Major Cold Shock Protein Research Articles

Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach

We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5′-GTCTTTG/T-3′. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA–protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function.

Transcription Antitermination by Translation Initiation Factor IF1

Bacterial translation initiation factor IF1 is an S1 domain protein that belongs to the oligomer binding (OB) fold proteins. Cold shock domain (CSD)-containing proteins such as CspA (the major cold shock protein of Escherichia coli) and its homologues also belong to the OB fold protein family. The striking structural similarity between IF1 and CspA homologues suggests a functional overlap between these proteins. Certain members of the CspA family of cold shock proteins act as nucleic acid chaperones: they melt secondary structures in nucleic acids and act as transcription antiterminators. This activity may help the cell to acclimatize to low temperatures, since cold-induced stabilization of secondary structures in nascent RNA can impede transcription elongation. Here we show that the E. coli translation initiation factor, IF1, also has RNA chaperone activity and acts as a transcription antiterminator in vivo and in vitro. We further show that the RNA chaperone activity of IF1, although critical for transcription antitermination, is not essential for its role in supporting cell growth, which presumably functions in translation. The results thus indicate that IF1 may participate in transcription regulation and that cross talk and/or functional overlap may exist between the Csp family proteins, known to be involved in transcription regulation at cold shock, and S1 domain proteins, known to function in translation.

Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA

Homocysteine (Hcy) is a thiol-containing amino acid that is considered to be medically important because it is linked to the development of several life-threatening diseases in humans, including cardiovascular disease and stroke. It inhibits the growth of Escherichia coli when supplied in the growth medium. Growth inhibition is believed to arise as a result of partial starvation for isoleucine, which occurs because Hcy perturbs the biosynthesis of this amino acid. This study attempted to further elucidate the inhibitory mode of action of Hcy by examining the impact of exogenously supplied Hcy on the transcriptome. Using gene macroarrays the transcript levels corresponding to 68 genes were found to be reproducibly altered in the presence of 0.5 mM Hcy. Of these genes, the biggest functional groups affected were those involved in translation (25 genes) and in amino acid metabolism (19 genes). Genes involved in protection against oxidative stress were repressed in Hcy-treated cells and this correlated with a decrease in catalase activity. The gene showing the strongest induction by Hcy was cspA, which encodes the major cold-shock protein CspA. RT-PCR and reporter fusion experiments confirmed that cspA was induced by Hcy. Induction of cspA by Hcy was not caused by nutritional upshift, a stimulus known to induce CspA expression, nor was it dependent on the presence of a functional CspA protein. The induction of cspA by Hcy was suppressed when isoleucine was included in the growth medium. These data suggest that the induction of CspA expression in the presence of Hcy occurs because of a limitation for isoleucine. The possibility that Hcy-induced cspA expression is triggered by translational stalling that occurs when the cells are limited for isoleucine is discussed.

Expression of Major Cold Shock Proteins and Genes by Yersinia enterocolitica in Synthetic Medium and Foods

Yersinia enterocolitica is a psychrotrophic foodborne pathogen that has been implicated in outbreaks of foodborne illness involving cold-stored foods, especially milk and pork. A major mechanism bacteria use to adapt to cold is expression of cold shock proteins. The objective of this research was to study the expression of major cold shock proteins of Y. enterocolitica in Luria-Bertani (LB) broth, milk, and pork following a temperature downshift from 30 to 4°C. Y. enterocolitica was inoculated into 10 ml of LB broth, sterile skim milk, or pork, and the samples were stored at 4°C (cold shock) or 30°C (control) for 0, 4, 8, 12, and 24 h. At each sampling time, total protein and total RNA were extracted from Y. enterocolitica harvested from LB broth, milk, and pork and subjected to two-dimensional gel electrophoresis and dot blot analysis. Two major cold shock proteins (CspA1 and CspA2) of ∼7 kDa and their genes were expressed by Y. enterocolitica following cold shock. However, the CspA1 and CspA2 proteins were not expressed by Y. enterocolitica at 30°C. Y. enterocolitica CspA1 and CspA2 were observed as early as 2 h of cold shock in cultures from LB broth and milk and at 8 h of cold shock in cultures from pork.

