Streptolysin S induces proinflammatory cytokine expression in calcium ion-influx-dependent manner.

Anginosus group streptococci (AGS) are opportunistic pathogens that reside in the human oral cavity. The ß-hemolytic strains of Streptococcus anginosus subsp. anginosus (SAA) produce streptolysin S (SLS), a streptococcal peptide hemolysin. In recent clinical scenarios, AGS, including this species, have frequently been isolated from infections and disorders beyond those in the oral cavity. Consequently, investigating this situation will reveal the potential pathogenicity of AGS to ectopic infections in humans. However, the precise mechanism underlying the cellular response induced by secreted SLS and its relevance to the pathogenicity of AGS strains remain largely unknown. This study aims to elucidate the mechanism underlying the host cellular response of the human acute monocytic leukemia cell line THP-1 to secreted SLS. In THP-1 cells incubated with the culture supernatant of ß-hemolytic SAA containing SLS as the sole cytotoxic factor, increased Ca2+ influx and elevated expression of proinflammatory cytokines were observed. Significantly reduced expression of SLS-dependent upregulated cytokine genes under Ca2+-chelating conditions suggests that Ca2+ influx triggers SLS-dependent cellular responses. Furthermore, SLS-dependent enhanced expression of IL-8 was also implicated in the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. The findings presented in this study are crucial for a comprehensive understanding of the real pathogenicity of SLS-producing ß-hemolytic AGS in the latest clinical situations.

Streptolysin S induces pronounced calcium-ion influx-dependent expression of immediate early genes encoding transcription factors.

Anginosus group streptococci (AGS) are opportunistic human pathogens of the oral cavity. The ß-hemolytic subgroup of Streptococcus anginosus subsp. anginosus secretes streptolysin S (SLS) and exhibits not only hemolytic activity but also cytotoxicity toward cultured human cell lines. However, the detailed mechanism of action of SLS and the cellular responses of host cells have not yet been fully clarified. To determine the pathogenic potential of SLS-producing ß-hemolytic S. anginosus subsp. anginosus, the SLS-dependent response induced in the human oral squamous cell carcinoma HSC-2 cells was investigated to determine the pathogenic potential of SLS-producing ß-hemolytic S. anginosus subsp. anginosus. This study revealed that the Ca2+ influx and the expression of immediate early genes (IEGs) encoding transcription factors such as early growth responses (EGRs) and activator protein-1 (AP-1) were greatly increased in HSC-2 cells incubated with the culture supernatant of SLS-producing ß-hemolytic S. anginosus subsp. anginosus. Moreover, this SLS-dependent increase in expression was significantly suppressed by Ca2+ chelation, except for jun. These results suggest that SLS caused Ca2+ influx into the cells following greatly enhanced expression of IEG-encoding transcription factors. The results of this study may help in understanding the pathogenicity of SLS-producing AGS.

Recognizability of heterologous co-chaperones with Streptococcus intermedius DnaK and Escherichia coli DnaK.

Streptococcus intermedius DnaK complements the temperature-sensitive phenotype of an Escherichia coli dnaK null mutant only when co-chaperones DnaJ and GrpE are co-expressed. Therefore, whether S. intermedius DnaK and E. coli DnaK can recognize heterologous co-chaperones in vitro was examined. Addition of heterologous GrpE to DnaK and DnaJ partially stimulated adenosine triphosphatase (ATPase) activity, and almost completely stimulated luciferase refolding activity. Addition of heterologous DnaJ to GrpE and DnaK also stimulated ATPase activity; however, significant luciferase refolding activity was not observed. Moreover, E. coli DnaJ had a negative effect on the luciferase refolding activity of the S. intermedius DnaK chaperone system. In E. coli chaperone mutants, with the exception of E. coli DnaJ, stronger expression of the heterologous co-chaperones partially or almost completely complemented the temperature-sensitive-phenotype. These results indicate that all heterologous co-chaperones can at least partially recognize DnaK of a distantly related species. A region of the ATPase domain that is present in the DnaK of gram-negative bacteria is absent from the DnaK of gram-positive bacteria. This region is believed to be important for recognition of co-chaperones from gram-negative bacteria. However, insertion of this segment into S. intermedius DnaK failed to increase its ability to recognize E. coli co-chaperones, implying that this region is unnecessary or insufficient for the recognition of E. coli co-chaperones. Thus, our data suggest that a basic structural similarity is conserved among the components of the S. intermedius and E. coli DnaK chaperone systems, allowing weak associations between heterologous components.

