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. 2005 Dec;187(23):8205–8210. doi: 10.1128/JB.187.23.8205-8210.2005

Vancomycin Stress Response in a Sensitive and a Tolerant Strain of Streptococcus pneumoniae

Wolfgang Haas 1,*, Deepak Kaushal 2, Jack Sublett 1, Caroline Obert 1, Elaine I Tuomanen 1
PMCID: PMC1291284  PMID: 16291696

Abstract

The vancomycin stress response was studied in Streptococcus pneumoniae strains T4 (TIGR4) and Tupelo. Vancomycin affected the expression of 175 genes, including genes encoding transport functions and enzymes involved in aminosugar metabolism. The two-component systems TCS03, TCS11, and CiaRH also responded to antibiotic treatment. We hypothesize that the three regulons are an important part of the bacterium's response to vancomycin stress.

A considerable number of strains of Streptococcus pneumoniae, the causative agent of pneumonia, bacteremia, otitis media, and meningitis, have become resistant to commonly used antibiotics. This has prompted the increased use of vancomycin, especially in cases of sepsis and meningitis, since no vancomycin-resistant isolates of S. pneumoniae have been reported to date. Vancomycin-sensitive strains stop growing in the presence of the antibiotic and rapidly undergo cell death through autolysis. Vancomycin-tolerant isolates have been described that cease to grow but do not undergo a significant degree of autolysis and retain the ability to grow once the antibiotic has dissipated (2, 10, 11, 17). This phenotype has been linked to treatment failure (15) and could foster the development of resistant strains that are able to not only survive but also grow in the presence of vancomycin. The recent emergence of vancomycin resistance in enterococci and staphylococci (1, 7, 22) is further cause for concern in this regard.

To gain insight into the effect of vancomycin on transcription in pneumococcus, we compared the stress response to vancomycin in two clinical isolates. The sequenced strain T4 (serotype 4) (21) is vancomycin sensitive, while strain Tupelo (serotype 14) is naturally vancomycin tolerant (17). Unlike T4, Tupelo does not undergo autolysis during stationary phase and lyses much slower in response to vancomycin, despite the presence of a fully functional LytA autolysin (17).

Experimental design.

Both strains were grown in C+Y medium to mid-logarithmic growth phase (optical density at 620 nm, 0.45 to 0.5) and exposed to 5 μg/ml vancomycin (equal to the 10-fold vancomycin MICs for each strain) for 10 and 20 min. The latter time point was chosen because it coincided with the cessation of growth and the onset of autolysis. RNA isolation and microarray analyses were performed as described previously (9). cDNA microarrays specific for strain T4 were received as a grant from the Pathogen Functional Genomics Resource Center (PFGRC; The Institute for Genomic Research, Rockville, MD). Three independent biological samples were used for each experiment. Genes that were differentially regulated by more than threefold and that had an analysis of variance P value of 0.001 or lower were considered further. The complete set of microarray data can be downloaded from St. Jude's web site (http://www.stjuderesearch.org/data/VancoT4Tupelo/).

Common themes in vancomycin stress response in strains T4 and Tupelo.

A number of transcripts exhibited similar expression patterns in both strains after vancomycin treatment. The hrcA-grpE-dnaK-dnaJ operon, encoding heat shock proteins, was induced in response to vancomycin in both strains, although expression levels fell to basal levels in strain T4 after 20 min (Table 1). The expression of other stress response genes, such as gor and htpX, increased in strain T4 by 3- to 6-fold in T4 but only 1.7- to 2.0-fold in Tupelo. A transcriptional regulator of the GntR family and the two-component systems TCS03 and TCS11 (13) were induced in both strains as well (see below). The choline binding proteins G and F were induced three- to fourfold within 10 min of vancomycin treatment. Other genes that were induced in both strains include a number of ABC transporters of unknown substrate specificity (i.e., SP1380/1, SP1715, and SP2003) and hypothetical proteins (i.e., SP0099, SP0385, and SP0910). Genes involved in aminosugar metabolism also responded in both strains: the glmS gene product catalyzes the synthesis of aminosugars, while the NagA and NagB proteins play a role in their catabolism. The expression of glmS was up to 20-fold reduced in vancomycin-treated T4 cultures, while nagA and nagB expression increased 15- and 18-fold, respectively, under the same conditions. In strain Tupelo, the genes followed the same overall expression pattern, although changes in transcription were only two- to fourfold. The expression of ribosomal proteins and translation factors decreased after the addition of the antibiotic, especially in strain T4. Transcripts of genes that play a role in the metabolism of nitrogen, polyamine, and purines were reduced in their expression as well.