Extended −10 Motif Is Critical for Activity of thecspAPromoter but Does Not Contribute to Low-Temperature Transcription

Bacterial promoters belonging to the extended -10 class contain a conserved TGn motif upstream of the -10 promoter consensus element. Open promoter complexes can be formed on some extended -10 Escherichia coli promoters at temperatures as low as 6 degrees C, when complexes on most promoters are closed. The promoter of cspA, a gene that codes for the major cold shock protein CspA of E. coli, contains an extended -10 motif. CspA is dramatically induced upon temperature downshift from 37 to 15 degrees C, and its cold shock induction has been attributed to transcription, translation, and mRNA stabilization effects. Here, we show that though the extended -10 motif is critical for high-level expression of cspA, it does not contribute to low-temperature expression. In fact, transcription from the wild-type cspA promoter is cold sensitive in vitro and in vivo. Thus, transcription appears to play little or no role in low-temperature induction of cspA expression.

Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces.

S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme.

Single-stranded DNA bound to bacterial cold-shock proteins: preliminary crystallographic and Raman analysis.

The cold-shock response has been described for several bacterial species. It is characterized by distinct changes in intracellular protein patterns whereby a set of cold-shock-inducible proteins become abundant. The major cold-shock proteins of Bacillus subtilis (Bs-CspB) and Bacillus caldolyticus (Bc-Csp) are small oligonucleotide/oligosaccharide-binding (OB) fold proteins that have been described as binding single-stranded nucleic acids. Bs-CspB (Mr = 7365) and Bc-Csp (Mr = 7333) were crystallized in the presence of the deoxyhexanucleotide (dT)6. Crystals of (dT)6 with Bs-CspB grew in the orthorhombic space group C222(1), with unit-cell parameters a = 49.0, b = 53.2, c = 77.0 A. Crystals with Bc-Csp grew in the primitive orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 74.3, b = 64.9, c = 31.2 A. These crystals diffract to maximal resolutions of 1.78 and 1.29 A, respectively. The presence of protein and DNA in the crystals was demonstrated by Raman spectroscopy.

The AGUAAA motif in cspA1/A2 mRNA is important for adaptation of Yersinia enterocolitica to grow at low temperature.

Acclimatization of the psychrotolerant Yersinia enterocolitica after a cold shock from 30 degrees C to 10 degrees C causes transcription of the major cold shock protein (CSP) bicistronic gene cspA1/A2 to increase by up to 300-fold. Northern blot analysis of cspA1/A2 using four probes that hybridize specifically to different regions of CSP mRNA revealed the appearance of a number of cspA1/A2 transcripts that are smaller than the original transcript and transiently visible at the end of the acclimation period. Primer extension and RNA protection experiments demonstrated that these smaller mRNAs have 5' ends located in the same core sequence (5'-AGUAAA-3') at five different places within the mRNA, indicating preferential cleavage of the CSP mRNA transcripts. A similar result was obtained for cspB of Escherichia coli, containing two such core sequences. Furthermore, this motif is present in the major CSP genes of a variety of Gram-negative and Gram-positive bacteria. We have therefore termed this sequence cold shock cut box (CSC-box). After inserting a CSC-box into a plasmid-bound lacZ gene in Y. enterocolitica, the mRNA of this construct was cleaved within the CSC-box, and a change in this CSC-box from AGUAAA to AGUCCC dramatically reduced cleavage of the mutated lacZ gene. Mutating all CSC-boxes in Y. enterocolitica of a plasmid bound cspA1/A2 dramatically increases the lag time after a cold shock before re-growth occurs. Based on these results, we suggest that the role of the CSC-box is related to downregulation of cspA mRNA after acclimation to low temperature.

Cold shock response and major cold shock proteins of Vibrio cholerae.

When exponentially growing Vibrio cholerae cells were shifted from 37 degrees C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15 degrees C, below which the growth was completely arrested. There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of V. cholerae. Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspA(VC)) and 7.5 kDa (CspV), and six other Csps, most of which were much larger. We cloned, sequenced, and analyzed the cspV gene encoding the CspV protein of V. cholerae O139 strain SG24. Although CspA(VC) and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes, cspA and cspV, which are located in the small chromosome, are not located in the same operon. A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock. Although both CspA(VC) and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37 degrees C, suggesting that this protein is probably necessary for adaptation at lower temperatures. Northern blot analysis confirmed that the cspV gene is cold shock inducible and is regulated tightly at the level of transcription. Interestingly, the cspV gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5' untranslated region is present in its mRNA transcript. Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including Escherichia coli. Our results suggest that V. cholerae may use an alternative pathway for regulation of gene expression during cold shock.

The major cold shock gene, cspA, is involved in the susceptibility of Staphylococcus aureus to an antimicrobial peptide of human cathepsin G.

