Leili Jia

Comparative Proteomics Analyses Reveal the virB of B. melitensis Affects Expression of Intracellular Survival Related Proteins

Backgound Brucella melitensis is a facultative, intracellular, pathogenic bacterium that replicates within macrophages. The type IV secretion system encoded by the virB operon (virB) is involved in Brucella intracellular survival. However, the underlying molecular mechanisms, especially the target proteins affected by the virB, remain largely unclear. Methodology/Principal Findings In order to define the proteins affected by virB, the proteomes of wild-type and the virB mutant were compared under in vitro conditions where virB was highly activated. The differentially expressed proteins were identified by MALDI-TOF-MS. Forty-four down-regulated and eighteen up-regulated proteins which exhibited a 2-fold or greater change were identified. These proteins included those involved in amino acid transport and metabolism, lipid metabolism, energy production, cell membrane biogenesis, translation, post-translational modifications and protein turnover, as well as unknown proteins. Interestingly, several important virulence related proteins involved in intracellular survival, including VjbR, DnaK, HtrA, Omp25, and GntR, were down-regulated in the virB mutant. Transcription analysis of virB and vjbR at different growth phase showed that virB positively affect transcription of vjbR in a growth phase dependent manner. Quantitative RT-PCR showed that transcription of these genes was also affected by virB during macrophage cell infection, consistent with the observed decreased survival of the virB mutant in macrophage. Conclusions/Significance These data indicated that the virB operon may control the intracellular survival of Brucella by affecting the expression of relevant proteins.

Emergence of a Novel Shigella flexneri Serotype 4s Strain That Evolved from a Serotype X Variant in China

ABSTRACT This paper describes the first isolation of a new Shigella flexneri serotype, designated 4s, in Beijing, China. Genotypic and phenotypic profiling suggests that this isolate is a clone of the S. flexneri serotype X variant reference strain. Of particular concern is the multidrug resistance exhibited by this isolate.

The type IV secretion system affects the expression of Omp25/Omp31 and the outer membrane properties of Brucella melitensis

The type IV secretion system (T4SS) contributes to Brucella intracellular survival through its effector proteins. Comparative proteomic analysis showed that intracellular survival proteins are expressed differentially in a virB mutant. Interestingly, several outer membrane proteins (OMPs) are also differentially expressed, implying that T4SS might affect the OM properties of Brucella. To further evaluate the impact of T4SS on OM, in the present study, the OM proteomes were isolated and compared. Many more products of OMPs, particularly different products of the Omp25/Omp31 family, were found to be altered in the virB mutant. The transcription profiles of Omp25/Omp31 were different from those of their protein products, implying their regulation by virB at both transcriptional and post-transcriptional levels. The virB mutant aggregates at a high cell density and produces exopolysaccharide, a phenotype resembling that of the vjbR mutant. The virB mutant showed increased sensitivity to polymyxin B and decreased survival under oxidative, high-salt and high-osmolarity stresses, indicating drastic membrane alterations. These results indicated that in addition to being an effector protein secretion system, T4SS affects OM properties that might be important for the adaptation of Brucella to both in vitro and in vivo hostile environments.

Emergence of a novel Shigella flexneri serotype 4s evolved from serotype X variant in China

ABSTRACT This paper describes the first isolation of a new Shigella flexneri serotype, designated 4s, in Beijing, China. Genotypic and phenotypic profiling suggests that this isolate is a clone of the S. flexneri serotype X variant reference strain. Of particular concern is the multidrug resistance exhibited by this isolate.

Removal of integrated hepatitis B virus DNA using CRISPR-Cas9

The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or “sterile” HBV cure.

Prevalence and antimicrobial resistance of Shigella flexneri serotype 2 variant in China

Shigella flexneri serotype 2 variant (II:3,4,7,8) was isolated in 2008 and first reported in China in 2013. In the present study, epidemiological surveillance from 2003 to 2013 in China suggested that this serotype first appeared in Guangxi in 2003; it then emerged in Shanghai and Xinjiang in 2004 and in Henan in 2008. Of the 1813 S. flexneri isolates, 58 S. flexneri serotype 2 variant strains were identified. Serotype 2 variant has emerged as a prominent serotype in recent years, with 2a (32.6%), X variant (25.2%), 1a (9.4%), X (6.3%), 2b (5.4%), and 1b (3.6%). According to phenotypic and genotypic analysis, the serotype 2 variant originated from 2a to 2b. A higher antibiotic resistance rate was observed between 2009 and 2013 than that between 2003 and 2008. Among 22 cephalosporin-resistant isolates, blaTEM-1, blaOXA-1, blaCTX-3, blaCTX-14, and blaCTX-79 were detected. Among 22 fluoroquinolone-resistant isolates, a Ser80Ile mutation in parC was present in all of the isolates. Moreover, 21 isolates had three gyrA point mutations (Ser83Leu, His211Tyr, Asp87Asn, or Gly) and one isolate had two gyrA point mutations (Ser83Leu and His211Tyr). The prevalence of His211Tyr in the fluoroquinolone-resistant isolates is concerning, and the mutation was first reported in China. Besides, 22 isolates harbored the aac(6′)-Ib-cr gene, and two isolates harbored qnrS1. In view of the increased epidemic frequency and multidrug-resistant strain emergence, continuous surveillance will be needed to understand the actual disease burden and provide guidance for shigellosis.

