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Virulence and the heat shock response
Int. J. Med. Microbiol. 292, 453 ± 461 (2003) ¹ Urban & Fischer Verlag http: // www.urbanfischer.de/journals/ijmm
Mini-Review Virulence and the heat shock response Uri Gophna, Eliora Z. Ron Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel 69978 Received December 19, 2001 ¥ Revision received February 26, 2002 ¥ Accepted March 6, 2002
Abstract The major adaptive response to elevation in temperature is the heat shock response that involves the induction of many proteins ± called heat shock proteins. These include chaperones, proteases, alternative sigma factors and other regulatory and structural proteins. The heat shock response is also turned on by other stress conditions, such as oxidative stress or pH changes. Bacterial entry into the host organism involves a significant environmental change, which is expected to induce the heat shock response. Indeed, some of the heat shock proteins are themselves virulence factors while others affect pathogenesis indirectly, by increasing bacterial resistance to host defenses or regulating virulence genes. The cross talk between heat shock and virulence genes is discussed. Key words: Heat shock ± stress response ± virulence ± pathogenesis ± chaperones ± proteases
Introduction Temperature increase constitutes one of the major environmental stresses encountered by bacteria. It is, therefore, not surprising that a large number of regulons are activated in response to temperature changes, and are often important in thermotolerance. The major adaptive response to elevation in temperature is the heat shock response that involves the induction of many proteins ± called heat shock proteins, or Hsps (VanBogelen et al., 1987). This is a global, highly conserved, regulatory network that exists in all living cells. The bacterial heat shock response is not limited to changes in temperature and is often a general stress response. Thus, many of the
heat shock proteins are also induced by other environmental changes, such as the addition of ethanol, heavy metals, high osmolarity, pollutants, starvation, or interaction with eukaryotic hosts (Blom et al., 1992; Hecker et al., 1996; Muffler et al., 1997; Van Dyk et al., 1995; VanBogelen et al., 1987). The genes belonging to this regulon are transcriptionally activated by specific heat shock regulators. Such regulators include alternative sigma factors that exist in many bacteria and recognize specific sequences in the promoter region, or repressors that recognize specific sequences upstream to heat shock genes. The heat shock proteins include chaperones and proteases that are presumably
Corresponding author: Eliora Z. Ron, Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel 69978. Fax: 972 3 641 4138, E-mail: [email protected]
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essential for overcoming changes that involve protein denaturation as well as other genes, such as transcriptional regulators. For further reading, the following reviews (among many excellent ones) deal with several aspects of bacterial heat shock response ± (Arsene et al., 2000; Bukau and Horwich, 1998; Lund, 2001; Narberhaus, 1999). Induction of the heat shock response improves thermotolerance, salt tolerance and tolerance to heavy metals (Inbar and Ron, 1993; Kusukawa and Yura, 1988; LaRossa and Van Dyk, 1991; Volker et al., 1992). Furthermore, in several bacterial species heat shock proteins have been shown to play a direct role in pathogenesis including heat shock proteins required for binding of Salmonella typhimurium to mucosal cells (Ensgraber and Loos, 1992), and chaperonin 10 responsible for the osteolytic activity of Mycobacterium tuberculosis in Pott's disease (Meghji et al., 1997). Pathogens of warm-blooded animals encounter temperature shifts upon contact with their hosts. In the case of Haemophilus influenza it was shown that an increase of 2 8C in body temperature of the animal restricted bacterial growth in vivo (O'Reilly and Zak, 1992). The contact with the host also involves exposure to additional stresses such as changes in pH or oxidative stress. All these types of stress are denaturing for many proteins and are therefore expected to elicit the heat shock response. This induction of the heat shock response probably affects pathogenesis indirectly by increasing bacterial resistance to host defenses or regulating virulence genes. Many studies on the potential correlation between pathogenesis and the heat shock response pointed out the existence of virulence-related proteins that are induced by shifts to higher temperature. However, in many cases the genes coding for these proteins are not part of the heat shock regulon. The induction of these proteins at increased temperature is mediated by other processes, or is regulated only indirectly by the heat shock response. Maybe the best known example is listeriolysin, an important toxin, required for escape of Listeria monocytogenes from phagosomes of macrophages. The expression of listeriolysin increases considerably after a rise in temperature (Sokolovic and Goebel, 1989), yet it is not part of the heat shock regulon in terms of transcriptional activators. Its expression at higher temperatures is affected by another protein ± the ClpP ATP-dependent protease (see below) ± that is part of the heat shock regulon (Gaillot et al., 2000). In this communication we review some of the interactions between the heat shock response and virulence. We will present evidence for virulence
genes the expression of which is affected by heat shock regulators or heat shock proteins. In addition, we will present evidence for changes in expression of heat shock genes resulting from host-pathogen interactions. This review is limited to interactions between the heat shock response and bacterial virulence and will not discuss the large number of genes with temperature-dependent expression. It is expected that heat shock genes will be important in virulence of bacteria that enter hosts with a body temperature higher than that of the environment. This expectation was supported by numerous reports on virulence genes ± like the one coding for listeriolysin ± that are induced at higher temperatures. However, a closer look at the data indicates that the number of cases in which virulence is heat shock dependent is much lower than expected.
Host response to bacterial heat shock proteins Heat shock proteins were assumed to play a role in infection since following bacterial diseases, sera often contained antibodies against them. Antibodies recognizing ClpC of Mycobacterium leprae were identified in patients infected with leprosy and tuberculosis alike (Misra et al., 1996). GroEL orthologs of various bacteria were shown to be immunogenic in diseased hosts, including that of Helicobacter pylori (Macchia et al., 1993), Ehrlichia risticii, the causative agent of Potomac horse fever (Vemulapalli et al., 1998), Ehrlichia chaffeensis, causative agent of human monocytic ehrlichiosis (Dumler et al., 1995), Pasteurella haemolytica, causing bovine pneumonic pasteurellosis (Mosier et al., 1998) and others (as reviewed in (Young and Elliott, 1989)). Another interesting host response was observed for Actinobacillus actinomycetemcomitans, an important pathogen in periodontitis, suggesting that higher expression levels of GroEL in the membrane induce proliferation of host epithelial cells (Paju et al., 2000). These findings indicate that heat shock proteins are important in the host response to bacteria. However, this does not necessarily imply they have a direct role in pathogenesis. Moreover, heat shock proteins, especially GroEL constitute a large portion of the bacterial proteome, so it is hardly surprising that they are often recognized by the host immune system.
