Intracellular trafficking of HIV-1 Gag via Syntaxin 6-positive compartments/vesicles: Involvement in tumor necrosis factor secretion.

HIV-1 Gag protein is synthesized in the cytosol and is transported to the plasma membrane, where viral particle assembly and budding occur. Endosomes are alternative sites of Gag accumulation. However, the intracellular transport pathways and carriers for Gag have not been clarified. We show here that Syntaxin6 (Syx6), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane fusion in post-Golgi networks, is a molecule responsible for Gag trafficking and also for tumor necrosis factor-α (TNFα) secretion and that Gag and TNFα are cotransported via Syx6-positive compartments/vesicles. Confocal and live-cell imaging revealed that Gag colocalized and cotrafficked with Syx6, a fraction of which localizes in early and recycling endosomes. Syx6 knockdown reduced HIV-1 particle production, with Gag distributed diffusely throughout the cytoplasm. Coimmunoprecipitation and pulldown show that Gag binds to Syx6, but not its SNARE partners or their assembly complexes, suggesting that Gag preferentially binds free Syx6. The Gag matrix domain and the Syx6 SNARE domain are responsible for the interaction and cotrafficking. In immune cells, Syx6 knockdown/knockout similarly impaired HIV-1 production. Interestingly, HIV-1 infection facilitated TNFα secretion, and this enhancement did not occur in Syx6-depleted cells. Confocal and live-cell imaging revealed that TNFα and Gag partially colocalized and were cotransported via Syx6-positive compartments/vesicles. Biochemical analyses indicate that TNFα directly binds the C-terminal domain of Syx6. Altogether, our data provide evidence that both Gag and TNFα make use of Syx6-mediated trafficking machinery and suggest that Gag expression does not inhibit but rather facilitates TNFα secretion in HIV-1 infection.

Prevalence of Diabetes in Tuberculosis Patients in Kathmandu Valley, Nepal.

In this descriptive cross-sectional study, the data on the prevalence of diabetes mellitus (DM) among tuberculosis (TB) patients at the Urban Directly Observed Treatment Centers in the Kathmandu, Bhaktapur, and Lalitpur districts of Nepal were collected. The prevalence of DM was assessed in 67 previously treated TB (PTTB) and 214 new TB patients. DM was diagnosed in 8 PTTB and 20 new TB patients. Clinical interviews identified 14 patients with DM, rapid blood glucose test was used to diagnose DM in 4 patients, and oral glucose tolerance test was used to diagnose DM in another 4 patients. Impaired glucose tolerance and impaired fasting glycemia were observed in 8 and 5 patients, respectively. The 18-24-year age group had the largest number of new TB patients (82, 38.3%). However, the incidence of DM among TB patients was higher in the >35-year age group. Moreover, DM was diagnosed in 24.2% of PTTB patients and in 23.1% of new TB patients. To determine the impact of DM screening in TB patients, a larger number of samples should be analyzed. DM screening for patients with TB is expected to start in developing countries. This should be initiated by conducting clinical interviews about DM and glucose tests using rapid kits.

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction.

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Adherence of Clostridium perfringens spores to human intestinal epithelial Caco-2 cells.

Clostridium perfringens is a gram-positive, spore-forming bacillus, and is a causative agent of foodborne infection, antibiotic-associated diarrhoea and sporadic diarrhoea in humans. In cases of antibiotic-associated and sporadic diarrhoea, C. perfringens colonises the intestine, proliferates and causes disease. However, bacterial colonisation of the intestine is not considered necessary in the pathogenesis of foodborne illness, because such pathogenesis can be explained by anchorage-independent production of diarrhoeic toxin by the bacterium in the intestine. In this study, we used an in vitro adherence assay to examine the adherence of C. perfringens spores to human intestinal Caco-2 cells. Adherence of spores from isolates of foodborne illness and nosocomial infection was observed within 15 min, and plateaued 60 min after inoculation. Electron microscopy revealed a tight association of spores with the surface of Caco-2 cells. The adherence of vegetative cells could not be confirmed by the same method, however. These results suggest that C. perfringens spores may adhere to intestinal epithelial cells in vivo, although its biological significance remains to be determined.

Sanitary evaluation of domestic water supply facilities with storage tanks and detection of Aeromonas, enteric and related bacteria in domestic water facilities in Okinawa Prefecture of Japan.

