Monitoring Quinolone Resistance Due to Mutations in GyrA and ParC in Haemophilus Influenzae（2012-17）

Knowing recent drug-resistant bacteria trends is important for proper antibacterial drug use to improve the prognosis of patients with infectious diseases and for public health. Because multiple quinolone antibacterial agents are simultaneously adopted in hospitals in Japan, we examined whether minimum inhibitory concentrations （MICs） against Haemophilus influenzae differ among quinolones. We determined MICs of six different quinolone antibacterial agents and performed molecular genetic analysis. We investigated β-lactamase-producing and β-lactamase-negative ampicillin-resistant（BLNAR）H. influenzae using the nitrocefin method in parallel. Overall, 144 clinical H. influenzae strains isolated at the Showa University Hospital between 2012 and 2017 were subjected to MIC determination for penicillin/quinolone antibacterial agents using the Clinical and Laboratory Standards Institute broth microdilution method. Amino acid mutations in the quinolone resistance-determining regions were analyzed in the isolates showing an MIC value ≥ 0.25µg/ml of quinolone antibacterial agents. BLNAR isolates increased from 2016 onward. Among quinolone antibacterial agents, all isolates remained susceptible to sitafloxacin. However, for moxifloxacin（MFLX）, strains with an MIC value＝0.5µg/ml were detected every year since 2013 except in 2015. Amino acid mutations were investigated in 17 isolates （11.8％） with MFLX MIC value≥ 0.25µg/ml and confirmed in 11 isolates （7.6％）, of which 9 contained GyrA mutations. The results demonstrated that MFLX was useful for predicting the presence of amino acid mutations and 0.25 was an appropriate MIC threshold for this purpose. This screening procedure may be effective for reducing the inappropriate use of quinolones and controlling the emergence of drug-resistant H. influenzae

Trans-complemented hepatitis C virus particles as a versatile tool for study of virus assembly and infection

AbstractIn this study, we compared the entry processes of trans-complemented hepatitis C virus particles (HCVtcp), cell culture-produced HCV (HCVcc) and HCV pseudoparticles (HCVpp). Anti-CD81 antibody reduced the entry of HCVtcp and HCVcc to almost background levels, and that of HCVpp by approximately 50%. Apolipoprotein E-dependent infection was observed with HCVtcp and HCVcc, but not with HCVpp, suggesting that the HCVtcp system is more relevant as a model of HCV infection than HCVpp. We improved the productivity of HCVtcp by introducing adapted mutations and by deleting sequences not required for replication from the subgenomic replicon construct. Furthermore, blind passage of the HCVtcp in packaging cells resulted in a novel mutation in the NS3 region, N1586D, which contributed to assembly of infectious virus. These results demonstrate that our plasmid-based system for efficient production of HCVtcp is beneficial for studying HCV life cycles, particularly in viral assembly and infection

A Pilot Study for the Detection of Protozoa Infections of the Gut at Autopsy

In order to investigate the incidence of latent infections by parasites within the human digestive tract, we examined fresh stool samples from the colon of 31 patients (37-90 years, median age 71; 26 men and 5 women) collected within 12 hours of death. These subjects had been admitted to a university hospital in Yokohama, Japan, and died between April 2007 and December 2008 from causes other than parasitosis. Stool samples were fixed and stained for microscopic analysis, and PCR analysis for Entamoeba histolytica was performed for the parasite-positive samples. Results showed that ten out of 31 subjects were infected by E. histolytica only, one subject was infected by Giardia intestinalis only, and four subjects were infected by both E. histolytica and G. intestinalis. These findings are in contrast to conventional theories concerning general parasite infection in Japan, and indicate continuous or latent infection within the human digestive tract. The presence of pathogens such as E. histolytica and G. intestinalis in elderly or immuno-compromised patients is a serious issue and warrants further attention as a public health issue, particularly in relation to its mode of transmission

Proteasomal Turnover of Hepatitis C Virus Core Protein Is Regulated by Two Distinct Mechanisms: a Ubiquitin-Dependent Mechanism and a Ubiquitin-Independent but PA28γ-Dependent Mechanism▿

We have previously reported on the ubiquitylation and degradation of hepatitis C virus core protein. Here we demonstrate that proteasomal degradation of the core protein is mediated by two distinct mechanisms. One leads to polyubiquitylation, in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is independent of the presence of ubiquitin. Gain- and loss-of-function analyses using lysineless mutants substantiate the hypothesis that the proteasome activator PA28γ, a binding partner of the core, is involved in the ubiquitin-independent degradation of the core protein. Our results suggest that turnover of this multifunctional viral protein can be tightly controlled via dual ubiquitin-dependent and -independent proteasomal pathways

E6AP Ubiquitin Ligase Mediates Ubiquitylation and Degradation of Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein