Characterization of a nuclear transport factor 2-like domain-containing protein in Plasmodium berghei.

BACKGROUND: Plasmodium lacks an mRNA export receptor ortholog, such as yeast Mex67. Yeast Mex67 contains a nuclear transport factor 2 (NTF2)-like domain, suggesting that NTF2-like domain-containing proteins might be associated with mRNA export in Plasmodium. In this study, the relationship between mRNA export and an NTF2-like domain-containing protein, PBANKA_1019700, was investigated using the ANKA strain of rodent malaria parasite Plasmodium berghei. METHODS: The deletion mutant Δ1019700 was generated by introducing gene-targeting vectors into the P. berghei ANKA genome, and parasite growth and virulence were examined. To investigate whether PBANKA_1019700 is involved in mRNA export, live-cell fluorescence imaging and immunoprecipitation coupled to mass spectrometry (IP-MS) were performed using transgenic parasites expressing fusion proteins (1019700::mCherry). RESULTS: Deletion of PBANKA_1019700 affected the sexual phase but not the asexual phase of malaria parasites. Live-cell fluorescence imaging showed that PBANKA_1019700 localizes to the cytoplasm. Moreover, IP-MS analysis of 1019700::mCherry indicated that PBANKA_1019700 interacts with ubiquitin-related proteins but not nuclear proteins. CONCLUSIONS: PBANKA_1019700 is a noncanonical NTF2-like superfamily protein.

An Experimental Adult Zebrafish Model for Shigella Pathogenesis, Transmission, and Vaccine Efficacy Studies.

Shigellosis has been a menace to society for ages. The absence of an effective vaccine against Shigella, improper sanitation, and unhygienic use of food and water allow the disease to flourish. Shigella can also be transmitted via natural water bodies. In the absence of a good animal model, the actual nature of pathogenesis and transmission remains unclear. Zebrafish larvae have previously been described as a model for Shigella pathogenesis. However, larval fish lack a mature intestinal microbiota and immune system. Here, the adult zebrafish was assessed as a potential model for Shigella pathogenesis. Their well-developed innate and adaptive immune responses mimic the mammalian immune system. Shigella showed a clear dose-, time-, and temperature-dependent colonization of the adult zebrafish gut. Efficacy of a three-dose immunization regime was tested using bath immunization with heat-killed trivalent Shigella immunogen. The present study demonstrates the efficacy of an adult zebrafish model for pathogenesis, transmission, and vaccine efficacy studies. IMPORTANCE Shigellosis is a diarrheal disease that is prevalent in developing countries and especially dangerous in young children. Currently, animal models for shigellosis are unable to model some aspects of the infectious cycle. Here, we describe a new shigellosis model in adult zebrafish, an increasingly common model organism for studying bacterial pathogens. The zebrafish model can be used to study Shigella colonization, transmission, and immune responses, as well as test vaccine efficacy.

The association between acute fatty liver disease and nitric oxide during malaria in pregnancy.

BACKGROUND: Liver disease is a common feature of malaria in pregnancy, but its pathogenesis remains unclear. METHODS: To understand the pathogenesis of liver disease during malaria in pregnancy, comparative proteomic analysis of the liver in a mouse model of malaria in pregnancy was performed. RESULTS: Decreased levels of mitochondrial and peroxisomal proteins were observed in the livers of pregnant mice infected with the lethal rodent malaria parasite Plasmodium berghei strain NK65. By contrast, increased levels of perilipin-2, amyloid A-1, and interferon (IFN)-γ signalling pathway-related proteins were observed in the livers of infected pregnant mice, suggesting that IFN-γ signalling may contribute to the development of liver disease during malaria in pregnancy. IFN-γ signalling is a potential trigger of inducible nitric oxide synthase (iNOS) expression. Liver disease associated with microvesicular fatty infiltration and elevated liver enzymes in pregnant wild-type mice infected with malaria parasites was improved by iNOS deficiency. CONCLUSIONS: In this study, a causative role of iNOS in liver disease associated with microvesicular fatty infiltration during malaria in pregnancy was demonstrated. These findings provide important insight for understanding the role of iNOS-mediated metabolic responses and the pathogenesis of high-risk liver diseases in pregnancy, such as acute fatty liver.