To provide for temporary restrictions of the public water supply system, storage tanks are commonly installed in the domestic water systems of houses and apartment buildings in Okinawa Prefecture of Japan. To learn more about the sanitary condition and management of these water supply facilities with storage tanks (hereafter called "storage tank water systems") and the extent of bacterial contamination of water from these facilities, we investigated their usage and the existence of Aeromonas, enteric and related bacteria. Verbal interviews concerning the use and management of the storage tank water systems were carried out in each randomly sampled household. A total of 54 water samples were collected for bacteriological and physicochemical examinations. Conventional methods were used for total viable count, fecal coliforms, identification of bacteria such as Aeromonas, Enterobacteriaceae and non-fermentative Gram-negative rods (NF-GNR), and measurement of residual chlorine. On Aeromonas species, tests for putative virulence factor and an identification using 16S rRNA and rpoB genes were also performed. Water from the water storage systems was reported to be consumed directly without boiling in 22 of the 54 houses (40.7%). 31 of the sampled houses had installed water storage tanks of more than 1 cubic meter (m3) per inhabitant, and in 21 of the sampled houses, the tank had never been cleaned. In all samples, the total viable count and fecal coliforms did not exceed quality levels prescribed by Japanese waterworks law. Although the quantity of bacteria detected was not high, 23 NF-GNR, 14 Enterobacteriaceae and 5 Aeromonas were isolated in 42.6%, 7.4% and 3.7% of samples respectively. One isolated A. hydrophila and four A. caviae possessed various putative virulence factors, especially A. hydrophila which had diverse putative pathogenic genes such as aer, hlyA, act, alt, ast, ser, and dam. Many bacteria were isolated when the concentration of residual chlorine was below 0.1 mg/l and the water temperature was above 20 °C. These results suggest that elevated water temperature and mismatch between tank size and water demand lead to loss of residual chlorine in tap water. Therefore, to minimize growth of aquatic bacteria such as Aeromonas spp. and Pseudomonas spp., we recommend that an appropriate size tank and/or volume of stored water is always used, and also suggest installation of some means of reducing water temperature such as shading.

Enhancement of adherence of Helicobacter pylori to host cells by virus: possible mechanism of development of symptoms of gastric disease.

It remains unclear why gastric disease does not develop in all cases of Helicobacter pylori infection. In this study, we analyzed whether simian virus 5 (SV5) enhanced adherence of H. pylori to adenocarcinoma epithelial cells (AGS). H. pylori in AGS (harboring SV5) and SV5-infected Vero cells, and an agglutination of H. pylori mixed with SV5 were observed by light microscopy, scanning and transmission electron microscopies. The adherent rate of H. pylori to SV5-infected Vero cells and treated with an anti-SV5 antibody was determined. H. pylori adhered to the surface of AGS cells near SV5 particles, as shown by scanning and transmission electron microscopies. The adherence of H. pylori to SV5-infected Vero cells was significantly enhanced compared with that to Vero cells. In contrast, the adherence of H. pylori to Vero cells was decreased by treatment with the anti-SV5 antibody. Agglutination of H. pylori mixed with SV5 was observed by scanning and transmission electron microscopies. Agglutination did not occur when SV5 was treated with the anti-SV5 antibody before mixing. These findings demonstrated that SV5 enhanced the adherence of H. pylori to host cells, suggesting that a persistently infected virus may be a factor enhancing the pathogenicity of H. pylori in humans.

Distribution of Aeromonas species in environmental water used in daily life in Okinawa Prefecture, Japan.

