Transposon mutagenesis of probiotic Lactobacillus casei identifies asnH, an asparagine synthetase gene involved in its immune-activating capacity.

Lactobacillus casei ATCC 27139 enhances host innate immunity, and the J1 phage-resistant mutants of this strain lose the activity. A transposon insertion mutant library of L. casei ATCC 27139 was constructed, and nine J1 phage-resistant mutants out of them were obtained. Cloning and sequencing analyses identified three independent genes that were disrupted by insertion of the transposon element: asnH, encoding asparagine synthetase, and dnaJ and dnaK, encoding the molecular chaperones DnaJ and DnaK, respectively. Using an in vivo mouse model of Listeria infection, only asnH mutant showed deficiency in their ability to enhance host innate immunity, and complementation of the mutation by introduction of the wild-type asnH in the mutant strain recovered the immuno-augmenting activity. AsnH protein exhibited asparagine synthetase activity when the lysozyme-treated cell wall extracts of L. casei ATCC 27139 was added as substrate. The asnH mutants lost the thick and rigid peptidoglycan features that are characteristic to the wild-type cells, indicating that AsnH of L. casei is involved in peptidoglycan biosynthesis. These results indicate that asnH is required for the construction of the peptidoglycan composition involved in the immune-activating capacity of L. casei ATCC 27139.

Salmonella type III effector SpvC, a phosphothreonine lyase, contributes to reduction in inflammatory response during intestinal phase of infection.

Salmonella phosphothreonine lyase SpvC inactivates the dual-phosphorylated host mitogen-activated protein kinases (MAPK) through ß-elimination. While SpvC can be secreted in vitro by both Salmonella pathogenicity island (SPI)-1 and SPI-2 type III secretion systems (T3SSs), translocation of this protein into the host cell cytosol has only been demonstrated by SPI-2 T3SS. In this study, we show that SpvC can be delivered into the host cell cytoplasm by both SPI-1 and SPI-2 T3SSs. Dephosphorylation of the extracellular signal-regulated protein kinases (ERK) was detected in an SPI-1 T3SS-dependent manner 2 h post infection. Using a mouse model for Salmonella enterocolitis, which was treated with streptomycin prior to infection, we observed that mice infected with Salmonella enterica serovar Typhimurium strains lacking the spvC gene showed pronounced colitis when compared with mice infected with the wild-type strain 1 day after infection. The effect of SpvC on the development of colitis was characterized by reduced mRNA levels of the pro-inflammatory cytokines and chemokines, and reduced inflammation with less infiltration of neutrophils. Furthermore, the reduction in inflammation by SpvC resulted in increased bacterial dissemination in spleen of mice infected with Salmonella. Collectively, our findings suggest that SpvC exerts as an anti-inflammatory effector and the attenuation of intestinal inflammatory response by SpvC is involved in systemic infection of Salmonella.

Evaluation of Salmonella enterica serovar Typhimurium and Choleraesuis slyA mutant strains for use in live attenuated oral vaccines.

SlyA protein plays a key role in virulence in Salmonella enterica. In this study, we evaluated the ability of the slyA mutant strain of S. enterica serovar Choleraesuis (S. choleraesuis) to protect against swine salmonellosis. Using a murine model infected with S. enterica serovar Typhimurium (S. typhimurium), we showed that the Salmonella strain with a deletion of slyA could be used as a highly immunogenic, effective and safe vaccine in mice. Based on these data, a slyA mutant of S. enterica serovar Choleraesuis strain RF-1 was constructed, and the ability of this mutant to protect immunized pigs from S. choleraesuis infection was examined. As with the S. typhimurium slyA mutant, immunization of pigs with the S. choleraesuis slyA mutant strain provided significant protection against subsequent challenge by the wild-type RF-1. These results demonstrate that SlyA is a potential target in the development of a novel live attenuated vaccine against S. enterica.

The Chromobacterium violaceum type III effector CopE, a guanine nucleotide exchange factor for Rac1 and Cdc42, is involved in bacterial invasion of epithelial cells and pathogenesis.

