Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence.

The ability of Listeria monocytogenes to tolerate low-pH environments is of particular importance because the pathogen encounters such environments in vivo, both during passage through the stomach and within the macrophage phagosome. In our study, L. monocytogenes was shown to exhibit a significant adaptive acid tolerance response following a 1-h exposure to mild acid (pH 5.5), which is capable of protecting cells from severe acid stress (pH 3.5). Susceptibility to pH 3.5 acid is growth phase dependent. Stationary-phase Listeria cultures are naturally resistant to the challenge pH (pH 3.5), while exponential-phase cultures require adaptation at pH 5.5 to induce acid tolerance. Adaptation requires protein synthesis, since treatment with chloramphenicol prevents the development of acid tolerance. Induction of the acid tolerance response also protects L. monocytogenes against the effect of other environmental stresses. Acid-adapted cells demonstrate increased tolerance toward thermal stress, osmotic stress, crystal violet, and ethanol. Following prolonged exposure of L. monocytogenes to pH 3.5, we isolated mutants which constitutively demonstrate increased acid tolerance at all stages of the growth cycle. These mutants do not display full acid tolerance, but their resistance to low pH can be further increased following adaptation to mild-acid conditions. The mutants demonstrated increased lethality for mice relative to that of the wild type when inoculated by the intraperitoneal route. When administered as lower inocula, the mutants reached higher levels in the spleens of infected mice than did the wild type. The data suggest that low-pH conditions may have the potential to select for L. monocytogenes mutants with increased natural acid tolerance and increased virulence.     

The glutamate decarboxylase acid resistance mechanism affects survival of Listeria monocytogenes LO28 in modified atmosphere-packaged foods

AIMS: The contribution of the glutamate decarboxylase (GAD) acid resistance system to survival and growth of Listeria monocytogenes LO28 in modified atmosphere-packaged foods was examined. METHODS AND RESULTS: The survival and growth of the wild-type LO28 and four GAD deletion mutants (DeltagadA, DeltagadB, DeltagadC, DeltagadAB) in packaged foods (minced beef, lettuce, dry coleslaw mix) during storage at 4, 8 and 15 degrees C were studied. Survival and growth patterns varied with strain, product type, gas atmosphere and storage temperature. In minced beef, the wild-type LO28 survived better (P < 0.05) than the GAD mutant strains at 8 and 15 degrees C. In both packaged vegetables at all storage temperatures, the wild-type strain survived better (P < 0.05) than the double mutant DeltagadAB. The requirement for the individual gad genes varied depending on the packaged food. In the case of lettuce, gadA played the most important role, while the gadB and gadC genes played the greatest role in packaged coleslaw (at 15 degrees C). CONCLUSIONS: This work demonstrates that elements of the GAD system play significant roles in survival of L. monocytogenes LO28 during storage in modified atmosphere-packaged foods. SIGNIFICANCE AND IMPACT OF THE STUDY: A better understanding of how L. monocytogenes behaves in modified atmosphere-packaged foods, and how it responds to elevated carbon dioxide atmospheres.     

Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28

The trimethylammonium compound glycine betaine (N,N, N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus sigmaB-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed Km value of 7.9 microM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL+ parent, signifying the role of the transporter in Listeria osmotolerance.     

Growth, morphology and surface properties of Listeria monocytogenes Scott A and LO28 under saline and acid environments

AIMS: The effect of salt and acid on the growth and surface properties of two strains of Listeria monocytogenes was investigated. METHODS AND RESULTS: Medium acidification and NaCl supplementation induced a marked increase in the lag and growth times (up to fivefold higher) and a decrease in the maximal optical density. Due to a strong synergic effect of pH and NaCl, growth was only detected after 280 h incubation for Scott A and not detected after 600 h for LO28 at pH 5.0 and 10% NaCl. Furthermore, the addition of NaCl in acidic conditions gave rise to cell filamentation and cell surfaces became strongly hydrophilic. CONCLUSIONS: Some L. monocytogenes strains subjected to high NaCl concentrations in acidic conditions are able to grow but may present altered adhesion properties due to modification of their surface properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted that L. monocytogenes do represent a hazard in acid and salted foods, such as soft cheese.     