Roles and Cellular Localization of GBP2 and NAB2 During the Blood Stage of Malaria Parasites.

The quality control and export of mRNA by RNA-binding proteins are necessary for the survival of malaria parasites, which have complex life cycles. Nuclear poly(A) binding protein 2 (NAB2), THO complex subunit 4 (THO4), nucleolar protein 3 (NPL3), G-strand binding protein 2 (GBP2) and serine/arginine-rich splicing factor 1 (SR1) are involved in nuclear mRNA export in malaria parasites. However, their roles in asexual and sexual development, and in cellular localization, are not fully understood. In this study using the rodent malaria parasite, Plasmodium berghei, we found that NAB2 and SR1, but not THO4, NPL3 or GBP2, played essential roles in the asexual development of malaria parasites. By contrast, GBP2 but not NPL3 was involved in male and female gametocyte production. THO4 was involved in female gametocyte production, but had a lower impact than GBP2. In this study, we focused on GBP2 and NAB2, which play important roles in the sexual and asexual development of malaria parasites, respectively, and examined their cellular localization. GBP2 localized to both the nucleus and cytoplasm of malaria parasites. Using immunoprecipitation coupled to mass spectrometry (IP-MS), GBP2 interacted with the proteins ALBA4, DOZI, and CITH, which play roles in translational repression. IP-MS also revealed that phosphorylated adapter RNA export protein (PHAX) domain-containing protein, an adaptor protein for exportin-1, also interacted with GBP2, implying that mRNA export occurs via the PHAX domain-containing protein pathway in malaria parasites. Live-cell fluorescence imaging revealed that NAB2 localized at the nuclear periphery. Moreover, IP-MS indicated that NAB2 interacted with transportin. RNA immunoprecipitation coupled to RNA sequencing revealed that NAB2 bound directly to 143 mRNAs, including those encoding 40S and 60S ribosomal proteins. Our findings imply that malaria parasites use an evolutionarily ancient mechanism conserved throughout eukaryotic evolution.

Malaria in the postpartum period causes damage to the mammary gland.

Mastitis is an inflammation of the mammary gland in the breast and is typically due to bacterial infection. In malaria-endemic areas, mastitis with accompanying fever can be challenging to differentiate from malaria. At the same time, it is unclear whether malaria infection is directly involved in the development of mastitis. In the present study, whether mastitis develops during infection with malaria parasites was investigated using a rodent malaria model with Plasmodium berghei (P. berghei; Pb) ANKA. The course of parasitemia in postpartum mice infected with Pb ANKA was similar to the course in infected virgin mice. However, infected postpartum mice died earlier than did infected virgin mice. In addition, the weight of pups from mice infected with Pb ANKA was significantly reduced compared with pups from uninfected mice. The macroscopic and histological analyses showed apparent changes, such as destruction of the alveolus wall and extensive presence of leukocytes, in mammary gland tissue in mice infected during the postpartum period. The findings suggest that women during the postpartum period are more vulnerable to complications when infected with malaria parasites, particularly women who do not acquire protective immunity against malaria parasites. Based on the proteomic analysis, IFN-γ signaling pathway-related proteins in mammary gland tissue of the infected postpartum mice were increased. Our results indicate that inflammation induced by IFN-γ, a proinflammatory cytokine, may contribute to negative histological changes in mammary gland tissue of postpartum mice infected with Pb ANKA. In IFN-γ receptor 1-deficient (IFNGR1-KO) mice, the histological changes in mammary gland tissue of the infected postpartum wild-type mice were improved to almost normal mammary gland structure. Furthermore, weight loss in pups delivered by infected IFNGR1-KO postpartum mice was not observed. Taken together, these findings indicate that inflammation induced by IFN-γ is associated with development of mastitis in postpartum mice infected with Pb ANKA. The present study results may increase our understanding of how disease aggravation occurs during postpartum malaria.

