Open Access

Streptococcus pneumoniaeearly response genes to human lung epithelial cells

  • Xin-Ming Song1Email author,
  • Wayne Connor1,
  • Karsten Hokamp2,
  • Lorne A Babiuk3 and
  • Andrew A Potter1
BMC Research Notes20081:64

https://doi.org/10.1186/1756-0500-1-64

Received: 04 July 2008

Accepted: 12 August 2008

Published: 12 August 2008

Abstract

Background

Streptococcus 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.

Findings

We 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.

Conclusion

These 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.

Findings

Background

As a major bacterial pathogen, Streptococcus pneumoniae infection starts from colonization of the human upper respiratory tract, causing respiratory tract diseases such as pneumonia, bronchitis, otitis media and sinusitis. Under certain circumstances, bacteria invade host cells and evade host immunity, causing systemic infections such as bacteremia, sepsis and meningitis. Therefore, the interaction of S. pneumoniae with host respiratory tract epithelial cells is an initial step for infection. Many factors that contribute to the colonization and/or invasion of host epithelial cells have been characterized in S. pneumoniae (recently reviewed by: [13]). However, it is becoming obvious that multiple factors are involved in this complex process [4].

Microarray-based transcriptome studies have been used in many pathogens, investigating their transcriptional responses to host cells [5]. However, they were rarely performed at an early stage of interaction time period, a stage that might be critical for microbes to establish an infection. This is likely due to the difficulty of obtaining sufficient bacterial RNA from a mixture of bacteria and host cells. In S. pneumoniae, transcriptome studies were initiated by Orihuela et al. [6] in which an unencapsulated derivative of TIGR4 was investigated following exposure to human pharyngeal epithelial cells (Detroit 562) for 3 h. By using self-spotted pneumococcal oligonucleotide (oligo) microarrays we have also examined gene expression changes of an encapsulated serotype 3 clinical isolate and one unencapsulated avirulent laboratory strain following incubation with human lung epithelial cells (A549) for 1 h and 3 h, respectively [7]. Nevertheless, a lack of information exists regarding pneumococcal gene expression at an early stage of interaction with host cells. The strain-specific gene regulation features of S. pneumoniae [8] also prompted our research interests on other serotype strains.

In this study, we have developed a system which can be used to isolate enough bacterial RNA for microarray analysis from encapsulated pathogenic strains following incubation with A549 cells for a short time period. By using TIGR microarrays, we performed transcriptome studies on an encapsulated wild-type strain TIGR4. This study highlighted the gene transcriptional profiles in S. pneumoniae and revealed the potential roles of some target genes during pathogen-host interactions.

Methods

Incubation of bacteria and host cells

Culturing and incubation of pneumococcal strain TIGR4 (provided by Dr. Caroline A. Obert, St. Jude Children's Research Hospital) and human lung epithelial cells A549 were performed as previously described [7] with minor modifications. Briefly, bacteria grown to early logarithmic-phase at OD600 0.3 were collected by centrifugation, re-suspended in antibiotic-free MEM complete medium supplemented with 1% fetal bovine serum (FBS), and incubated with host cells in T75 flasks at a multiplicity of infection 120:1. After incubation, non-adherent bacteria were removed by washing 3 times with 5 ml of antibiotic-free cell culture medium. Host cells were removed by incubation with a host cell lysis buffer containing guanidine thiocyanate (Sigma), β-mercaptoethanol, phenol and ethanol at room temperature for 10 min. Bacterial samples were collected by centrifugation for RNA isolation. Bacteria incubated with cell culture medium for different time points, treated with RNALater (Ambion), were collected as medium control samples.

Preparation of bacterial RNA

Isolation of bacterial RNA was performed with RiboPure™-Bacteria Kit (Ambion) or a modified method using RNeasy MiniKit (Qiagen) as previously described [7]. From each flask of cell infection, about 2~4 μg bacterial total RNA with less than 10% of eukaryotic RNA contamination could be generated. Medium control RNA samples at each incubation time point were generated by pooling RNAs isolated from 3 separate assays. Genomic DNA contamination was removed by the treatment with RNase-free of DNase I (Ambion).

