Construction of in vitro reconstitution system for inverse autotransporter Intimin / 中华微生物学和免疫学杂志

Objective:To construct an in vitro reconstitution system for inverse autotransporters in order to further investigate their translocation mechanism. Methods:Intimin from Escherichi coli was used as a model substrate. Spheroplasts were prepared from Escherichi coli strains overexpressing Intimin to induce the expression of Intimin. Recombinant β-barrel assembly machinery (BAM) complex was obtained and purified, and then proteoliposomes containing BAM were prepared. Following the digestion with proteinase K, the translocation was detected by SDS-PAGE. Results:Spheroplasts were induced to express Intimin, and then BAM-containing proteoliposomes were added to the system. Compared with control and liposomes groups, the experimental group showed that Intimin was resistant to proteinase K treatment, indicating that Intimin was successfully translocated.Conclusions:The translocation of Intimin required the participation of BAM complex. An in vitro reconstitution system for inverse autotransporters was constructed in this study, providing a method to study the translocation mechanism of inverse autotransporters.

Surface display of HPV16L1 by autotransporter Ag43 / 中华微生物学和免疫学杂志

Objective:To construct a surface display system containing various lengths of the Ag43 passenger domain for an optimal bacterial surface display of foreign protein HPV16L1.Methods:(1) Ag43 gene sequences of different lengths were inserted into pET22b vector to construct four Ag43 surface display vectors (Ag43/138, Ag43/551, Ag43/552 and Ag43/700) using PCR and subcloning strategy. (2) The generation of four HPV16L1-Ag43 fusion constructs was completed by PCR and subcloning methods. (3) HPV16L1-Ag43 fusion proteins were expressed and analyzed by SDS-PAGE. (4) The surface exposure of HPV-16L1 was verified using trypsin digestion.Results:PCR analysis and sequencing results showed that Ag43 surface display vectors and HPV16L1-Ag43 fusions were constructed successfully. SDS-PAGE showed that the expression of HPV16L1-Ag43 fusion proteins could be induced with 0.2 mmol/L IPTG and the protein content was reduced after the cells were treated with trypsin, especially the content of Ag43/700-HPV16L1 that showed a drastic reduction.Conclusions:The Ag43 surface display system was successfully constructed and could be used for a successful display of HPV16L1. This study also showed that Ag43/700 comprising only the α-helix and the β-barrel of Ag43 provided an optimal surface display for HPV16L1.

Molecular Characterization of sca Genes Found in Orientia tsutsugamushi Genome

Scrub typhus is an acute, febrile illness caused by Orientia tsutsugamushi infection and it is one of the main causes of acute febrile illness in the Asian-Pacific region. The incidence of scrub typhus has been significantly increased in Korea during last 10 years. Although early diagnosis and proper antibiotic treatment are important to prevent severe complications, the clinical discrimination of scrub typhus from other undifferentiated fevers, such as leptospirosis or dengue fever, is often very difficult. In addition, an effective vaccine has not yet been developed. As a novel diagnostic and vaccine target for scrub typhus, we described surface cell antigen (sca) family genes encoding autotransporter proteins found in the genome of O. tsutsugamushi. The molecular characteristics and recent findings on the bacterial genes were introduced in this letter.