Additional Og-Typing PCR Techniques Targeting Escherichia coli-Novel and Shigella-Unique O-Antigen Biosynthesis Gene Clusters.

The O-serogrouping of pathogenic Escherichia coli is a standard method for subtyping strains for epidemiological studies and controls. O-serogroup diversification shows a strong association with the genetic diversity in some O-antigen biosynthesis gene clusters. Through genomic studies, in addition to the types of O-antigen biosynthesis gene clusters (Og-types) from conventional O-serogroup strains, a number of novel Og-types have been found in E. coli isolates. To assist outbreak investigations and surveillance of pathogenic E. coli at inspection institutes, in previous studies, we developed PCR methods that could determine almost all conventional O-serogroups and some novel Og-types. However, there are still many Og-types that may not be determined by simple genetic methods such as PCR. Thus, in the present study, we aimed to develop an additional Og-typing PCR system. Based on the novel Og-types, including OgN32, OgN33, and OgN34, presented in this study, we designed an additional 24 PCR primer pairs targeting 14 novel and 2 diversified E. coli Og-types and 8 Shigella-unique Og-types. Subsequently, we developed 5 new multiplex PCR sets consisting of 33 primers, including the aforementioned 24 primers and 9 primers reported in previous studies. The accuracy and specificity of the PCR system was validated using approximately 260 E. coli and Shigella O-serogroup and Og-type reference strains. The Og-typing PCR system reported here can determine a wide range of Og-types of E. coli and may help epidemiological studies, in addition to the surveillance of pathogenic E. coli.

Superstructure formation by RodZ hexamers of Shigella sonnei maintains the rod shape of bacilli.

The rod shape of bacilli is maintained by bacterial cytoskeletal protein MreB, an actin homolog that acts in concert with the inner membrane protein RodZ. We previously reported RodZ binds RNA to control the posttranscriptional regulation of invE (virB), which controls the type III secretion system essential for the virulence of Shigella. Here, we show that purified RodZ forms "superstructures" of high molecular mass that dissociate into a midsized "basal complex" in the presence of nonionic detergent, or to a monomer in the presence of dithiothreitol. We used mass spectrometry to show that the basal complex was a hexamer. Electrophoresis mobility shift assays combined with gel filtration detected the RNA-binding activity in fractions containing molecules larger than the basal hexamer. The superstructure was consistently detected with MreB in crude cell lysates of S. sonnei that were fractionated using gel filtration. Immunofluorescence microscopy using two different super-resolution settings showed that wild-type RodZ was distributed in cells as separate dots. Consistent with the superstructure comprising homohexamers, majority of the dots distributed among areas of discrete values. In addition, simultaneous immunodetection of MreB provided the first evidence of colocalization with RodZ as larger patch like signals. These findings indicate that native RodZ forms clusters of various sizes, which may correspond to a superstructure comprising multiple hexamers required for the RNA-binding activity.

An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

Intracellular fate of Ureaplasma parvum entrapped by host cellular autophagy.

Genital mycoplasmas, including Ureaplasma spp., are among the smallest human pathogenic bacteria and are associated with preterm birth. Electron microscopic observation of U. parvum showed that these prokaryotes have a regular, spherical shape with a mean diameter of 146 nm. U. parvum was internalized into HeLa cells by clathrin-mediated endocytosis and survived for at least 14 days around the perinuclear region. Intracellular U. parvum reached endosomes in HeLa cells labeled with EEA1, Rab7, and LAMP-1 within 1 to 3 hr. After 3 hr of infection, U. parvum induced the cytosolic accumulation of galectin-3 and was subsequently entrapped by the autophagy marker LC3. However, when using atg7-/- MEF cells, autophagy was inadequate for the complete elimination of U. parvum in HeLa cells. U. parvum also colocalized with the recycling endosome marker Rab11. Furthermore, the exosomes purified from infected HeLa cell culture medium included U. parvum. In these purified exosomes ureaplasma lipoprotein multiple banded antigen, host cellular annexin A2, CD9, and CD63 were detected. This research has successfully shown that Ureaplasma spp. utilize the host cellular membrane compartments possibly to evade the host immune system.

