Effect of RSV and HPIV3 infection on the adherence of S. pneumoniae to human pharyngeal cells
D562 cells were permissive to infection with RSV and HPIV3, with discrete cytopathic effect (CPE) after 72 h but without much detachment of the monolayers hence allowing for pneumococcal adherence. We then investigated whether pneumococci adhered in greater numbers to virus-infected D562 cells than to mock-infected cells. The optimal incubation period of virus-infected cells required for maximum bacterial adherence was determined to be 72 h in a preliminary time-course experiment (data not shown). The adherence of pneumococcal strains TIGR4 and G54 to D562 cells increased significantly with prior viral infection (p
0.05) (Figure ). The degree of enhanced attachment ranged from 1.3- to 2-fold and was dependent on the dose of initial viral infection. Maximal adherence due to RSV infection was obtained at a viral titer of 6.5
/ml, and maximal adherence to HPIV3 was obtained at a viral titer of 8.3
/ml. This influence of viral dose on bacterial adherence was similar for both pneumococcal strains, although the strains varied markedly in their baseline adherence capacity without viral infection (Figure ).
Figure 1 Binding of S. pneumoniae TIGR4 and G54 to D562 cells infected with HPIV3 (A) or RSV (B). The number of bacteria adhering to mock-infected cells (gray) and to viral-infected cells (black) is shown. The number of bacteria is calculated as the mean (± (more ...)
Gene expression patterns of S. pneumoniae in contact with either virus- or mock-infected human pharyngeal cells
To limit the number of variables being compared for the gene expression analysis, we restricted the experiments to one strain of pneumococcus, TIGR4, and one respiratory virus – HPIV3. RNA was isolated from TIGR4 in contact with HPIV3- or mock-infected D562 cells.
Compared to controls (pneumococci in cell culture medium), the gene regulation ratios for pneumococci in contact with virus- or mock-infected pharyngeal cells varied from 0.1 to 14.4. Among the 77 genes that were up-regulated, 51 were regulated in both virus-infected and mock-infected conditions (Additional file 1
: Table S3). These included genes involved in adhesive functions (psaA
, pilus islet), choline uptake and incorporation (lic
operon), transport and binding (SP_1855-SP_1857) and fatty acid metabolism (SP_0421-SP_0427). The most highly represented genes were those coding for hypothetical proteins (25%), some of which are putative membrane proteins. Of the 98 down-regulated pneumococcal genes, 62 were down-regulated in both virus-infected and mock-infected conditions and these included choline transporters (proWX, proV
) and purine biosynthesis genes (pur
operon) (Additional file 1
: Table S3). Among these 51 up-regulated and 62 down-regulated genes, the degree of regulation was remarkably consistent in the two adherence conditions (Additional file 1
: Figure S1). These genes underpin the common set of cell functions that are induced by exposure to pharyngeal cells – regardless of whether those cells are viral-infected.
Eight pneumococcal genes appeared in the list of genes that were up-regulated after contact with HPIV3-infected cells but were absent in the list of genes up-regulated after contact with mock-infected cells. These were: glnQ, a fibronectin-binding/glutamine transport gene, SP_0204-SP_0206, ribonucleotide reductase genes, SP_1428 and SP_2005, genes encoding hypothetical proteins, and SP_1765-SP_1767, glycosyl transferase genes. Upon further examination, we found that these genes were absent from the list of genes up-regulated by TIGR4 in contact with mock-infected cells because they did not meet our criteria for inclusion in the analysis (see Methods). We used qRT-PCR to verify the expression levels of these genes in both conditions, adherence to HPIV3-infected and mock-infected cells. The 8 genes had comparable Ct values between the two conditions. Eighteen genes, all encoding hypothetical proteins, were up-regulated by TIGR4 in contact with mock-infected cells only. It is likely that the expression of these 18 genes is inhibited by the viral infection.
Effect of HPIV3 and RSV on the expression of human cell adhesion molecules
Given the very large number of human genes and the particular hypothesis under test in this study, we focused our analysis on genes encoding cell adhesion molecules. HPIV3 infection induced the up-regulation of 55 human genes (Table A). Four of these were also up-regulated in RSV-infected cells (Table panels A and B, CD47, AADACL1, ZC3HAV1 and cIAP2, an apoptosis inhibitor). Other genes that were up-regulated by HPIV3-infected cells included interferon stimulated genes (ISGs), plasminogen activator urokinase (PLAU), collagen type XVII alpha 1, claudin 1, laminin alpha 3, matrix metallopeptidase 14, and molecules of the carcinoembryonic antigen-related cell adhesion molecules (CEACAM1). Infection with RSV induced the up-regulation of 20 genes, including proteins of the zinc finger family (ZNF23, ZNF318), protocadherin beta 19, Rho family GTPase 1, fibronectin type III domain containing protein, and caspase 10.
Differential expression of cell adhesion molecules
Since the results above suggested that the enhanced expression of various adhesion molecules following HPIV3 and RSV infection may increase the level of adherence of pneumococci, we sought to describe the transcription profiles of cell adhesion molecules exposed to the TIGR4 strain and compared them to the virus-induced transcription profiles. We found that genes that were regulated in the presence of HPIV3 and RSV were not significantly regulated in TIGR4; rather, infection with TIGR4 alone resulted in the up-regulation of a unique set of genes (Table C) which included selenium-binding protein 1 (SELENBP1), granulocyte colony stimulating factor receptor (CSF3R), and ADAM metallopeptidases. We also analyzed the transcription of host cells exposed to concurrent stimulation by both viral and bacterial pathogens and found that infection of D562 cells with HPIV3 or RSV followed by TIGR4 induced a similar response to that of infection with HPIV3 or RSV only (Table panels A, B and C).
Confirmation of microarray results by qRT-PCR
A subset of differentially expressed genes from the human microarray experiments was selected and qRT-PCR analysis was undertaken to confirm the relative levels of gene expression. The experiments were performed on the same RNA samples used in microarray experiments. The correlation coefficient (R) between qRT-PCR and microarray results for pneumococcal transcription in contact with either virus-infected or mock-infected epithelial cells was 0.92 and 0.84, respectively. On the host side, the correlation coefficient between the two conditions in HPIV-3 infected cells and RSV-infected cells were 0.87 and 0.65, respectively (Additional file 1
: Figure S2).