Virulence and the heat shock response
Heat shock regulators affecting virulence Transcriptional modulators Sigma 32 In Gram negative bacteria s32 (product of the rpoH gene) is the major transcriptional activator of heat shock genes and recognizes a specific heat shock promoter (Yura and Nakahigashi, 1999). This sigma factor was shown to play a role in the control of several virulence genes, mostly by controlling transcription of genes encoding regulators, chaperones and proteases. For example, in the intracellular pathogen Legionella pneumophila, the global stress protein gene (gspA) is induced during the intracellular infection of macrophages. One of the promoters controlling transcription of gspA is regulated by s32. However, although gspA transcription is induced during intracellular growth, a gspA mutant was not impaired in cytotoxicity or replication within amoebae and macrophages (Abu Kwaik et al., 1997). RpoH also affects transcription of leuX, encoding tRNA5Leu, important for the expression of virulence factors encoded by the pathogenicity island PAI II536 of the uropathogenic E. coli strain 536 (Dobrindt and Hacker, 2001). ToxR is an important positive regulator of several virulence genes in Vibrio cholerae. The expression of toxR is reduced at 37 8C compared to 22 8C, attributed to the binding of sigma 32 to the neighboring divergently transcribed heat shock gene htpG, at this temperature, blocking access of sigma 70 RNA polymerase to the toxR promoter (Parsot and Mekalanos, 1990). Sigma E Sigma E is a regulator of the extreme heat shock response. The mucoid phenotype, having a major role in the pathogenesis of Pseudomonas aeruginosa in cystic fibrosis respiratory infections, is controlled by the alternative sigma factor AlgU, a homologue of sE in E. coli. In Salmonella enterica serovar typhimurium an rpoE mutant was defective in proliferation within macrophages and could not colonize livers and spleens of mice infected i.v. (Humphreys et al., 1999). This result is not simply due to inability of mutants to grow at the higher temperatures of the host, since rpoE is not required for growth of Salmonella at elevated temperatures, indicating a possible role in infection. Sigma B An important regulator of the heat shock response of Gram-positive bacteria is the transcriptional activa-
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tor sB (Hecker et al., 1996; Schulz and Schumann, 1996; Zuber and Schumann, 1994). In Staphylococcus aureus a role in virulence was suggested for the general stress response sigma factor sB. A comparative analysis of 2-D protein gels of a sB mutant and wild-type S. aureus was conducted to identify proteins influenced by the alternative sigma factor sB regulon. Only three proteins identified were transcribed solely by sB. Of the new proteins identified, several were putative dehydrogenases which may be required to defend the bacterium against oxidative damage within the host (Gertz et al., 2000). An indirect effect of sB was suggested to take place via the sar regulatory locus, that partly controls several extracellular virulence determinants of S. aureus. One of the promoters of the sar locus is recognized by sB, leading to the assumption that this transcriptional activator will affect virulence (Deora et al., 1997; Manna et al., 1998). This is supported by in vitro data showing strong sigma B expression to be correlated to growth-dependent sar expression (Bischoff et al., 2001). However, it was shown that in a sB mutant the levels of SarA and the transcription of various virulence factors in a mouse subcutaneous abscess model of S. aureus pathogenesis were not affected (Chan et al., 1998). Moreover, additional findings indicate that sB may downregulate virulence genes, because increased production of alpha-hemolysin was observed in a sigB mutant with elevated sarA expression (Cheung et al., 1999).
Control of chaperone expression In Gram-positive bacteria and several Gram-negative bacteria the expression of all or some of the chaperones (see below) is controlled by a repressor (HrcA) that binds to a conserved inverted repeat (CIRCE Conserved Inverted Repeat of Chaperone Expression) and downregulates the operons under non-heat-shock conditions. In Gram-positive bacteria, these elements modulate the expression of the two operons coding for chaperones ± the groE operon and the dnaK operon, that are transcribed by the vegetative sigma factor, sA (Schulz and Schumann, 1996; Zuber and Schumann, 1994). In contrast, in the Gram-negative bacteria the operons coding for the chaperones are transcribed by a specific heat shock sigma factor ± a sigma 32-like activator ± and the CIRCE-HrcA control affects only the groESL operon (Nakahigashi et al., 1998, 1999; Segal and Ron, 1995, 1996a, 1998). These control elements affect virulence through their critical role in regulating the expression of chaperones, the role of which in virulence is discussed later.
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ATP-dependent proteases Bacteria possess many energy-dependent proteolytic enzymes. More than 90% of the protein degradation occurring in the bacteria is energy-dependent and requires ATP (Maurizi, 1992). The major ATPdependent proteases are Lon (La), Clp and the recently discovered HslVU (ClpQY) (Kanemori et al., 1997, 1999), which are responsible for 70 ± 80% of the energy-dependent degradation of proteins in vivo. Additional important ATP-dependent proteases include the serine protease HtrA (DegP) (Pallen and Wren, 1997) that degrades denatured proteins formed in the cellular envelope (SkorkoGlonek et al., 1999). As the structure of proteins is affected by temperature, it is clear that elevation of temperature brings about unfolding and misfolding of many proteins. Since ATP-dependent proteases are essential for degradation of damaged proteins, it is not surprising that their concentration in the cell increases upon temperature shift-up. Moreover, all of the above mentioned ATP-dependent proteases were found to be activated by the heat shock response, via s32 in Gram-negative bacteria. Lon (La) The Lon (La) ATP-dependent protease (Goldberg, 1990; Gottesman, 1996) is probably the major ATPdependent protease in bacteria. In lon mutants the level of energy-dependent proteolysis decreases by more than 50%. So far, Lon was linked with virulence in Brucella abortus, where lon mutants showed reduced survival in cultured murine macrophages and significant attenuation in BALB/c mice (Robertson et al., 2000). In E. coli K-12 it has been shown that Lon regulates capsule production through degradation of the positive regulator RcsA (Torres-Cabassa and Gottesman, 1987) and lon mutants are heavily capsulated. Although capsule production is an important virulence property that could affect many pathogenic extraintestinal E. coli strains, it appears that Group 1 capsule-producing E. coli strains have a different regulation of capsule production. This assumption is based on finding that although they possess conserved rcs homologs, rcs null mutants of these strains are encapsulated (Jayaratne et al., 1993). The family of Clp proteases Signature-tagged mutagenesis identifies specific gene products necessary for bacterial survival in the host (Hensel et al., 1995). The gene encoding ClpP of Salmonella enterica serovar typhimurium was isolated by signature-tagged mutagenesis, demonstrating its importance for virulence in the mouse model. Signature-tagged mutagenesis also
