The relationship between acid stress responses and virulence in Salmonella typhimurium and Listeria monocytogenes

International Journal of Food Microbiology 50 (1999) 93–100

The relationship between acid stress responses and virulence in Salmonella typhimurium and Listeria monocytogenes Cormac G.M. Gahan, Colin Hill* National Food Biotechnology Centre and Department of Microbiology, University College Cork, Cork, Ireland

Abstract All pathogenic bacteria possess the ability to evade or surmount body defenses (stresses, as experienced by the bacterium) long enough to cause a sufficient reaction, which is then manifested as a disease or illness. While opportunistic pathogens will only cause illness in the event of a predisposing weakness in these defenses, many pathogens must take on and overcome intact defenses. This is particularly true of gastrointestinal pathogens such as Listeria monocytogenes and Salmonella spp., which must circumvent many different stresses in order to arrive at the site of infection. These include the acid barrier of the stomach, the physical barrier of the epithelial cells lining the gastrointestinal tract, and various immune defenses including the initial onslaught of macrophages. Thus, these organisms have developed elaborate systems for sensing stress, and for responding to those stresses in a self-protective fashion. One well characterised adaptive response is to acid stress, the so-called acid tolerance response (ATR). The ATR is a complex phenomenon, involving a number of changes in the levels of different proteins and presumably, many allied events at the level of gene regulation. A number of molecular approaches have identified numerous interesting chromosomal loci involved both in sensing and responding to stress and in virulence. The identity of some of these genes, and their impact on stress responses and virulence will be discussed.  1999 Elsevier Science B.V. All rights reserved. Keywords: Listeria; Salmonella; Stress; Virulence; Acid tolerance response

1. Introduction Foodborne bacterial pathogens have evolved intricate systems for evading immune responses and colonising the host. For example, Listeria monocytogenes utilises a unique set of virulence factors to facilitate cell invasion, escape from the host cell phagosome, move within the cytoplasm and sub*Corresponding author. Tel. 1 353-21-902-397; fax 1 353-21903-101. E-mail address: [email protected] (C. Hill)

sequently spread to neighbouring cells. Salmonella typhimurium also induces a range of virulence factors to allow invasion of target cells and subsequently prevents the maturation of infected phagosomes to inhospitable phagolysosomes. However, whilst the intracellular lifestyles of these pathogens differ greatly, both organisms share the ability to survive the unfavourable, stressful growth environments encountered in the host. For both L. monocytogenes and S. typhimurium, the passage from the environment to the host is long and tortuous. Foodborne pathogens may encounter

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stressful environments during food production, preparation and storage. Following consumption, they are exposed to the low pH of the stomach. Survivors will subsequently encounter volatile fatty acids, bile and low oxygen in the small intestine. Competition with the gut flora at this stage is intense, but survivors will invade through the gut epithelial cells (possibly via M cells). Uptake by macrophages results in internalisation within phagosomes, specialised organelles which prevent bacterial multiplication by means of acidic pH and production of defensins (oxygen-independent mechanisms), as well as through production of hydrogen peroxide and superoxide radicals (oxygen-dependent mechanisms). The purpose of this review is to investigate the connection between the ability of bacteria to sense and respond to stress, and their virulence capabilities. The review will focus on two foodborne pathogens, namely S. typhimurium and L. monocytogenes, with particular reference to the survival of bacteria exposed to suboptimal low pH host environments.

2. Intracellular expression of bacterial stress proteins Examination of bacterial protein synthesis following macrophage internalisation has demonstrated significant alterations in protein profiles of both L. monocytogenes and S. typhimurium, indicative of a shift to less favourable growth conditions. Some variation in the results points to the difficulties encountered in using different models to investigate expression during intracellular growth and survival. In S. typhimurium, the levels of 40 proteins were seen to increase during growth within macrophagelike U937 cells, while synthesis of approximately 100 proteins was downregulated under the same conditions (Abshire and Neidhardt, 1993). Many of those proteins subject to in vivo regulation could also be regulated by individual stress responses (low pH, oxidative stress, heat shock) during extracellular growth. It was also observed that the heat shock proteins GroEL and DnaK were not induced by intracellular growth, a finding that conflicts with an earlier study using a different macrophage cell line (Buchmeier and Heffron, 1990). Similarly, the synthesis of at least 32 proteins is increased in L. monocytogenes during intracellular growth in J774-1 macrophage cells (Hanawa et al.,