Microarray experiment and analysis

TIGR (J. Craig Venter Institute) S. pneumoniae 70-mer oligo microarray (version 6), provided by the Pathogen Functional Genomics Resource Center (PFGRC), was used in this study. The cDNA synthesis, Cy-dye labelling, and microarray hybridization were carried out according to TIGR's standard operating procedures (SOPs) http://www.tigr.org. Hybridization signals were captured with a GenePix 4200A scanner (Axon Instruments) and the data were processed and analyzed through ArrayPipe http://www.pathogenomics.ca/arraypipe[9]. This includes flagging of marker spots, background correction, printTip Loess normalization with Limma, and statistical analysis with Limma's eBayes moderated t-test [10]. Gene expressions of fold change ≥ 2.0 (bacteria incubated with host cells vs. bacteria incubated with media) with statistical significance (p ≤ 0.05) were classified as being significantly changed. In this study, eight independent hybridizations, including four labelled in dye flips, using RNA samples isolated from eight separate assays were performed for each incubation time point.

Quantitative real-time PCR (qRT-PCR) analysis

The oligo primers used for qRT-PCR analysis (Table 1) were designed from S. pneumoniae TIGR4 genome sequences by using Clone Manager Suite 7 (Scientific & Educational Software) and synthesized by Invitrogen. The qRT-PCR reaction and analysis were performed as previously described [7]. For each gene, duplicate reactions were performed on the RNA samples isolated from at least two separate assays for each incubation time point.
Table 1

Oligonucleotide primers used for qRT-PCR analysis

Gene name

TIGR4 genome acc. No.

Oligonucleotide primers 5' to 3'

Amplified product (bp)

purH

SP0050

Sense: TCAAGCAACCAATGCGTTACGGTGAG

110

  

Anti-sense: TTTCCCGTTGAGCTGTTTGGCTGAAG

 

strH

SP0057

Sense: GTGTCAGCCCAAGCAGCTACCATACCAC

128

  

Anti-sense: GGCCAAGGCTGGTACAATCTCGATCAGG

 

cbpI

SP0069

Sense: GCTATGAAGACAGGCTGGTACAAG

133

  

Anti-sense: TCACAGCCAAAGCTCCTGAAC

 

nrdD

SP0202

Sense: TGCAACCAAGCGGATGTATCCAGACG

99

  

Anti-sense: TGAAGGAAAGAACGGCAGCCCATAGG

 
 

SP0287

Sense: CAGTCGGTGCCATTGCAGGTACTTCAAAC

103

  

Anti-sense: GCTACAACCAAGGCTGTCAAACCAGTACG

 

caps4A

SP0346

Sense: GTCAGAGTATCCAGACTACGCATCGAAG

159

  

Anti-sense: TCTGATCGCGACACCGAACTAATAGG

 

bgaA

SP0648a

Sense: CAAGCCAGCCGTGAACGCTATAAGG

128

  

Anti-sense: GAGTGGGCAGTCAGGGTGAATTTCC

 

gyrA

SP1219a

Sense: GTGCTGCCGCTCAACGTTATACCGAGG

142

  

Anti-sense: AAACGCGCTGGCAAGACCAAGGGTTCC

 

pyrR

SP1278

Sense: GACAGACCGCGAAGTTATCTTGGTGG

115

  

Anti-sense: AACTGCTAAACTCACACGCGCAGGAC

 
 

SP1679

Sense: GGACAGGGGATTACAGTTGATGAGATGG

149

  

Anti-sense: GCAGTTGCAGCTACCCTACTTAAGATCG

 
 

SP1680

Sense: GCCTGCATAACCATTTGGCTGATGTG

127

  

Anti-sense: AGCATTCGACGAAGCGAGTGACATTG

 
 

SP1688

Sense: AAGTGAACGAAGGGCTACTGCTACTGTC

136

  

Anti-sense: GCTACCGATTGTAGCACCAGGTATTG

 

nanA

SP1693

Sense: GACATATTCGAAAGCGGGCGTAACGG

117

  

Anti-sense: GCGTTCATCTGCACCTGCGATCAAAG

 

purR

SP1979a

Sense: AGGCAGCCGTGTCTTGATTGTGG

120

  

Anti-sense: TTGTCCGCAAAGACCGCTACACC

 

a. Obtained from [7].

