Xin-Ming Song

The growth phase-dependent regulation of the pilus locus genes by two-component system TCS08 in Streptococcus pneumoniae.

The two-component system TCS08 of Streptococcus pneumoniae contributes to the virulence in vivo and regulates phosphotransferase system (PTS) genes in an avirulent strain. However, its role in pathogenic strains and virulence mechanism are largely unknown. In this study, we constructed TCS08 knockout mutants in a serotype 4 encapsulated pathogenic strain TIGR4, and investigated target genes regulated by TCS08 through transcriptional profile analysis. Compared to TIGR4, expression of the rlrA islet genes (SP0461-SP0468) encoding pneumococcal pili was found to be up-regulated in the rr08 mutant (Deltarr08). Further quantitative real-time PCR (qRT-PCR) analysis revealed that such induction was more significant when the strains were grown to late-logarithmic phase. In phenotype analyses, disruption of both hk08 and rr08 genes (DeltaTCS08) resulted in increased adherence to human lung epithelial cells (A549) at 3 h at late-logarithmic and stationary phases. However, the invasion level of DeltaTCS08 was reduced at different growth phases. Similar phenotype changes, though less significant, were also observed when the assays were performed on human nasopharyngeal epithelial cells (Detroit 562). These data suggest that TCS08 is involved in adhesion and invasion of host epithelial cells, which is likely mediated via regulation of the pilus locus genes in a growth phase-dependent manner.

Streptococcus pneumoniae early response genes to human lung epithelial cells

BackgroundStreptococcus pneumoniae infection starts from colonization of the host respiratory tract where interaction with host respiratory tract epithelial cells occurs. To investigate pneumococcal genes that are involved in the early stage of interaction with host epithelial cells, transcriptional responses of an encapsulated pathogenic pneumococcal strain TIGR4 upon exposure to human lung epithelial cells A549 for 0.5 h and 1 h time periods were investigated by using TIGR (JCVI) microarray technology. Gene expression changes were validated by quantitative real-time PCR (qRT-PCR) analysis.FindingsWe observed different transcriptional profiles at two incubation time periods in which most gene expressions were down-regulated at 0.5 h but up-regulated at 1 h. Many genes associated with ribonucleotide biosynthesis were down-regulated at both time points, whereas the genes associated with cell envelope, energy metabolism, transport and protein synthesis were mostly up-regulated at 1 h. Furthermore, these profiles were compared to the transcriptomes of a TIGR4-derived strain in response to human macrophages for the same time periods. We found one set of genes that exhibited similar expression changes upon exposure to both types of host cells, including cell envelope-associated bgaA (SP0648) and nanA (SP1693), and uncharacterized gene clusters such as SP1677–SP1680 and SP1688–SP1690.ConclusionThese data indicate that at the early stage of interaction with host epithelial cells, a complex gene regulation and expression change occur in bacteria. Some of them might play an essential role during pathogen-host interactions and for the establishment of infection.

Surface-expressed Mig protein protects Streptococcus dysgalactiae against phagocytosis by bovine neutrophils

ABSTRACT The mig gene of Streptococcus dysgalactiae, a major bovine mastitis pathogen, encodes two plasma protein-binding receptors, α2-macroglobulin (α2-M) and immunoglobulin G (IgG). In this study, the mig gene from oneS. dysgalactiae isolate was cloned and expressed inEscherichia coli. The IgG receptor region encoded bymig was conserved in 16 S. dysgalactiae strains. An isogenic mig mutant was constructed by allele replacement mutagenesis of the wild-type gene inS. dysgalactiae. The IgG-binding activity was lost in the mig mutant strain, whereas the α2-M receptor activity was still expressed but was detected only in the culture supernatant. In flow cytometry phagocytosis and bacterial-colony-counting bactericidal assays, the wild-type strain was found to be significantly more resistant to phagocytosis and killing by bovine neutrophils (PMNs) than the mig mutant strain when bacteria were preincubated with bovine serum. We therefore speculate that the Mig protein of S. dysgalactiaeplays a role in virulence of the bacteria by binding to the plasma protein α2-M or IgG and thus preventing phagocytosis by bovine PMNs.