1995). These authors also failed to detect an increase in synthesis of GroEL or DnaK during intracellular growth (Hanawa et al., 1995). Indeed, synthesis of other stress inducible proteins was not observed during intramacrophage growth in this study. The authors examined bacterial protein synthesis 5 h after infection of the monolayer, during which time Listeria cells were observed to be growing rapidly. It is possible that induction of specific stress proteins occurs at an earlier stage of infection. An earlier study also showed that neither DnaK nor GroEL are induced, even during the early stages of macrophage uptake (Hevin et al., 1993). However, this does not rule out a role for other stress inducible proteins during transit through the phagosome. It has also been observed that up to 53 proteins were either induced or downregulated in L. monocytogenes LO28 following acid adaptation in vitro (O’Driscoll et al., 1997) It is clear that cells of both Listeria and Salmonella adapt to the intracellular growth environment by regulating the synthesis of specific proteins. In S. typhimurium, these proteins represent some components of regulons involved in adaptation to heat shock, oxidative stress and acid stress (Abshire and Neidhardt, 1993). In L. monocytogenes, escape from the phagosome to the less stressful cytoplasm may result in only a transient requirement for stress proteins. Some of the techniques developed to identify the nature of in vivo induced genes will be discussed in a later section.

3. Effect of adaptation prior to infection (stress response) Bacterial cells can adapt to normally unfavourable growth conditions following a brief exposure to mild stress. Examples of this phenomenon include the heat shock response and the adaptive acid tolerance response (ATR, Fig. 1) (Hill et al., 1995). A number of studies have attempted to determine whether adaptation of pathogens (‘‘stress hardening’’) prior to use in virulence studies can affect the outcome of infection. In Salmonella enterica, acid tolerant bacterial cells (stationary phase) demonstrate similar virulence potential to acid sensitive, chilled log phase cells (Humphrey et al., 1998). We have similarly shown that an increase in acid tolerance following acid adaptation of L. monocytogenes fails

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Fig. 1. Adaptive acid tolerance response in Listeria monocytogenes LO28. Exponential-phase cells were transferred directly from pH 7.0 to pH 3.5 (lactic acid) (s) or were adapted at pH 5.5 (lactic acid) for 60 min prior to exposure to pH 3.5 (d).

to alter the virulence of the pathogen (Fig. 2a). This may simply be a consequence of the ability of Listeria cells to naturally develop acid tolerance following uptake by macrophages (Fig. 2b), suggesting that natural stress adaptation during infection eliminates any advantage of prior adaptation. In contrast, two separate studies have demonstrated that L. monocytogenes cells grown at refrigeration temperatures display significantly increased virulence for mice (Czuprynski et al., 1989; Stephens et al., 1991). This phenomenon suggests that alterations in cell physiology, possibly affecting cell membrane constitution, cold shock protein synthesis or synthesis of virulence factors, can influence virulence even though these physiological changes may be inducible and transitory.

4. Regulation of the ATR and virulence In S. typhimurium, acid stress responses are subject to regulation by the alternative sigma factor s s encoded by rpoS, the two-component sensor regulator PhoP/ Q and the ferric uptake regulator Fur. s s

Fig. 2. (a) Effect of acid adaptation on virulence of Listeria monocytogenes. Exponential-phase cells of L. monocytogenes LO28 were inoculated intraperitoneally into Balb / c mice (LO28) or were exposed to pH 5.5 (lactic acid) for 60 min prior to inoculation (LO28-adapted). Levels of Listeria in the spleens of infected mice 3 days postinfection are shown (n 5 4). Repeat experiments showed similar results. (b) Acid tolerance of cells internalised by macrophages. Exponential-phase cells were used to infect either J774 macrophage cells (j), or fresh peritoneal exudate cells (d), for 30 min, macrophage monolayers were washed three times with phosphate buffered saline (PBS) and lysed with cold sterile distilled water to release internalised bacteria. Bacteria were then exposed to BHI broth at pH 3.5 (lactic acid). Controls (s), consisted of exponential-phase cells transferred to tissue culture medium (DMEM) for 30 min, centrifuged, washed in PBS and resuspended in sterile distilled water. Repeat experiments showed similar results.