Results and discussion

Transcriptional responses of S. pneumoniaeto host epithelial cells

Microarray analysis revealed many gene expression changes following exposure to A549 cells (Table 2). At 0.5 h, most gene expressions were down-regulated (35 vs. 16) and a smaller number of genes changed (Fig. 1). At 1 h, more genes were changed and most of them were up-regulated (50 vs. 25) (Fig. 2). Furthermore, most of those changed genes were only defined at a certain incubation time period (Fig. 3). These data indicate divergent transcriptional profiles between 0.5 h and 1 h incubation time periods. Repressed transcriptional profiles at 0.5 h (Fig. 1) suggest that the interaction with human respiratory tract epithelial cells, a natural reservoir for S. pneumoniae, might be a favourable situation for pneumococci. This is in contrast to the S. pneumoniae transcriptomes to macrophages, where most genes that showed transcriptional changes at the early stage of interactions were up-regulated (Song XM, Connor W, Hokamp K, Babiuk LA, Potter AA: Transcriptome studies on Streptococcus pneumoniae, illustration of early response genes to THP-1 human macrophages, submitted). When incubated for 1 h, bacterial survival, growth and virulence mechanisms appear to be activated, apparent from an induced expression of genes in cell envelope, energy metabolism, transport, protein synthesis, and hypothetical proteins (Fig. 2).
Table 2

Microarray identified genes in pneumococcal strain TIGR4 upon exposure to A549 cells for 0.5 h and 1 h time periods

Function/gene name

Protein

TIGR4 genome acc. No.

Incubation time

   

0.5 h

1 h

Cell envelope

cbpI a

choline binding protein I

SP0069

 

2.8

cps4A

capsular polysaccharide biosynthesis protein Cps4A

SP0346

 

2.9

cps4B

capsular polysaccharide biosynthesis protein Cps4B

SP0347

 

2.0

cps4C

capsular polysaccharide biosynthesis protein Cps4C

SP0348

 

3.3

cps4E

capsular polysaccharide biosynthesis protein Cps4E

SP0350

 

2.9

cps4I

UDP-N-acetylglucosamine-2-epimerase

SP0357

 

2.4

bgaA

β-galactosidase

SP0648

 

17.0

nanA a

neuraminidase A, authentic frameshift

SP1693

 

16.5

Energy metabolism

agaS

sugar isomerase domain protein AgaS

SP0065

 

5.6

pyk

pyruvate kinase

SP0897

-2.7

 

glgA

glycogen synthase

SP1124

 

3.8

 

acetoin dehydrogenase complex, E2 component, dihydrolipoamide acetyltransferase, putative

SP1162

 

2.7

zwf

glucose-6-phosphate 1-dehydrogenase

SP1243

-2.7

 

scrB

sucrose-6-phosphate hydrolase

SP1724

3.0

4.4

galT

galactose-1-phosphate uridylyltransferase

SP1852

 

2.7

galK

galactokinase

SP1853

 

2.3

recP

transketolase

SP2030

 

-3.6

arcA

arginine deiminase

SP2148

4.6

 

gplK

glycerol kinase

SP2186

 

3.0

Hypothetical proteins

 

conserved hypothetical protein

SP0024

 

-2.6

 

hypothetical protein

SP0026

-2.3

 
 

hypothetical protein

SP0052

-3.5

-5.6

 

hypothetical protein

SP0067

2.4

2.1

 

conserved hypothetical protein

SP0095

-2.4

 
 

conserved hypothetical protein

SP0159

-2.3

 
 

hypothetical protein

SP0190

2.3

 
 

hypothetical protein

SP0203

-2.5

 
 

conserved hypothetical protein

SP0207

-2.1

 
 

conserved hypothetical protein

SP0288

-4.2

-2.2

 

conserved hypothetical protein

SP0742

 

-2.9

 

conserved hypothetical protein

SP0951

 