OBJECTIVES: The genus Aeromonas is known to causes diseases such as food poisoning, sepsis, and wound infection. However, the mode of Aeromonas transmission from environment to humans is not clearly understood. To evaluate the health risks of Aeromonas spp. in environmental freshwater, the number, proportion and putative virulence factors of Aeromonas species were investigated in Okinawa Prefecture, Japan. METHODS: Environmental freshwater samples were collected from three dams, two springs and three private wells. Aeromonas strains were identified by the biochemical method and the viable count was calculated. The production of extracellular enzymes and the virulence genes were investigated for possessing putative virulence factors using representative isolates. RESULTS: At least seven species of already-known Aeromonas isolates as well as unidentified Aeromonas spp. with/without arginin dehydrolase (ADH) exist in water at these sites. Aeromonas spp. was found to exist at over 1000 CFU/100 ml in one spring and two wells. A. veronii biovar sobria and A. jandaei were the predominant species in dams, and A. hydrophila and/or A. eucrenophila were predominant in wells. Almost all the sampled Aeromonas species produced protease, gelatinase, lipase, esterase and DNase, but A. caviae, A. caviae-like bacteria, and A. eucrenophila had low hemolytic activity. Most sampled A. hydrophila strains possessed both aerolysin gene (aer) and hemolysin gene (hlyA), but A. caviae and A. eucrenophila strains did not possess either gene. CONCLUSIONS: Since these results indicated that several Aeromonas species having potential pathogenicity exist in environmental water in Okinawa, surveys are recommended as a public health measure.

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication.

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.

ATP-association to intrabacterial nanotransportation system in Vibrio cholerae.

Vibrio cholerae colonizes the lumen of the proximal small intestine, which has an alkaline environment, and secretes cholera toxin (CT) through a type II secretion machinery. V. cholerae possesses the intrabacterial nanotransportation system (ibNoTS) for transporting CT from the inner portion toward the peripheral portion of the cytoplasm, and this system is controlled by extrabacterial pH. Association of ATP with ibNoTS has not yet been examined in detail. In this study, we demonstrated by immunoelectron microscopy that ibNoTS of V. cholerae under the extrabacterial alkaline condition was inhibited by ATP inhibitors, 2,4-dinitrophenol (DNP), a protonophore, or 8-amino-adenosine which produces inactive form of ATP. The inhibition of CT transport can be reversed by neutralization of DNP. Those inhibitions were associated with decrease of CT secretion by which ibNoTS followed. We propose that ATP closely associates with V. cholerae ibNoTS for transporting CT.

Disinfection potential of electrolyzed strongly acidic water against Mycobacteria: conditions of disinfection and recovery of disinfection potential by reelectrolysis.

We attempted to clarify in detail the conditions of disinfection using electrolyzed strongly acidic water (ESW) against Mycobacteria, and the recovery of the disinfection potential of inactivated ESW by re-electrolysis. We mixed ESW containing 10, 20, and 30 ppm free chlorine with M. bovis cells (10(5)-10(8) CFU/mL) for 0-7 min. The disinfection potential of ESW positively correlated with free chlorine concentration, and negatively correlated with the initial density of bacterial cells. To clarify the recovery of the disinfection potential of inactivated ESW by re-electrolysis, we mixed ESW containing 10 ppm free chlorine with M. bovis cells (10(7) CFU/mL) for 1 min. The number of viable cells decreased to 1/10(3), but the cells were still detected. After re-electrolysis for 7 min, viable cells were not detected. Moreover, we confirmed by reusing the re-electrolyzed water against M. bovis cells that it regained its disinfection potential. These findings indicate that ESW once inactivated during disinfection can be re-activated by re-electrolysis. In conclusion, we were able to clarify in detail the conditions of ESW against Mycobacteria, and found the recovery of the disinfection potential of inactivated ESW by re-electrolysis.

Route of intrabacterial nanotransportation system for CagA in Helicobacter pylori.

Helicobacter pylori (H. pylori) possesses an intrabacterial nanotransportation system (ibNoTS) for transporting CagA and urease within the bacterial cytoplasm; this system is controlled by the extrabacterial environment. The transportation routes of the system have not yet been studied in detail. In this study, we demonstrated by immunoelectron microscopy that CagA localizes closely with the MreB filament in the bacterium, and MreB polymerization inhibitor A22 obstructs ibNoTS for CagA. These findings indicate that the route of ibNoTS for CagA is closely associated with the MreB filament. Because these phenomena were not observed in ibNoTS for urease, the route of ibNoTS for CagA is different from that of ibNoTS for urease as previously suggested. We propose that the route of ibNoTS for CagA is associated with the MreB filament in H. pylori.

Phosphorylation of human immunodeficiency virus type 1 capsid protein at serine 16, required for peptidyl-prolyl isomerase-dependent uncoating, is mediated by virion-incorporated extracellular signal-regulated kinase 2.