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Is an important regulator of subsets of both stationary phase and exponential phase proteins involved both in stress adaptation and virulence. Mutations in the rpoS gene of virulent S. typhimurium strains render them unable to develop a full ATR and significantly reduce virulence potential (Fang et al., 1992; Lee et al., 1995). Indeed, the attenuated virulence of the common laboratory strain, S. typhimurium LT2, has been mapped to a mutation in rpoS (Bearson et al., 1997; Wilmes-Riesenberg et al., 1997). However, since s s is known to control expression of a number of specific virulence factors including components of the spv operon (Heiskanen et al., 1994), it is not possible to attribute the diminished virulence of rpoS mutants to a reduction in stress resistance alone. The S. typhimurium PhoP/ Q system has been shown to influence the expression of a number of genes involved in virulence. These include a number of PhoP/ Q-activated genes ( pags) and PhoP/ Q-repressed genes ( prgs). Disruption of specific pags and prgs genes can attenuate virulence of S. typhimurium. Recently, PhoP/ Q has been shown to be regulated by low pH and plays an important role in the development of acid tolerance (Bearson et al., 1998). This indicates that an important regulator of virulence gene expression in Salmonella is itself regulated by environmental stress conditions. In addition to the regulatory circuits mentioned above, it is known that the ferric uptake regulator Fur plays an important role in the regulation of acid adaptation in S. typhimurium. Disruption of Fur in virulent S. typhimurium strains attenuates virulence for mice suggesting a role for this regulator both in acid adaptation and virulence (Wilmes-Riesenberg et al., 1996). In L. monocytogenes, relatively little is known of the circuits involved in regulation of stress responsive genes. Recently, the alternative sigma factor s B has been identified and sequenced in L. monocytogenes (Wiedmann et al., 1998). This sigma factor appears to regulate the synthesis of a number of stress responsive proteins. Indeed, BetL, a glycine betaine transport system linked to salt tolerance of L. monocytogenes has recently been shown to possess a consensus s B -dependent promotor binding site (Sleator et al., 1999). An in-frame deletion of a portion of the s B gene eliminates the ability to tolerate acid stress. However, this mutation does not appear to influence the virulence potential of this

strain. Similarly, mutating the s B homologue in Staphylococcus aureus fails to influence virulence of the pathogen in a mouse abscess model of infection (Chan et al., 1998). To date, the data indicate that s B -influenced gene expression may not play a significant role in the in vivo survival of gram-positive pathogens. Nonetheless, further studies will be required to investigate any potential role for s B in virulence. We have recently identified an operon in L. monocytogenes with significant sequence homology to two-component regulatory systems of Group A streptococci, Lactococcus lactis and Bacillus subtilis (Cotter et al., unpublished data). This two-component signal transduction system, designated LisRK, appears to play a role in the regulation of acid resistance in L. monocytogenes. In addition, mutation of either the histidine kinase component (lisK) or the response regulator (lisR) results in a significant attenuation of virulence potential as evidenced by an inability to survive during the early stages of infection in the mouse model (Fig. 3). A more detailed analysis of this regulatory system is in progress in

Fig. 3. Virulence of a LisK mutant of L. monocytogenes. Mutation was due to an in-frame deletion in lisK, encoding a histidine kinase. Balb / c mice were inoculated with 3 ? 10 6 Listeria cells of either the wild-type LO28 (s), or the mutant (d), by the intraperitoneal route. The arrow indicates the inoculum level. Each datum point represents the mean log 10 number of viable Listeria cells per spleen for at least four mice (6standard deviation).