identified the regulatory Clp subunit gene clpC of Streptococcus pneumoniae (Polissi et al., 1998) and ClpX of S. aureus (Mei et al., 1997) to be involved in virulence. The ClpC ATPase is required for early bacterial escape from the phagosome of macrophages and is important for intracellular survival of L. monocytogenes (Rouquette et al., 1998) and its virulence in mice (Rouquette et al., 1996). In addition a clpP mutant of L. monocytogenes also exhibited restricted intracellular survival and virulence in mice, due to reduced listeriolysin O production; and complementation of the mutant restored the hemolytic and virulent phenotype (Gaillot et al., 2000). An additional protein important for in vivo growth of L. monocytogenes is the homologue of ctsR, the first gene in the clpC operon, which is a repressor of stress response genes. It was shown that when this gene is constitutively expressed the bacteria were attenuated in vivo (Derre et al., 1999; Kruger and Hecker, 1998; Nair et al., 2000a). In Yersinia enterocolitica expression of Ail, a 17-kDa cell-surface protein conferring resistance to serum killing and the ability to attach to cells and invade them in vitro, is repressed by ClpP at lower (nonhost) temperatures (Pederson et al., 1997). L. monocytogenes mutants in clpE were shown to exhibit reduced virulence, which may be due to its role in cell division (Nair et al., 1999). In these bacteria, ClpC is required for adhesion and invasion by modulating expression of the virulence factors InlA, InlB, and ActA, which are required for entry into various cells, unlike ClpE which does not affect the expression of these virulence factors (Nair et al., 2000b). HtrA HtrA (DegP) is a heat shock-induced serine protease found in bacteria and eukaryotes. This widely conserved heat shock protein DegP (HtrA) also acts as a molecular chaperone. At low temperatures the chaperone function dominates, whereas at elevated temperatures the protein acts as a protease (Spiess et al., 1999). In bacteria its chief role is to degrade misfolded membrane proteins (Pallen and Wren, 1997). The expression of htrA is regulated by a complex set of signal transduction pathways, which includes RpoE, RseA (an anti-sigma factor), a two-component regulatory system (CpxRA) and two phosphoprotein phosphatases (PrpA and PrpB). This serine protease appears to play an important role in scavenging oxidatively damaged proteins from the cell before they reach toxic levels. Mutations in the htrA genes of Salmonella, Brucella and Yersinia cause decreased survival in mice and/or macrophages. It was therefore believed that htrA mutants can act as vaccines.
Virulence and the heat shock response
In S. enterica serovar typhimurium an htrA mutant exhibited diminished survival in macrophages (Baumler et al., 1994). In Brucella melitensis, a goat pathogen, a mutant with a deletion in htrA exhibited increased sensitivity to H2O2 and grew poorly in liquid broth. The number of mutant bacteria recovered from spleens and livers of infected BALB/c mice was significantly lower than for the wild type one week post infection. However, by 3 weeks post-infection, numbers were similar, indicating a possible role in early stages of infection (Phillips et al., 1995). Recent re-examination on a new htrA mutant has shown that it is probably not required for wild-type virulence in BALB/c mice, attributing the previously observed reduction in virulence to a secondary mutation (Phillips and Roop, 2001). A recent study on the role of HtrA in pregnant goats, a natural host of this pathogen, has shown that its contribution to virulence is probably not very high (Roop et al., 2001). An htrA homologue in Yersinia enterocolitica, gsrA, has been shown by transposon mutagenesis to be required for both growth at high temperature and survival in macrophages. The gsrA gene was found to be induced by macrophage phagocytosis and is also regulated by sE (Yamamoto et al., 1997).
Chaperones Chaperones play a role in many processes in bacterial cells involving protein folding. Among them is assisting the folding of newly synthesized proteins, preventing aggregation of proteins during heat shock, and repairing proteins that have been damaged or misfolded by heat shock and other stresses. The two major bacterial chaperones are GroE and DnaK. GroE acts by providing a protective micro-environment for protein folding, whereas DnaK acts by binding and protecting exposed regions on unfolded or partially folded protein chains from proteolysis and aggregation (Lund, 2001; Segal and Ron, 1996b). DnaK DnaK may have a role in the virulence of Vibrio cholerae, through its effect on expression of ToxR, an important regulator of several virulence genes in this bacterium. In a dnaK mutant, a decrease in toxR transcription was observed, followed by a reduction in expression of cholera toxin and the major subunit of the toxin-coregulated pilus. These results were obtained in vitro. However, in this mutant in vivo, toxR was transcribed at wild-type levels leading to the conclusion that in vivo regulation of toxR is
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more complex and may not be DnaK dependent (Chakrabarti et al., 1999). GroESL As mentioned earlier, bacterial life within the host is stressful, and even more so for bacteria growing within macrophages. In L. monocytogenes an increase in transcription of groEL during growth within J774 mouse macrophage cells was demonstrated using GFP fusions and RT-PCR, which also showed a clear increase in expression of dnaK. Using a fusion of the hemolysin gene hly to the gro promoter in an hly-deficient mutant, expression of groESL in vivo was assessed by the level of bacteria recovered from the spleen of infected mice. High numbers of bacteria were recovered though lower than observed with a L. monocytogenes wild-type strain, indicating the groESL level in vivo may not be very high (Gahan et al., 2001).
Heat shock genes affected by interaction with the host SarA, a positive regulator of extracellular virulence proteins in S. aureus was shown to up-regulate sB (Chan et al., 1998). In P. aeruginosa, sigma 32 is transcribed by AlgU containing RNA polymerase from one of its three promoters, upon exposure to extreme heat shock, so the mucoidy and heat-shock responses are in fact co-regulated (Schurr and Deretic, 1997). The pleiotropic regulatory factor, PrfA, (Mengaud et al., 1991) of L. monocytogenes, controlling several virulence factors including listeriolysin, was shown to downregulate clpC at the level of transcription (Ripio et al., 1998). In a differential display study in Streptococcus gordonii, an early colonizer of the clean enamel surface of teeth, a clpE homologue was identified as upregulated in saliva (Du and Kolenbrander, 2000). In L. pneumophila GroEL synthesis was induced during intracellular growth in human monocyte and mouse L929 cells and was the dominant protein (along with OmpS) 17 hours post-infection (Fernandez et al., 1996). The finding that heat shock genes and virulence genes are concurrently upregulated does not necessarily indicate that one of them controls the other ± it is possible that both are independently affected by the same regulator or signal. For example, in H. pylori, a stress-responsive operon (sro) with a promoter activated by an upshift from 37 8C to 45 8C was found to contain the flagellar motor switch gene cheY, as well as genes assumed to be
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involved in heat shock adjustment such as ftsH (Beier et al., 1997). This gene localization results in co-regulation of motility, which is highly important for H. pylori virulence, with the heat shock response.