Construction of Anti-HER2 Recombinants as Targeting Modules for a Drug-delivery System Against HER2-positive Cells.

BACKGROUND/AIM: Recombinant antibodies have been investigated and used in applications such as targeting modules of drug-delivery systems (DDS) against cancers. This study aimed to prepare recombinant antibodies against HER2, containing sortase A (SrtA) recognition sequence, that are applicable as targeting modules in DDS after linkage with the drug-carrier containing oligoglycine-acceptor peptide by SrtA transpeptidation. MATERIALS AND METHODS: The recombinant trastuzumab fragment antibodies (scFvs and Fab) with the SrtA-recognition motif (LPXTG) at their C-terminal were constructed and expressed in Escherichia coli and Chinese hamster ovary (CHO) cells, respectively. The reactivity of the purified recombinant antibodies towards HER2-expressing cells was also evaluated via immunofluorescent staining. RESULTS: Fab demonstrated higher yield and purity and better reactivity towards HER2-expressing cells (HCT-15 and HeLa) when compared to scFvs. CONCLUSION: The CHO expression system possesses superior yield and purity when compared to the E. coli expression system with respect to the preparation of recombinant antibodies applicable in targeting modules for DDS (DDS-TM). Moreover, a Fab variant prepared in this study demonstrated the potential to be a DDS-TM against HER2-expressing cancer cells.

Positive- and Negative-Control Pathways by Blood Components for Intermedilysin Production in Streptococcus intermedius.

Streptococcus intermedius is an opportunistic bacterial pathogen secreting a human-specific cytolysin called intermedilysin (ILY) as a major pathogenic factor. This bacterium can degrade glycans into monosaccharides using two glycosidases, multisubstrate glycosidase A (MsgA) and neuraminidase (NanA). Here, we detected a stronger hemolytic activity mediated by ILY when S. intermedius PC574 was cultured in fetal bovine serum (FBS) than when it was grown in the standard culture medium. FBS-cultured cells also showed higher MsgA and NanA activity, although overproduction of ILY in FBS was undetectable in mutants nanA-null and msgA-null. Addition of purified MsgA and NanA to the FBS resulted in a release of 2.8 mM galactose and 4.3 mM N-acetylneuraminic acid; these sugar concentrations were sufficient to upregulate the expression of ILY, MsgA, and NanA. Conversely, when strain PC574 was cultured in human plasma, no similar increase in hemolytic activity was observed. Moreover, addition of human plasma to the culture in FBS appeared to inhibit the stimulatory effect of FBS on ILY, MsgA, and NanA, although there were individual differences among the plasma samples. We confirmed that human plasma contains immunoglobulins that can neutralize ILY, MsgA, and NanA activities. In addition, human plasma had a neutralizing effect on cytotoxicity of S. intermedius toward HepG2 cells in FBS, and a higher concentration of human plasma was necessary to reduce the cytotoxicity of an ILY-high-producing strain than an ILY-low-producing strain. Overall, our data show that blood contains factors that stimulate and inhibit ILY expression and activity, which may affect pathogenicity of S. intermedius.

Development of a Sortase A-mediated Peptide-labeled Liposome Applicable to Drug-delivery Systems.

BACKGROUND/AIM: In order to develop an efficient drug-delivery system (DDS), a lipopeptide-loaded liposome that functions as a platform for the transpeptidase reaction mediated by sortase A (SrtA) was constructed and its stability, as well as cell-specific targeting were evaluated in the present study. MATERIALS AND METHODS: Several lipopeptides possessing an acceptor peptide sequence (oligoglycine ≥ three residues) or donor peptide sequence (LPETG) for the SrtA-mediated reaction were chemically synthesized and then inserted into the liposome membrane composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (DPPC-Chol-lipo) to obtain the lipopeptide-loaded liposomes. The transpeptidase reaction mediated by recombinant SrtA (His-ΔN59SrtA) was employed to modify the peptide moiety on the liposomal surface using a fluorescently-labeled substrate peptide corresponding to the species of each loaded lipopeptide. Furthermore, lung tumor-binding peptide (LTBP)-labeled liposomes, prepared by this transpeptidase reaction, were investigated for selective targeting to lung cancer cells in vitro. RESULTS AND DISCUSSION: The His-ΔN59SrtA-mediated transpeptidation of fluorescently-labeled peptide on the lipopeptide-loaded DPPC-Chol-lipo was confirmed. The selective targeting of LTBP-labeled liposomes to the lung cancer cell line A549 was also observed in vitro. These results suggest that the labeling of acceptor or donor lipopeptide-loaded liposomes with the transpeptidase SrtA could be a useful method for developing a platform applicable to a cancer-targeting DDS.

Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells.

Estrogenic compounds include endogenous estrogens such as estradiol as well as soybean isoflavones, such as daidzein and its metabolite equol, which are known phytoestrogens that prevent osteoporosis in postmenopausal women. Indeed, mineralization of MC3T3-E1 cells, a murine osteoblastic cell line, was significantly decreased in medium containing fetal bovine serum treated with charcoal-dextran to deplete endogenous estrogens, but estradiol and these soybean isoflavones dose-dependently restored the differentiation of MC3T3-E1 cells; equol was tenfold more effective than daidzein. These differentiation-promoting effects were inhibited by the addition of fulvestrant, which is a selective downregulator of estrogen receptors. Analysis of the expression pattern of bone-related genes by reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qRT-PCR), which focused on responsiveness to the estrogen stimuli, revealed that the transcription of PACE4, a subtilisin-like proprotein convertase, was tightly linked with the differentiation of MC3T3-E1 cells induced by estrogen stimuli. Moreover, treatment with RNAi of PACE4 in MC3T3-E1 cells resulted in a drastic decrease of mineralization in the presence of estrogen stimuli. These results strongly suggest that PACE4 participates in bone formation at least in osteoblast differentiation, and estrogen receptor-mediated stimuli induce osteoblast differentiation through the upregulation of PACE4 expression.

Investigation on the reaction conditions of Staphylococcus aureus sortase A for creating surface-modified liposomes as a drug-delivery system tool.

BACKGROUND/AIM: This study aimed to determine the preferred conditions for the transpeptidase reaction of sortase A from Staphylococcus aureus, for the purpose of creating functional liposomes useful for a drug-delivery system (DDS). MATERIALS AND METHODS: His-tagged recombinant sortase A with 59 amino acids deleted from the N-terminus (His-ΔN59SrtA) was prepared using an Escherichia coli expression system. The pH dependency and sorting signal sequence dependency of the transpeptidase reaction of His-ΔN59SrtA were analyzed by monitoring the transfer of model donor-substrates (i.e. His-tagged mutant green fluorescent proteins with a C-terminal LPxTG sorting signal) to model acceptor-beads with a GGGGGC peptide. In addition, using preferred conditions, the sortase A reaction was used to modify liposome surface. RESULTS AND DISCUSSION: The transpeptidase reaction of His-ΔN59SrtA was enhanced under weakly acidic conditions. Transfer efficiency, based on sorting signal recognition by His-ΔN59SrtA, was similar to or higher than that obtained using several substrates with amino acids other than Glu in the sorting signal position "x". Furthermore, liposomes containing GGGGGC peptide-linked dipalmitoylphosphatidylethanolamine were successfully modified using the preferred conditions for His-ΔN59SrtA determined in this study. CONCLUSION: Preferred conditions for the transpeptidase reaction of His-ΔN59SrtA, especially in a weakly acidic environment to enhance reaction, was established and successfully used to create functional liposomes applicable to DDS.

LacR mutations are frequently observed in Streptococcus intermedius and are responsible for increased intermedilysin production and virulence.