Development of a cost-effective vaccine candidate with outer membrane vesicles of a tolA-disrupted Shigella boydii strain.

Our previous studies on outer membrane vesicles based vaccine development against shigellosis, revealed the inability of Shigella to release significant amount of vesicles naturally, during growth. Disruption of tolA, one of the genes of the Tol-Pal system of Gram negative bacterial membrane, has increased the vesicle release rate of a Shigella boydii type 4 strain to approximately 60% higher. We also noticed the vesicles, released from tolA-disrupted strain captured more OmpA protein and lipopolysaccharide, compared to the vesicles released from its wild type prototype. Six to seven weeks old BALB/c mice, immunized with 25 µg of three oral doses of the vesicles, released by tolA mutant, conferred 100% protection against lethal homologous challenge through nasal route, compared to only 60% protection after the same dose of wild type immunogen. Mice, immunized with the vesicles from tolA-mutant, manifested significant secretion of mucosal IgG and IgA. A sharp and significant response of pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) were also observed in the lung lavage of these groups of mice, within 6h post challenge; but at 24h, these inflammatory cytokines showed the sign of subsidence and the system was taken over by the release of anti-inflammatory cytokines (IL-4 and IL-10). Studies with naïve peritoneal macrophages, proved further, the potency of these vesicles to stimulate nitric oxide and TNF-α, IL-12p70, IL-6 and IL-10 productions in-vitro. The ability of these vesicles to trigger polarization of CD4(+) T cells toward Th1 adaptive immune response, had also been observed along with the presence of anti-inflammatory cytokines in the system. Our study demonstrated, the vesicles from tolA-disrupted Shigella were able to suppress Shigella-mediated inflammation in the host and could balance between inflammation and anti-inflammation, promoting better survival and health of the infected mice. Outer membrane vesicles from tolA-mutant, could be a potential cost-effective vaccine candidate against shigellosis.

Characterization of enterohemorrhagic Escherichia coli O111 and O157 strains isolated from outbreak patients in Japan.

In April and May 2011, there was a serious food-poisoning outbreak in Japan caused by enterohemorrhagic Escherichia coli (EHEC) strains O111:H8 and O157:H7 from raw beef dishes at branches of a barbecue restaurant. This outbreak involved 181 infected patients, including 34 hemolytic-uremic syndrome (HUS) cases (19%). Among the 34 HUS patients, 21 developed acute encephalopathy (AE) and 5 died. Patient stool specimens yielded E. coli O111 and O157 strains. We also detected both EHEC O111 stx2 and stx-negative E. coli O111 strains in a stock of meat block from the restaurant. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem-repeat analysis (MLVA) showed that the stx-negative E. coli O111 isolates were closely related to EHEC O111 stx2 isolates. Although the EHEC O157 strains had diverse stx gene profiles (stx1, stx2, and stx1 stx2), the PFGE and MLVA analyses indicated that these isolates originated from a single clone. Deletion of the Stx2-converting prophage from the EHEC O111 stx2 isolates was frequently observed during in vitro growth, suggesting that strain conversion from an EHEC O111 stx2 to an stx-negative strain may have occurred during infection.

Regulation of natural competence by the orphan two-component system sensor kinase ChiS involves a non-canonical transmembrane regulator in Vibrio cholerae.