Conclusions Entry of a pathogen into a warm-blooded host is usually accompanied by a shift-up in temperature. It is therefore expected that heat shock genes play an important role in bacterial infection, an assumption supported by the findings that following bacterial infections, sera of patients contain high titres of antibodies against heat-shock proteins. It is assumed that heat shock genes are induced upon infection in order to prevent misfolding and aggregation of damaged proteins, by refolding or proteolysis. These processes are probably essential for the survival of the pathogen within the host. Induction of heat shock genes is by no means limited to the host entry stage, and preliminary colonization. We see induction of some heat-shock genes at a later stage of infection, namely during growth of intracellular bacteria within macrophages. This probably reflects the role of heat-shock genes in protection of the bacterium against a variety of other stresses, such as oxidative stress or low pH. Since the temperature up-shift at host entry provides a distinct signal for the bacterium, it can be assumed that many genes required by the pathogen for adaptation to the host and for virulence would be regulated by heat shock proteins. However, to date, the number of virulence genes which fit this paradigm, is smaller than expected. This review describes several cases where heatshock genes are regulated by virulence factors. This co-regulation may be beneficial to the pathogen's survival, especially when heat shock genes are induced, since the onset of induction of toxins and other virulence factors often leads to an increase in temperature and oxidative stress within the host. Several experiments demonstrated that mutants in heat shock genes have reduced virulence. Some of these findings were taken as indications for the essential role of heat shock genes in pathogenesis. Yet, it should be remembered that mutations in certain heat shock genes severely impair the ability of bacteria to grow under any conditions and their effect on virulence is secondary. Since heat shock genes are essential for bacterial survival under a variety of stresses, it was often suggested that bacteria deficient in heat shock genes would be attenuated and could therefore be used as
live vaccines. However, it was found that in many cases the disruption of one heat shock gene is not enough to reduce virulence sufficiently enough to produce a safe vaccine. In order to construct good vaccine strains, one would have to ™knock out∫ several heat shock genes, while ensuring the antigenic potential is preserved. Development of such attenuated strains is not trivial, since some heat shock proteins are themselves antigenic and in addition, their loss may result in the reduced display of various antigenic determinants belonging to virulence proteins the expression of which is regulated by heat shock genes. Acknowledgements. This work was supported, in part, by the German Israeli Science Foundation (GIF), by the Manja and Morris Leigh Chair for Biophysics and Biotechnology, and by the Center of Emerging Diseases, Israel. U. Gophna was partially supported by the Wolf doctoral fellowship.
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signature-tagged mutagenesis. Mol. Microbiol. 26, 399 ± 407 (1997). Mengaud, J., Dramsi, S., Gouin, E., Vazquez-Boland, J.A., Milon, G., Cossart, P.: Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated. Mol. Microbiol. 5, 2273 ± 2283 (1991). Misra, N., Habib, S., Ranjan, A., Hasnain, S.-E., Nath, I.: Expression and functional characterisation of the clpC gene of Mycobacterium leprae: ClpC protein elicits human antibody response. Gene 172, 99 ± 104 (1996). Mosier, D., Iandolo, J., Rogers, D., Uhlich, G., Crupper, S.: Characterization of a 54-kDa heat-shock-inducible protein of Pasteurella haemolytica. Vet. Microbiol. 60, 67 ± 73 (1998). Muffler, A., Barth, M., Marschall, C., Hengge-Aronis, R.: Heat shock regulation of sigmaS turnover: a role for DnaK and relationship between stress responses mediated by sigmaS and sigma32 in Escherichia coli. J. Bacteriol. 179, 445 ± 452 (1997). Nair, S., Derre, I., Msadek, T., Gaillot, O., Berche, P.: CtsR controls class III heat shock gene expression in the human pathogen Listeria monocytogenes. Mol. Microbiol. 35, 800 ± 811 (2000a). Nair, S., Frehel, C., Nguyen, L., Escuyer, V., Berche, P.: ClpE, a novel member of the HSP100 family, is involved in cell division and virulence of Listeria monocytogenes. Mol. Microbiol. 31, 185 ± 196 (1999). Nair, S., Milohanic, E., Berche, P.: ClpC ATPase is required for cell adhesion and invasion of Listeria monocytogenes. Infect. Immun. 68, 7061 ± 7068 (2000b). Nakahigashi, K., Ron, E.-Z., Yanagi, H., Yura, T.: Differential and independent roles of a sigma(32) homolog (RpoH) and an HrcA repressor in the heat shock response of Agrobacterium tumefaciens. J. Bacteriol. 181, 7509 ± 7515 (1999). Nakahigashi, K., Yanagi, H., Yura, T.: Regulatory conservation and divergence of sigma32 homologs from gram-negative bacteria: Serratia marcescens, Proteus mirabilis, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. J. Bacteriol. 180, 2402 ± 2408 (1998). Narberhaus, F.: Negative regulation of bacterial heat shock genes. Mol. Microbiol. 31, 1 ± 8 (1999). O'Reilly, T., Zak, O.: Elevated body temperature restricts growth of Haemophilus influenzae type b during experimental meningitis. Infect. Immun. 60, 3448 ± 3451 (1992). Paju, S., Goulhen, F., Asikainen, S., Grenier, D., Mayrand, D., Uitto, V.: Localization of heat shock proteins in clinical Actinobacillus actinomycetemcomitans strains and their effects on epithelial cell proliferation. FEMS Microbiol. Lett. 182, 231 ± 235 (2000). Pallen, M.-J., Wren, B.-W.: The HtrA family of serine proteases. Mol. Microbiol. 26, 209 ± 221 (1997). Parsot, C., Mekalanos, J.-J.: Expression of ToxR, the transcriptional activator of the virulence factors in
Vibrio cholerae, is modulated by the heat shock response. Proc. Natl. Acad. Sci. USA 87, 9898 ± 9902 (1990). Pederson, K.-J., Carlson, S., Pierson, D.-E.: The ClpP protein, a subunit of the Clp protease, modulates ail gene expression in Yersinia enterocolitica. Mol. Microbiol. 26, 99 ± 107 (1997). Phillips, R.-W., Elzer, P.-H., Roop, R.-M., II: A Brucella melitensis high temperature requirement A (htrA) deletion mutant demonstrates a stress response defective phenotype in vitro and transient attenuation in the BALB/c mouse model. Microb. Pathog. 19, 227 ± 284 (1995). Phillips, R.-W., Roop, R.-M., 2nd: Brucella abortus HtrA functions as an authentic stress response protease but is not required for wild-type virulence in BALB/c mice. Infect. Immun. 69, 5911 ± 5913 (2001). Polissi, A., Pontiggia, A., Feger, G., Altieri, M., Mottl, H., Ferrari, L., Simon, D.: Large-scale identification of virulence genes from Streptococcus pneumoniae. Infect. Immun. 66, 5620 ± 5629 (1998). Ripio, M.-T., Vazquez-Boland, J.-A., Vega, Y., Nair, S., Berche, P.: Evidence for expressional crosstalk between the central virulence regulator PrfA and the stress response mediator ClpC in Listeria monocytogenes. FEMS Microbiol. Lett. 158, 45 ± 50 (1998). Robertson, G.-T., Kovach, M.-E., Allen, C.-A., Ficht, T.A., Roop, R.-M., 2nd: The Brucella abortus Lon functions as a generalized stress response protease and is required for wild-type virulence in BALB/c mice. Mol. Microbiol. 35, 577 ± 588 (2000). Roop, R. M., 2nd, Phillips, R.-W., Hagius, S., Walker, J.V., Booth, N.-J., Fulton, W. T., Edmonds, M.-D., Elzer, P.-H.: Re-examination of the role of the Brucella melitensis HtrA stress response protease in virulence in pregnant goats. Vet. Microbiol. 82, 91 ± 95. (2001). Rouquette, C., de Chastellier, C., Nair, S., Berche, P.: The ClpC ATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages. Mol. Microbiol. 27, 1235 ± 1245 (1998). Rouquette, C., Ripio, M.-T., Pellegrini, E., Bolla, J.-M., Tascon, R.-I., Vazquez-Boland, J.-A., Berche, P.: Identification of a ClpC ATPase required for stress tolerance and in vivo survival of Listeria monocytogenes. Mol. Microbiol. 21, 977 ± 987 (1996). Schulz, A., Schumann, W.: hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes. J. Bacteriol. 178, 1088 ± 1093 (1996). Schurr, M.-J., Deretic, V.: Microbial pathogenesis in cystic fibrosis: co-ordinate regulation of heat-shock response and conversion to mucoidy in Pseudomonas aeruginosa. Mol. Microbiol. 24, 411 ± 420 (1997). Segal, G., Ron, E.-Z.: The dnaKJ operon of Agrobacterium tumefaciens: transcriptional analysis and evidence for a new heat shock promoter. J. Bacteriol. 177, 5952 ± 5958 (1995).