2.4

 

conserved hypothetical protein

SP1003

 

2.1

 

hypothetical protein

SP1049

2.0

 
 

hypothetical protein

SP1059

 

4.4

 

conserved hypothetical protein

SP1174

 

2.4

 

hypothetical protein

SP1198

2.7

2.6

 

hypothetical protein

SP1199

2.9

2.0

 

conserved hypothetical protein

SP1601

 

2.4

 

hypothetical protein

SP1677

 

10.3

 

hypothetical protein

SP1678

2.9

6.1

 

hypothetical protein

SP1679

4.6

9.6

 

conserved hypothetical protein

SP1680

5.3

11.5

 

hypothetical protein

SP2183

2.7

4.1

Others

 

bacteriocin, putative

SP0109

2.3

 
 

lactose phosphotransferase system repressor, degenerate

SP0169

 

2.2

 

dihydropteroate synthase

SP0289

-2.2

-2.1

acpP

acyl carrier protein

SP0418

-2.0

 

fabF

3-oxoacyl-(acyl-carrier-protein) synthase II

SP0422

-2.4

 

accD

acetyl-CoA carboxylase, carboxyl transferase subunit beta

SP0426

-2.4

 

accA

acetyl-CoA carboxylase, carboxyl transferase subunit alpha

SP0427

-3.4

 

ilvB

acetolactate synthase, large subunit, biosynthetic type

SP0445

-2.8

 

zmpB

zinc metalloprotease ZmpB

SP0664

-2.1

 

ilvE

branched-chain amino acid aminotransferase

SP0856

-2.0

 
 

preprotein translocase, SecG subunit, putative

SP0974

 

2.5

asd

aspartate-semialdehyde dehydrogenase

SP1013

-2.0

 

bta

bacterocin transport accessory protein

SP1499

-2.7

-2.4

 

transcriptional regulator, MerR family

SP1856

2.0

 

groEL

chaperonin, 60 kDa

SP1906

 

-2.4

Protein synthesis

rpsD

ribosomal protein S4

SP0085

 

2.7

rpsJ

ribosomal protein S10

SP0208

 

4.1

rplW

ribosomal protein L23

SP0211

 

2.9

rpsC

ribosomal protein S3

SP0215

 

2.0

infA

translation initiation factor IF-1

SP0232

 

2.4

valS

valyl-tRNA synthetase

SP0568

-2.1

 

rplK

ribosomal protein L11

SP0630

 

2.5

infC

translation initiation factor IF-3

SP0959

 

2.5

rpml

ribosomal protein L35

SP0960

 

3.9

rpsR

ribosomal protein S18

SP1539

 

2.8

rpsF

ribosomal protein S6

SP1541

2.9

3.0

rpmH

ribosomal protein L34

SP1993

 

2.4

rpmG

ribosomal protein L33

SP2135

 

2.1

yfiA

ribosomal subunit interface protein

SP2206

 

-3.9

Purine and pyrimidine ribonucleotide biosynthesis

purA

adenylosuccinate synthetase

SP0019

-2.5

 

purC a

phosphoribosylaminoimidazole-succinocarboxamide synthase

SP0044

-5.1

-4.7

purH

phosphoribosylaminoimidazolecarboxamide formyltransferase-IMP cyclohydrolase

SP0050

-15.3

-4.1

purE a

phosphoribosylaminoimidazole carboxylase, catalytic subunit

SP0053

-6.4

-8.5

purK a

phosphoribosylaminoimidazole carboxylase, ATPase subunit

SP0054

 

-2.4

nrdD

anaerobic ribonucleoside-triphosphate reductase

SP0202

-4.4

-4.3

nrdG

anaerobic ribonucleoside-triphosphate reductase activating protein

SP0205

-3.4

-2.8

thyA

thymidylate synthase

SP0669

-2.2

 

pyrK

dihydroorotate dehydrogenase, electron transfer subunit

SP0963

 

-3.6

nrdH

NrdH-redoxin

SP1178

 

-2.1

carB

carbamoyl-phosphate synthase, large subunit

SP1275

 