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order to determine the signal(s) which are being sensed and the identity of the genes which are under the control of this system. Coordinate regulation of virulence gene expression in L. monocytogenes is carried out by the transcriptional activator, PrfA. The regulation of virulence factor expression has been the subject of much study and has been reviewed elsewhere (Brehm et al., 1996). In general, virulence gene expression is upregulated by increased temperature and downregulated by mono- and disaccharides, including cellobiose. The evidence to date suggests that this environmental regulation of virulence gene expression in L. monocytogenes functions to signal the transition from external environment to the host. There is also some evidence that expression of the PrfA regulated virulence factor, listeriolysin, is downregulated by low pH (Datta, 1994). This reduction in listeriolysin expression at low pH is surprising considering the apparent requirement for acidic pH for activity of this virulence factor in the host cell phagosome (Beauregard et al., 1997).

5. Mutations in stress tolerance mechanisms, effect on virulence In S. typhimurium, numerous genes have been identified that play a role in the management of stress responses in vitro and also appear to contribute to virulence. A mutation in the major proton translocating ATPase (atp) in virulent S. typhimurium increases acid sensitivity, eliminates the ability to induce an ATR and significantly decreases virulence in the mouse typhoid model (Portillo et al., 1993). As mentioned previously, virulent S. typhimurium strains contain wild-type rpoS. Inactivation of single genes known to contribute to acid tolerance in the attenuated strain LT2 had only a marginal effect on acid tolerance in virulent Salmonella (WilmesRiesenberg et al., 1996). In a virulent S. typhimurium background, mutation of two or more genes was required to eliminate acid resistance and the ability to induce an ATR. Double and triple mutants containing an atrC ( polA) mutation lacked an ATR and were highly attenuated in mouse and macrophage tissue culture models. S. typhimurium mutants can be isolated which demonstrate constitutively increased resistance to

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acid without prior adaptation. In the murine model of infection, such mutants demonstrate marginally increased survival potential in host tissues (WilmesRiesenberg et al., 1996). However, studies using S. enterica have shown that a stress tolerant strain is significantly more virulent for mice and more invasive in chickens than a nonresistant strain (Humphrey et al., 1996). We have also isolated a number of mutants of L. monocytogenes which strongly suggest a link between stress management systems and virulence in this organism. As has been shown for S. typhimurium, naturally acid tolerant mutants of L. monocytogenes can be readily isolated following consecutive passages at low pH (O’Driscoll et al., 1996). We have shown that two of these mutants demonstrate increased virulence for mice inoculated by the peritoneal route (O’Driscoll et al., 1996). These mutants appear to be particularly resistant to the initial influx of neutrophils and macrophages that occurs early following infection, with the result that larger numbers of mutant cells are apparent in the spleen 24 h postinoculation. Subsequent growth rates in the spleens of infected mice are similar to the wild-type. Based on these growth kinetics in the spleen, it appears that the innate acid resistance of these mutants enhances survival during their initial encounter with highly bactericidal phagocytes in the peritoneal cavity. This leads to increased survival during the early stages of infection. It is interesting to note that such mutants also demonstrate significantly improved survival potential relative to the wild-type during long term storage of foods (Gahan et al., 1996). Using Tn917 mutagenesis we have selected a number of mutants which demonstrate acid sensitivity relative to the wild-type. These mutants are currently being characterised at the genetic level. One mutant selected using a Tn917 -lac screening system shows an inability to develop an ATR but is not affected in its heat shock response (Marron et al., 1997). This mutant demonstrates a significant reduction in virulence relative to the wild-type. Again, this mutant appears to be particularly sensitive to early infiltrating peritoneal neutrophils and macrophages, and reaches the murine spleen at much lower levels than a similar wild-type infection. Once present in the spleen, the mutant is capable of growth at rates similar to the wild-type. Collectively, our data suggest that acid stress resistance mechanisms are of