In Vibrio cholerae, 41 chitin-inducible genes, including the genes involved in natural competence for DNA uptake, are governed by the orphan two-component system (TCS) sensor kinase ChiS. However, the mechanism by which ChiS controls the expression of these genes is currently unknown. Here, we report the involvement of a novel transcription factor termed 'TfoS' in this process. TfoS is a transmembrane protein that contains a large periplasmic domain and a cytoplasmic AraC-type DNA-binding domain, but lacks TCS signature domains. Inactivation of tfoS abolished natural competence as well as transcription of the tfoR gene encoding a chitin-induced small RNA essential for competence gene expression. A TfoS fragment containing the DNA-binding domain specifically bound to and activated transcription from the tfoR promoter. Intracellular TfoS levels were unaffected by disruption of chiS and coexpression of TfoS and ChiS in Escherichia coli recovered transcription of the chromosomally integrated tfoR::lacZ gene, suggesting that TfoS is post-translationally modulated by ChiS during transcriptional activation; however, this regulation persisted when the canonical phosphorelay residues of ChiS were mutated. The results presented here suggest that ChiS operates a chitin-induced non-canonical signal transduction cascade through TfoS, leading to transcriptional activation of tfoR.

RodZ regulates the post-transcriptional processing of the Shigella sonnei type III secretion system.

The expression of the type III secretion system-a main determinant of virulence in Shigella-is controlled by regulator cascades VirF-InvE (VirB) and CpxAR two-component system. A screen for mutants that restore virulence in the cpxA background led to the isolation of a mutant of rodZ, a cytoskeletal protein that maintains the rod-shaped morphology of bacilli. InvE is normally repressed at 30 °C because of decreased messenger RNA (mRNA) stability, but rodZ mutants markedly increase invE-mRNA stability. Importantly, the inhibition of InvE production by RodZ can be genetically separated from its role in cell-shape maintenance, indicating that these functions are distinguishable. Thus, we propose that RodZ is a new membrane-bound RNA-binding protein that provides a scaffold for post-transcriptional regulation.

Identification of a chitin-induced small RNA that regulates translation of the tfoX gene, encoding a positive regulator of natural competence in Vibrio cholerae.

The tfoX (also called sxy) gene product is the central regulator of DNA uptake in the naturally competent bacteria Haemophilus influenzae and Vibrio cholerae. However, the mechanisms regulating tfoX gene expression in both organisms are poorly understood. Our previous studies revealed that in V. cholerae, chitin disaccharide (GlcNAc)2 is needed to activate the transcription and translation of V. cholerae tfoX (tfoX(VC)) to induce natural competence. In this study, we screened a multicopy library of V. cholerae DNA fragments necessary for translational regulation of tfoX(VC). A clone carrying the VC2078-VC2079 intergenic region, designated tfoR, increased the expression of a tfoX(VC)::lacZ translational fusion constructed in Escherichia coli. Using a tfoX(VC)::lacZ reporter system in V. cholerae, we confirmed that tfoR positively regulated tfoX(VC) expression at the translational level. Deletion of tfoR abolished competence for exogenous DNA even when (GlcNAc)2 was provided. The introduction of a plasmid clone carrying the tfoR(+) gene into the tfoR deletion mutant complemented the competence deficiency. We also found that the tfoR gene encodes a 102-nucleotide small RNA (sRNA), which was transcriptionally activated in the presence of (GlcNAc)2. Finally, we showed that this sRNA activated translation from tfoX(VC) mRNA in a highly purified in vitro translation system. Taking these results together, we propose that in the presence of (GlcNAc)2, TfoR sRNA is expressed to activate the translation of tfoX(VC), which leads to the induction of natural competence.

Inhibition of Streptococcus mutans biofilm formation by Streptococcus salivarius FruA.