-4.2

pyrR

pyrimidine operon regulatory protein

SP1278

-2.1

-7.8

guaA a

GMP synthase

SP1445

-2.4

 

purR

pur operon repressor

SP1979

-2.7

-2.3

Transport

 

PTS system, IIA component

SP0064

2.2

3.4

 

PTS system, mannose-specific IID component

SP0282

-3.7

 
 

xanthine-uracil permease family protein

SP0287

-8.4

-5.3

 

O-antigen transporter RfbX, putative

SP0356

 

2.5

 

PTS system, IIC component, putative

SP0647

 

4.3

 

sugar ABC transporter, ATP-binding protein

SP0846

-2.1

 
 

ABC transporter, permease protein

SP1688

 

5.3

 

ABC transporter, permease protein

SP1689

 

2.8

 

ABC transporter, substrate-binding protein

SP1690

 

2.1

msmE

sugar ABC transporter, sugar-binding protein

SP1897

 

2.1

malD

maltodextrin ABC transporter, permease protein

SP2110

 

2.6

Unknown function

 

vanZ protein, putative

SP0049

-2.9

 
 

ACT domain protein

SP0238

-2.1

-3.4

 

HIT family protein

SP0521

 

-2.4

gid

Gid protein

SP0943

 

-2.2

 

flavoprotein

SP1231

 

-2.0

usp45

secreted 45 kd protein

SP2216

2.1

 

a genes that are also involved in pathogenesis according to TIGR genome annotation

Figure 1

Transcriptional profiles of functional categories of genes identified in microarray analysis at 0.5 h incubation time period. The number of differentially regulated genes (x-axis) identified in microarray analysis in S. pneumoniae TIGR4 following incubation with A549 cells for 0.5 h time period. They are represented in different functional categories (y-axis) and marked with up-regulated (open bars) and down-regulated (grey bars) expressions. No cell envelope genes were identified.

Figure 2

Transcriptional profiles of functional categories of genes identified in microarray analysis at 1 h incubation time period. The number of differentially regulated genes (x-axis) identified in microarray analysis in S. pneumoniae TIGR4 following incubation with A549 cells for 1 h time period. They are represented in different functional categories (y-axis) and marked with up-regulated (open bars) and down-regulated (grey bars) expressions.

Figure 3

Venn diagrams of microarray identified genes. The up-regulated (A) and down-regulated (B) genes in S. pneumoniae TIGR4 following incubation with A549 cells for 0.5 h (grey circles) and 1 h (open circles), respectively.

We also observed a common change between two incubation time points, that more than 10 purine and pyrimidine ribonucleotide biosynthesis genes, including purine and pyrimidine regulatory genes purR and pyrR, were consistently down-regulated (Table 2; Figs. 1, 2). The roles of ribonucleotide biosynthesis and their gene regulation mechanism in S. pneumoniae are largely unknown. However, down-regulation of these genes in pneumococci appears to occur only at an early stage of interaction with host epithelial cells, but not at 3 h [6, 7]. It also might be specific to the pneumococcal strains and the types of host cells because most of those ribonucleotide biosynthesis genes were unchanged in a serotype 3 strain [7] or when the TIGR4-derived strain was exposed to the host macrophages (Song XM, Connor W, Hokamp K, Babiuk LA, Potter AA: Transcriptome studies on Streptococcus pneumoniae, illustration of early response genes to THP-1 human macrophages, submitted). Perhaps this is the shift of bacteria to parasitism enabling the uptake of substrates from the host cells [11], or the indication of metabolic changes in different pneumococcal strains in different host environment.

Microarray data have been deposited in the ArrayExpress microarray database http://www.ebi.ac.uk/arrayexpress under accession No. E-FPMI-15.