A Tn551 insertional library of Staphylococcus aureus strain ISP479 was challenged with an antimicrobial peptide (CG 117-136) derived from human neutrophil cathepsin G (CG). After repeated selection and screening of surviving colonies, a mutant was identified that expressed increased resistance to CG 117-136. Southern hybridization analysis revealed that the Tn551 insert in this mutant (SK1) was carried on a 10.6-kb EcoRI chromosomal DNA fragment. Subsequent physical mapping of this Tn551 insert revealed that it was positioned between a putative promoter sequence and the translational start codon of the cspA gene, which encodes a protein (CspA) highly similar to the major cold shock proteins CspA and CspB of Escherichia coli and Bacillus subtilis, respectively. This Tn551 insertion as well as a separate deletion-insertion mutation in cspA decreased the capacity of S. aureus to respond to the stress of cold shock and increased resistance to CG 117-136. The results indicate for the first time that a physiologic link exists between bacterial susceptibility to an antimicrobial peptide and a stress response system.

Effect of insertional mutation in the cspA gene encoding the major cold-shock protein on radiation resistance of Escherichia coli

Plasmid pCspA::Km carrying a cloned mutant allele of the cspA gene for the major Escherichia coli cold-shock protein CspA with an insertion of the kanamycin resistance gene cassette from transposon Tn903 into the core region of the coding sequence causes a 2.3-fold increase in radioresistance of wild-type E. coli cells (cspA+). The radioprotective effect of this plasmid is abolished or drastically reduced in mutants recA13 and rpoH15 defective in RecA protein and in induction of the heat-shock protein regulon, respectively. Plasmid pCspA::Km causes a 1.3-fold elevation in the resistance to gamma-irradiation of E. coli mutants with an intermediate level of radioresistance (Gamr445 and KS0160) but slightly diminishes resistance of a highly radiation-resistant Gamr445 mutant. In the chromosome of E. coli with normal DNA repair systems, the cspA::Km mutation in the homozygous state enhances resistance to the lethal effect of gamma-rays and UV light 2.9 and 1.4 times, respectively. These data suggest that the system of cold-shock proteins can modulate resistance of E. coli cells to the lethal effect of gamma-rays and UV light.

Proteomic study of cold shock protein in Bacillus stearothermophilus P1: Comparison of temperature downshifts.

The thermophilic bacterium Bacillus stearothermophilus P1 is unique in its ability to thrive in extreme environments such as high temperatures or high pH conditions. The study of cold shock response is very interesting and interpreted as a shock response to express the genes involved in synthesis of specific proteins. This study investigated the study of cold shock protein of B. stearothermophilus P1 when the cell culture temperature shifted from 65 degrees C to 37 degrees C and 25 degrees C. Cell growth at 37 degrees C weakly increased in the previous 3 h and then slowly decreased. In contrast, cell growth at 25 degrees C was slowly decreased. The protein contents after temperature downshifts were analyzed by proteomic techniques using protein chip and two-dimensional (2-D) electrophoresis that are highly effective and useful for protein separation and identification. The different proteins after a temperature decrease from 65 degrees C to 37 degrees C and 25 degrees C were expressed on 2-D gel patterns and the cold shock protein was detected in the acidic area with the isoelectric point and molecular mass approximately 4.5 and 7.3 kDa, respectively. The NH(2)-terminal sequence of a major cold shock protein from B. stearothermophilus P1 was MQRGKVKWFNNEKGFGFIEVEGGSD, similar to other cold shock proteins from Bacillus sp. up to 96% identity, but different from the other bacteria with homology less than 80% identity.

Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures.

Trehalose accumulates dramatically in microorganisms during heat shock and osmotic stress and helps protect cells against thermal injury and oxygen radicals. Here we demonstrate an important role of this sugar in cold-adaptation of bacteria. A mutant Escherichia coli strain unable to produce trehalose died much faster than the wild type at 4 degrees C. Transformation of the mutant with the otsA/otsB genes, responsible for trehalose synthesis, restored trehalose content and cell viability at 4 degrees C. After temperature downshift from 37 degrees C to 16 degrees C ("cold shock"), trehalose levels in wild-type cells increased up to 8-fold. Although this accumulation of trehalose did not influence growth at 16 degrees C, it enhanced cell viability when the temperature fell further to 4 degrees C. Before the trehalose build-up, levels of mRNA encoding OtsA/OtsB increased markedly. This induction required the sigma factor, RpoS, but was independent of the major cold-shock protein, CspA. otsA/B mRNA was much more stable at 16 degrees C than at 37 degrees C and contained a "downstream box," characteristic of cold-inducible mRNAs. Thus, otsA/otsB induction and trehalose synthesis are activated during cold shock (as well as during heat shock) and play an important role in resistance of E. coli (and probably other organisms) to low temperatures.

