| Streptococcus agalactiae, commonly known as group B streptococcus (GBS), has a broad host range. It has been reported to cause neonatal pneumonia and meningitis in humans, mastitis in cows and meningoencephalitis in fish. Recently, numerous outbreaks of S1. agalactiae infections have been described in multiple fish farms, especially tilapia farms. The pathogenesis of S. agalactiae infection in tilapias is not yet fully understood. In this study, selective capture of transcribed sequences (SCOTS) was used to investigate microbial gene expression associated with murine macrophages infection, and the biological functions of genes including aroA, clpE, msrB and pulA were described. This study will lead to a better understanding of the pathogenicity of the piscine S.agalactiae.1. Interaction of S. agalactiae with murine macrophages and brain microvascular endothelial cellsMurine macrophages (RAW264.7) and murine brain microvascular endothelial cells (bEnd.3) were used as in vitro cell infection models to evaluate the pathogenetic characteristics of GD201008-001. The results showed that entry of S. agalactiae into RAW264.7 macrophages occurred in a dose-dependent manner. To explore the interaction of S. agalactiae with RAW264.7 and bEnd.3, piscine strain GD201008-001, bovine strain 13813 and human strain A909 were used, and some characteristics were analyzed, including the antiphagocytosis to RAW264.7 and intracellular survival, and cytotoxicity activity to RAW264.7 and bEnd.3. The results showed that the antiphagocytosis of GD201008-001 was significantly higher than those of 13813 and A909 (P<0.05), and the phagocytosis rates were 12.77%,28.48% and 32.58%, respectively. All the strains could survival for more than 24h within RAW264.7, and the survival rates were 16.18% (GD201008-001),6.00%(13813) and 94.87%(A909) at 24h post infection, respectively. In addition, the cytotoxicity of GD201008-001 to RAW264.7 and bEnd.3 was significantly higher than those of 13813 and A909 (P<0.05). Our result indicated that the virulence of GD201008-001 was higher than those of 13813 and A909.2. Identification of genes preferentially expressed by highly virulent piscine S. agalactiae upon interaction with macrophagesSelective capture of transcribed sequences (SCOTS) was used to investigate microbial gene expression associated with intracellular survival. This positive cDNA selection technique identified 60 distinct genes that could be characterized into 7 functional categories:(1) Nine genes were involved in cell envelope biogenesis/outer membrane synthesis, responsible for the construction of peptidoglycans, cell surface proteins; (2)Thirty-two genes were involved in metabolism/stress response, including enzymes involved in the biosynthesis and metabolism of sugar, fatty acids, proteins, and nucleic acids; (3) Four transporters genes were identified, including the components of ABC-type transport systems and chloride channels; (4) Five genes were involved in cell division/replication, including genes associated with the replication and genetic regulation of the bacteria; (5) Three genes were involved in protein sorting, including molecular chaperones and signal peptidase; (6) Four genes possessed regulatory functions, including transcriptional regulation, and four genes showed no significant similarities to any other previously described genes. More than 50% of the differentially expressed genes were involved in metabolic adaptation. Some genes have previously been described as associated with virulence in other bacteria. This study constitutes the first step in further gene expression analyses that will lead to a better understanding of the molecular mechanisms used by S. agalactiae to survive in macrophages and to cause infection.3. Construction of the deleted mutants of genes preferentially expressed by S. agalactiae upon interaction with macrophagesTo investigate the roles of genes preferentially expressed by S. agalactiae upon interaction with macrophages by SCOTS, four GD201008-001 isogenic mutants (△aroA, △clpE, △msrB and △pulA) and complementation strains (C△aroA, C△clpE, C△msrB and C△pulA) were respectively constructed by temperature-sensitive Streptococcus-Escherichia coli shuttle vector pSET4s and pSET2. Then the mutants and complementation strains were further verified by PCR, sequencing and qRT-PCR analysis. The mutant and complementation strains showed good genetic stability. There were no significant differences in morphology. However, the growth rate of ApulA was significantly lower than that of the wild strain. For the other mutants, there were no significant differences in the growth characteristics relative to wild strain.4. Characterizations of the deleted mutants of genes preferentially expressed by S. agalactiae upon interaction with macrophagesTo explore the roles of genes related to interaction with macrophages, including aroA, clpE, msrB and pulA in the pathogenesis of S. agalactiae, the following characteristics were analyzed, including the antiphagocytosis to RAW264.7 and intracellular survival, tolerance to the acidic and oxidizing environment, adhesion to bEnd.3, cytotoxicity to RAW264.7 and bEnd.3, virulence (LD50) to zebrafish and mice, colonization in blood, spleen and brain tissues of mice and the capacity of stimulating the release of pro-inflammatory cytokines in macrophages and mice. The results showed that the antiphagocytosis of clpE mutant was significantly decreased. The intracellular survivals as well as tolerances to oxidizing environment of aroA, clpE and msrB mutants were decreased. Moreover, clpE and msrB mutants were significantly more sensitive to acidic environment. The adhesion of, clpE and pulA mutants to bEnd.3 was significantly decreased as well as the cytotoxicity to RAW264.7 and bEnd.3. The virulence of aroA mutant was significantly attenuated in the zebrafish and ICR mice, and its LD50 values were 1.84×103 and 1.56×106 folds of the wild type, respectively. LD50 values of clpE, msrB and pulA mutants were elevated slightly in zebrafish, while in ICR mice, there was no difference between the mutant and wild strains, but the time of death was delayed. At 16h post infection, the colonization capacities of the aroA, clpE and pulA mutants in blood, spleen and brain tissues of mice significantly decreased as compared to the wild type. However, the colonization capacity of the msrB mutant only decreased in brain tissue. The aroA mutant stimulated a significantly higher release of pro-inflammatory cytokines, such as IL-1βp, IL-6 and TNF-a, than the wild-type in macrophages. Conversely, The aroA mutant stimulated the release of pro-inflammatory cytokines at a low level 15 hours post infection in mice.5. Immune efficacy of S. agalactiae aroA mutant as a live attenuated vaccine in miceAn aroA mutant strain of S. agalactiae was used as a live attenuated vaccine and evaluated for its protective effect compared with inactivated vaccine in mice.The result showed that, a single vaccination with different doses (101-103CFU) conferred a protective immunity. A single vaccination with103 CFU produced a relative percentage survival (RPS) of 90% against an i.p. challenge with a dose of 104 CFU (>103LD50) of the S. agalactiae wild strain. The IgG antibody level of attenuated vaccine and inactivated vaccine groups were significantly higher than that of PBS control group, and the IgG antibody level of attenuated vaccine group was higher than that of inactivated vaccine group.Likewise, The percentages of CD4+ and CD8+ in spleen lymphocyte were increased at the same trend as well as the mRNA expression levels of IL-4 and IFN-y, and the increased estent of attenuated vaccine group was higher than that of inactivated vaccine immunization group. The attenuated vaccine and inactivated vaccine conferred the RPSs of 100% and 60% for the challenged mice, respectively. It suggested that the attenuated vaccine could stimulate the humoral and cellular immune responses, and provide better protection in mice against S. agalactiae infection, relative to the inactivated vaccine. |
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