Microarray data validation

To confirm gene expression changes identified in microarray analysis, we performed qRT-PCR analysis on 16 selected genes at different incubation time point, most of them associated with cell envelope, ribonucleotide biosynthesis, SP1677-SP1680 and SP1688-SP1690 gene clusters. Except for the unchanged SP1680 at 0.5 h, all the other gene expressions changed in accordance to the microarray data, but at a greater average fold change in the qRT-PCR analysis (Figs. 4, 5). Expression change of SP0057 at 1 h was only obtained from qRT-PCR assay because the strain-specific oligo probes were absent on the microarrays (Fig. 5).
Figure 4

Validation of up-regulated genes by qRT-PCR. The up-regulated genes identified in microarray (open bars) and qRT-PCR (grey bars) analyses. The characterized genes incubated with A549 cells for different time periods (0.5 h or 1 h) are marked on the x-axis. For consistency, each gene is indicated by the TIGR4 genome accession number (SP), not the gene name. The fold changes (mean) from all the repeated assays with standard deviations are marked on the y-axis. Scales on the y-axis (0~5, 5~250) are not continuous due to large changes for some genes.

Figure 5

Validation of down-regulated genes by qRT-PCR. The down-regulated genes identified in microarray (open bars) and qRT-PCR (grey bars) analyses. The characterized genes incubated with A549 cells for different time periods (0.5 h or 1 h) are marked on the x-axis. For consistency, each gene is indicated by the TIGR4 genome accession number (SP), not the gene name. The fold changes (mean) from all the repeated assays with standard deviations are marked on the y-axis. Scales on the y-axis (0~-5, -5~-80) are not continuous due to large changes for some genes.

Common response genes to host cells

In a separate transcriptome study, we have investigated gene expression changes of a TIGR4-derived unencapsulated strain following incubation with human THP-1 derived macrophages for different time points (0.5 h, 1 h and 3 h) (Song XM, Connor W, Hokamp K, Babiuk LA, Potter AA: Transcriptome studies on Streptococcus pneumoniae, illustration of early response genes to THP-1 human macrophages, submitted). As similar experimental procedures and microarray technology were applied, we compared these two studies and revealed many common response genes at early interaction time periods, including well characterized virulence genes such as bgaA and nanA, and uncharacterized gene clusters such as SP1677-SP1680 (hypothetical) and SP1688-SP1690 (ABC transporter) (Table 3). It indicates common features in pneumococcal gene responses to different types of host cells. Although the interactions with host epithelial cells and macrophages are mainly associated with different pathogenesis processes, reflected by the colonization of host epithelial cells and the survival from host phagocytic cells, we assume these processes are closely related and some of those genes might be assigned with multiple functions.
Table 3

Common response genes to both A549 cells and THP-1 derived macrophages at 0.5 h and 1 h incubation time periods

Function/gene name

Protein

TIGR4 genome acc. No.

A549a

THP-1b

   

0.5 h

1 h

0.5 h

1 h

Cell envelope

cbpI c

choline binding protein I

SP0069

 

2.8

 

8.4

bgaA

beta-galactosidase

SP0648

 

17.0

3.4

26.9

nanA c

neuraminidase A, authentic frameshift

SP1693

 

16.5

3.9

47.1

Energy metabolism

agaS

sugar isomerase domain protein AgaS

SP0065

 

5.6

 

10.3

glgA

glycogen synthase

SP1124

 

3.8

 

5.4

 

acetoin dehydrogenase complex, E2 component, dihydrolipoamide acetyltransferase, putative

SP1162

 

2.7

4.9

6.0

scrB

sucrose-6-phosphate hydrolase

SP1724

3.0

4.4

2.4

4.7

galT

galactose-1-phosphate uridylyltransferase

SP1852

 

2.7

2.6

4.4

galK

galactokinase

SP1853

 

2.3

2.3

2.8

recP

transketolase

SP2030

 

-3.6

-2.0

-2.5

gplK

glycerol kinase

SP2186

 

3.0

 

4.1

Hypothetical proteins

      
 

hypothetical protein

SP0052

-3.5

-5.6

-2.6

-3.5

 

hypothetical protein

SP0067

2.4

2.1

 

4.1

 

conserved hypothetical protein

SP0159

-2.3

 

-2.0

 
 

conserved hypothetical protein

SP0742

 

-2.9

-6.5

-3.2

 

conserved hypothetical protein

SP1003

 

2.1

2.1

3.4

 

hypothetical protein

SP1059

 

4.4

56.3

16.0

 

conserved hypothetical protein

SP1174

 