TABLE 1.

Genes that are differentially expressed in S. pneumoniae strains T4 and Tupelo 10 and 20 min after the addition of vancomycina

Identity and function Annotation Gene T4 with vancomycin for:
Tupelo with vancomycin for:
10 min 20 min 10 min 20 min
Stress response
    SP0515 Heat-inducible transcription repressor HrcA hrcA 5.8 −1.0 s 4.7 3.3
    SP0516 Heat shock protein GrpE grpE 6.1 1.2 s 4.8 3.1
    SP0517 DnaK protein dnaK 6.6 2.0 4.6 3.5
    SP0519 DnaJ protein dnaJ 3.9 1.6 s 3.8 3.1
    SP0766 Superoxide dismutase, manganese dependent sodA 1.3 s 1.3 s −1.1 s −3.4
    SP0784 Glutathione reductase gor 2.0 3.3 1.6 s 1.9
    SP1283 Heat shock protein HtpX htpX 1.7 3.5 1.4 s 1.7
    SP1284 LemA protein lemA 1.7 3.5 1.5 s 1.8
    SP1996 Universal stress protein family uspA 2.1 6.0 −1.6 s −2.5
    SP2206 Ribosomal subunit interface protein yfiA 1.7 s 3.3 −1.7 s −2.7
Transcription factors and two-component systems
    SP0386 Sensor histidine kinase HK03 5.3 5.5 4.1 5.3
    SP0387 DNA-binding response regulator RR03 4.7 4.4 4.4 5.1
    SP0727 Transcriptional repressor, putative 1.4 s 4.3 1.4 s 1.3 s
    SP1714 Transcriptional regulator, GntR family 6.8 27.6 9.8 8.1
    SP2000 DNA-binding response regulator RR11 2.7 2.5 3.6 3.3
    SP2001 Sensor histidine kinase HK11 3.4 2.8 3.8 3.7
Aminosugar and cell wall metabolism
    SP0266 Glucosamine-fructose-6-phosphate aminotransferase glmS −8.3 −20.1 −4.2 −3.9
    SP1415 Glucosamine-6-phosphate isomerase nagB 7.8 18.5 3.0 2.0
    SP1975 SpolllJ family protein 2.3 2.3 2.6 3.1
    SP2056 N-Acetylglucosamine-6-phosphate deacetylase nagA 7.1 15.6 2.6 2.4
    SP2217 Rod shape-determining protein MreD, putative −1.5 s −3.1 −1.0 #s −1.1 s
    SP2218 Rod shape-determining protein MreC mreC −1.4 s −2.6 1.1 #s 1.1 s
Nitrogen, purine, and polyamine metabolism
    SP0044 Phosphoribosylaminoimidazole-succinocarboxamide synthase purC −2.7 −3.5 1.1 s −6.5
    SP0045 Phosphoribosylformylglycinamidine synthase, putative −2.0 s −3.1 1.2 s −3.7
    SP0231 Adenylate kinase adk −1.8 s −5.1 −1.6 s −1.6 s
    SP0287 Xanthine/uracil permease family protein −2.4 −6.2 −1.3 s −3.1
    SP0502 Glutamine synthetase, type I glnA −2.2 −3.3 −1.4 s −1.4 s
    SP0916 Lysine decarboxylase cad −2.5 −6.9 −1.7 #s −1.2 #s
    SP0918 Spermidine synthase speE −2.4 −7.8 −2.4 −2.1 s
    SP0920 Carboxynorspermidine decarboxylase nspC −1.9 s −7.2 −2.1 s −2.2
    SP0922 Carbon-nitrogen hydrolase family protein −2.0 −7.0 −2.1 s −2.2
    SP1180 Ribonucleoside-diphosphate reductase 2, beta subunit nrdF −1.7 −3.4 −1.3 s −1.7
    SP1847 Xanthine phosphoribosyltransferase xpt −2.3 −10.9 1.2 s −3.0
    SP1848 Xanthine permease pbuX −2.4 −11.3 1.2 s −2.3
Fermentation, polysaccharide, and sugar metabolism
    SP0285 Alcohol dehydrogenase, zinc containing 2.3 3.1 −3.4 −3.5
    SP0459 Formate acetyltransferase pfl 1.7 1.9 −2.1 −4.2
    SP1118 Pullulanase, putative −1.6 s −3.9 −1.1 s −1.1 s
    SP1122 Glucose-1-phosphate adenylyltransferase glgC 1.2 s 1.5 s −2.3 s −3.2
    SP1123 Glycogen biosynthesis protein GlgD glgD 1.2 s 1.3 s −2.1 s −3.1
    SP1329 N-Acetylneuraminate lyase 1.7 #s 1.6 #s −2.4 s −4.0
    SP1330 N-Acetylmannosamine-6-P epimerase, putative nanE 1.4 #s 1.7 #s −2.7 s −4.4
    SP1382 Alpha-amylase amy 3.1 6.0 1.2 #s 2.1 #s
    SP1852 Galactose-1-phosphate uridylyltransferase galT 1.0 s −1.0 s −1.9 s −3.3
    SP2026 Alcohol dehydrogenase, iron-containing adhE 2.8 1.4 s −3.4 −3.2
    SP2106 Glycogen phosphorylase family protein 3.3 3.4 −1.0 s −1.2 s
    SP2107 4-Alpha-glucanotransferase malQ 4.3 3.9 −1.0 s −1.2 s
    SP2157 Alcohol dehydrogenase, iron-containing fucO 3.7 5.3 −1.2 #s −1.2 #s
    SP2167 l-Fuculose kinase FucK, putative fucK 2.4 #s 4.1 1.2 #s 1.5 #s
Capsule biosynthesis
    SP0346 Capsular polysaccharide biosynthesis protein Cps4A cps4A −1.4 s −1.8 1.0 s 1.1 s
    SP0347 Capsular polysaccharide biosynthesis protein Cps4B cps4B −1.3 s −1.5 1.1 s 1.1 s
    SP0348 Capsular polysaccharide biosynthesis protein Cps4C cps4C −1.5 s −2.4 −1.0 s 1.1 s
    SP0349 Capsular polysaccharide biosynthesis protein Cps4D cps4D −1.7 −3.1 −1.2 s −1.2 s
    SP0350 Capsular polysaccharide biosynthesis protein Cps4E cps4E −1.9 −4.0 −1.0 #s 1.2 #s
    SP0351 Capsular polysaccharide biosynthesis protein Cps4F cps4F −1.9 −4.3 1.4 #s −1.4 #s
    SP0352 Capsular polysaccharide biosynthesis protein Cps4G cps4G −1.9 s −4.0 1.1 #s 1.1 #s
    SP0353 Capsular polysaccharide biosynthesis protein Cps4H cps4H −1.6 −1.5 1.1 #s −1.0 #s
    SP0357 UDP-N-acetylglucosamine-2-epimerase cps4I −2.0 −6.6 −1.0 #s −1.0 #s
    SP0358 Capsular polysaccharide biosynthesis protein Cps4J cap4J −1.8 s −6.3 1.7 #s 1.0 #s
    SP0359 Capsular polysaccharide biosynthesis protein Cps4K cps4K −1.8 −6.4 1.1 #s −1.3 #s
    SP0360 UDP-N-acetylglucosamine-2-epimerase cps4L −1.7 −5.8 1.2 #s 1.2 #s
Surface proteins
    SP0390 Choline binding protein G cbpG 3.9 4.4 3.4 4.3
    SP0391 Choline binding protein F cbpF 3.2 4.2 3.0 4.5
    SP0462 Cell wall surface anchor family protein −1.5 s −2.6 1.0 #s 1.2 #s
    SP0463 Cell wall surface anchor family protein −1.7 s −3.4 1.5 #s 1.3 #s
    SP0464 Cell wall surface anchor family protein −1.5 s −3.7 1.1 #s −1.3 #s
    SP1002 Adhesion lipoprotein lmb −1.8 s −4.3 1.0 s −1.3 s
Translation and ribosomal proteins
    SP0085 Ribosomal protein S4 rpsD −2.6 −5.0 −1.6 s −2.0
    SP0232 Translation initiation factor IF-1 infA −1.8 −3.0 −1.5 s −1.7
    SP0233 Ribosomal protein L36 rpmJ −1.8 −2.9 −1.6 s −1.9 s
    SP0234 Ribosomal protein S13 rpsM −1.8 −3.3 −1.5 s −1.7
    SP0235 Ribosomal protein S11 rpsK −1.8 −3.3 −1.6 −1.6
    SP0271 Ribosomal protein S12 rpsL −1.8 −3.3 −1.4 s −1.5 s
    SP0272 Ribosomal protein S7 rpsG −1.7 −3.1 −1.4 s −1.4 s
    SP0294 Ribosomal protein L13 rplM −1.8 −3.1 −1.4 s −1.5
    SP0775 Ribosomal protein S16 rpsP −1.7 −3.8 −1.2 s −2.3
    SP0862 Ribosomal protein S1 rpsA −2.2 −3.5 −1.4 s −1.8
    SP0959 Translation initiation factor IF-3 infC −1.8 −4.9 −1.3 s −1.1 s
    SP0960 Ribosomal protein L35 rpmI −1.6 s −3.2 −1.3 s −1.4 s
    SP0961 Ribosomal protein L20 rplT −1.8 −3.2 −1.4 s −1.5 s
    SP1354 Ribosomal protein L7/L12 rplL −1.6 −3.0 −1.6 s −1.6
    SP1355 Ribosomal protein L10 rplJ −1.5 −3.3 −1.5 s −1.2 s
    SP2214 Translation elongation factor Ts tsf −1.2 −3.2 −1.2 s −1.3 s