Nonsense Mutations in cspA Cause Ribosome Trapping Leading to Complete Growth Inhibition and Cell Death at Low Temperature in Escherichia coli

CspA, the major cold shock protein of Escherichia coli, is dramatically induced immediately after cold shock. CspA production is transient and reduces to a low basal level when cells become adapted. Here we show that expression from multicopy plasmids of mutant cspA mRNAs bearing nonsense mutations in the coding region caused sustained high levels of the mutant mRNAs at low temperature, resulting in complete inhibition of cell growth ultimately leading to cell death. We demonstrate that the observed growth inhibition was caused by largely exclusive occupation of cellular ribosomes by the mutant cspA mRNAs. Such sequestration of ribosomes even occurs without a single peptide bond formation, implying that the robust translatability of the cspA mRNA is determined at the step of initiation. Further analysis demonstrated that the downstream box of the cspA mRNA was dispensable for the effect, whereas the upstream box of the mRNA was essential. Our system may offer a novel means to study sequence or structural elements involved in the translation of the cspA mRNA and may also be utilized to regulate bacterial growth at low temperature.

Induction of CspA, an E. coli major cold-shock protein, upon nutritional upshift at 37 degrees C.

The synthesis of CspA, the major cold-shock protein of Escherichia coli, is dramatically induced upon cold shock. It was recently reported that there is massive presence of CspA under nonstress conditions, and it is thus claimed that CspA as the cold-shock protein is a misnomer. Here, we re-examined and confirmed that CspA is induced upon culture dilution at 37 degrees C. However, its induction level is one-sixth of the cold-shock-induced level, clearly indicating that the major stress that induces CspA is cold shock. It was further found that CspA induction can be achieved not only by culture dilution but also by the simple addition of nutrients, and that it was almost completely abolished in the presence of rifampicin or nalidixic acid. Nutritional upshift causes the induction of only CspA but not other cold-shock-inducible CspA homologues. The amount of cspA mRNA rapidly and transiently increased by culture dilution, but its stability was not significantly changed. These results suggest that CspA is a nutritional-upshift stress protein as well as a cold-shock stress protein, and that CspA induction following nutritional upshift may be due to transcriptional activation.

Response and tolerance of toxigenic Vibro cholerae O1 to cold temperatures.

Survival and tolerance at cold temperatures, the differentially expressed cellular proteins, and cholera toxin (CTX) production were evaluated in Vibrio cholerae O1. Rapid loss of culturability and change to distinct coccoid morphology occurred when cultures of V cholerae 01 were exposed to 5 degrees C directly from 35 degrees C. Also, cultures of V. cholerae first exposed to 15 degrees C for 2 h and then maintained at 5 degrees C failed to exhibit an adaptive response, instead a rapid loss of viable plate count was noticed. Results from Western blot experiments revealed the absence of a major cold shock protein, CS7.4. Also, a decreased level of CTX was noticed in V. cholerae O1 cultures exposed to 5 or 15 degrees C after first being exposed to 15 degrees C for 2 h, followed by transfer to 5 degrees C. Reduced expression of CTX at cold temperatures, compared to the cultures maintained at 35 degrees C, may be a result of decreased cellular metabolic activity. When V. cholerae O1 cultures were exposed to 15 degrees C for 2 h, elevated expressions of 8, 26 and 194 kDa, and decreased expression of 28 and 183 kDa proteins occurred. It is suggested that these differentially expressed cold-responsive proteins are involved in regulating culturability and conversion to a coccoid cell morphology in V cholerae O1.

Acquisition of double-stranded DNA-binding ability in a hybrid protein between Escherichia coli CspA and the cold shock domain of human YB-1.

