bowman et al., 2010; Cacace et al., 2010). Oxidative stress is deleterious to various molecular processes and cellular components such as membranes, proteins, nucleic acids, and enzymes. To cope against oxidative stress, molecular detoxification of ROS, as well as protein, membrane, and nucleic acid damage repair mechanisms must be activated.

Gorski et al. (2008) found that best colonizing L. monocytogenes strains in oxidative food environments were those with an enhanced ability to tolerate oxidative stress exposure. Bacterial ROS detoxification systems include superoxide dismutase, catalase (Kat), and alkyl hydroperoxide (AhpCF) enzyme systems (Helmann et al., 2003). The Kat and superoxide dismutase have been shown to be important in oxidative stress protection of L. monocytogenes cells. The sod and kat null L. monocytogenes strains display poor aerobic growth phenotypes as well as increased oxidative stress sensitivity, poor macrophage survival, and virulence (Archambaud et al., 2006; Azizoglu and Kathariou, 2010b). The Dps ferritin has also been shown to be important in oxidative stress protection of L. monocytogenes. The cells of this bacterium that are deleted in the ferritin protein encoding gene fri exhibit increased sensitivity to oxidative stress and poor macrophage infectivity (Dussurget et al., 2005). A ferric transcriptional regulator per deleted L. monocytogenes mutant was reported to exhibit increased sensitivity to hydrogen peroxide and poor aerobic growth (Rea et al., 2005). In addition, Per regulon determination in this study unveiled Per-dependent repression of numerous ROS detoxification genes, including the kat, trxB, fur, fri, and hemA genes. The increased peroxide sensitivity in per mutant cells was suggested to be a potential consequence of increased expression of some ROS defense genes that are otherwise normally controlled through Per repression.

Role of σ Factors in L. monocytogenes Stress Adaptation

Proteins encoded and governed by global general stress response regulator σ factors play vital roles in protecting L. monocytogenes cells against various stress stimuli. σ^B is a positive regulator of several general and cold stress response genes, including fri, oppA, opuCA, and ltrC genes, with genetically confirmed cold stress adaptation roles in this bacterium (Chan et al., 2007). While recently defective cold growth phenotypes were also documented in L. monocytogenes mutant strains lacking genes for the σ^C, σ^H, and σ^L (RpoN) alternative sigma factors, indicating that besides σ^B, these alternative sigma factors are also involved in regulation of cold adaptation processes in this bacterium (Chan et al., 2008; Raimann et al., 2009). The L. monocytogenes class II Hsps are positively controlled by σ^B, and increased heat stress sensitivity in a σ^B deletion mutant is consistent with σ^B-dependent transcriptional activation of the class II stress response genes in this bacterium (van der Veen et al., 2007). Apart from σ^B, the loss of σ^c function also increases L. monocytogenes sensitivity to a thermal treatment (Zhang et al., 2005).

Both σ^B and σ^L in L. monocytogenes are transcriptionally upregulated in response to elevated NaCl concentrations, whereas σ^B- and σ^L-deleted mutants of this bacterium are NaCl salt-stress sensitive (Okada et al., 2008; Raimann et al., 2009). Based on transcriptomic profiling, σ^B-regulated genes in osmotic stress adaptation are associated with general stress responses, transcriptional regulation, cell transport, envelope modification, metabolism, protein synthesis and modification, as well as virulence-associated functions (Raengpradub et al., 2008). Genes positively controlled through σ^B also include osmolyte transporter genes (opuCA and gbuA) as well as clpC and hfq genes, which have all been genetically associated with osmotic stress adaptation (Christiansen et al., 2004; Abram et al., 2008; Raengpradub et al., 2008). The general stress response protein Ctc is vital in NaCl salt stress tolerance and the ctc gene in L. monocytogenes is preceded by a putative σ^B-dependent promoter (Gardan et al., 2003b).