    SP2215 Ribosomal protein S2 rpsB −1.3 −3.8 −1.2 s −1.3 s
PTS systems and ABC transporters
    SP0090 ABC transporter, permease protein 3.2 5.2 1.3 #s 1.6 #s
    SP0091 ABC transporter, permease protein 2.0 s 3.1 1.2 #s −1.1 #s
    SP0092 ABC transporter, substrate-binding protein 2.9 4.8 1.4 #s 1.3 #s
    SP0282 PTS system, mannose-specific IID component manN −1.2 −1.3 −2.2 s −3.4
    SP0283 PTS system, mannose-specific IIC component manM −2.6 −3.2 −2.3 −3.0
    SP0284 PTS system, mannose-specific IIAB components manL −2.8 −3.8 −1.9 s −3.3
    SP0786 ABC transporter, ATP-binding protein 2.0 3.0 3.2 2.2
    SP0912 ABC transporter, ATP-binding protein 3.2 5.5 1.8 s 1.9 s
    SP0913 ABC transporter, permease protein, putative 3.1 5.0 1.8 s 2.2
    SP0957 ABC transporter, ATP-binding protein 1.2 s 1.2 s −1.9 s −3.1
    SP1032 Iron compound ABC transporter, iron compound-binding protein piuB −1.5 s −2.3 −1.2 s −1.3 s
    SP1033 Iron compound ABC transporter, permease protein piuC −2.0 s −4.3 1.1 #s 1.1 s
    SP1034 Iron compound ABC transporter, permease protein piuD −2.0 s −4.6 −1.1 #s 1.1 s
    SP1035 Iron compound ABC transporter, ATP-binding protein piuA −2.0 s −4.6 −1.1 s −1.1 s
    SP1380 Putative permease 2.0 3.0 2.1 3.3
    SP1381 ABC transporter, ATP-binding protein 2.4 s 5.8 2.0 #s 3.3
    SP1580 Sugar ABC transporter, ATP-binding protein msmK −1.1 s −1.1 s −3.5 −6.6
    SP1688 ABC transporter, permease protein 1.8 s 3.8 1.2 #s −1.5 #s
    SP1689 ABC transporter, permease protein 2.0 s 2.9 1.1 #s −1.1 #s
    SP1690 ABC transporter, substrate-binding protein 2.1 s 3.6 1.5 #s 1.6 #s
    SP1715 ABC transporter, ATP-binding domain and permease 7.9 27.2 9.7 12.2
    SP2002 Putative permease 4.5 4.4 1.8 #s 1.7 #s
    SP2003 ABC transporter, ATP-binding protein 10.3 9.2 4.4 4.9
    SP2108 Maltose/maltodextrin ABC transporter, maltose-binding protein malX 1.7 2.6 −2.7 −4.0
    SP2109 Maltodextrin ABC transporter, permease protein malC 1.6 s 2.1 −2.6 s −3.9
    SP2110 Maltodextrin ABC transporter, permease protein malD 1.6 s 2.4 −1.5 #s −2.5
ClaRH regulon
    SP0282 PTS system, mannose-specific IID component manN −1.2 −1.3 −2.2 s −3.4
    SP0283 PTS system, mannose-specific IIC component manM −2.6 −3.2 −2.3 −3.0
    SP0284 PTS system, mannose-specific IIAB components manL −2.8 −3.8 −1.9 s −3.3
    SP0798 DNA-binding response regulator CiaR ciaR 2.7 2.5 −1.1 s −1.3 s
    SP0799 Sensor histidine kinase CiaH ciaH 2.8 2.2 −1.1 s −1.2
    SP0879 Hypothetical protein 9.9 14.1 1.5 #s 1.2 s
    SP1027 Conserved hypothetical protein 6.1 9.7 1.2 s −1.2 s
    SP2206 Ribosomal subunit interface protein yfiA 1.7 s 3.3 −1.7 s −2.7
    SP2239 HtrA serine protease htrA 5.4 4.1 −1.2 s −1.7
    SP2240 SpoJ protein parB 4.7 3.6 −1.1 s −1.6
Miscellaneous functions
    SP0006 Transcription-repair coupling factor mfd −1.6 s −3.9 −1.1 s −1.3 s
    SP0109 Bacteriocin, putative 1.9 s 3.5 −1.3 s −1.6
    SP0356 O-antigen transporter RfbX, putative −1.8 s −6.5 1.8 # −1.0 #s
    SP0962 Lactoylglutathione lyase gloA −1.8 s −3.6 −1.3 s −1.3
    SP1117 DNA ligase, NAD dependent ligA −1.8 s −4.3 1.0 s −1.0 s