The oral microbial flora consists of many beneficial species of bacteria that are associated with a healthy condition and control the progression of oral disease. Cooperative interactions between oral streptococci and the pathogens play important roles in the development of dental biofilms in the oral cavity. To determine the roles of oral streptococci in multispecies biofilm development and the effects of the streptococci in biofilm formation, the active substances inhibiting Streptococcus mutans biofilm formation were purified from Streptococcus salivarius ATCC 9759 and HT9R culture supernatants using ion exchange and gel filtration chromatography. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed, and the results were compared to databases. The S. salivarius HT9R genome sequence was determined and used to indentify candidate proteins for inhibition. The candidates inhibiting biofilms were identified as S. salivarius fructosyltransferase (FTF) and exo-beta-d-fructosidase (FruA). The activity of the inhibitors was elevated in the presence of sucrose, and the inhibitory effects were dependent on the sucrose concentration in the biofilm formation assay medium. Purified and commercial FruA from Aspergillus niger (31.6% identity and 59.6% similarity to the amino acid sequence of FruA from S. salivarius HT9R) completely inhibited S. mutans GS-5 biofilm formation on saliva-coated polystyrene and hydroxyapatite surfaces. Inhibition was induced by decreasing polysaccharide production, which is dependent on sucrose digestion rather than fructan digestion. The data indicate that S. salivarius produces large quantities of FruA and that FruA alone may play an important role in multispecies microbial interactions for sucrose-dependent biofilm formation in the oral cavity.

Involvement of RNA-binding protein Hfq in the osmotic-response regulation of invE gene expression in Shigella sonnei.

BACKGROUND: The expression of Type III secretion system (TTSS) in Shigella is regulated in response to changes in environmental osmolarity and temperature. Temperature-dependent regulation of virF, the master regulator of TTSS synthesis, is believed to occur at the transcriptional level. We recently demonstrated, however, that TTSS synthesis also involves post-transcriptional regulation of the synthesis of InvE, a target of virF and key regulator of TTSS synthesis. The mRNA levels of invE (virB) are stable at 37 degrees C, but mRNA stability markedly decreases at low temperatures where the TTSS synthesis is tightly repressed. Deletion of hfq, which encodes an RNA chaperone in Gram-negative bacteria, results in the restoration of expression of invE and other TTSS genes at low temperature due to an increase in the stability of invE mRNA. To date, the molecular details of the regulation of TTSS expression in response to osmotic pressure are not known. In the current study, we investigated the mechanism of regulation of TTSS by osmotic pressure. RESULTS: Transcription of virF, which encodes the master regulator of TTSS expression, was partially repressed under low osmotic conditions. Several lines of evidence indicated that osmolarity-dependent changes in TTSS synthesis are controlled at the post-transcriptional level, through the regulation of InvE synthesis. First, the expression InvE protein was tightly repressed under low osmotic growth conditions, even though invE mRNA transcripts were readily detectable. Second, under low osmotic conditions, invE mRNA was rapidly degraded, whereas deletion of hfq, which encodes an RNA chaperone, resulted in increased invE mRNA stability and the production of InvE protein. Third, the binding of purified Hfq in vitro to invE RNA was stronger in low-salt buffer, as assessed by gel-shift analysis and surface plasmon resonance (Biacore analysis). CONCLUSION: Osmolarity-dependent changes in TTSS synthesis in Shigella involve the post-transcriptional regulation of InvE expression, in addition to partial transcriptional activation by virF. The stability of invE mRNA is reduced under low osmotic conditions, similar to the effect of temperature. Deletion of an RNA chaperone gene (hfq) abolished the repression of TTSS synthesis at low osmolarity through a mechanism that involved increased stability of invE mRNA. We propose that the expression of Shigella virulence genes in response to both osmolarity and temperature involves the post-transcriptional regulation of expression of InvE, a critical regulator of TTSS synthesis.

Differential expression of the Smb bacteriocin in Streptococcus mutans isolates.

The two-component lantibiotic Smb is produced by Streptococcus mutans GS5. In the present study, we identified seven strains of S. mutans containing the smb gene cluster. These strains could be classified into high- and low-level Smb producers relative to the levels of Smb production by indicator strains in vitro. This classification was dependent upon the transcription levels of the structural smbA and smbB genes. Sequence analysis upstream of smbA in the high- and low-level Smb-producing strains revealed differences at nucleotide position -46 relative to the smbA start codon. Interestingly, the transcription start site was present upstream of the point mutation, indicating that both groups of strains have the same promoter constructs and that the differential expression of smbA and smbB mRNA occurred subsequent to transcription initiation. In addition, smbA::lacZ fusion expression was higher when it was regulated by the sequences of strains with high-level Smb activity than when it was regulated by the comparable region from strains with low-level Smb activity. Taken together, we conclude that high- or low-level Smb expression is dependent on the presence of a G or a T nucleotide at position -46 relative to the smbA translational start site in S. mutans Smb producers.