Both σ^B and σ^L have also been functionally linked to regulation of the acid stress adaptation responses in this bacterium. L. monocytogenes cells deleted on σ^B and its regulators rsBT and rsBV, as well as σ^L, have been found to display acid stress sensitive phenotypes (Chaturongakul and Boor, 2004; Wemekamp-Kamphuis et al., 2004; Raimann et al., 2009). σ^B is also a positive regulator of genes of the GAD and ADI acid stress response systems and its deletion diminishes expression of GAD and ADI genes (Cotter et al., 2001; Ryan et al., 2009). σ^B regulons in this bacterium have also been shown to include several oxidative stress response genes and σ^B-deleted mutant strains are significantly impaired in oxidative stress tolerance (Ferreira et al., 2001; Hain et al., 2008; Oliver et al., 2010). It has been recently described that the σ^B contribution to oxidative stress might also depend on strain genotype. Oliver et al. (2010) found that although σ^B loss induced oxidative stress sensitivity in strains of lineage I, II, and IIIB, this was not the case in a lineage IIIA strain. Such differences between the L. monocytogenes genomic lineages lead to a conclusion that such strain-specific differences might therefore also influence σ^B-dependent oxidative stress response gene regulation, in different genomic lineages of this bacterium.

Cross Protective Stress Responses

It has been shown through several studies that the exposure of L. monocytogenes to sublethal stress induces the development of stress-conditioned organisms, which are physiologically more tolerant to increased levels of the same or different stresses. For example, L. monocytogenes cells exposed to sublethal acid stress display increased resistance to higher acid stress levels as well as become more tolerant to heat and osmotic stress (Gahan et al., 1996). Acid- and cold-adapted cells of this bacterium are more protected from the effects of high hydrostatic pressure stress (Wemekamp-Kamphuis et al., 2002). L. monocytogenes cells preadapted by exposure to sublethal heat stress show enhanced osmotic and ethanol stress protection (Lou and Yousef, 1997). Taormina and Beuchat (2002) showed that alkaline stress-adapted L. monocytogenes cross-contaminating food products are more resistant to thermal food safety measures in comparison to those not previously exposed to sublethal alkaline stress conditions.

To date, investigators have identified various molecular mechanisms that appear to be crucial in mediation of cross protective stress responses in this bacterium. For instance, molecular adaptive challenge envisaged in cold environments, particularly freezing, include intracellular accumulation of ROS due to cold stress-induced metabolic alteration. A catalase-deficient L. monocytogenes F2365 mutant was recently described, which is impaired during cold growth (Azizoglu and Kathariou, 2010b). In addition, mutants lacking some csp genes are also characterized by increased peroxide stress sensitivity and reduced host cell infectivity (Loepfe et al., 2010). Moreover, L. monocytogenes mechanisms to combat osmotic stress seem to also involve functions of the cold shock domain family proteins. A diminished osmotic stress tolerance phenotype is induced by cspD deletion in this bacterium (Schmidt et al., 2009). A cstR null mutant was described vital to osmotic stress adaptation responses of L. monocytogenes cells, indicating that some CtsR-repressed heat stress proteins also contribute toward osmotic stress (Nair et al., 2000). Another heat stress repressor protein, HtrA, is also presumed to degrade misfolded or aggregated proteins that accumulate when L. monocytogenes cells are exposed to harsh environmental conditions such as high osmolarity and low pH in addition to high temperatures (Wilson et al., 2006; van der Veen et al., 2007). Giotis et al. (2008b) reported induced expression DnaK and GroEL chaperones by alkaline stress and as such these proteins have very well-defined role in heat stress adaptation. Thus, based on above examples it is evident that a large number of proteins and regulatory mechanisms are involved in L. monocytogenes cross protective responses.

Conclusion and Future Perspectives

The development of more effective food preservation methods depend on an improved understanding of fundamental changes that are instituted at the gene expression level in cells of this bacterium when challenged with adverse environmental stress conditions (Tasara and Stephan, 2006). Although considerable progress has been made so far in understating the L. monocytogenes stress response, one major draw back is that the majority of current information is based on broth models and not with actual food substrates. We therefore propose that more efforts should be invested in understanding the cellular stress response of L. monocytogenes at the molecular level in the presence of different food substrates and varying environmental conditions. It is hoped that with this further knowledge, the complexity and hierarchical nature of stress adaptation response mechanisms can be understood better, paving the way to the development of novel strategies that are more effective in combating the stress resistance properties of L. monocytogenes. Such future breakthroughs may include (1) novel ways to block the expression of transporter proteins that govern cold/osmotic stress adaptation to prevent the uptake of osmolyte molecules from food substrates to make L. monocytogenes cells susceptible to both cold and osmotic stress stimuli, and (2) identifying the antimicrobial compounds that suppress the induction of stress proteins of L. monocytogenes in food products.