The Mig protein of Streptococcus dysgalactiae inhibits bacterial internalization into bovine mammary gland epithelial cells

The role of the Mig protein of Streptococcus dysgalactiae in bacterial adhesion and internalization of bovine mammary gland epithelial cells (MAC-T) was investigated with the wild-type and isogenic mig mutant strains. While there was no difference in adhesion between the strains, the wild-type strain exhibited a significantly lower level of invasion than the mutants. The lower level of internalization of the Mig(+) strain is likely due to Mig-mediated interference with uptake of the microorganisms rather than the host protein binding properties of Mig. Avoidance of intimate interactions with the host cells might be an alternative strategy for S. dysgalactiae to survive and persist in the bovine mammary glands.

Transcriptome studies on Streptococcus pneumoniae, illustration of early response genes to THP-1 human macrophages

Pathogen-host interaction plays an essential role in pathogenicity. In this study, we investigated transcriptomes of one Streptococcus pneumoniae TIGR4-derived unencapsulated strain upon exposure to THP-1 human macrophage-like cells for 0.5 h, 1 h and 3 h, respectively. Expression of most genes was up-regulated and the changes of selected genes were validated by qRT-PCR. To characterize the functions of the identified genes, one locus of genes (SP1057-SP1063) was deleted in TIGR4 by insertion replacement mutagenesis. Compared to the wild-type strain, the constructed mutant exhibited lower binding and internalization activities to the THP-1 macrophages at early incubation time periods (0.5 h and/or 1 h) but not at 3 h. However, no change was observed in the intracellular survival assays. These data indicate that the SP1057-SP1063 locus is involved in the early stage of interaction with host macrophages. Further sequence and PCR analyses suggest that the SP1057-SP1063 locus was acquired by lateral transfer.

Bovine immunoglobulin A (IgA)-binding activities of the surface-expressed Mig protein of Streptococcus dysgalactiae

The Mig protein of Streptococcus dysgalactiae is a type III immunoglobulin G (IgG)-binding protein, expressing IgG- and alpha2-macroglobulin (alpha2-M)-binding receptors. This study showed that the Mig protein also displays binding activities to bovine immunoglobulin A (B-IgA). Biotin-labelled bovine serum IgA bound immobilized recombinant Mig and alpha2-M receptors derived from Mig, as well as the native Mig extracted from the surface of S. dysgalactiae strain SDG8 and the alpha(2)-M receptor released from the isogenic mig mutant strain Mig8-Mt, as determined by Western blotting and ELISA. There was no B-IgA binding activity to the immobilized IgG receptor derived from Mig or the proteins in the culture supernatant from the mig mutant strain Mig7-Mt, in which expression of Mig or Mig-related peptides on the cell surface was completely abolished. In a reciprocal experiment, biotin-labelled Mig was found to bind immobilized bovine serum IgA but not human IgA (H-IgA). The binding of Mig to bovine serum IgA was competitively inhibited by unlabelled Mig, intact and truncated alpha(2)-M receptors, and bovine serum IgA, but not by the Mig-IgG receptor, H-IgA or B-IgG. The binding of Mig and partially purified bovine secretory IgA (B-sIgA) was also characterized by Western blotting. Membrane-immobilized B-sIgA did not react with the biotin-labelled Mig, whereas soluble B-sIgA showed binding activity to the immobilized alpha2-M receptor of Mig. It is therefore concluded that the 11 kDa N-terminal region of the alpha2-M receptor of the S. dysgalactiae Mig protein specifically binds soluble and immobilized bovine serum IgA, as well as soluble B-sIgA. This is believed to be the first report of a B-IgA-binding protein in S. dysgalactiae.