Molecular characterization of the InvE regulator in the secretion of type III secretion translocases in Salmonella enterica serovar Typhimurium.

The type III secretion systems (T3SSs) are exploited by many Gram-negative pathogenic bacteria to deliver a set of effector proteins into the host cytosol during cell entry. The T3SS of Salmonella enterica serovar Typhimurium is composed of more than 20 proteins that constitute the membrane-associated base, the needle and the tip complex at the distal end of the T3SS needle. Membrane docking and piercing between the T3SS and host cells is followed by the secretion of effector proteins. Therefore, a secretion hierarchy among the substrates of the T3SS is required. The secretion of the pore-forming translocase proteins SipB, SipC and SipD is controlled by the T3SS regulator protein, InvE. During an attempt to identify the regions of InvE that are involved in T3SS regulation, it was observed that the secretion of SipB, SipC and SipD was inhibited when the C-terminal 52 amino acids were removed from InvE. In addition, InvE derivatives lacking the N-terminal 30 and 100 residues were unable to secrete translocases into the culture medium. Interestingly, in the absence of the N-terminal 180 residues of InvE, SipD is unstable, resulting in the hypersecretion of SipB. We also found that both the type III secretion signals of SipB and SptP were functionally interchangeable with the first 30 amino acids of InvE, which could allow the secretion of a reporter protein. These results indicate that InvE may have two functional domains responsible for regulating the secretion of translocases: an N-terminal secretion signal and a C-terminal regulatory domain.

Effect of iacP mutation on flagellar phase variation in Salmonella enterica serovar typhimurium strain UK-1.

Flagella are surface appendages that are important for bacterial motility and invasion of host cells. Two flagellin subunits in Salmonella enterica serovar Typhimurium, FliC and FljB, are alternatively expressed by a site-specific DNA inversion mechanism called flagellar phase variation. Although this inversion mechanism is understood at the molecular level, the key factor controlling the expression of the two flagellin subunits has not been determined. In this study, we found that a putative acyl carrier protein, IacP, affects flagellar phase variation in S. Typhimurium strain UK-1 under Salmonella pathogenicity island 1 (SPI1)-inducing conditions. Liquid chromatography-mass spectrometry analysis of the secreted proteins from S. Typhimurium determined that the amount of FljB secreted was significantly higher in the iacP mutant strain, a finding confirmed by Western blot analysis. Northern blotting, quantitative PCR, and microarray data showed that the level of FljB in the iacP mutant strain was regulated at the transcriptional level, although the transcription and expression of the fliC gene were independent of IacP. FljB production was abolished by the deletion of the Hin DNA invertase but could be restored by the introduction of a plasmid carrying the hin gene. We also found that in the iacP mutant strain, the orientation of the invertible H segment is in the FljB-expressing phase. Furthermore, electron microscopy observations indicated that the iacP mutant strain had more flagella per cell than the wild-type strain. These results suggest that IacP is associated with flagellar phase switching under SPI1-inducing conditions.

Expression and delivery of tetanus toxin fragment C fused to the N-terminal domain of SipB enhances specific immune responses in mice.

Live attenuated bacteria can be used as a carrier for the delivery of foreign antigens to a host's immune system. The N-terminal domain of SipB, a translocon protein of the type III secretion system of Salmonella enterica serovar Typhimurium, is required for secretion and outer membrane localization. In the present study, vaccine plasmids for antigen delivery in which the non-toxic tetanus toxin fragment C (TTFC), which contains a T cell epitope, is fused to the N-terminal 160 amino acids of SipB were developed. It was found that the recombinant proteins are secreted into the culture media and localized to the bacterial surface. TTFC-specific antibody responses are significantly increased in mice orally immunized with attenuated S. Typhimurium BRD509 strains carrying TTFC delivery plasmids. When the TTFC delivery cassettes were introduced into a low copy vector, the plasmid was stably maintained in the BRD509 strain and induced an immune response to the TTFC antigen in mice. These results suggest that expression and delivery of heterologous antigens fused to the N-terminus of SipB enhance the induction of antigen-specific immune responses, and that the N-terminal domain of SipB can be used as a versatile delivery system for foreign antigens.

Role of Salmonella Pathogenicity Island 1 protein IacP in Salmonella enterica serovar typhimurium pathogenesis.