Escherichia coli CspA, a major cold shock protein, is dramatically induced upon temperature downshift. As it binds co-operatively to single-stranded DNA (ssDNA) and RNA without apparent sequence specificity, it has been proposed that CspA acts as an RNA chaperone to facilitate transcription and translation at low temperature. CspA consists of a five-stranded beta-barrel structure containing two RNA-binding motifs, RNP1 and RNP2. Eukaryotic Y-box proteins, such as human YB-1, are a family of nucleic acid-binding proteins that share a region of high homology with CspA (43% identity), termed the cold shock domain (CSD). Their cellular functions are very diverse and are associated with growth-related processes. Here, we replaced the six-residue loop region of CspA between the beta3 and beta4 strands with the corresponding region of the CSD of human YB-1 protein. The resulting hybrid protein became capable of binding to double-stranded DNA (dsDNA) in addition to ssDNA and RNA. The dsDNA-binding ability of an RNP1 point mutant (F20L) of the hybrid was almost unchanged. On the other hand, the dsDNA-binding ability of the hybrid protein was abolished in high salt concentrations in contrast to its ssDNA-binding ability. These results indicate that the loop region between the beta3 and beta4 strands of Y-box proteins, which is a little longer and more basic than that of CspA, plays an important role in their binding to dsDNA.

Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus.

The identification of target structures is a prerequisite for the development of new treatment options, like antibody based therapy, against methicillin resistant Staphylococcus aureus (MRSA). In this study we identified immunodominant structures which were expressed in vivo during sepsis caused by MRSA. Using human sera we compared the immune response of humans with MRSA sepsis with the immune response of normal individuals and asymptomatically colonized individuals. We identified and characterized four staphylococcal specific antigenic structures. One target is a staphylococcal protein of 29 kDa that exhibited 29% identity to secreted protein SceA precursor of Staphylococcus carnosus. The putative function of this protein, which was designated IsaA (immunodominant staphylococcal antigen), is unknown. The second target is an immunodominant protein of 17 kDa that showed no homology to any currently known protein. This immunodominant protein was designated IsaB. The third and fourth antigens are both immunodominant proteins of 10 kDa. One of these proteins showed 100% identity to major cold shock protein CspA of S. aureus and the other protein was identified as the phosphocarrier protein Hpr of S. aureus. The identified immunodominant proteins may serve as potential targets for the development of antibody based therapy against MRSA.

Spermidine Acetyltransferase Is Required To Prevent Spermidine Toxicity at Low Temperatures in Escherichia coli

Polyamines are required for optimal growth in most cells; however, polyamine accumulation leads to inhibition of cellular growth. To reduce intracellular polyamine levels, spermidine is monoacetylated in both prokaryotes and eukaryotes. In Escherichia coli, the speG gene encodes the spermidine acetyltransferase, which transfers the acetyl group to either the N-1 or N-8 position. In addition to polyamine accumulation, stress conditions, such as cold shock, cause an increase in the level of spermidine acetylation, suggesting an adaptive role for reduced polyamine levels under stressful growth conditions. The effect of spermidine accumulation on the growth of E. coli at low temperature was examined using a speG mutant. At 37 degrees C, growth of the speG mutant was normal in the presence of 0. 5 or 1 mM spermidine. However, following a shift to 7 degrees C, the addition of 0.5 or 1 mM spermidine resulted in inhibition of cellular growth or cell lysis, respectively. Furthermore, at 7 degrees C, spermidine accumulation resulted in a decrease in total protein synthesis accompanied by an increase in the synthesis of the major cold shock proteins CspA, CspB, and CspG. However, the addition of 50 mM Mg(2+) restored growth and protein synthesis in the presence of 0.5 mM spermidine. The results indicate that the level of spermidine acetylation increases at low temperature to prevent spermidine toxicity. The data suggest that the excess spermidine replaces the ribosome-bound Mg(2+), resulting in ribosome inactivation at low temperatures.

Human antibody response during sepsis against targets expressed by methicillin resistant Staphylococcus aureus

The identification of target structures is a prerequisite for the development of new treatment options, like antibody based therapy, against methicillin resistant Staphylococcus aureus (MRSA). In this study we identified immunodominant structures which were expressed in vivo during sepsis caused by MRSA. Using human sera we compared the immune response of humans with MRSA sepsis with the immune response of normal individuals and asymptomatically colonized individuals. We identified and characterized four staphylococcal specific antigenic structures. One target is a staphylococcal protein of 29 kDa that exhibited 29% identity to secreted protein SceA precursor of Staphylococcus carnosus. The putative function of this protein, which was designated IsaA (immunodominant staphylococcal antigen), is unknown. The second target is an immunodominant protein of 17 kDa that showed no homology to any currently known protein. This immunodominant protein was designated IsaB. The third and fourth antigens are both immunodominant proteins of 10 kDa. One of these proteins showed 100% identity to major cold shock protein CspA of S. aureus and the other protein was identified as the phosphocarrier protein Hpr of S. aureus. The identified immunodominant proteins may serve as potential targets for the development of antibody based therapy against MRSA.