Objective      To lay a foundation for further exploration of host defense responses to DnaJ through the construction of dnaJ-defecient mutant strain of Streptococcus pneumoniae (S. pn) and the preliminary study of its virul

ence.      Methods      The dnaJ gene was replaced with an erythromycin resistance gene (erm) by long flanking homology polymerase chain reaction (LFH-PCR)

and the recombinant fragment was transformed into S. pn. The dnaJ-defecient mutant strain of S.pn was screened out from the blood plates containing erythromycin and was verified by PCR. The growth of dnaJ-defecient mutant strain in vitro was observed based on its OD value (600 nm). Forty-four BALB/c mice were randomly divided into 2 groups

a wild-type strain group and a dnaJ-deficient mutant strain group. Each group was subdivided into a high dose (2×10

8

cfu) and a low dose (2×10

6

cfu) group (11 mice in each subgroup). The mice of the two groups were intraperitoneally given a high and a low dose of 100 μl D39 and △dnaJ bacteria liquid separately. The mice death time was recorded

and the survival rate was calculated. Sixty BALB/c mice were randomly divided into 2 groups

the mice of which were intranasally given D39 and △dnaJ bacteria liquid (30 μl

2×10

7

cfu) separately. After 6 h

12 h

24 h

36 h and 48 h infection

the mice were put to death (5 mice of each time point). The mice nasopharyngeal lavage fluid

lung tissue (homogenate) and heart blood were collected and cultured seperately. The colonization curve of the bacteria was drawn according to the average colony counting in each group.      Results      The PCR result showed that the dnaJ gene was completely replaced by the erm gene

and the dnaJ-deficient mutant strain was successfully constructed. The growth of the single colony in culture medium showed that the dnaJ-deficient bacteria growth was inhibited by heat shock in vitro. The virulence test suggested that the survival rate of mice with abdominal infection of the deficient strain was 100%

while that of mice with abdominal infection of wild-type strain was 0% (P0.01). In the intranasal infection experiment

the loading amount of the deficient strain

which could be cleared in the blood within 24 h after infect

ion

was significantly lower than that of the wild-type strain in the nasopharyngeal lavage fluid and lung tissue at each time point (P0.01).      Conclusion      It is a convenient way to completely substitute for dnaJ gene using LFH-PCR. The dnaJ deficiency inhibits the growth of dnaJ-deficient bacteria under the heat shock in vitro and simultaneously reduces the bacterial virulence and colonization in vivo.