Gram-negative bacteria, including Salmonella enterica serovar Typhimurium, exploit type III secretion systems (T3SSs) through which virulence proteins are delivered into the host cytosol to reinforce invasive and replicative niches in their host. Although many secreted effector proteins and membrane-bound structural proteins in the T3SS have been characterized, the functions of many cytoplasmic proteins still remain unknown. In this study, we found that IacP, encoded by Salmonella pathogenicity island 1, was important for nonphagocytic cell invasion and bacterial virulence. When the iacP gene was deleted from several Salmonella serovar Typhimurium strains, the invasion into INT-407 epithelial cells was significantly decreased compared to that of their parental strains, and retarded rearrangements of actin fibers were observed for the iacP mutant-infected cells. Although IacP had no effect on the secretion of type III translocon proteins, the levels of secretion of the effector proteins SopB, SopA, and SopD into the culture medium were decreased in the iacP mutant. In a mouse infection model, mice infected with the iacP mutant exhibited alleviated pathological signs in the intestine and survived longer than did wild-type-infected mice. Taken together, IacP plays a key role in Salmonella virulence by regulating the translocation of T3SS effector proteins.

Inhibition of Bacillus cereus Growth and Toxin Production by Bacillus amyloliquefaciens RD7-7 in Fermented Soybean Products.

Bacillus cereus is a gram-positive, rod-shaped, spore-forming bacterium that has been isolated from contaminated fermented soybean food products and from the environment. B. cereus produces diarrheal and emetic toxins and has caused many outbreaks of foodborne diseases. In this study, we investigated whether B. amyloliquefaciens RD7-7, isolated from rice doenjang (Korean fermented soybean paste), a traditional Korean fermented soybean food, shows antimicrobial activity against B. cereus and regulates its toxin gene expression. B. amyloliquefaciens RD7-7 exhibited strong antibacterial activity against B. cereus and inhibited the expression of B. cereus toxin-related genes (groEL, nheA, nheC, and entFM). We also found that addition of water extracts of soybean and buckwheat soksungjang (Korean fermented soybean paste made in a short time) fermented with B. amyloliquefaciens RD7-7 significantly reduced the growth and toxin expression of B. cereus. These results indicate that B. amyloliquefaciens RD7-7 could be used to control B. cereus growth and toxin production in the fermented soybean food industry. Our findings also provide a basis for the development of candidate biological control agents against B. cereus to improve the safety of fermented soybean food products.

Protective Effects of a Novel Probiotic Strain of Lactobacillus plantarum JSA22 from Traditional Fermented Soybean Food Against Infection by Salmonella enterica Serovar Typhimurium.

Lactobacillus species have been shown to enhance intestinal epithelial barrier function, modulate host immune responses, and suppress the growth of pathogenic bacteria, yeasts, molds, and viruses. Thus, lactobacilli have been used as probiotics for treating various diseases, including intestinal disorders, and as biological preservatives in the food and agricultural industries. However, the molecular mechanisms used by lactobacilli to suppress pathogenic bacterial infections have been poorly characterized. We previously isolated Lactobacillus plantarum JSA22 from buckwheat sokseongjang, a traditional Korean fermented soybean food, which possessed high enzymatic, fibrinolytic, and broad-spectrum antimicrobial activity against foodborne pathogens. In this study, we investigated the effects of L. plantarum JSA22 on the growth of S. Typhimurium and S. Typhimurium-induced cytotoxicity by stimulating the host immune response in intestinal epithelial cells. The results showed that coincubation of S. Typhimurium and L. plantarum JSA22 with intestinal epithelial cells suppressed S. Typhimurium infection, S. Typhimurium-induced NF-kappaB activation, and IL-8 production, and lowered the phosphorylation of both Akt and p38. These data indicated that L. plantarum JSA22 has probiotic properties, and can inhibit S. Typhimurium infection of intestinal epithelial cells. Our findings can be used to develop therapeutic and prophylactic agents against pathogenic bacteria.

Biogenic Amine Degradation by Bacillus Species Isolated from Traditional Fermented Soybean Food and Detection of Decarboxylase-Related Genes.

Biogenic amines in some food products present considerable toxicological risks as potential human carcinogens when consumed in excess concentrations. In this study, we investigated the degradation of the biogenic amines histamine and tyramine and the presence of genes encoding histidine and tyrosine decarboxylases and amine oxidase in Bacillus species isolated from fermented soybean food. No expression of histidine and tyrosine decarboxylase genes (hdc and tydc) were detected in the Bacillus species isolated (B. subtilis HJ0-6, B. subtilis D'J53-4, and B. idriensis RD13-10), although substantial levels of amine oxidase gene (yobN) expression were observed. We also found that the three selected strains, as non-biogenic amineproducing bacteria, were significantly able to degrade the biogenic amines histamine and tyramine. These results indicated that the selected Bacillus species could be used as a starter culture for the control of biogenic amine accumulation and degradation in food. Our study findings also provided the basis for the development of potential biological control agents against these biogenic amines for use in the food preservation and food safety sectors.