Isolation and characterization of a Listeria monocytogenes mutant strain hyperproducing virulence factors

A mutant strain characterized by hyperproduction of a number of cell wall and supernatant proteins was isolated after N-methyl-N′-nitro-N-nitrosoguanidine treatment of Listeria monocytogenes strain NCTC10527. Several of these proteins were identified as virulence factors. When a wild-type strain was grown in the presence of activated charcoal it was shown to exhibit the same protein pattern as the isolated mutant.     

A hypermutator phenotype attenuates the virulence of Listeria monocytogenes in a mouse model

The integrity of the genetic material of bacteria is guaranteed by a set of distinct repair mechanisms. The participation of these repair systems in bacterial pathogenicity has been addressed only recently. Here, we study for the first time the participation in virulence of the MutSL mismatch repair system of Listeria monocytogenes. The mutS and mutL genes, which are contiguous in the L. monocytogenes chromosome, were identified after in silico analysis. The deduced MutS shares 62% identity with MutS of Bacillus subtilis and 50% identity with HexA, its homologue in Streptococcus pneumoniae; MutL shares 59% identity with MutL of B. subtilis and 47% identity with HexB of S. pneumoniae. Functional analysis of the mutSL locus was studied by constructing a double knock-out mutant. We showed that the deletion DeltamutSL induces: (i) a 100- to 1000-fold increase in the spontaneous mutation rate; and (ii) a 10- to 15-fold increase in the frequency of transduction, thus demonstrating the role of mutSL of L. monocytogenes in both mismatch repair and homologous recombination. We found that the deletion DeltamutSL moderately affected bacterial virulence, with a 1-log increase in the lethal dose 50% (LD50) in the mouse. Strikingly, repeated passages of the mutant strain in mice reduced virulence further. Competition assays between wild-type and mutant strains showed that the deletion DeltamutSL reduced the capacity of L. monocytogenes to survive and multiply in mice. These results thus demonstrate that, for the intracellular pathogen L. monocytogenes, a hypermutator phenotype is more deleterious than profitable to its virulence.     

Two-Dimensional Polyacrylamide Gel Electrophoresis Analysis of the Acid Tolerance Response in Listeria monocytogenes LO28

Listeria monocytogenes is capable of withstanding low pH after initial exposure to sublethal acidic conditions, a phenomenon termed the acid tolerance response (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1996). Treatment of L. monocytogenes LO28 with chloramphenicol during acid adaptation abrogated the protective effect, suggesting that de novo protein synthesis is required for the acid tolerance response. Analysis of protein expression during acid adaptation by two-dimensional gel electrophoresis revealed changes in the levels of 53 proteins. Significant protein differences were also evident between nonadapted L. monocytogenes LO28 and a constitutively acid-tolerant mutant, ATM56. In addition, the analysis[S_TABC] revealed differences in protein expression between cells induced with a weak acid (lactic acid) and those induced with a strong acid (HCl). Comparison of both acid-adapted LO28 and ATM56 revealed that both are capable of maintaining their internal pH (pH(infi)) at higher levels than nonadapted control cells during severe acid stress. Collectively, the data demonstrate the profound alterations in protein synthesis which take place during acid adaptation in L. monocytogenes and ultimately lead to an increased ability to survive severe stress conditions.     

The response regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria monocytogenes

Listeria monocytogenes is a versatile bacterial pathogen that is able to accommodate to diverse environmental and host conditions. Presently, we have identified a L. monocytogenes two-component response regulator, ResD that is required for the repression of virulence gene expression known to occur in the presence of easily fermentable carbohydrates not found inside host organisms. Structurally and functionally, ResD resembles the respiration regulator ResD in Bacillus subtilis as deletion of the L. monocytogenes resD reduces respiration and expression of cydA, encoding a subunit of cytochrome bd. The resD mutation also reduces expression of mptA encoding the EIIABman component of a mannose/glucose-specific PTS system, indicating that ResD controls sugar uptake. This notion was supported by the poor growth of resD mutant cells that was alleviated by excess of selected carbohydrates. Despite the growth deficient phenotype of the mutant in vitro the mutation did not affect intracellular multiplication in epithelial or macrophage cell lines. When examining virulence gene expression we observed traditional induction by charcoal in both mutant and wild-type cells whereas the repression observed in wild-type cells by fermentable carbohydrates did not occur in resD mutant cells. Thus, ResD is a central regulator of L. monocytogenes when present in the external environment.     