    SP1214 Transulfuration enzyme family protein, authentic point mutation 2.5 s 3.3 1.0 #s 1.3 #s
    SP1325 Oxidoreductase, Gfo/Idh/MocA family 1.9 #s 1.8 # −2.3 s −4.0
    SP1326 Neuraminidase, putative 1.3 #s 2.1 #s −2.6 s −3.7
    SP1328 Sodium:solute symporter family protein 1.4 s 1.4 #s −2.1 s −3.2
    SP1343 Prolyl oligopeptidase family protein 1.9 s 3.2 1.5 #s 1.4 #s
    SP1402 NOL1/NOP2/sun family protein −2.1 s −4.4 −1.1 s −1.7 s
    SP1466 Hemolysin 2.8 6.1 −1.4 s −2.5
    SP1513 ATP synthase F0, A subunit atpB 1.2 s 1.3 s 1.8 s 3.2
    SP1586 ATP-dependent RNA helicase, putative −1.3 s −4.4 1.4 s 1.2 s
    SP1687 Neuraminidase B nanB 2.1 s 3.4 −1.1 #s 1.4 #s
    SP1807 Acetyltransferase, GNAT family 2.1 s 3.3 1.3 #s −1.1 s
Hypothetical proteins
    SP0034 Membrane protein 1.6 1.7 s 2.2 s 3.9
    SP0088 Hypothetical protein 1.5 s 3.2 1.4 #s 1.1 #s
    SP0096 Hypothetical protein 1.8 s 3.2 2.0 #s 1.6 #s
    SP0097 Conserved domain protein 2.0 2.3 3.5 2.3 s
    SP0098 Hypothetical protein 3.2 2.9 3.5 2.6
    SP0099 Hypothetical protein 3.6 3.5 3.8 2.9
    SP0100 Conserved hypothetical protein 3.4 3.6 3.7 2.3
    SP0189 Conserved hypothetical protein 2.9 5.4 2.1 1.7 s
    SP0191 Hypothetical protein 3.3 4.0 2.3 s 2.8
    SP0288 Conserved hypothetical protein −3.2 −5.4 −1.0 #s −1.6 #s
    SP0293 Hypothetical protein 2.2 3.5 1.5 s 1.9
    SP0298 Conserved hypothetical protein 2.2 s 3.1 1.4 #s −1.1 #s
    SP0355 Hypothetical protein −2.0 −6.4 1.2 #s 1.0 #s
    SP0385 Conserved hypothetical protein 5.5 5.9 3.9 5.1
    SP0389 Hypothetical protein 3.4 4.0 2.8 s 3.6
    SP0430 Hypothetical protein −1.5 s −3.3 −1.3 s −1.6 s
    SP0595 Hypothetical protein 1.0 s −3.1 1.1 #s 1.8 #s
    SP0728 Hypothetical protein 1.5 s 3.6 1.3 s 1.2 s
    SP0742 Conserved hypothetical protein 1.9 s 1.6 s −2.2 −3.7
    SP0785 Conserved hypothetical protein 2.2 3.0 3.3 2.4
    SP0787 Conserved hypothetical protein 2.0 s 3.2 3.5 2.2
    SP0879 Hypothetical protein 9.9 14.1 1.5 #s 1.2 s
    SP0910 Conserved hypothetical protein 4.1 8.0 3.2 3.4
    SP0919 Conserved hypothetical protein −2.3 s −6.0 −1.9 s −2.0 #
    SP0921 Conserved hypothetical protein −1.9 s −6.9 −2.3 −2.3
    SP0925 Conserved hypothetical protein 1.7 s 3.2 1.5 #s 1.9 s
    SP0958 Hypothetical protein 1.1 s 1.1 s −2.3 −4.1
    SP1004 Conserved hypothetical protein −1.3 s −4.0 2.4 s 1.3
    SP1027 Conserved hypothetical protein 6.1 9.7 1.2 s −1.2 s
    SP1327 Conserved hypothetical protein 1.3 #s 1.4 #s −2.3 s −3.6
    SP1344 Conserved hypothetical protein 3.1 #s 4.9 1.7 #s 1.2 #s
    SP1465 Hypothetical protein 3.2 7.3 −1.4 s −2.9
    SP1532 Conserved domain protein, authentic frameshift −1.7 s −3.8 −1.1 s −1.2 s
    SP1612 Conserved domain protein 1.2 #s −1.2 s 1.3 #s 3.2
    SP1685 Conserved hypothetical protein 1.1 s 1.5 s −2.2 −3.3
    SP1691 Conserved hypothetical protein 1.8 #s 5.4 1.4 #s 2.2 s
    SP1716 Conserved hypothetical protein natB 3.3 4.2 1.0 s 1.1 s
    SP1972 Membrane protein 2.7 3.4 1.2 s 1.1 s
    SP2004 Hypothetical protein 4.6 4.3 1.7 #s 1.3 #s
    SP2005 Hypothetical protein 6.6 9.5 1.8 #s 1.1 #s
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a