Footnotes

Acknowledgments

This research was supported in part by Food Safety Initiative Award to R.N. by the Mississippi Agricultural and Forestry Experiment Station under project MIS-401100.

Disclosure Statement

No competing financial interests exist.

References

Abram

, Starr

, Karatzas

, Matlawska-Wasowska

, Boyd

, Wiedmann

, Boor

, Connally

, O'Byrne

. Identification of components of the sigmaB regulon in Listeria monocytogenes that contribute to acid and salt tolerance. Appl Environ Microbiol, 2008; 74:6848–6858.

Agoston

, Soni

, Jesudhasan

, Russell

, Mohacsi-Farkas

, Pillai

. Differential expression of proteins in Listeria monocytogenes under thermotolerance-inducing, heat shock, and prolonged heat shock conditions. Foodborne Pathog Dis, 2009; 6:1133–1140.

Angelidis

, Smith

. Role of the glycine betaine and carnitine transporters in adaptation of Listeria monocytogenes to chill stress in defined medium. Appl Environ Microbiol, 2003a. 69:7492–7498.

Angelidis

, Smith

. Three transporters mediate uptake of glycine betaine and carnitine by Listeria monocytogenes in response to hyperosmotic stress. Appl Environ Microbiol, 2003b. 69:1013–1022.

Archambaud

, Nahori

, Pizarro-Cerda

, Cossart

, Dussurget

. Control of Listeria superoxide dismutase by phosphorylation. J Biol Chem, 2006; 281:31812–31822.

Azizoglu

, Kathariou

. Inactivation of a cold-induced putative rna helicase gene of Listeria monocytogenes is accompanied by failure to grow at low temperatures but does not affect freeze-thaw tolerance. J Food Prot, 2010a. 73:1474–1479.

Azizoglu

, Kathariou

. Temperature-dependent requirement for catalase in aerobic growth of Listeria monocytogenes F2365. Appl Environ Microbiol, 2010b. 76:6998–7003.

Borezee

, Pellegrini

, Berche

. OppA of Listeria monocytogenes, an oligopeptide-binding protein required for bacterial growth at low temperature and involved in intracellular survival. Infect Immun, 2000; 68:7069–7077.

Bowman

, Lee Chang

, Pinfold

, Ross

. Transcriptomic and phenotypic responses of Listeria monocytogenes strains possessing different growth efficiencies under acidic conditions. Appl Environ Microbiol, 2010; 76:4836–4850.

10.

Cacace

, Mazzeo

, Sorrentino

, Spada

, Malorni

, Siciliano

. Proteomics for the elucidation of cold adaptation mechanisms in Listeria monocytogenes. J Proteomics, 2010; 73:2021–2030.

11.

Chan

, Hu

, Chaturongakul

, Files

, Bowen

, Boor

, Wiedmann

. Contributions of two-component regulatory systems, alternative sigma factors, and negative regulators to Listeria monocytogenes cold adaptation and cold growth. J Food Prot, 2008; 1:420–425.

12.

Chan

, Raengpradub

, Boor

, Wiedmann

. Microarray-based characterization of the Listeria monocytogenes cold regulon in log- and stationary-phase cells. Appl Environ Microbiol, 2007; 73:6484–6498.

13.

Chan

, Wiedmann

. Physiology and genetics of Listeria monocytogenes survival and growth at cold temperatures. Crit Rev Food Sci Nutr, 2009; 49:237–253.

14.

Chassaing

, Auvray

. The lmo1078 gene encoding a putative UDP-glucose pyrophosphorylase is involved in growth of Listeria monocytogenes at low temperature. FEMS Microbiol Lett, 2007; 275:31–37.

15.

Chaturongakul

, Boor

. RsbT and RsbV contribute to sigmaB-dependent survival under environmental, energy, and intracellular stress conditions in Listeria monocytogenes. Appl Environ Microbiol, 2004; 70:5349–5356.

16.

Chen

, Jiang

, Chen

, Zhao

, Luo

, Chen

, Fang

. lmo0038 is involved in acid and heat stress responses and specific for Listeria monocytogenes lineages I and II, and Listeria ivanovii. Foodborne Pathog Dis, 2009; 6:365–376.

17.

Christiansen

, Larsen

, Ingmer

, Søgaard-Andersen

, Kallipolitis

. The RNA-binding protein Hfq of Listeria monocytogenes: role in stress tolerance and virulence. J Bacteriol, 2004; 186:3355–3362.