Virulence and the heat shock response Segal, G., Ron, E.-Z.: Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat. J. Bacteriol. 178, 3634 ± 3640 (1996a). Segal, G., Ron, E.-Z.: Regulation of heat-shock response in bacteria. Ann. N.Y. Acad. Sci. 851, 147 ± 151 (1998). Segal, R., Ron, E.-Z.: Regulation and organization of the groE and dnaK operons in Eubacteria. FEMS Microbiol. Lett. 138, 1 ± 10 (1996b). Skorko-Glonek, J., Zurawa, D., Kuczwara, E., Wozniak, M., Wypych, Z., Lipinska, B.: The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress. Mol. Gen. Genet. 262, 342 ± 350 (1999). Sokolovic, Z., Goebel, W.: Synthesis of listeriolysin in Listeria monocytogenes under heat shock conditions. Infect. Immun. 57, 295 ± 298 (1989). Spiess, C., Beil, A., Ehrmann, M.: A temperaturedependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97, 339 ± 347 (1999). Torres-Cabassa, A.-S., Gottesman, S.: Capsule synthesis in Escherichia coli K-12 is regulated by proteolysis. J. Bacteriol. 169, 981 ± 989 (1987). Van Dyk, T.-K., Reed, T.-R., Vollmer, A.-C., LaRossa, R.-A.: Synergistic induction of the heat shock response in Escherichia coli by simultaneous treatment
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with chemical inducers. J. Bacteriol. 177, 6001 ± 6004 (1995). VanBogelen, R.-A., Kelley, P.-M., Neidhardt, F.-C.: Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli. J. Bacteriol. 169, 26 ± 32 (1987). Vemulapalli, R., Biswas, B., Dutta, S.-K.: Cloning and molecular analysis of genes encoding two immunodominant antigens of Ehrlichia risticii. Microb. Pathog. 24, 361 ± 372 (1998). Volker, U., Mach, H., Schmid, R., Hecker, M.: Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis. J. Gen. Microbiol. 138, 2125 ± 2135 (1992). Yamamoto, T., Hanawa, T., Ogata, S., Kamiya, S.: The Yersinia enterocolitica GsrA stress protein, involved in intracellular survival, is induced by macrophage phagocytosis. Infect. Immun. 65, 2190 ± 2196 (1997). Young, R.-A., Elliott, T.-J.: Stress proteins, infection, and immune surveillance. Cell 59, 5 ± 8 (1989). Yura, T., Nakahigashi, K.: Regulation of the heat-shock response. Curr. Opin. Microbiol. 2, 153 ± 158 (1999). Zuber, U., Schumann, W.: CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J. Bacteriol. 176, 1359 ± 1363 (1994).
Mini-Review Virulence and the heat shock response Uri Gophna, Eliora Z. Ron Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel 69978 Received December 19, 2001 ¥ Revision received February 26, 2002 ¥ Accepted March 6, 2002
Abstract The major adaptive response to elevation in temperature is the heat shock response that involves the induction of many proteins ± called heat shock proteins. These include chaperones, proteases, alternative sigma factors and other regulatory and structural proteins. The heat shock response is also turned on by other stress conditions, such as oxidative stress or pH changes. Bacterial entry into the host organism involves a significant environmental change, which is expected to induce the heat shock response. Indeed, some of the heat shock proteins are themselves virulence factors while others affect pathogenesis indirectly, by increasing bacterial resistance to host defenses or regulating virulence genes. The cross talk between heat shock and virulence genes is discussed. Key words: Heat shock ± stress response ± virulence ± pathogenesis ± chaperones ± proteases
Introduction Temperature increase constitutes one of the major environmental stresses encountered by bacteria. It is, therefore, not surprising that a large number of regulons are activated in response to temperature changes, and are often important in thermotolerance. The major adaptive response to elevation in temperature is the heat shock response that involves the induction of many proteins ± called heat shock proteins, or Hsps (VanBogelen et al., 1987). This is a global, highly conserved, regulatory network that exists in all living cells. The bacterial heat shock response is not limited to changes in temperature and is often a general stress response. Thus, many of the
heat shock proteins are also induced by other environmental changes, such as the addition of ethanol, heavy metals, high osmolarity, pollutants, starvation, or interaction with eukaryotic hosts (Blom et al., 1992; Hecker et al., 1996; Muffler et al., 1997; Van Dyk et al., 1995; VanBogelen et al., 1987). The genes belonging to this regulon are transcriptionally activated by specific heat shock regulators. Such regulators include alternative sigma factors that exist in many bacteria and recognize specific sequences in the promoter region, or repressors that recognize specific sequences upstream to heat shock genes. The heat shock proteins include chaperones and proteases that are presumably
Corresponding author: Eliora Z. Ron, Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel 69978. Fax: 972 3 641 4138, E-mail: [email protected]
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essential for overcoming changes that involve protein denaturation as well as other genes, such as transcriptional regulators. For further reading, the following reviews (among many excellent ones) deal with several aspects of bacterial heat shock response ± (Arsene et al., 2000; Bukau and Horwich, 1998; Lund, 2001; Narberhaus, 1999). Induction of the heat shock response improves thermotolerance, salt tolerance and tolerance to heavy metals (Inbar and Ron, 1993; Kusukawa and Yura, 1988; LaRossa and Van Dyk, 1991; Volker et al., 1992). Furthermore, in several bacterial species heat shock proteins have been shown to play a direct role in pathogenesis including heat shock proteins required for binding of Salmonella typhimurium to mucosal cells (Ensgraber and Loos, 1992), and chaperonin 10 responsible for the osteolytic activity of Mycobacterium tuberculosis in Pott's disease (Meghji et al., 1997). Pathogens of warm-blooded animals encounter temperature shifts upon contact with their hosts. In the case of Haemophilus influenza it was shown that an increase of 2 8C in body temperature of the animal restricted bacterial growth in vivo (O'Reilly and Zak, 1992). The contact with the host also involves exposure to additional stresses such as changes in pH or oxidative stress. All these types of stress are denaturing for many proteins and are therefore expected to elicit the heat shock response. This induction of the heat shock response probably affects pathogenesis indirectly by increasing bacterial resistance to host defenses or regulating virulence genes. Many studies on the potential correlation between pathogenesis and the heat shock response pointed out the existence of virulence-related proteins that are induced by shifts to higher temperature. However, in many cases the genes coding for these proteins are not part of the heat shock regulon. The induction of these proteins at increased temperature is mediated by other processes, or is regulated only indirectly by the heat shock response. Maybe the best known example is listeriolysin, an important toxin, required for escape of Listeria monocytogenes from phagosomes of macrophages. The expression of listeriolysin increases considerably after a rise in temperature (Sokolovic and Goebel, 1989), yet it is not part of the heat shock regulon in terms of transcriptional activators. Its expression at higher temperatures is affected by another protein ± the ClpP ATP-dependent protease (see below) ± that is part of the heat shock regulon (Gaillot et al., 2000). In this communication we review some of the interactions between the heat shock response and virulence. We will present evidence for virulence