Data represent fold increase or decrease in gene expression after treatment. Only genes whose expression changed by at least threefold are shown. Each datum point represents three biological samples and a total of 12 cDNA hybridization spots. A “#” marks data that are based on low signal intensities for the sample and the reference. Data that had analysis of variance P values larger than 0.001 are indicated by an “s.”

Responses to vancomycin stress which are unique to strain T4 or Tupelo.

While some genes responded to vancomycin treatment similarly in both strains, other genes were induced or repressed in one strain but not the other. Expression of the cps4 genes, which are responsible for synthesis of the type 4 capsule, and of a locus that encodes three cell wall surface anchor family proteins was reduced in strain T4. No significant signal was obtained in the case of strain Tupelo, because the loci are either divergent or missing (data not shown). The expression of stress response genes, such as uspA, yfiA, and adhE, increased in T4 but decreased in Tupelo. Expression of superoxide dismutase decreased by 3.4-fold in Tupelo but remained steady in T4. The pfl and adhE genes, whose products are involved in mixed acid fermentation, as well as two other genes encoding alcohol dehydrogenases were induced in strain T4 but remained unchanged or were repressed in Tupelo. Transcripts for glycolytic enzymes were increased in strain T4, while the expression of genes involved in glycogen biosynthesis was decreased in Tupelo. Several ABC transporters and hypothetical proteins were also differentially expressed in one strain but not the other.

