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Journal of Bacteriology, December 2005, p. 8205-8210, Vol. 187, No. 23
0021-9193/05/$08.00+0     doi:10.1128/JB.187.23.8205-8210.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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 and Elaine I. Tuomanen1

Departments of Infectious Diseases,1 Functional Genomics, Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children's Research Hospital, Memphis, Tennessee2

Received 13 July 2005/ Accepted 15 September 2005


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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.


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TEXT
 
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.


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  		TABLE 1. Genes that are differentially expressed in S. pneumoniae strains T4 and Tupelo 10 and 20 min after the addition of vancomycina

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.


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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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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Center for Oral Biology, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Phone: (585) 275-7722. Fax: (585) 276-0190. E-mail: wolfgang_haas{at}urmc.rochester.edu. Back


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Journal of Bacteriology, December 2005, p. 8205-8210, Vol. 187, No. 23
0021-9193/05/$08.00+0     doi:10.1128/JB.187.23.8205-8210.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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