The type III secretion system (T3SS) encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a) is critical for Chromobacterium violaceum pathogenesis. T3SS-dependent virulence is commonly characterized by type III effector virulence function, but the full repertoire of the effector proteins of Cpi-1/-1a T3SS is unknown. In this study, we showed that expression of Cpi-1/-1a T3SS is controlled by the master regulator CilA. We used transcriptional profiling with DNA microarrays to define CilA regulon and identified genes encoding T3SS effectors whose translocation into host cells was dependent on Cpi-1/-1a T3SS. From these effectors, we found that CopE (CV0296) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases in its C-terminal portion. The N-terminal portions (1-81 amino acids) of CopE and a CivB as a putative chaperone were required for its translocation. CopE specifically activates Rac1 and Cdc42 followed by the induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades human epithelial HeLa cells in a Cpi-1/-1a-encoded T3SS- and CopE-dependent manner. Finally, C. violaceum strains lacking copE and expressing a CopE-G168V deficient in GEF activity were attenuated for virulence in mice, suggesting that CopE contributes to the virulence of this pathogen.

Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages.

BACKGROUND: The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium), several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. RESULTS: Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. CONCLUSIONS: A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.

Oral administration of heat-killed Lactobacillus plantarum strain b240 protected mice against Salmonella enterica Serovar Typhimurium.

The purpose of this study was to investigate the effects of heat-killed Lactobacillus plantarum strain b240 (b240) on systemic infection by Salmonella enterica serovar Typhimurium (S. Typhimurium) and to determine the mechanism by which b240 protects against infection. Mice were administered either b240 or saline orally for 3 weeks, and then inoculated with S. Typhimurium. The mice treated with b240 were significantly protected against S. Typhimurium as compared to those fed saline. Moreover, translocation of S. Typhimurium into each organ tested in the mice that received b240 tended to be less than in the control mice. An important mechanism of protection against infection was demonstrated by the ability of b240 to inhibit both binding by and invasion of S. Typhimurium into cells. These results indicate that nonviable lactic acid bacteria also play important roles in preventing infection by enteric pathogens.

Chromobacterium pathogenicity island 1 type III secretion system is a major virulence determinant for Chromobacterium violaceum-induced cell death in hepatocytes.

Chromobacterium violaceum is a Gram-negative bacterium that causes fatal septicaemia in humans and animals. C. violaceum ATCC 12472 possesses genes associated with two distinct type III secretion systems (T3SSs). One of these systems is encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi-1/-1a), another is encoded by Chromobacterium pathogenicity island 2 (Cpi-2). Here we show that C. violaceum causes fulminant hepatitis in a mouse infection model, and Cpi-1/-1a-encoded T3SS is required for its virulence. In addition, using C. violaceum strains with defined mutations in the genes that encode the Cpi-1/-1a or Cpi-2 locus in combination with cultured mammalian cell lines, we found that C. violaceum is able to induce cytotoxicity in a Cpi-1/-1a-dependent manner. Characterization of Chromobacterium-induced cytotoxicity revealed that cell lysis by C. violaceum infection involves the formation of pore structures on the host cell membrane, as demonstrated by protection by cytotoxicity in the presence of osmoprotectants. Finally, we demonstrated that CipB, a Cpi-1/-1a effector, is implicated in translocator-mediated pore formation and the ability of CipB to form a pore is essential for Chromobacterium-induced cytotoxicity. These results strongly suggest that Cpi-1/-1a-encoded T3SS is a virulence determinant that causes fatal infection by the induction of cell death in hepatocytes.

Phytohabitans suffuscus gen. nov., sp. nov., an actinomycete of the family Micromonosporaceae isolated from plant roots.

An actinomycete strain, K07-0523(T), was isolated from the roots of an orchid collected in Okinawa prefecture, Japan. 16S rRNA gene sequence analysis indicated that the new strain belonged to the family Micromonosporaceae and the similarity values between strain K07-0523(T) and the type species of 24 genera in the family Micromonosporaceae were 93.3-97.7 %. Strain K07-0523(T) contained D-glutamic acid, glycine, D-alanine, meso-diaminopimelic acid, hydroxydiaminopimelic acid and L-lysine in the cell wall. The major menaquinones were MK-9(H(6)), MK-10(H(4)) and MK-10(H(6)). Galactose, glucose, mannose, ribose and xylose were present in the whole-cell sugars. The acyl type of the peptidoglycan was glycolyl. Major fatty acids were anteiso-C(17 : 0), iso-C(17 : 0), iso-C(16 : 0) and iso-C(15 : 0). Phosphatidylethanolamine was detected as the major phospholipid and corresponded to phospholipid type II. The G+C content of the genomic DNA was 73 mol%. On the basis of phylogenetic and chemotaxonomic data, the new strain represents a member of a new genus and novel species, namely Phytohabitans suffuscus gen. nov., sp. nov., in the family Micromonosporaceae. The type strain of the type species is K07-0523(T) (=DSM 45306(T)=NBRC 105367(T)).