Streptococcus intermedius secretes a human-specific cytolysin, intermedilysin (ILY), which is considered to be the major virulence factor of this pathogen. We screened for a repressor of ily expression by using random gene disruption in a low-ILY-producing strain (PC574). Three independent high-ILY-producing colonies that had plasmid insertions within a gene that has high homology to lacR were isolated. Validation of these observations was achieved through disruption of lacR in strain PC574 with an erythromycin cassette, which also led to higher hemolytic activity, increased transcription of ily, and higher cytotoxicity against HepG2 cells, compared to the parental strain. The direct binding of LacR within the ily promoter region was shown by a biotinylated DNA probe pulldown assay, and the amount of ILY secreted into the culture supernatant by PC574 cells was increased by adding lactose or galactose to the medium as a carbon source. Furthermore, we examined lacR nucleotide sequences and the hemolytic activity of 50 strains isolated from clinical infections and 7 strains isolated from dental plaque. Of the 50 strains isolated from infections, 13 showed high ILY production, 11 of these 13 strains had one or more point mutations and/or an insertion mutation in LacR, and almost all mutations were associated with a marked decline in LacR function. These results strongly suggest that mutation in lacR is required for the overproduction of ILY, which is associated with an increase in pathogenicity of S. intermedius.

Construction of an improved drug delivery system tool with enhanced versatility in cell-targeting.

BACKGROUND/AIM: The aim of this study was to develop an improved drug delivery system (DDS) tool with enhanced versatility in the cell-targeting step using as Z-domain, a modified IgG binding domain of protein A from Staphylococcus aureus, as an IgG adapter domain. MATERIALS AND METHODS: The chimera protein expression system composed of the Z-domain and chimeric cholesterol-dependent cytolysin mutant named His-Z-CDC(ss)(IS) was constructed in Escherichia coli. His-Z-CDC(ss)(IS) was purified by Ni-affinity chromatography, and its abilities for controlled pore formation, membrane binding, IgG binding, and target cell-specific delivery of liposomes carrying medicine were investigated. RESULTS AND DISCUSSION: His-Z-CDC(ss)(IS) purified by Ni-affinity chromatography indicated pore-forming activity only under disulfide bond reducing conditions. His-Z-CDC(ss)(IS) also demonstrated an ability to bind both IgG and cholesterol-embedded liposomes via its Z-domain and domain 4, respectively. Furthermore, anticarcinoembryonic antigen (CEA) IgG-bound His-Z-CDC(ss)(IS) indicated effective delivery of liposomes carrying drugs to CEA-expressing cells. CONCLUSION: His-Z-CDC(ss)(IS) was revealed to be an improved DDS tool with enhanced versatility in cell targeting.

Investigation of a bacterial pore-forming chimera toxin for application as a novel drug-delivery system tool.

BACKGROUND/AIM: Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins from Gram-positive bacteria. The aim of this study was to investigate the potential of a CDC, intermedilysin, as a drug-delivery system (DDS) for clinical application. MATERIALS AND METHODS: Intermedilysin was modified by the addition of a disulfide bridge to regulate pore formation, by swapping domain 4 to provide cholesterol-binding capacity, and by the introduction of a targeting domain. The resultant chimera protein, His-LTBP-CDC(ss)(IP), was investigated for its use as a DDS tool in vitro. RESULTS: His-LTBP-CDC(ss)(IP) exhibited a regulated pore-forming capacity under reducing conditions. This chimera protein was able to deliver a drug-carrier liposome specifically to the target cell, to be endocytosed into the cell with subsequent release of the components into the cytoplasm. CONCLUSION: A chimera protein derived from the bacterial pore-forming toxin intermedilysin (His-LTBP-CDC(ss)(IP)) forms the basis for a novel DDS tool.

Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity.

Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that causes purulent infections and abscess formation. The DnaK chaperone system has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity. However, the role of DnaK in S. intermedius remains unclear. Therefore, we constructed a dnaK knockout mutant that exhibited slow growth, thermosensitivity, accumulation of GroEL in the cell, and reduced cytotoxicity to HepG2 cells. The level of secretion of a major pathogenic factor, intermedilysin, was not affected by dnaK mutation. We further examined the function and property of the S. intermedius DnaK chaperone system by using Escherichia coli ΔdnaK and ΔrpoH mutant strains. S. intermedius DnaK could not complement the thermosensitivity of E. coli ΔdnaK mutant. However, the intact S. intermedius DnaK chaperone system could complement the thermosensitivity and acid sensitivity of E. coli ΔdnaK mutant. The S. intermedius DnaK chaperone system could regulate the activity and stability of the heat shock transcription factor σ(32) in E. coli, although S. intermedius does not utilize σ(32) for heat shock transcription. The S. intermedius DnaK chaperone system was also able to efficiently eliminate the aggregated proteins from ΔrpoH mutant cells. Overall, our data showed that the S. intermedius DnaK chaperone system has important functions in quality control of cellular proteins but has less participation in the modulation of expression of pathogenic factors.