18.

Cotter

, Emerson

, Gahan

, Hill

. Identification and disruption of lisRK, a genetic locus encoding a two-component signal transduction system involved in stress tolerance and virulence in Listeria monocytogenes. J Bacteriol, 1999; 181:6840–6843.

19.

Cotter

, Gahan

, Hill

. Analysis of the role of the Listeria monocytogenes F₀F₁-ATPase operon in the acid tolerance response. Int J Food Microbiol, 2000; 60:137–146.

20.

Cotter

, Gahan

, Hill

. A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid. Mol Microbiol, 2001; 40:465–475.

21.

Duche

, Tremoulet

, Glaser

, Labadie

. Salt stress proteins induced in Listeria monocytogenes. Appl Environ Microbiol, 2002a. 68:1491–1498.

22.

Duche

, Tremoulet

, Namane

, Labadie

. A proteomic analysis of the salt stress response of Listeria monocytogenes. FEMS Microbiol Lett, 2002b. 215:183–188.

23.

Dussurget

, Dumas

, Archambaud

, Chafsey

, Chambon

, Hébraud

, Cossart

. Listeria monocytogenes ferritin protects against multiple stresses and is required for virulence. FEMS Microbiol Lett, 2005; 15:253–261.

24.

Ferreira

, O'Byrne

, Boor

. Role of sigma(B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes. Appl Environ Microbiol, 2001; 67:4454–4457.

25.

Fraser

, Sue

, Wiedmann

, Boor

, O'Byrne

. Role of sigmaB in regulating the compatible solute uptake systems of Listeria monocytogenes: osmotic induction of opuC is sigmaB dependent. Appl Environ Microbiol, 2003; 69:2015–2022.

26.

Gahan

, O'Driscoll

, Hill

. Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation. Appl Environ Microbiol, 1996; 62:3128–3132.

27.

Gardan

, Cossart

, Labadie

. European Listeria Genome Consortium. Identification of Listeria monocytogenes genes involved in salt and alkaline-pH tolerance. Appl Environ Microbiol, 2003a. 69:3137–3143.

28.

Gardan

, Duche

, Leroy-Setrin

, Labadie

. Role of ctc from Listeria monocytogenes in osmotolerance. Appl Environ Microbiol, 2003b. 69:154–161.

29.

Giotis

, Julotok

, Wilkinson

, Blair

, McDowell

. Role of sigma B factor in the alkaline tolerance response of Listeria monocytogenes 10403S and cross-protection against subsequent ethanol and osmotic stress. J Food Prot, 2008a. 71:1481–1485.

30.

Giotis

, McDowell

, Blair

, Wilkinson

. Role of branched-chain fatty acids in pH stress tolerance in Listeria monocytogenes. Appl Environ Microbiol, 2007; 73:997–1001.

31.

Giotis

, Muthaiyan

, Blair

, Wilkinson

, McDowell

. Genomic and proteomic analysis of the Alkali-Tolerance Response (AlTR) in Listeria monocytogenes 10403S. BMC Microbiol, 2008b. 8:102.

32.

Giotis

, Muthaiyan

, Natesan

, Wilkinson

, Blair

, McDowell

. Transcriptome analysis of alkali shock and alkali adaptation in Listeria monocytogenes 10403S. Foodborne Pathog Dis, 2010; 7:1147–1157.

33.

Gorski

, Flaherty

, Duhé

. Comparison of the stress response of Listeria monocytogenes strains with sprout colonization. J Food Prot, 2008; 71:1556–1562.

34.

Hain

, Hossain

, Chatterjee

, Machata

, Volk

, Wagner

, Brors

, Haas

, Kuenne

, Billion

, Otten

, Pane-Farre

, Engelmann

, Chakraborty

. Temporal transcriptomic analysis of the Listeria monocytogenes EGD-e sigmaB regulon. BMC Microbiol, 2008; 8:20.

35.

Hebraud

, Guzzo

. The main cold shock protein of Listeria monocytogenes belongs to the family of ferritin-like proteins. FEMS Microbiol Lett, 2000; 190:29–34.

36.

Helmann

, Wu

, Gaballa

, Kobel

, Morshedi

, Fawcett

, Paddon

. The global transcriptional response of Bacillus subtilis to peroxide stress is coordinated by three transcription factors. J Bacteriol, 2003; 185:243–253.