Functional characterization of SsaE, a novel chaperone protein of the type III secretion system encoded by Salmonella pathogenicity island 2.

The type III secretion system (T3SS) encoded by Salmonella pathogenicity island 2 (SPI-2) is involved in systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. In this study, we investigated the function of SsaE, a small cytoplasmic protein encoded within the SPI-2 locus, which shows structural similarity to the T3SS class V chaperones. An S. enterica serovar Typhimurium ssaE mutant failed to secrete SPI-2 translocator SseB and SPI-2-dependent effector PipB proteins. Coimmunoprecipitation and mass spectrometry analyses using an SsaE-FLAG fusion protein indicated that SsaE interacts with SseB and a putative T3SS-associated ATPase, SsaN. A series of deleted and point-mutated SsaE-FLAG fusion proteins revealed that the C-terminal coiled-coil domain of SsaE is critical for protein-protein interactions. Although SseA was reported to be a chaperone for SseB and to be required for its secretion and stability in the bacterial cytoplasm, an sseA deletion mutant was able to secrete the SseB in vitro when plasmid-derived SseB was overexpressed. In contrast, ssaE mutant strains could not transport SseB extracellularly under the same assay conditions. In addition, an ssaE(I55G) point-mutated strain that expresses the SsaE derivative lacking the ability to form a C-terminal coiled-coil structure showed attenuated virulence comparable to that of an SPI-2 T3SS null mutant, suggesting that the coiled-coil interaction of SsaE is absolutely essential for the functional SPI-2 T3SS and for Salmonella virulence. Based on these findings, we propose that SsaE recognizes translocator SseB and controls its secretion via SPI-2 type III secretion machinery.

Genome-wide identification of novel genomic islands that contribute to Salmonella virulence in mouse systemic infection.

Salmonella pathogenicity islands are inserted into the genome by horizontal gene transfer and are required for expression of full virulence. Here, we performed tRNA scanning of the genome of Salmonella enterica serovar Typhimurium and compared it with that of nonpathogenic Escherichia coli in order to identify genomic islands that contribute to Salmonella virulence. Using deletion analysis, we identified four genomic islands that are required for virulence in the mouse infection model. One of the newly identified pathogenicity islands was the pheV-tRNA-located genomic island, which is comprised of 26 126 bp, and encodes 22 putative genes, including STM3117-STM3138. We also showed that the pheV tRNA-located genomic island is widely distributed among different nontyphoid Salmonella serovars. Furthermore, genes including STM3118-STM3121 were identified as novel virulence-associated genes within the pheV-tRNA-located genomic island. These results indicate that a Salmonella-specific pheV-tRNA genomic island is involved in Salmonella pathogenesis among the nontyphoid Salmonella serovars.

DsbA directs efficient expression of outer membrane secretin EscC of the enteropathogenic Escherichia coli type III secretion apparatus.

The formation of disulfide bond is essential for the folding, activity, and stability of many secreted proteins of Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into exported proteins from the cytoplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we investigated the role of DsbA in enteropathogenic Escherichia coli. Here, we show that the DsbA is required for stable expression of outer membrane secretin EscC. DsbA has no effect on LEE transcription as measured with LEE-lacZ fusions. Replacement of either cysteine residue 136 or 155 of EscC with a serine resulted in reduced level of EscC, similar to the effect of the dsbA mutation. These results demonstrate the role of DsbA in assembly of the type III secretion apparatus.

Identification of amino acid residues of Salmonella SlyA that are critical for transcriptional regulation.

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2) is essential for the intracellular survival and replication of Salmonella enterica. The expression of SPI-2 genes is dependent on a two-component regulatory system, SsrA (SpiR)/SsrB, encoded in the SPI-2 region. This paper shows that SlyA regulates transcription of the sensor kinase SsrA by binding to the ssrA promoter, indicating that SlyA is directly involved in the regulation of SPI-2 gene expression. A structure model of the SlyA dimer in complex with DNA was constructed. The model of SlyA indicated that its structure is very similar to that of other MarR family proteins. Based on this model, site-directed mutagenesis of residues located in the winged-helix region required for DNA binding and in the alpha-helices of the N-terminal and C-terminal regions required for dimerization of the SlyA protein was performed to identify the residues that are critical for SlyA function. Nine mutants of SlyA with single substitutions were unable to activate ssrA transcription in vivo. These mutant SlyA proteins revealed that the residues Leu-63, Val-64, Arg-65, Leu-67, Leu-70, Arg-86 and Lys-88 within the winged-helix region are required for DNA binding, and residues Leu-12 and Leu-126 within the alpha-helices of the N-terminal and C-terminal regions are required for efficient dimer formation. A Salmonella slyA mutant strain carrying a plasmid expressing SlyA derivatives containing mutations at these amino acid positions did not exhibit restored SlyA function in infected HeLa cells, thereby confirming the structural and functional relationships of the SlyA protein.