A significant difference in gene expression was observed for the CiaRH regulon. The CiaRH two-component system has been shown to regulate various functions in S. pneumoniae, such as autolysis, competence, virulence, and beta-lactam susceptibility (5, 8, 20, 24). Several screens have identified a number of genes that could be regulated by the CiaRH system (15, 19). Some of these were differentially regulated in response to vancomycin, including the manLMN mannose-specific phosphotransferase (PTS) system, the ciaRH two-component system itself, the hypothetical proteins SP0879 and SP1027, the iron compound ABC transporter piuBCDA, the two-component system TCS11, the ribosomal subunit interface protein YfiA, the serine protease HtrA, and the Spo0J-like protein ParB. Most of the genes listed above are up-regulated (or derepressed) in vancomycin-treated cultures of strain T4 but down-regulated or not differentially expressed in strain Tupelo.

Similarities to the vancomycin stress response in Staphylococcus aureus and Bacillus subtilis.

Work with S. aureus has shown that the VraSR two-component system is upregulated in response to treatment with vancomycin and other inhibitors of cell wall synthesis (12, 18). In B. subtilis, exposure to vancomycin results in the activation of alternate sigma factors and two-component systems, including LiaRS (YvqCE) (16). A BLASTP search revealed that the pneumococcal two-component system TCS03 (SP0386 and SP0387), which was induced in T4 and Tupelo after vancomycin treatment, has significant similarity to VraSR and to LiaRS. The histidine kinases share 38 to 40% identical and 62 to 65% similar residues with HK03, while the response regulators share 51% identical and 73 to 76% conserved amino acids with RR03. The loci encoding the two-component systems in the three species are preceded by predicted membrane proteins that share 24 to 29% identical and 51 to 53% conserved residues. The three proteins are 232 to 241 amino acids in size and contain the conserved domain COG1458 (14).

The HtrA serine protease, which is part of the CiaRH regulon in pneumococcus, was also induced in all three bacterial species in response to vancomycin stress (4, 12), although the corresponding gene was not induced in S. pneumoniae strain Tupelo.

A protein with similarity to phage shock protein A, LiaH (YvqH), has been shown to play a role in the vancomycin stress response in B. subtilis (12). In S. pneumoniae, the open reading frame SP0910 is induced by vancomycin and encodes a conserved hypothetical protein that contains a phage shock protein C domain. The pspABCDE operon from Escherichia coli is induced in response to ethanol, heat, osmotic shock, and bacteriophage infection (3). Phage shock proteins A and C play a role in the repression and activation (6, 23) of stress-responsive genes, respectively. Whether the SP0910 gene product has a similar function in S. pneumoniae remains to be determined.

Conclusions.

The data presented here demonstrate that the vancomycin-sensitive strain T4 and the vancomycin-tolerant strain Tupelo have a number of genes in common that are differentially expressed in response to vancomycin stress. The two-component systems TCS03 and TCS11 were induced in both strains, of which the former shares sequence similarity with a vancomycin-induced two-component system from S. aureus and B. subtilis. Genes that responded to vancomycin in one pneumococcal strain but not the other were also observed in large numbers. The CiaRH regulon, which has been shown to play a role in autolysis, was induced in strain T4 but not Tupelo. It will be interesting to ascertain if lack of induction of this regulon is the reason for the tolerant phenotype of strain Tupelo.

Acknowledgments

This work was supported by grant 5R01AI039482-07 from the National Institute of Allergy and Infectious Diseases, Cancer Center grant P30 CA21765, and the American Lebanese Syrian Associated Charities.

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