We reported previously that Pin1 facilitates human immunodeficiency virus type 1 (HIV-1) uncoating by interacting with the capsid core through the phosphorylated Ser(16)-Pro(17) motif. However, the specific kinase responsible for Ser(16) phosphorylation has remained unknown. Here, we showed that virion-associated extracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser(16). The characterization of immature virions produced by exposing chronically HIV-1LAV-1-infected CEM/LAV-1 cells to 10 µM saquinavir indicated that Ser(16) is phosphorylated after the initiation of Pr55(Gag) processing. Furthermore, a mass spectrometry-based in vitro kinase assay demonstrated that ERK2 specifically phosphorylated the Ser(16) residue in the Ser(16)-Pro(17) motif-containing substrate. The treatment of CEM/LAV-1 cells with the ERK2 inhibitor sc-222229 decreased the Ser(16) phosphorylation level inside virions, and virus partially defective in Ser(16) phosphorylation showed impaired reverse transcription and attenuated replication owing to attenuated Pin1-dependent uncoating. Furthermore, the suppression of ERK2 expression by RNA interference in CEM/LAV-1 cells resulted in suppressed ERK2 packaging inside virions and decreased the Ser(16) phosphorylation level inside virions. Interestingly, the ERK2-packaging-defective virus showed impaired reverse transcription and attenuated HIV-1 replication. Taken together, these findings provide insights into the as-yet-obscure processes in Pin1-dependent HIV-1 uncoating.

A new type of intrabacterial nanotransportation system for VacA in Helicobacter pylori.

Helicobacter pylori possesses intrabacterial nanotransportation systems (ibNoTSs) for CagA and urease. Both systems are UreI-dependent and urea-independent, and activated by extrabacterial acid. The activation occurs/appears within 15 min after exposure to extrabacterial acid stimulation. Although it has been clarified that VacA is secreted via the type-V secretion machinery, it remains unclear how this toxin is transported toward the machinery. To clarify the intrabacterial nanotransportation system for H. pylori VacA, immunoelectron microscopic analysis was performed in this study. VacA shifted to the periphery of the bacterial cytoplasm at 30 min after the extracellular pH change, whereas CagA and urease did so within 15 min. Studies using an ureI-deletion mutant revealed that unlike CagA and urease transport, VacA transport was not UreI-dependent. VacA secretion was accelerated without an increase in the production of VacA 30 min after the exposure to extrabacterial acid. These findings indicated that H. pylori possesses a novel type of ibNoTS for VacA, which is different from that for CagA or urease, in response time and dependency of UreI.

Application of electrolysis for inactivation of an antiviral drug that is one of possible selection pressure to drug-resistant influenza viruses.

The recent development of antiviral drugs has led to concern that the release of the chemicals in surface water due to expanded medical use could induce drug-resistant mutant viruses in zoonosis. Many researchers have noted that the appearance of an oseltamivir (Tamiflu(®))-resistant avian influenza mutant virus, which may spread to humans, could be induced by oseltamivir contamination of surface water. Although past studies have reported electrolysis as a possible method for degradation of antineoplastics and antibacterials in water, the validity of the method for treatment of antiviral drugs is unknown. In this study, electrolysis was used to degrade an antiviral prodrug, oseltamivir, and a stable active form, oseltamivir carboxylate, and the degradation process was monitored with HPLC-UV and the neuraminidase inhibitory assay. HPLC-UV-detectable oseltamivir and oseltamivir carboxylate were decomposed by electrolysis within 60 min, and inhibitory activity of neuraminidase decreased below the detection limit of the assay used. Cytotoxic and genotoxic activity were not detected in electrolyzed fluid. These results indicate that electrolysis is a possible treatment for inactivation of the antiviral drug oseltamivir.

Application of electrolysis to inactivation of antibacterials in clinical use.

Contamination of surface water by antibacterial pharmaceuticals (antibacterials) from clinical settings may affect aquatic organisms, plants growth, and environmental floral bacteria. One of the methods to decrease the contamination is inactivation of antibacterials before being discharged to the sewage system. Recently, we reported the novel method based on electrolysis for detoxifying wastewater containing antineoplastics. In the present study, to clarify whether the electrolysis method is applicable to the inactivation of antibacterials, we electrolyzed solutions of 10 groups of individual antibacterials including amikacin sulfate (AMK) and a mixture (MIX) of some commercial antibacterials commonly prescribed at hospitals, and measured their antibacterial activities. AMK was inactivated in its antibacterial activities and its concentration decreased by electrolysis in a time-dependent manner. Eighty to ninety-nine percent of almost all antibacterials and MIX were inactivated within 6h of electrolysis. Additionally, cytotoxicity was not detected in any of the electrolyzed solutions of antibacterials and MIX by the Molt-4-based cytotoxicity test.