Probiotic Lactobacillus casei activates innate immunity via NF-kappaB and p38 MAP kinase signaling pathways.

Probiotic bacteria are microorganisms that benefit the host through improvement of the balance of intestinal microflora and possibly by augmentation of host defense systems. We examined the mechanisms for the up-regulation of innate immune responses by a probiotic Lactobacillus casei ATCC27139, in vivo. Using mouse models of systemic Listeria monocytogenes infection and MethA fibrosarcoma tumorigenesis in combination with BALB/c and SCID mice, we found that parenteral administration of L. casei ATCC27139 confers a protective effect against L. monocytogenes infection and anti-tumor activity against MethA fibrosarcoma by activation of innate immunity, while L. casei ATCC27139-J1R strains, which are J1 phage-resistant strains that have been selected from MNNG-treated clones, lacked these activities. Substantial differences between ATCC27139 and ATCC27139-J1R strains were observed in the capacity to induce innate cytokines such as TNF-alpha, IL-12, IL-18, and IFN-gamma, and pathogen-associated molecular pattern receptors, TLR2 and Nod2, by spleen cells. In addition, although phosphorylation of NF-kappaB p65 in spleen was equally enhanced in the ATCC27139- and the ATCC27139-J1R-treated groups, phosphorylation of both p38 MAPK and MAPKAPK-2 was significantly induced only by ATCC27139. Furthermore, inhibitors of NF-kappaB (sulfasalazine) and p38 MAPK (SB203580) significantly reduced cytokine production by the spleen cells of the mice treated with L. casei ATCC27139, suggesting that both NF-kappaB and p38 MAPK signaling pathways play important roles in the augmentation of innate immunity by the probiotic L. casei.

Identification of the outer-membrane protein PagC required for the serum resistance phenotype in Salmonella enterica serovar Choleraesuis.

Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteraemia, by many pathogenic bacteria. Salmonella enterica serovar Choleraesuis is an important enteric pathogen that causes serious systemic infections in swine and humans. Here, it was found that, when introduced into Escherichia coli, a recombinant plasmid carrying the pagC gene from a plasmid-based genomic library of S. enterica serovar Choleraesuis conferred a high-level resistance to the bactericidal activity of pooled normal swine serum. The resistance was equal to the level conferred by rck, a gene encoding a 17 kDa outer-membrane protein which promotes the serum resistance phenotype in S. enterica serovar Typhimurium. Insertional mutagenesis of the cloned pagC gene generated a mutation that resulted in the loss of the serum resistance phenotype in E. coli. When this mutation was introduced into the chromosome of S. enterica serovar Choleraesuis by homology recombination with the wild-type allele, the resulting strain could not produce PagC, and it showed a decreased level of resistance to complement-mediated killing. The mutation could be restored by introduction of the intact pagC gene on a plasmid, but not by introduction of the point-mutated pagC gene. In addition, PagC was able to promote serum resistance in the S. enterica serovar Choleraesuis LPS mutant strain, which is highly sensitive to serum killing. Although PagC is not thought to confer serum resistance directly, these results strongly suggest that PagC is an important outer-membrane protein that plays an important role in the serum resistance of S. enterica serovar Choleraesuis.

Sequence analysis and characterization of sulfonamide resistance plasmid pRF-1 from Salmonella enterica serovar Choleraesuis.

The nucleotide sequence of a small plasmid, designated pRF-1, isolated from Salmonella enterica serovar Choleraesuis, was determined. We identified seven open reading frames (ORFs) encoded by 6066 nucleotides with a total G + C content of 53.6%. Analysis of the complete nucleotide sequence revealed a replicon of pRF-1 to have high similarity to the p15A origin of replication, with a possible cer-like region. ORF1, which is composed of 816 nucleotides, shows a high degree of similarity to dihydropteroate synthetase encoded by the sulII gene from plasmids in several enteropathogenic bacteria, which functions as the sulfonamide resistance determinant. In fact, Salmonella and Escherichia coli strains carrying pRF-1 were found to show strong resistance to sulfathiazole, suggesting that orf1 is a functional gene. Four of seven ORFs were found to encode putative proteins of unknown function.

Characterization of Salmonella pathogenicity island 1 type III secretion-dependent hemolytic activity in Salmonella enterica serovar Typhimurium.

A number of bacteria that are pathogenic for animals and plants possess a type III secretion system (TTSS) to translocate virulence-associated proteins into host cells. In several bacteria, it has been reported that the TTSS is correlated with an ability to cause contact-dependent hemolysis in vitro. Here, we showed that the Salmonella enterica serovar Typhimurium strain SL1344 exhibited Salmonella pathogenicity island 1 type III secretion-dependent, contact-mediated, hemolytic activity. Mutations with a single deletion in genes encoding putative pore-forming proteins, SipB and SipC, secreted by the TTSS abolished hemolytic activity. In addition, the osmoprotection studies revealed that molecules larger than PEG2000 conferred significant protection against lysis induced by Salmonella. These results indicate that the hemolysis generated by Salmonella is due to the formation of pores within the erythrocyte membrane.

Two periplasmic disulfide oxidoreductases, DsbA and SrgA, target outer membrane protein SpiA, a component of the Salmonella pathogenicity island 2 type III secretion system.

The formation of disulfide is essential for the folding, activity, and stability of many proteins secreted by Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into proteins exported from the cytoplasm to periplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we examined the role of the Dsb enzymes in Salmonella pathogenesis, and we demonstrated that DsbA, but not DsbC, is required for the full expression of virulence in a mouse infection model of Salmonella enterica serovar Typhimurium. Salmonella strains carrying a dsbA mutation showed reduced function mediated by type III secretion systems (TTSSs) encoded on Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2). To obtain a more detailed understanding of the contribution of DsbA to both SPI-1 and SPI-2 TTSS function, we identified a protein component of the SPI-2 TTSS apparatus affected by DsbA. Although we found no substrate protein for DsbA in the SPI-1 TTSS apparatus, we identified SpiA (SsaC), an outer membrane protein of SPI-2 TTSS, as a DsbA substrate. Site-directed mutagenesis of the two cysteine residues present in the SpiA protein resulted in the loss of SPI-2 function in vitro and in vivo. Furthermore, we provided evidence that a second disulfide oxidoreductase, SrgA, also oxidizes SpiA. Analysis of in vivo mixed infections demonstrated that a Salmonella dsbA srgA double mutant strain was more attenuated than either single mutant, suggesting that DsbA acts in concert with SrgA in vivo.

Intracellular expression of the Salmonella plasmid virulence protein, SpvB, causes apoptotic cell death in eukaryotic cells.

The spv genes carried on the Salmonella virulence plasmid are commonly associated with severe systemic infection in experimental animals. The SpvB virulence-associated protein has been shown to ADP-ribosylate actin, and this enzymatic activity is essential for virulence in mice. Here, we present evidence that intracellular expression of SpvB protein induces not only disruption of actin filaments but also apoptotic cell death in eukaryotic cells.

Extracellular secretion of the virulence plasmid-encoded ADP-ribosyltransferase SpvB in Salmonella.

Nontyphoid Salmonella enterica requires the plasmid-encoded spv genes to establish successful systemic infection in experimental animals. The SpvB virulence-associated protein has recently been shown to contain the ADP-ribosyltransferase domain. SpvB ADP-ribosilates actin and depolymerizes actin filaments when expressed in cultured epithelial cells. However, spontaneous secretion or release of SpvB has not been observed under in vitro growth conditions. In the present study we investigated the secretion of SpvB from Salmonella using in vitro and in vivo assay systems. We showed that SpvB is secreted into supernatant from Salmonella strains that contain the cloned spvB gene on a plasmid when they grew in intracellular salts medium (ISM), a minimal medium mimicing the intracellular iron concentrations of eukaryotic cells. A series of mutant SpvB proteins revealed that an N-terminal region of SpvB located at amino acids 1-229 was sufficient to promote secretion into extracellular milieu. Confocal immunofluorescence microscopy also demonstrated efficient localization of the N-terminal domain of SpvB(1-360) tagged with biotinylated peptide within infected host cell cytosol but not truncated SpvB(1-179) fusion protein. In addition, mutations that inactivate genes within Salmonella pathogenicity island 1 or Salmonella pathogenicity island 2 that encode type III secretion systems (TTSS) could secrete the SpvB protein into the culture medium. These results indicate that SpvB protein is transported from the bacteria and into the host cytoplasm independent of TTSS.

Protection against Shiga toxin 1 challenge by immunization of mice with purified mutant Shiga toxin 1.

Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.