Molecular characterization of enterohemorrhagic Escherichia coli O157:H7 isolates dispersed across Japan by pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis.

We identified seven distinct subtypes of enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were derived from sporadic cases and outbreaks from multiple prefectures in Japan in 2005. A surveillance system utilizing pulsed-field gel electrophoresis (PFGE), PulseNet Japan, was used. Some strains showed indistinguishable PFGE patterns using another restriction enzyme (BlnI or SpeI) in each subtype of EHEC O157:H7 isolates that were routinely subtyped by the XbaI PFGE pattern. In order to examine the genotypic relatedness of these strains, we carried out a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). By using the MLVA system, we found that three of seven subtypes of EHEC O157:H7 strains that were isolated from sporadic cases dispersed across multiple prefectures within a few months showed indistinguishable PFGE patterns and identical MLVA types. Strains belonging to the other four subtypes of EHEC O157:H7 in the PFGE analysis were further classified into different clusters of EHEC O157:H7. Therefore, compared to PFGE, MLVA showed greater discriminatory power with respect to analysis of the isolates in this study.

Involvement of RNA-binding protein Hfq in the post-transcriptional regulation of invE gene expression in Shigella sonnei.

The temperature-dependent regulation of Shigella virulence genes is believed to be accomplished at the transcriptional stage by the regulators VirF and InvE. Several lines of evidence herein described indicate that post-transcriptional regulation of InvE expression plays a key role in the temperature-dependent regulation of virulence gene expression: (i) a considerable amount of invE mRNA continues to be transcribed under low temperature conditions, where the production of InvE protein is tightly repressed; (ii) the stability of invE mRNA markedly decreases, because its decay rate is significantly increased under the repressing conditions. Strikingly, in the hfq mutant of Shigella sonnei, a considerable amount of InvE protein was produced even at low temperature. This increase in the InvE level was found to be associated with the improved stability of invE mRNA, in agreement with the finding that the RNA chaperon Hfq influences post-transcriptional regulations of various genes. Consistently, overexpression of the Hfq protein decreased the production of InvE protein even under the expressing condition at 37 degrees C. The binding in vitro of purified Hfq protein to invE RNA was shown to be stronger at 30 degrees C than at 37 degrees C in two experiments, gel shift analysis and surface plasmon resonance (Biacore) analysis. These results altogether suggest that Hfq plays an important role in the temperature-dependent regulation of invE expression at the post-transcriptional step.

Effectiveness of pulsed-field gel electrophoresis for the early detection of diffuse outbreaks due to Shiga toxin-producing Escherichia coli in Japan.

We applied pulsed-field gel electrophoresis (PFGE) to the investigation of diffuse outbreaks of illness due to Shiga toxin?producing Escherichia coli O157:H7 (STEC O157) in Japan and used these data to develop a database of STEC O157 PFGE patterns and associated clinical and microbiologic information to facilitate the recognition of geographic and temporal clusters of cases based on their PFGE profiles. This project has evolved into a subtyping network called PulseNet Japan that is cooperatively run by National Institute of Infectious Diseases (NIID) and the local Health Institutes and the Ministry of Health, Labor and Welfare. Although our domestic PFGE network that utilized locally developed PFGE protocols was effective in recognizing diffuse outbreaks of STEC O157 within Japan, we decided to adopt the standardized PFGE protocols from PulseNet USA and collaborate closely with the Centers for Disease Control and Prevention (CDC) in the United States to facilitate recognition of international clusters of STEC O157 and their investigations.