The DnaK chaperone machinery converts the native FlhD2C2 hetero-tetramer into a functional transcriptional regulator of flagellar regulon expression in Salmonella.

The DnaK chaperone binds non-specifically to many unfolded polypeptides and also binds selectively to specific substrates. Although its involvement in targeting the unfolded polypeptides to assist proper folding is well documented, less is known about its role in targeting the folded polypeptides. We demonstrate that DnaK regulates the expression of the Salmonella flagellar regulon by modulating the FlhD and FlhC proteins, which function as master regulators at the apex of a transcription hierarchy comprising three classes of genes. FlhD and FlhC form an FlhD2C2 complex that activates sigma70 promoter of class 2 genes. In DeltadnaK cells, FlhD and FlhC proteins seemed to be assembled into hetero-tetrameric FlhD2C2 but the complex was not fully active in class 2 gene transcription, suggesting that the DnaK chaperone is involved in activating native FlhD2C2 complex into a regulator of flagellar regulon expression. This is the first time that involvement of the DnaK chaperone machinery in activating folded oligomerized proteins has been demonstrated.

Derepression of Salmonella pathogenicity island 1 genes within macrophages leads to rapid apoptosis via caspase-1- and caspase-3-dependent pathways.

Salmonella enterica serovar Typhimurium has been reported to induce apoptosis in infected macrophages within 14 h from the time of infection by a caspase-1-dependent mechanism. Here, we demonstrate that depletion of Lon protease in serovar Typhimurium induces rapid and massive apoptosis in macrophages by a mechanism involving both caspases-1 and -3. This excessive induction of apoptosis was abrogated by disruption of invF, which is required for the expression of the Salmonella pathogenicity island 1 (SPI1) genes. Expression of hilA, a central regulator of SPI1 transcription, was repressed in the macrophages after phagocytosis, but this gene was continuously expressed when the DeltaLon mutant grew within the macrophages, so the SPI1 proteins accumulated. Thus, the increase in macrophage apoptosis induced by the DeltaLon mutant could result from continued expression of SPI1 genes under conditions where they are normally repressed. Once Salmonella has established a systemic infection, excess apoptosis of macrophages cells upon which the organism is reliant would be detrimental to the pathogen. Therefore, the Lon protease may be required to suppress apoptosis sufficiently to allow time for the bacterium to replicate, escape and invade new macrophages.

The DnaK/DnaJ chaperone machinery of Salmonella enterica serovar Typhimurium is essential for invasion of epithelial cells and survival within macrophages, leading to systemic infection.

Salmonella enterica serovar Typhimurium, similar to various facultative intracellular pathogens, has been shown to respond to the hostile conditions inside macrophages of the host organism by inducing stress proteins, such as DnaK. DnaK forms a chaperone machinery with the cochaperones DnaJ and GrpE. To elucidate the role of the DnaK chaperone machinery in the pathogenesis of S. enterica serovar Typhimurium, we first constructed an insertional mutation in the dnaK-dnaJ operon of pathogenic strain chi3306. The DnaK/DnaJ-depleted mutant was temperature sensitive for growth, that is, nonviable above 39 degrees C. We then isolated a spontaneously occurring revertant of the dnaK-dnaJ-disrupted mutant at 39 degrees C and used it for infection of mice. The mutant lost the ability to cause a lethal systemic disease in mice. The impaired ability for virulence was restored when a functional copy of the dnaK-dnaJ operon was provided, suggesting that the DnaK/DnaJ chaperone machinery is required by Salmonella for the systemic infection of mice. This result also indicates that with respect to the DnaK/DnaJ chaperone machinery, the cellular requirements for growth at a high temperature are not identical to the cellular requirements for the pathogenesis of Salmonella. Macrophage survival assays revealed that the DnaK/DnaJ-depleted mutant could not survive or proliferate at all within macrophages. Of further interest are the findings that the mutant could neither invade cultured epithelial cells nor secrete any of the invasion proteins encoded within Salmonella pathogenicity island 1. This is the first time that the DnaK/DnaJ chaperone machinery has been shown to be involved in bacterial invasion of epithelial cells.

Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones.

BACKGROUND: It is well known that expression of certain bacterial genes responds rapidly to such stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins encoded in these genes are important for successful survival of the organism under the hostile conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed to affect the organisms' susceptibility to these agents. RESULTS: We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX), a fluoroquinolone (FQ), induce the syntheses of heat shock proteins and RecA. To examine whether the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK, groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The increased susceptibility of the lon mutant was corroborated by experiments in which the gene encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the depletion of Lon and the continuous induction of the SOS response in the presence of FQs. CONCLUSION: The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.

Turnover of FlhD and FlhC, master regulator proteins for Salmonella flagellum biogenesis, by the ATP-dependent ClpXP protease.

In enterobacteria such as Salmonella, flagellar biogenesis is dependent upon the master operon flhDC at the apex of the flagellar gene hierarchy, which is divided into three classes 1, 2 and 3. Previously we reported that depletion of the ClpXP ATP-dependent protease results in dramatic enhancement of class 2 and class 3 gene transcription, whereas the transcription level of the flhDC operon remains normal in Salmonella enterica serovar Typhimurium. This suggests that the ClpXP protease may be responsible for the turnover of the FlhD and FlhC master regulators (Tomoyasu, T., Ohkishi, T., Ukyo, Y., Tokumitsu, A., Takaya, A., Suzuki, M. et al., 2002, J Bacteriol 184:645-653). In this study, to establish the role of the ClpXP protease in the turnover of FlhD and FlhC proteins, we analysed levels of the FlhD and FlhC proteins in wild-type and ClpXP mutant cells using anti-FlhD and anti-FlhC antibodies. The results show that both FlhD and FlhC proteins are markedly accumulated in ClpXP mutant cells and the half-life of FlhC is approximately fivefold longer in the ClpXP mutant, suggesting that the ClpXP protease is responsible for the degradation of FlhD and FlhC. The results also show that the ClpXP protease degrades both proteins in FlhD2FlhC2 complex but does not seem to recognize the respective subunits synthesized individually. Taken together, it is suggested that the cellular concentration of the FlhD2FlhC2 master regulator is tightly controlled at the post-translational level by the ClpXP protease. We also examined the role of other members of the ATP-dependent protease family in the regulation of flagellar biogenesis and concluded that only ClpXP in this family functions as a negative regulator for flagellar biogenesis in Salmonella.

Lon, a stress-induced ATP-dependent protease, is critically important for systemic Salmonella enterica serovar typhimurium infection of mice.

Studies on the pathogenesis of Salmonella enterica serovar Typhimurium infections in mice have revealed the presence of two prominent virulence characteristics-the invasion of the nonphagocytic cells to penetrate the intestinal epithelium and the proliferation within host phagocytic cells to cause a systemic spread and the colonization of host organs. We have recently demonstrated that the ATP-dependent Lon protease of S. enterica serovar Typhimurium negatively regulates the efficiency of invasion of epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). This study was performed to reveal the contribution of the Lon protease to the virulence of S. enterica serovar Typhimurium in mice. Determination of 50% lethal doses for the lon disruption mutant and wild-type strain revealed that the mutant was highly attenuated when administered either orally or intraperitoneally to BALB/c mice. The mutant was also found to be able to reach extraintestinal sites but unable to proliferate efficiently within the spleen and cause lethal systemic disease of mice. Macrophage survival assays revealed that the lon disruption mutant could not survive or proliferate within murine macrophages. In addition, the mutant showed extremely increased susceptibility to hydrogen peroxide, which contributes to the bactericidal capacity of phagocytes. The mutant also showed increased sensitivity to acidic conditions. Taken together, the impaired ability of the lon disruption mutant to survive and grow in macrophages could be due to the enhanced susceptibility to the oxygen-dependent killing mechanism associated with respiratory burst and the low phagosomal pH. These results suggest that the Lon protease is essentially involved in the systemic infection of mice with S. enterica serovar Typhimurium, which can be fatal. Of further interest is the finding that the lon disruption mutant persists in the BALB/c mice for long periods without causing an overwhelming systemic infection.

Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium.

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.