Disinfective process of strongly acidic electrolyzed product of sodium chloride solution against Mycobacteria.

Electrolyzed acid water (EAW) has been studied for its disinfective potential against pathogenic microbes; however, the bactericidal process against Mycobacteria has not been clearly presented. In this study, to clarify the disinfective process against Mycobacteria, EAW-treated bacteria were examined against laboratory strains of Mycobacterium bovis (M. bovis), Mycobacterium smegmatis (M. smegmatis), and Mycobacterium terrae (M. terrae) by recovery culture and observation of morphology, enzymatic assay, and the detection of DNA. All experiments were performed with the use of EAW containing 30 ppm free chlorine that kills Mycobacteria, including three pathogenic clinical isolates of Mycobacterium tuberculosis (M. tuberculosis) and six isolates of other Mycobacteria, within 5 min. In morphology, the bacterial surface became rough, and a longitudinal concavity-like structure appeared. The intrabacterial enzyme of EAW-contacted bacteria was inactivated, but chromosomal DNA was not totally denatured. These results suggest that the bactericidal effect of EAW against Mycobacteria occurs by degradation of the cell wall, followed by denaturation of cytoplasmic proteins, but degeneration of the nucleic acid is not always necessary.

Fine visualization of filamentous structures in the bacterial cytoplasm.

A new method was established for fine visualization of bacterial subcelluar filamentous structures by freezing the bacterial cells to displace cytoplasmic matrix granules to the periphery. This method was successfully applied in immunoelectron microscopy and electron microscopic tomography, and should be applicable for further studies of bacterial architecture and nanotransportation.

Detection of dengue virus genome in urine by real-time reverse transcriptase PCR: a laboratory diagnostic method useful after disappearance of the genome in serum.

The reemergence of dengue virus (DENV) infection has created a requirement for improved laboratory diagnostic procedures. In this study, DENV genome detection in urine was evaluated as a diagnostic method. The DENV genome was detected by real-time reverse transcriptase PCR (RT-PCR) in urine and serum of dengue patients. The detection rate of DENV genome in urine was 25% (2/8) on disease days 0 to 3 and 32% (7/22) on days 4 to 5. The rate was 50% or higher on days 6 to 16, 52% (11/21) on days 6 to 7, 78% (7/9) on days 8 to 9, 80% (4/5) on days 10 to 11, 50% (2/4) on days 12 to 13, and 60% (3/5) on days 14 to 16. The last positive urine sample was on day 16. The detection rates in serum were highest on days 0 to 3 and were greater than 50% on days 0 to 7. Detection rates decreased thereafter, and the last positive detection was on day 11. These results indicate that the time frames for positive detection differ between urine and serum samples, whereby detection rates of 50% or higher are evident between days 6 to 16 for urine samples and days 0 to 7 for serum samples. Nucleotide sequences of PCR products were identical between urine and serum samples. The detection of DENV genome in urine samples by real-time RT-PCR is useful to confirm DENV infection, particularly after viremia disappears.

Regulation of hepatitis C virus secretion by the Hrs-dependent exosomal pathway.

The molecular mechanisms of assembly and budding of hepatitis C virus (HCV) remain poorly understood. The budding of several enveloped viruses requires an endosomal sorting complex required for transport (ESCRT), which is part of the cellular machinery used to form multivesicular bodies (MVBs). Here, we demonstrated that Hrs, an ESCRT-0 component, is critical for the budding of HCV through the exosomal secretion pathway. Hrs depletion caused reduced exosome production, which paralleled with the decrease of HCV replication in the host cell, and that in the culture supernatant. Sucrose-density gradient separation of the culture supernatant of HCV-infected cells revealed the co-existence of HCV core proteins and the exosome marker. Furthermore, both the core protein and an envelope protein of HCV were detected in the intraluminal vesicles of MVBs. These results suggested that HCV secretion from host cells requires Hrs-dependent exosomal pathway in which the viral assembly is also involved.