Shift in serotype distribution of Shigella species in China, 2003–2013

We identified 2912 Shigella isolates from diarrhoeal patients in China during 2003-2013. The most common species was Shigella flexneri (55.3%), followed by Shigella sonnei (44.1%); however, S. sonnei is becoming increasingly prevalent. Among the S. flexneri isolates, serotypes 2a and X variant (-:7,8, E1037) were the two most prevalent serotypes, and serologically atypical isolates were also commonly identified. Overall, S. sonnei, S. flexneri 2a and S. flexneri X variant (-:7,8, E1037) accounted for 76.1% of all Shigella isolates, and their prevalence increased from 54.0% during 2003-2004 to 84.1% during 2011-2013. A change was observed in the serotype distribution of Shigella in China during this period, and we propose an ideal strategy to inform the development of a broadly effective Shigella vaccine candidate.

An Outbreak of Mycoplasma pneumoniae Caused by a Macrolide-Resistant Isolate in a Nursery School in China

ABSTRACT Eighteen out of 45 children were reported to have a respiratory illness during an outbreak at a temporary dormitory in a nursery school in China in 2011. To study the outbreak and to determine the risk factors for infection, an epidemiological investigation was performed. A standardized questionnaire was completed for a total of 45 children with the help of their guardians and parents. In addition, acute- and convalescent-phase serum samples and throat swabs from the children were taken for laboratory diagnosis. The diagnosis of a Mycoplasma-like illness was based on the following clinical criteria. The criteria were onset of illness after 31 May 2011, characterized by a cough, fever(>37.5°C), or at least 3 of the following symptoms: fever, sore throat, cough or expectoration, and runny or stuffy nose. PCR-restriction fragment length polymorphism (PCR-RFLP), determination of MICs, and sequencing were performed to determine the genotype, antibiotic resistance, and sequence polymorphisms of the isolated strains, respectively. The paired sera revealed that 15 patients were infected with Mycoplasma pneumoniae. Epidemiology confirmed that this was a point source outbreak, characterized by a short incubation period, a high secondary attack rate, and a long period of hospitalization. PCR-RFLP analysis revealed that the 12 isolated strains of M. pneumoniae shared the same subtype P1 gene, and 23S rRNA sequence analysis showed that these strains harbored two macrolide-resistant gene-related point mutations at position 2063 and 2617. In this outbreak, the major risk factor was the distance between the bed of the first patient and the beds of close contacts (beds less than three meters apart). The strains isolated in this study were found to harbor two point mutations conferring macrolide resistance, indicating the importance of pathogen and drug resistance surveillance systems.

Stable Nanocomposite Based on PEGylated and Silver Nanoparticles Loaded Graphene Oxide for Long-Term Antibacterial Activity

The increasing occurrence of antibiotic-resistant pathogens, especially superbugs, is compromising the efficacy of traditional antibiotics. Silver nanoparticles (AgNPs) loaded graphene oxide (GO) nanocomposite (GO-Ag) has drawn great interest as a promising alternative antibacterial material. However, GO-Ag nanocomposite often irreversibly aggregates in physiological solutions, severely influencing its antibacterial capacity and practical application. Herein, a PEGylated and AgNPs loaded GO nanocomposite (GO-PEG-Ag) is synthesized through a facile approach utilizing microwave irradiation, while avoiding extra reducing agents. Through PEGylation, the synthesized GO-PEG-Ag nanocomposite dispersed stably over one month in a series of media and resisted centrifugation at 10 000×g for 5 min, which would benefit effective contact between the nanocomposite and the bacteria. In contrast, GO-Ag aggregated within 1 h of dispersion in physiological solutions. In comparison with GO-Ag, GO-PEG-Ag showed stronger bactericidal capability toward not only normal Gram-negative/positive bacteria such as E. coli and S. aureus (∼100% of E. coli and ∼95.3% of S. aureus reduction by 10 μg/mL nanocomposite for 2.5 h), but also superbugs. Moreover, GO-PEG-Ag showed lower cytotoxicity toward HeLa cells. Importantly, GO-PEG-Ag presented long-term antibacterial effectiveness, remaining ∼95% antibacterial activity after one-week storage in saline solution versus <35% for GO-Ag. The antibacterial mechanisms of GO-PEG-Ag were evidenced as damage to the bacterial structure and production of reactive oxygen species, causing cytoplasm leakage and metabolism decrease. The stable GO-PEG-Ag nanocomposite with powerful and long-term antibacterial capability provides a more practical and effective strategy for fighting superbugs-including pathogen threats in biomedicine and public health.

An Effective Molecular Target Site in Hepatitis B Virus S Gene for Cas9 Cleavage and Mutational Inactivation.

Chronic hepatitis B infection remains incurable because HBV cccDNA can persist indefinitely in patients recovering from acute HBV infection. Given the incidence of HBV infection and the shortcomings of current therapeutic options, a novel antiviral strategy is urgently needed. To inactivate HBV replication and destroy the HBV genome, we employed genome editing tool CRISPR/Cas9. Specifically, we found a CRISPR/Cas9 system (gRNA-S4) that effectively targeted the HBsAg region and could suppress efficiently viral replication with minimal off-target effects and impact on cell viability. The mutation mediated by CRISPR/Cas9 in HBV DNA both in a stable HBV-producing cell line and in HBV transgenic mice had been confirmed and evaluated using deep sequencing. In addition, we demonstrated the reduction of HBV replication was caused by the mutation of S4 site through three S4 region-mutated monoclonal cells. Besides, the gRNA-S4 system could also reduce serum surface-antigen levels by 99.91 ± 0.05% and lowered serum HBV DNA level below the negative threshold in the HBV hydrodynamics mouse model. Together, these findings indicate that the S4 region may be an ideal target for the development of innovative therapies against HBV infection using CRISPR/Cas9.