Bacillus subtilis HJ18-4 from traditional fermented soybean food inhibits Bacillus cereus growth and toxin-related genes.

Bacillus subtilis HJ18-4 isolated from buckwheat sokseongjang, a traditional Korean fermented soybean food, exhibits broad-spectrum antimicrobial activity against foodborne pathogens, including Bacillus cereus. In this study, we investigated the antibacterial efficacy and regulation of toxin gene expression in B. cereus by B. subtilis HJ18-4. Expression of B. cereus toxin-related genes (groEL, nheA, nheC, and entFM) was downregulated by B. subtilis HJ18-4, which also exhibited strong antibacterial activity against B. cereus. We also found that water extracts of soy product fermented with B. subtilis HJ18-4 significantly inhibited the growth of B. cereus and toxin expression. These results indicate that B. subtilis HJ18-4 could be used as an antimicrobial agent to control B. cereus in the fermented soybean food industry. Our findings also provide an opportunity to develop an efficient biological control agent against B. cereus.

Functional insight from the tetratricopeptide repeat-like motifs of the type III secretion chaperone SicA in Salmonella enterica serovar Typhimurium.

SicA functions both as a class II chaperone for SipB and SipC of the type III secretion system (T3SS)-1 and as a transcriptional cofactor for the AraC-type transcription factor InvF in Salmonella enterica subsp. enterica serovar Typhimurium. Bioinformatic analysis has predicted that SicA possesses three tetratricopeptide repeat (TPR)-like motifs, which are important for protein-protein interactions and serve as multiprotein complex mediators. To investigate whether the TPR-like motifs in SicA are critical for its transcriptional cofactor function, the canonical residues in these motifs were mutated to glutamate (SicAA44E , SicAA78E , and SicAG112E ). None of these mutants except SicAA44E were able to activate the expression of the sipB and sigD genes. SicAA44E still has a capacity to interact with InvF in vitro, and despite its instability in cell, it could activate the sigDE operon. This suggests that TPR motifs are important for the transcriptional cofactor function of the SicA chaperone.

Enhancement of host immune responses by oral vaccination to Salmonella enterica serovar Typhimurium harboring both FliC and FljB flagella.

Flagellin, the structural component of the flagellar filament in various motile bacteria, can contribute to the activation of NF-κB and proinflammatory cytokine expression during the innate immune response in host cells. Thus, flagellin proteins represent a particularly attractive target for the development of vaccine candidates. In this study, we investigated the immune response by increasing the flagella number in the iacP mutant strain and the adjuvant activity of the flagellin component FljB of Salmonella enterica serovar Typhimurium. We found that the iacP mutant strain expresses two flagellin proteins (FliC and FljB), which result in increased NF-κB-dependent gene expression in bone marrow derived macrophages. Using an oral immunization mouse model, we observed that the administration of a live attenuated S. typhimurium BRD509 strain expressing the FliC and FljB flagellins induced significantly enhanced flagellin-specific IgG responses in the systemic compartment. The mice immunized with the recombinant attenuated S. typhimurium strain that has two types of flagella were protected from lethal challenge with the Salmonella SL1344 strain. These results indicate that overexpression of flagella in the iacP mutant strain enhance the induction of an antigen-specific immune responses in macrophage cell, and both the FliC and FljB flagellar filament proteins-producing S. typhimurium can induce protective immune responses against salmonellosis.

N-terminal residues of SipB are required for its surface localization on Salmonella enterica serovar Typhimurium.

SipB, one of the invasion proteins encoded in Salmonella pathogenicity island 1 (SPI-1), is known to be secreted outside the cell, where it functions as a translocon by assembling into a host-cell plasma membrane-integral structure. Here, we confirmed that wild-type SipB could be localized to the bacterial outer membrane, and further showed that its localization was dependent on extracellular secretion, and was independent of the presence of the SipD protein. Proteinase K susceptibility and immunofluorescence assays indicated that SipB was not incorporated into the outer membrane, but rather was displayed on the bacterial surface. Finally, mutation studies revealed that the N-terminal 100-140 aa (especially amino acids 135-138) of SipB were required for its localization on the bacterial outer membrane.