37.

, Oliver

, Raengpradub

, Palmer

, Orsi

, Wiedmann

, Boor

. Transcriptomic and phenotypic analyses suggest a network between the transcriptional regulators HrcA and sigmaB in Listeria monocytogenes. Appl Environ Microbiol, 2007a. 73:7981–7991.

38.

, Raengpradub

, Schwab

, Loss

, Orsi

, Wiedmann

, Boor

. Phenotypic and transcriptomic analyses demonstrate interactions between the transcriptional regulators CtsR and Sigma B in Listeria monocytogenes. Appl Environ Microbiol, 2007b. 73:7967–7980.

39.

Kallipolitis

, Ingmer

. Listeria monocytogenes response regulators important for stress tolerance and pathogenesis. FEMS Microbiol Lett, 2001; 204:111–115.

40.

Kim

, Gorski

, Reynolds

, Orozco

, Fielding

, Park

, Borucki

. Role of uvrA in the growth and survival of Listeria monocytogenes under UV radiation and acid and bile stress. J Food Prot, 2006; 69:3031–3036.

41.

Liberek

, Marszalek

, Ang

, Georgopoulos

, Zylicz

. Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. Proc Natl Acad Sci U S A, 1991; 88:2874–2878.

42.

Liu

, Bayles

, Mason

, Wilkinson

. A cold-sensitive Listeria monocytogenes mutant has a transposon insertion in a gene encoding a putative membrane protein and shows altered (p)ppGpp levels. Appl Environ Microbiol, 2006; 72:3955–3959.

43.

Liu

, Graham

, Bigelow

, Morse

2nd , Wilkinson

. Identification of Listeria monocytogenes genes expressed in response to growth at low temperature. Appl Environ Microbiol, 2002; 68:1697–1705.

44.

Loepfe

, Raimann

, Stephan

, Tasara

. Reduced host cell invasiveness and oxidative stress tolerance in double and triple csp gene family deletion mutants of Listeria monocytogenes. Foodborne Pathog Dis, 2010; 7:775–783.

45.

Lou

, Yousef

. Adaptation to sublethal environmental stresses protects Listeria monocytogenes against lethal preservation factors. Appl Environ Microbiol, 1997; 63:1252–1255.

46.

Mbandi

, Phinney

, Whitten

, Shelef

. Protein variations in Listeria monocytogenes exposed to sodium lactate, sodium diacetate, and their combination. J Food Prot, 2007; 70:58–64.

47.

Mead

, Slutsker

, Dietz

, McCaig

, Bresee

, Shapiro

, Griffin

, Tauxe

. Food-related illness and death in the United States. Emerg Infect Dis, 1999; 5:607–625.

48.

Morange

, Hevin

, Fauve

. Differential heat-shock protein synthesis and response to stress in three avirulent and virulent Listeria species. Res Immunol, 1993; 144:667–677.

49.

Nair

, Derre

, Msadek

, Gaillot

, Berche

. CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenes. Mol Microbiol, 2000; 35:800–811.

50.

Okada

, Makino

, Okada

, Asakura

, Yamamoto

, Igimi

. Identification and analysis of the osmotolerance associated genes in Listeria monocytogenes. Food Addit Contam, 2008; 15:1–6.

51.

Okada

, Okada

, Makino

, Asakura

, Yamamoto

, Igimi

. The sigma factor RpoN (sigma54) is involved in osmotolerance in Listeria monocytogenes. FEMS Microbiol Lett, 2006; 263:54–60.

52.

Oliver

, Orsi

, Wiedmann

, Boor

. Listeria monocytogenes {sigma}B has a small core regulon and a conserved role in virulence but makes differential contributions to stress tolerance across a diverse collection of strains. Appl Environ Microbiol, 2010; 76:4216–4232.

53.

Phan-Thanh

, Jansch

. Elucidation of mechanisms of acid stress in Listeria monocytogenes by proteomic analysis. Methods Biochem Anal, 2006; 49:75–88.

54.

Raengpradub

, Wiedmann

, Boor

. Comparative analysis of the sigma B-dependent stress responses in Listeria monocytogenes and Listeria innocua strains exposed to selected stress conditions. Appl Environ Microbiol, 2008; 74:158–171.

55.