2.4

2.7

4.4

 

hypothetical protein

SP1198

2.7

2.6

 

2.8

 

hypothetical protein

SP1199

2.9

2.0

 

2.2

 

hypothetical protein

SP1677

 

10.3

 

14.6

 

hypothetical protein

SP1678

2.9

6.1

 

6.9

 

hypothetical protein

SP1679

4.6

9.6

 

6.6

 

conserved hypothetical protein

SP1680

5.3

11.5

2.0

14.6

Others

 

lactose phosphotransferase system repressor, degenerate

SP0169

 

2.2

15.4

6.0

acpP

acyl carrier protein

SP0418

-2.0

 

-2.3

 

fabF

3-oxoacyl-(acyl-carrier-protein) synthase II

SP0422

-2.4

  

-5.1

bta

bacterocin transport accessory protein

SP1499

-2.7

-2.4

-4.2

-2.2

Protein synthesis

rpsD

ribosomal protein S4

SP0085

 

2.7

 

2.4

rpsJ

ribosomal protein S10

SP0208

 

4.1

 

2.9

rpsC

ribosomal protein S3

SP0215

 

2.0

2.2

 

infC

translation initiation factor IF-3

SP0959

 

2.5

2.2

2.3

rpmI

ribosomal protein L35

SP0960

 

3.9

2.2

 

rpsF

ribosomal protein S6

SP1541

2.9

3.0

 

2.2

yfiA

ribosomal subunit interface protein

SP2206

 

-3.9

-2.6

-2.5

Purine and pyrimidine ribonucleotide biosynthesis

purC c

phosphoribosylaminoimidazole-succinocarboxamide synthase

SP0044

-5.1

-4.7

-2.4

-7.8

purH

phosphoribosylaminoimidazolecarboxamide formyltransferase-IMP cyclohydrolase

SP0050

-15.3

-4.1

-4.3

-5.5

purE c

phosphoribosylaminoimidazole carboxylase, catalytic subunit

SP0053

-6.4

-8.5

 

-4.2

carB

carbamoyl-phosphate synthase, large subunit

SP1275

 

-4.2

 

-2.6

pyrR

pyrimidine operon regulatory protein

SP1278

-2.1

-7.8

-2.3

-4.4

Transport

 

PTS system, IIA component

SP0064

2.2

3.4

 

6.5

 

PTS system, mannose-specific IID component

SP0282

-3.7

 

-2.4

 
 

xanthine-uracil permease family protein

SP0287

-8.4

-5.3

 

-2.3

 

PTS system, IIC component, putative

SP0647

 

4.3

3.5

8.2

 

ABC transporter, permease protein

SP1688

 

5.3

2.4

13.6

 

ABC transporter, permease protein

SP1689

 

2.8

3.7

18.9

 

ABC transporter, substrate-binding protein

SP1690

 

2.1

3.9

21.6

msmE

sugar ABC transporter, sugar-binding protein

SP1897

 

2.1

 

4.1

malD

maltodextrin ABC transporter, permease protein

SP2110

 

2.6

2.1

6.4

Unknown function

 

vanZ protein, putative

SP0049

-2.9

 

-3.4

-4.9

 

ACT domain protein

SP0238

-2.1

-3.4

 

-2.3

 

HIT family protein

SP0521

 

-2.4

 

-2.1

 

flavoprotein

SP1231

 

-2.0

-2.3

 

a Genes identified in this study.

b Genes identified in Song XM, Connor W, Hokamp K, Babiuk LA, Potter AA: Transcriptome studies on Streptococcus pneumoniae, illustration of early response genes to THP-1 human macrophages, submitted.

c Genes that are also involved in pathogenesis according to TIGR genome annotation.

The exoglycosidase family genes

In S. pneumoniae, the bgaA-encoded β-galactosidase (BgaA) and the nanA-encoded neuraminidase (NanA) belong to a family of exoglycosidases exposed on the bacterial surface. Studies have demonstrated that both enzymes, especially NanA, are involved in adherence to host respiratory tract epithelial cells, possibly by clearing host cell surface structures and secreted components to enhance pathogen-host interactions [1215]. Recently, it was demonstrated that BgaA and NanA, together with StrH (β-N-aceylglucosaminidase), act sequentially to remove sialic acid, galactose and N-acetylglucosamine [15]. These reports demonstrated the importance of S. pneumoniae to deglycosylate human targets during colonization and/or pathogenesis.

