Biology Reference
In-Depth Information
which would challenge our future investigation. Moreover, it seems to be a paradox
that compound 4 have less bactericidal effects in the time- and concentration-depen-
dent antibacterial assays, but demonstrated signifi cant therapeutic effects in mice in-
fected by
S. pneumoniae
. However, due to the VicK' is not essential in
S. pneumoniae
,
this chemical may have a possibility to interrupt the invasion and virulence rather than
cause numerous death of the bacterium, which decreases the selection pressure and
contributes to the maintenance of species diversity, thus reduces the emergence of
drug-resistant strains. Anyway, the subtle mechanisms need our future work.
MATERIAL AND METHODS
Bacterial Strains, Media, and Reagents
S. pneumoniae
(D39) ATCC7466 was purchased from the American Type Culture col-
lection (ATCC, USA).
S. pneumoniae
D39 was grown in C + Y medium. Plasmids
were transformed into
Escherichia coli
(
E. coli
) strains that were grown in Luria-
Bertani (LB) broth. For selection of
E. coli
transformants, kanamycin (50 μg/ml, final
concentration) was added to the growth medium.
All compounds screened out in our study were purchased from the SPECS Com-
pany in the Netherlands. Stock solutions of the compounds were prepared in Dimethyl
Sulfoxide (DMSO). Other chemicals were purchased from Sigma.
Bioinformatics Analysis
Domain analysis was performed based on the SMART database. The complete ge-
nome sequences of the
S. pneumoniae
strain ATCC7466 were accessed from the
National center for Biotechnology information (NCBI) genome database. For the ho-
mologous sequences with the VicK HATPase_c domain of
S. pneumoniae
ATCC7466,
the PDB was searched by using the Blastp program. ClustalX was used to align the
protein sequences.
3D Structure Modeling of the VicK HATPase_c Domain
The sequence of
S. pneumoniae
VicK was retrieved from GenBank (accession num-
ber: AAK75332.1). The Align123 module in Insight II was used in the pairwise se-
quence alignment. Using the secondary structure information of
Thermotoga maritima
(PDB entry: 2c2a), the sequence alignment was adjusted manually to obtain a fine
alignment for 3D structure construction. The 3D model of the VicK HATPase_c do-
main was generated by using the MODELLER module in Insight II. Several structural
analysis programs such as Prostat and Profile-3D were used to check the structure
quality. The Prostat module of Insight II was used to analyze the properties of bonds,
angles, and torsions. The profile-3D program was used to check the structure and se-
quence compatibility.
Structure-based Virtual Screening
The SBVS was performed as described previously [36], with modification. Briefly, the
binding pocket of the VicK HATPase_c domain was used as a target for screening the
SPECS database by using the docking approach. A primary screening was conducted