Raimann

, Schmid

, Stephan

, Tasara

. The alternative sigma factor sigma(L) of L. monocytogenes promotes growth under diverse environmental stresses. Foodborne Pathog Dis, 2009; 6:583–591.

56.

Rea

, Hill

, Gahan

. Listeria monocytogenes PerR mutants display a small-colony phenotype, increased sensitivity to hydrogen peroxide, and significantly reduced murine virulence. Appl Environ Microbiol, 2005; 71:8314–8322.

57.

Rea

, Gahan

, Hill

. Disruption of putative regulatory loci in Listeria monocytogenes demonstrates a significant role for Fur and PerR in virulence. Infect Immun, 2004; 72:717–727.

58.

Ryan

, Begley

, Gahan

, Hill

. Molecular characterization of the arginine deiminase system in Listeria monocytogenes: regulation and role in acid tolerance. Environ Microbiol, 2009; 11:432–445.

59.

Schmid

, Klumpp

, Raimann

, Loessner

, Stephan

, Tasara

. Role of cold shock proteins in growth of Listeria monocytogenes under cold and osmotic stress conditions. Appl Environ Microbiol, 2009; 75:1621–1627.

60.

Sleator

, Gahan

, Hill

. Identification and disruption of the proBA locus in Listeria monocytogenes: role of proline biosynthesis in salt tolerance and murine infection. Appl Environ Microbiol, 2001; 67:2571–2577.

61.

Sleator

, Hill

. A novel role for the LisRK two-component regulatory system in Listerial osmotolerance. Clin Microbiol Infect, 2005; 11:599–601.

62.

Sokolovic

, Fuchs

, Goebel

. Synthesis of species-specific stress proteins by virulent strains of Listeria monocytogenes. Infect Immun, 1990; 58:3582–3587.

63.

Taormina

, Beuchat

. Survival of Listeria monocytogenes in commercial food-processing equipment cleaning solutions and subsequent sensitivity to sanitizers and heat. J Appl Microbiol, 2002; 92:71–80.

64.

Tasara

, Stephan

. Cold stress tolerance of Listeria monocytogenes: a review of molecular adaptive mechanisms and food safety implications. J Food Prot, 2006; 69:1473–1484.

65.

van der Veen

, Abee

, de Vos

, Wells-Bennik

. Genome-wide screen for Listeria monocytogenes genes important for growth at high temperatures. FEMS Microbiol Lett, 2009; 295:195–203.

66.

van der Veen

, Hain

, Wouters

, Hossain

, de Vos

, Abee

, Chakraborty

, Wells-Bennik

. The heat-shock response of Listeria monocytogenes comprises genes involved in heat shock, cell division, cell wall synthesis, and the SOS response. Microbiology, 2007; 153:3593–3607.

67.

Wemekamp-Kamphuis

, Karatzas

, Wouters

, Abee

. Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure. Appl Environ Microbiol, 2002; 68:456–463.

68.

Wemekamp-Kamphuis

, Wouters

, de Leeuw

, Hain

, Chakraborty

, Abee

. Identification of sigma factor sigma B-controlled genes and their impact on acid stress, high hydrostatic pressure, and freeze survival in Listeria monocytogenes EGD-e. Appl Environ Microbiol, 2004; 70:3457–3466.

69.

Wilson

, Brown

, Kirkwood-Watts

, Warren

, Lund

, King

, Jones

, Hruby

. Listeria monocytogenes 10403S HtrA is necessary for resistance to cellular stress and virulence. Infect Immun, 2006; 74:765–768.

70.

Wonderling

, Wilkinson

, Bayles

. The htrA (degP) gene of Listeria monocytogenes 10403S is essential for optimal growth under stress conditions. Appl Environ Microbiol, 2004; 70:1935–1943.

71.

Zhang

, Nietfeldt

, Zhang

, Benson

. Functional consequences of genome evolution in Listeria monocytogenes: the lmo0423 and lmo0422 genes encode sigmaC and LstR, a lineage II-specific heat shock system. J Bacteriol, 2005; 187:7243–7253.

72.

Zhu

, Bayles

, Xiong

, Jayaswal

, Wilkinson

. Precursor and temperature modulation of fatty acid composition and growth of Listeria monocytogenes cold-sensitive mutants with transposon-interrupted branched-chain alpha-keto acid dehydrogenase. Microbiology, 2005; 151:615–623.