In this study, expression of bgaA (SP0648) and nanA (SP1693) was highly induced when incubated with A549 cells for 1 h in both microarray and qRT-PCR analyses (Table 2; Fig. 4). Further qRT-PCR assay revealed an unchanged expression of strH (SP0057) (Fig. 5), correlated to the previous observation that StrH was not involved in the adherence [15]. The enhanced expression of bgaA and nanA was also observed in a TIGR4-derived strain when exposed to human macrophages for 0.5 h and 1 h time periods (Table 3). It suggests that both bgaA and nanA belong to a family of conserved early response genes. Clearing host cell surface components and accessing to the host cells are a priority for bacteria at the early stage of pathogen-host interactions.

Other genes

The cbpI (SP0069), encoding choline binding protein I, was also up-regulated in expression (Table 2; Fig. 4). The choline binding proteins (CBPs) are a family of surface proteins, many of them are involved in colonization of nasopharynx [16]. However, cbpI was the only CBP gene that was identified in this study. The function of CbpI is still unclear but its crystal structure has been solved [17]. Whether it is important in colonization, most CBPs might not be required at the early stage of interaction with host epithelial cells.

Because of strain-specific gene regulations in S. pneumoniae [7, 8], different microarray technologies and experimental conditions, some potential gene targets might be missed in our transcriptome studies. For example, the pspC (SP1417) gene was reported to be up-regulated in a serotype 2 strain D39 within 1 h post-infection in mice [18]. However, expression change of pspC was not identified in our assays, despite of a degenerated PspC carried by the TIGR4 genome (TIGR). Another unchanged gene cluster was the rlrA pathogenicity islet genes (SP0461-SP0468) encoding pneumococcal pili [19, 20]. All of these TIGR4-specific oligo probes were carried by the TIGR microarrays, and they were clearly identified in our studies of the regulation mechanisms for the pilus locus genes (Song XM, Connor W, Hokamp K, Babiuk LA, Potter AA: The growth phase-dependent regulation of the pilus locus genes by two-component system TCS08 in Streptococcus pneumoniae, submitted). We could therefore exclude the technical concern for these genes in our microarray analysis. Earlier studies suggested that pneumococcal pili were mainly involved in the host cell adhesion [21]. Recently, Rosch, et al. defined the restricted functions of pili in invasion of host lung epithelial cells [22], suggesting its roles at a late stage of pathogen-host interactions. If this is the case, also supported by our negative findings, the rlrA pilus locus genes are not likely to be involved in the early stage of interaction with host epithelial cells.

Conclusion

The data presented here provide the first assessment of S. pneumoniae early response genes to human lung epithelial cells. It revealed gene expression changes that might be associated with bacterial adaptation, survival, growth and colonization. Up-regulation of several cell envelope genes, such as bgaA and nanA, and the genes with unknown functions, is likely required for a successful colonization. The specific roles of the identified genes and the functions of coordinated regulation of multiple genes have yet to be further investigated.

Declarations

Acknowledgements

We thank Dr. Caroline A. Obert, St. Jude Children's Research Hospital, Memphis, for providing S. pneumoniae strain TIGR4.

This work was gratefully supported by the Saskatchewan Health Research Foundation (SHRF) and the Delfari Bridging Fund of the University of Saskatchewan. Microarray slides and experimental protocols were kindly provided by the Pathogen Functional Genomics Resource Center (PFGRC) through NIAID. We also acknowledge the support of Genome Canada, Genome BC and Genome Prairie for the "Pathogenomics of Innate Immunity" research program.

Published with permission of the Director of VIDO as journal series No. 489

Authors’ Affiliations

(1)
Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan
(2)
Smurfit Institute of Genetics, Trinity College Dublin
(3)
University of Alberta

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© Song et al; licensee BioMed Central Ltd. 2008

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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