genes the expression of which is affected by heat shock regulators or heat shock proteins. In addition, we will present evidence for changes in expression of heat shock genes resulting from host-pathogen interactions. This review is limited to interactions between the heat shock response and bacterial virulence and will not discuss the large number of genes with temperature-dependent expression. It is expected that heat shock genes will be important in virulence of bacteria that enter hosts with a body temperature higher than that of the environment. This expectation was supported by numerous reports on virulence genes ± like the one coding for listeriolysin ± that are induced at higher temperatures. However, a closer look at the data indicates that the number of cases in which virulence is heat shock dependent is much lower than expected.
Host response to bacterial heat shock proteins Heat shock proteins were assumed to play a role in infection since following bacterial diseases, sera often contained antibodies against them. Antibodies recognizing ClpC of Mycobacterium leprae were identified in patients infected with leprosy and tuberculosis alike (Misra et al., 1996). GroEL orthologs of various bacteria were shown to be immunogenic in diseased hosts, including that of Helicobacter pylori (Macchia et al., 1993), Ehrlichia risticii, the causative agent of Potomac horse fever (Vemulapalli et al., 1998), Ehrlichia chaffeensis, causative agent of human monocytic ehrlichiosis (Dumler et al., 1995), Pasteurella haemolytica, causing bovine pneumonic pasteurellosis (Mosier et al., 1998) and others (as reviewed in (Young and Elliott, 1989)). Another interesting host response was observed for Actinobacillus actinomycetemcomitans, an important pathogen in periodontitis, suggesting that higher expression levels of GroEL in the membrane induce proliferation of host epithelial cells (Paju et al., 2000). These findings indicate that heat shock proteins are important in the host response to bacteria. However, this does not necessarily imply they have a direct role in pathogenesis. Moreover, heat shock proteins, especially GroEL constitute a large portion of the bacterial proteome, so it is hardly surprising that they are often recognized by the host immune system.
Virulence and the heat shock response
Heat shock regulators affecting virulence Transcriptional modulators Sigma 32 In Gram negative bacteria s32 (product of the rpoH gene) is the major transcriptional activator of heat shock genes and recognizes a specific heat shock promoter (Yura and Nakahigashi, 1999). This sigma factor was shown to play a role in the control of several virulence genes, mostly by controlling transcription of genes encoding regulators, chaperones and proteases. For example, in the intracellular pathogen Legionella pneumophila, the global stress protein gene (gspA) is induced during the intracellular infection of macrophages. One of the promoters controlling transcription of gspA is regulated by s32. However, although gspA transcription is induced during intracellular growth, a gspA mutant was not impaired in cytotoxicity or replication within amoebae and macrophages (Abu Kwaik et al., 1997). RpoH also affects transcription of leuX, encoding tRNA5Leu, important for the expression of virulence factors encoded by the pathogenicity island PAI II536 of the uropathogenic E. coli strain 536 (Dobrindt and Hacker, 2001). ToxR is an important positive regulator of several virulence genes in Vibrio cholerae. The expression of toxR is reduced at 37 8C compared to 22 8C, attributed to the binding of sigma 32 to the neighboring divergently transcribed heat shock gene htpG, at this temperature, blocking access of sigma 70 RNA polymerase to the toxR promoter (Parsot and Mekalanos, 1990). Sigma E Sigma E is a regulator of the extreme heat shock response. The mucoid phenotype, having a major role in the pathogenesis of Pseudomonas aeruginosa in cystic fibrosis respiratory infections, is controlled by the alternative sigma factor AlgU, a homologue of sE in E. coli. In Salmonella enterica serovar typhimurium an rpoE mutant was defective in proliferation within macrophages and could not colonize livers and spleens of mice infected i.v. (Humphreys et al., 1999). This result is not simply due to inability of mutants to grow at the higher temperatures of the host, since rpoE is not required for growth of Salmonella at elevated temperatures, indicating a possible role in infection. Sigma B An important regulator of the heat shock response of Gram-positive bacteria is the transcriptional activa-
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tor sB (Hecker et al., 1996; Schulz and Schumann, 1996; Zuber and Schumann, 1994). In Staphylococcus aureus a role in virulence was suggested for the general stress response sigma factor sB. A comparative analysis of 2-D protein gels of a sB mutant and wild-type S. aureus was conducted to identify proteins influenced by the alternative sigma factor sB regulon. Only three proteins identified were transcribed solely by sB. Of the new proteins identified, several were putative dehydrogenases which may be required to defend the bacterium against oxidative damage within the host (Gertz et al., 2000). An indirect effect of sB was suggested to take place via the sar regulatory locus, that partly controls several extracellular virulence determinants of S. aureus. One of the promoters of the sar locus is recognized by sB, leading to the assumption that this transcriptional activator will affect virulence (Deora et al., 1997; Manna et al., 1998). This is supported by in vitro data showing strong sigma B expression to be correlated to growth-dependent sar expression (Bischoff et al., 2001). However, it was shown that in a sB mutant the levels of SarA and the transcription of various virulence factors in a mouse subcutaneous abscess model of S. aureus pathogenesis were not affected (Chan et al., 1998). Moreover, additional findings indicate that sB may downregulate virulence genes, because increased production of alpha-hemolysin was observed in a sigB mutant with elevated sarA expression (Cheung et al., 1999).
Control of chaperone expression In Gram-positive bacteria and several Gram-negative bacteria the expression of all or some of the chaperones (see below) is controlled by a repressor (HrcA) that binds to a conserved inverted repeat (CIRCE Conserved Inverted Repeat of Chaperone Expression) and downregulates the operons under non-heat-shock conditions. In Gram-positive bacteria, these elements modulate the expression of the two operons coding for chaperones ± the groE operon and the dnaK operon, that are transcribed by the vegetative sigma factor, sA (Schulz and Schumann, 1996; Zuber and Schumann, 1994). In contrast, in the Gram-negative bacteria the operons coding for the chaperones are transcribed by a specific heat shock sigma factor ± a sigma 32-like activator ± and the CIRCE-HrcA control affects only the groESL operon (Nakahigashi et al., 1998, 1999; Segal and Ron, 1995, 1996a, 1998). These control elements affect virulence through their critical role in regulating the expression of chaperones, the role of which in virulence is discussed later.
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ATP-dependent proteases Bacteria possess many energy-dependent proteolytic enzymes. More than 90% of the protein degradation occurring in the bacteria is energy-dependent and requires ATP (Maurizi, 1992). The major ATPdependent proteases are Lon (La), Clp and the recently discovered HslVU (ClpQY) (Kanemori et al., 1997, 1999), which are responsible for 70 ± 80% of the energy-dependent degradation of proteins in vivo. Additional important ATP-dependent proteases include the serine protease HtrA (DegP) (Pallen and Wren, 1997) that degrades denatured proteins formed in the cellular envelope (SkorkoGlonek et al., 1999). As the structure of proteins is affected by temperature, it is clear that elevation of temperature brings about unfolding and misfolding of many proteins. Since ATP-dependent proteases are essential for degradation of damaged proteins, it is not surprising that their concentration in the cell increases upon temperature shift-up. Moreover, all of the above mentioned ATP-dependent proteases were found to be activated by the heat shock response, via s32 in Gram-negative bacteria. Lon (La) The Lon (La) ATP-dependent protease (Goldberg, 1990; Gottesman, 1996) is probably the major ATPdependent protease in bacteria. In lon mutants the level of energy-dependent proteolysis decreases by more than 50%. So far, Lon was linked with virulence in Brucella abortus, where lon mutants showed reduced survival in cultured murine macrophages and significant attenuation in BALB/c mice (Robertson et al., 2000). In E. coli K-12 it has been shown that Lon regulates capsule production through degradation of the positive regulator RcsA (Torres-Cabassa and Gottesman, 1987) and lon mutants are heavily capsulated. Although capsule production is an important virulence property that could affect many pathogenic extraintestinal E. coli strains, it appears that Group 1 capsule-producing E. coli strains have a different regulation of capsule production. This assumption is based on finding that although they possess conserved rcs homologs, rcs null mutants of these strains are encapsulated (Jayaratne et al., 1993). The family of Clp proteases Signature-tagged mutagenesis identifies specific gene products necessary for bacterial survival in the host (Hensel et al., 1995). The gene encoding ClpP of Salmonella enterica serovar typhimurium was isolated by signature-tagged mutagenesis, demonstrating its importance for virulence in the mouse model. Signature-tagged mutagenesis also
identified the regulatory Clp subunit gene clpC of Streptococcus pneumoniae (Polissi et al., 1998) and ClpX of S. aureus (Mei et al., 1997) to be involved in virulence. The ClpC ATPase is required for early bacterial escape from the phagosome of macrophages and is important for intracellular survival of L. monocytogenes (Rouquette et al., 1998) and its virulence in mice (Rouquette et al., 1996). In addition a clpP mutant of L. monocytogenes also exhibited restricted intracellular survival and virulence in mice, due to reduced listeriolysin O production; and complementation of the mutant restored the hemolytic and virulent phenotype (Gaillot et al., 2000). An additional protein important for in vivo growth of L. monocytogenes is the homologue of ctsR, the first gene in the clpC operon, which is a repressor of stress response genes. It was shown that when this gene is constitutively expressed the bacteria were attenuated in vivo (Derre et al., 1999; Kruger and Hecker, 1998; Nair et al., 2000a). In Yersinia enterocolitica expression of Ail, a 17-kDa cell-surface protein conferring resistance to serum killing and the ability to attach to cells and invade them in vitro, is repressed by ClpP at lower (nonhost) temperatures (Pederson et al., 1997). L. monocytogenes mutants in clpE were shown to exhibit reduced virulence, which may be due to its role in cell division (Nair et al., 1999). In these bacteria, ClpC is required for adhesion and invasion by modulating expression of the virulence factors InlA, InlB, and ActA, which are required for entry into various cells, unlike ClpE which does not affect the expression of these virulence factors (Nair et al., 2000b). HtrA HtrA (DegP) is a heat shock-induced serine protease found in bacteria and eukaryotes. This widely conserved heat shock protein DegP (HtrA) also acts as a molecular chaperone. At low temperatures the chaperone function dominates, whereas at elevated temperatures the protein acts as a protease (Spiess et al., 1999). In bacteria its chief role is to degrade misfolded membrane proteins (Pallen and Wren, 1997). The expression of htrA is regulated by a complex set of signal transduction pathways, which includes RpoE, RseA (an anti-sigma factor), a two-component regulatory system (CpxRA) and two phosphoprotein phosphatases (PrpA and PrpB). This serine protease appears to play an important role in scavenging oxidatively damaged proteins from the cell before they reach toxic levels. Mutations in the htrA genes of Salmonella, Brucella and Yersinia cause decreased survival in mice and/or macrophages. It was therefore believed that htrA mutants can act as vaccines.