Thiamine plays a critical role in the acid tolerance of Listeria monocytogenes

Understanding the molecular basis of acid tolerance in the food-borne pathogen Listeria monocytogenes is important as this property contributes to survival in the food-chain and enhances survival within infected hosts. The aim of this study was to identify genes contributing to acid tolerance in L.?monocytogenes using transposon mutagenesis and subsequently to elucidate the physiological role of these genes in acid tolerance. One mutant harboring a Tn917 insertion in the thiT gene (formerly lmo1429), which encodes a thiamine (vitamin B1) uptake system, was found to be highly sensitive to acid. The acid-sensitive phenotype associated with loss of this gene was confirmed with an independently isolated mutant, from which the thiT gene was deleted (?thiT). Cells of both wild-type and ?thiT mutant that were thiamine depleted were found to be significantly more acid sensitive than control cultures. Thiamine-depleted cultures failed to produce significant concentrations of acetoin, consistent with the known thiamine dependence of acetolactate synthase, an enzyme required for acetoin synthesis from pyruvate. As acetoin synthesis is a proton-consuming process, we suggest that the acid sensitivity observed in thiamine-depleted cultures may be owing to an inability to produce acetoin.     

Different assembly of acid and salt tolerance response in two dairy Listeria monocytogenes wild strains

A lack on the association between acid tolerance response (ATR) and osmotolerance response (OTR) among Listeria monocytogenes dairy isolates was found. In order to evaluate how wild L. monocytogenes isolates mount tolerance responses under a sub-lethal pH and a low sodium chloride concentration (pH 5.5 and 3.5 % [w/v] NaCl), a proteomic approach was used. The ATR and OTR of two L. monocytogenes cheese dairy isolates (strain T8, serotype 4b and A9, serotype 1/2b or 3b) were determined. The proteomes of the adapted and non-adapted cultures were evaluated by 2-DE. One strain displayed an ATR, but not an OTR and the other displayed an OTR, but not an ATR. The ATR positive strain showed the over-production of proteins related with protein synthesis, protein folding, attainment of reduction power, ribose production and cell wall. In contrast, in the OTR-positive-strain proteins related with glycolysis, general stress and detoxification were identified.     

Enhanced levels of cold shock proteins in Listeria monocytogenes LO28 upon exposure to low temperature and high hydrostatic pressure

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37 degrees C to 10 degrees C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37 degrees C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.     

Enhanced Levels of Cold Shock Proteins in Listeria monocytogenes LO28 upon Exposure to Low Temperature and High Hydrostatic Pressure

Listeria monocytogenes is a psychrotrophic food-borne pathogen that is problematic for the food industry because of its ubiquitous distribution in nature and its ability to grow at low temperatures and in the presence of high salt concentrations. Here we demonstrate that the process of adaptation to low temperature after cold shock includes elevated levels of cold shock proteins (CSPs) and that the levels of CSPs are also elevated after treatment with high hydrostatic pressure (HHP). Two-dimensional gel electrophoresis combined with Western blotting performed with anti-CspB of Bacillus subtilis was used to identify four 7-kDa proteins, designated Csp1, Csp2, Csp3, and Csp4. In addition, Southern blotting revealed four chromosomal DNA fragments that reacted with a csp probe, which also indicated that a CSP family is present in L. monocytogenes LO28. After a cold shock in which the temperature was decreased from 37°C to 10°C the levels of Csp1 and Csp3 increased 10- and 3.5-fold, respectively, but the levels of Csp2 and Csp4 were not elevated. Pressurization of L. monocytogenes LO28 cells resulted in 3.5- and 2-fold increases in the levels of Csp1 and Csp2, respectively. Strikingly, the level of survival after pressurization of cold-shocked cells was 100-fold higher than that of cells growing exponentially at 37°C. These findings imply that cold-shocked cells are protected from HHP treatment, which may affect the efficiency of combined preservation techniques.     

Bioenergetic mechanism for nisin resistance, induced by the acid tolerance response of Listeria monocytogenes

This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR(+)) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR(+) cells had lower transmembrane pH (DeltapH) and electric potential (Deltapsi) than the control (ATR(-)) cells. The decreased proton motive force (PMF) of ATR(+) cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR(+) cells were approximately 7-fold higher than those in ATR(-) cells. This suggested a role for the F(o)F(1) ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the F(o)F(1) ATPase enzyme complex by N'-N'-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR(-) but not in ATR(+) cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR(+) listeriae, the downregulation of the proton-translocating c subunit of the F(o)F(1) ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of F(o)F(1) ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin.     