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some importance in survival of the organism during infection of bactericidal infiltrating phagocytes. In addition, we have recently demonstrated the presence of a glutamate decarboxylase operon in L. monocytogenes. Inducible decarboxylase systems have been shown to play a role in the maintenance of intracellular pH homeostasis in enteric bacteria (Bearson et al., 1997). We have created a deletion mutant in the gadC gene in L. monocytogenes and this mutant is undergoing analysis for its ability to induce an ATR and also for its virulence potential. Another locus, clpC, which encodes a heat shock inducible ATPase is known to play a role in both stress tolerance and virulence of L. monocytogenes. Disruption of clpC impairs tolerance of L. monocytogenes to various stress conditions including low pH, iron limitation, elevated temperature and salt stress (Rouquette et al., 1996; Ripio et al., 1998). In addition, ClpC mutants demonstrate impaired virulence in the mouse model and in a tissue culture model of infection. ClpC mutants exhibit a significantly increased 50% lethal dose (LD 50 ) for mice relative to the wild-type, but can grow, albeit at a decreased rate, in spleen and liver cells initially following intravenous inoculation. Recent work indicates that ClpC plays a role in permitting escape of the bacterium from the host phagosome, but does not appear to protect L. monocytogenes from intraphagosomal killing (Rouquette et al., 1998). Interestingly, ClpC expression is negatively controlled at the transcriptional level by PrfA, possibly to downregulate synthesis of this stress protein in the relatively ‘‘nonstressful’’ environment of the cytoplasm (Ripio et al., 1998).

6. Selection strategies for in vivo induced stress genes Mahan et al. (1993) have developed an elaborate strategy for the selection of in vivo induced (ivi) genes in S. typhimurium. This strategy, termed in vivo expression technology (IVET) was initially based on the use of an avirulent pur mutant of S. typhimurium. Random integration of the pur gene back into the mutant resulted in a bank in which integration of pur downstream of properly positioned promoters resulted in expression of pur and complementation of virulence. Mutants of interest were

those that expressed pur in vivo and therefore survived screening in a mouse model of infection, yet were Pur 2 in vitro. These represented fusions of pur to promoters that were induced exclusively during infection. Recent characterisation of S. typhimurium ivi genes isolated using IVET has revealed many loci that are regulated by in vitro signals including low pH and low Mg 21 that are also encountered by pathogens in vivo (Heithoff et al., 1999). These low pH-inducible loci are all activated through the PhoP/ Q regulatory system. Collectively the data generated thus far using the IVET system in S. typhimurium indicate that this system will prove invaluable for the study of stress responsive genes and their role in virulence. While, to date, no such IVET strategy has been described for Listeria, five in vivo expressed genes have been identified using a Tn917 -lac screening strategy. Mutants expressing b-galactosidase during intracellular growth in J774 cells but not during normal growth were subjected to genetic analysis. Three genes involved in nucleotide biosynthesis, purH, purD and pyrE, were seen to be induced in vivo, a finding consistent with low levels of free nucleotides in the host cell cytoplasm. In addition, a L. monocytogenes locus encoding an ATP binding cassette (ABC)-type arginine transport system, was seen to be induced in vivo and was necessary for full virulence. This suggests a requirement for an arginine transport system during infection (Klarsfeld et al., 1994). The authors acknowledge that the limitations of this screening system include disruption of essential virulence genes by Tn917 -lac insertion leading to reduced intracellular growth and an apparent reduction in b-galactosidase expression. In addition, the existence of preferential transposon insertion sites may influence the randomness of the insertion bank.

7. Conclusions Whilst their strategies for intracellular survival may differ, there are many similarities between stress and virulence systems studied in S. typhimurium and in L. monocytogenes. Regulatory systems such as PhoP/ Q and RpoS in Salmonella and PrfA in Listeria influence the expression of both specific virulence factors and also effectors involved in stress

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resistance. In all cases, these regulatory controls are themselves influenced by environmental stress conditions encountered in vivo. It appears therefore, that the ability of intracellular pathogens to sense and adapt to environmental conditions encountered in vivo is an essential component of virulence. Both Salmonella and Listeria cells respond to in vivo stress by regulating systems for coping with exposure to such environments and by regulating virulence gene expression. In this way, both organisms have evolved mechanisms to overcome initial host defenses and use these hostile conditions encountered during infection as signals to regulate synthesis of genes specific for virulence.

Acknowledgements CGMG was supported by an Irish Health Research Board Post-Doctoral Research Fellowship.

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