Virulence and the heat shock response
In S. enterica serovar typhimurium an htrA mutant exhibited diminished survival in macrophages (Baumler et al., 1994). In Brucella melitensis, a goat pathogen, a mutant with a deletion in htrA exhibited increased sensitivity to H2O2 and grew poorly in liquid broth. The number of mutant bacteria recovered from spleens and livers of infected BALB/c mice was significantly lower than for the wild type one week post infection. However, by 3 weeks post-infection, numbers were similar, indicating a possible role in early stages of infection (Phillips et al., 1995). Recent re-examination on a new htrA mutant has shown that it is probably not required for wild-type virulence in BALB/c mice, attributing the previously observed reduction in virulence to a secondary mutation (Phillips and Roop, 2001). A recent study on the role of HtrA in pregnant goats, a natural host of this pathogen, has shown that its contribution to virulence is probably not very high (Roop et al., 2001). An htrA homologue in Yersinia enterocolitica, gsrA, has been shown by transposon mutagenesis to be required for both growth at high temperature and survival in macrophages. The gsrA gene was found to be induced by macrophage phagocytosis and is also regulated by sE (Yamamoto et al., 1997).
Chaperones Chaperones play a role in many processes in bacterial cells involving protein folding. Among them is assisting the folding of newly synthesized proteins, preventing aggregation of proteins during heat shock, and repairing proteins that have been damaged or misfolded by heat shock and other stresses. The two major bacterial chaperones are GroE and DnaK. GroE acts by providing a protective micro-environment for protein folding, whereas DnaK acts by binding and protecting exposed regions on unfolded or partially folded protein chains from proteolysis and aggregation (Lund, 2001; Segal and Ron, 1996b). DnaK DnaK may have a role in the virulence of Vibrio cholerae, through its effect on expression of ToxR, an important regulator of several virulence genes in this bacterium. In a dnaK mutant, a decrease in toxR transcription was observed, followed by a reduction in expression of cholera toxin and the major subunit of the toxin-coregulated pilus. These results were obtained in vitro. However, in this mutant in vivo, toxR was transcribed at wild-type levels leading to the conclusion that in vivo regulation of toxR is
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more complex and may not be DnaK dependent (Chakrabarti et al., 1999). GroESL As mentioned earlier, bacterial life within the host is stressful, and even more so for bacteria growing within macrophages. In L. monocytogenes an increase in transcription of groEL during growth within J774 mouse macrophage cells was demonstrated using GFP fusions and RT-PCR, which also showed a clear increase in expression of dnaK. Using a fusion of the hemolysin gene hly to the gro promoter in an hly-deficient mutant, expression of groESL in vivo was assessed by the level of bacteria recovered from the spleen of infected mice. High numbers of bacteria were recovered though lower than observed with a L. monocytogenes wild-type strain, indicating the groESL level in vivo may not be very high (Gahan et al., 2001).
Heat shock genes affected by interaction with the host SarA, a positive regulator of extracellular virulence proteins in S. aureus was shown to up-regulate sB (Chan et al., 1998). In P. aeruginosa, sigma 32 is transcribed by AlgU containing RNA polymerase from one of its three promoters, upon exposure to extreme heat shock, so the mucoidy and heat-shock responses are in fact co-regulated (Schurr and Deretic, 1997). The pleiotropic regulatory factor, PrfA, (Mengaud et al., 1991) of L. monocytogenes, controlling several virulence factors including listeriolysin, was shown to downregulate clpC at the level of transcription (Ripio et al., 1998). In a differential display study in Streptococcus gordonii, an early colonizer of the clean enamel surface of teeth, a clpE homologue was identified as upregulated in saliva (Du and Kolenbrander, 2000). In L. pneumophila GroEL synthesis was induced during intracellular growth in human monocyte and mouse L929 cells and was the dominant protein (along with OmpS) 17 hours post-infection (Fernandez et al., 1996). The finding that heat shock genes and virulence genes are concurrently upregulated does not necessarily indicate that one of them controls the other ± it is possible that both are independently affected by the same regulator or signal. For example, in H. pylori, a stress-responsive operon (sro) with a promoter activated by an upshift from 37 8C to 45 8C was found to contain the flagellar motor switch gene cheY, as well as genes assumed to be
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involved in heat shock adjustment such as ftsH (Beier et al., 1997). This gene localization results in co-regulation of motility, which is highly important for H. pylori virulence, with the heat shock response.
Conclusions Entry of a pathogen into a warm-blooded host is usually accompanied by a shift-up in temperature. It is therefore expected that heat shock genes play an important role in bacterial infection, an assumption supported by the findings that following bacterial infections, sera of patients contain high titres of antibodies against heat-shock proteins. It is assumed that heat shock genes are induced upon infection in order to prevent misfolding and aggregation of damaged proteins, by refolding or proteolysis. These processes are probably essential for the survival of the pathogen within the host. Induction of heat shock genes is by no means limited to the host entry stage, and preliminary colonization. We see induction of some heat-shock genes at a later stage of infection, namely during growth of intracellular bacteria within macrophages. This probably reflects the role of heat-shock genes in protection of the bacterium against a variety of other stresses, such as oxidative stress or low pH. Since the temperature up-shift at host entry provides a distinct signal for the bacterium, it can be assumed that many genes required by the pathogen for adaptation to the host and for virulence would be regulated by heat shock proteins. However, to date, the number of virulence genes which fit this paradigm, is smaller than expected. This review describes several cases where heatshock genes are regulated by virulence factors. This co-regulation may be beneficial to the pathogen's survival, especially when heat shock genes are induced, since the onset of induction of toxins and other virulence factors often leads to an increase in temperature and oxidative stress within the host. Several experiments demonstrated that mutants in heat shock genes have reduced virulence. Some of these findings were taken as indications for the essential role of heat shock genes in pathogenesis. Yet, it should be remembered that mutations in certain heat shock genes severely impair the ability of bacteria to grow under any conditions and their effect on virulence is secondary. Since heat shock genes are essential for bacterial survival under a variety of stresses, it was often suggested that bacteria deficient in heat shock genes would be attenuated and could therefore be used as
live vaccines. However, it was found that in many cases the disruption of one heat shock gene is not enough to reduce virulence sufficiently enough to produce a safe vaccine. In order to construct good vaccine strains, one would have to ™knock out∫ several heat shock genes, while ensuring the antigenic potential is preserved. Development of such attenuated strains is not trivial, since some heat shock proteins are themselves antigenic and in addition, their loss may result in the reduced display of various antigenic determinants belonging to virulence proteins the expression of which is regulated by heat shock genes. Acknowledgements. This work was supported, in part, by the German Israeli Science Foundation (GIF), by the Manja and Morris Leigh Chair for Biophysics and Biotechnology, and by the Center of Emerging Diseases, Israel. U. Gophna was partially supported by the Wolf doctoral fellowship.
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