Influence of the natural microbial flora on the acid tolerance response of Listeria monocytogenes in a model system of fresh meat decontamination fluids

Depending on its composition and metabolic activity, the natural flora that may be established in a meat plant environment can affect the survival, growth, and acid tolerance response (ATR) of bacterial pathogens present in the same niche. To investigate this hypothesis, changes in populations and ATR of inoculated (10(5) CFU/ml) Listeria monocytogenes were evaluated at 35 degrees C in water (10 or 85 degrees C) or acidic (2% lactic or acetic acid) washings of beef with or without prior filter sterilization. The model experiments were performed at 35 degrees C rather than lower (8.0 log CFU/ml) by day 1. The pH of inoculated water washings decreased or increased depending on absence or presence of natural flora, respectively. These microbial and pH changes modulated the ATR of L. monocytogenes at 35 degrees C. In filter-sterilized water washings, inoculated L. monocytogenes increased its ATR by at least 1.0 log CFU/ml from days 1 to 8, while in unfiltered water washings the pathogen was acid tolerant at day 1 (0.3 to 1.4 log CFU/ml reduction) and became acid sensitive (3.0 to >5.0 log CFU/ml reduction) at day 8. These results suggest that the predominant gram-negative flora of an aerobic fresh meat plant environment may sensitize bacterial pathogens to acid.     

Identification and Disruption of BetL, a Secondary Glycine Betaine Transport System Linked to the Salt Tolerance of Listeria monocytogenes LO28

The trimethylammonium compound glycine betaine (N,N,N-trimethylglycine) can be accumulated to high intracellular concentrations, conferring enhanced osmo- and cryotolerance upon Listeria monocytogenes. We report the identification of betL, a gene encoding a glycine betaine uptake system in L. monocytogenes, isolated by functional complementation of the betaine uptake mutant Escherichia coli MKH13. The betL gene is preceded by a consensus ς^B-dependent promoter and is predicted to encode a 55-kDa protein (507 amino acid residues) with 12 transmembrane regions. BetL exhibits significant sequence homologies to other glycine betaine transporters, including OpuD from Bacillus subtilis (57% identity) and BetP from Corynebacterium glutamicum (41% identity). These high-affinity secondary transporters form a subset of the trimethylammonium transporter family specific for glycine betaine, whose substrates possess a fully methylated quaternary ammonium group. The observed K_m value of 7.9 μM for glycine betaine uptake after heterologous expression of betL in E. coli MKH13 is consistent with values obtained for L. monocytogenes in other studies. In addition, a betL knockout mutant which is significantly affected in its ability to accumulate glycine betaine in the presence or absence of NaCl has been constructed in L. monocytogenes. This mutant is also unable to withstand concentrations of salt as high as can the BetL⁺ parent, signifying the role of the transporter in Listeria osmotolerance.     

A homolog of Bacillus subtilis trigger factor in Listeria monocytogenes is involved in stress tolerance and bacterial virulence

Molecular chaperones play an essential role in the folding of nascent chain polypeptides, as well as in the refolding and degradation of misfolded or aggregated proteins. They also assist in protein translocation and participate in stress functions. We identified a gene, designated tig, encoding a protein homologous to trigger factor (TF), a cytosolic ribosome-associated chaperone, in the genome of Listeria monocytogenes. We constructed a chromosomal Delta tig deletion and evaluated the impact of the mutation on bacterial growth in broth under various stress conditions and on pathogenesis. The Delta tig deletion did not affect cell viability but impaired survival in the presence of heat and ethanol stresses. We also identified the ffh gene, encoding a protein homologous to the SRP54 eukaryotic component of the signal recognition particle. However, a Delta ffh deletion was not tolerated, suggesting that Ffh is essential, as it is in Bacillus subtilis and Escherichia coli. Thus, although dispensable for growth, TF is involved in the stress response of L. monocytogenes. The Delta tig mutant showed no or very modest intracellular survival defects in eukaryotic cells. However, in vivo it showed a reduced capacity to persist in the spleens and livers of infected mice, revealing that TF has a role in the pathogenicity of L. monocytogenes.