Induction of DnaK and GroEL in Brucella Ovis under Various Stress Conditions

Brucella ovis is a facultative intracellular pathogen, able to survive and replicate within macrophages. The role of molecular chaperones in Brucella intracellular survival has been previously mentioned, but nothing was published on B. ovis in this respect, as far as we know. In the present work, in order to understand any possible contribution of the molecular chaperones DnaK and GroEL for Brucella ovis stress survival and intracellular persistance, the expression on different culture media under different stress conditions were analysed at the transcriptional and translational levels and compared with that of bacteria grown inside murine J774A.1 cells. The transcription analyses showed different levels of dnaK and groEL induction under various stress conditions (e.g., macrophages survival, acidic pH shock, high temperature, oxidative and osmotic shock) and a significant influence of the media composition. These results were consistent with the level of DnaK and GroEL expression analysed by the traditional proteomic methods. Here we demonstrate the molecular chaperones involvement in macrophage-B. ovis interaction, their expression under stress conditions suggesting the chaperones role in the bacterial resistance to such unfavourable environmental conditions.


Introduction
Brucella ovis is a facultative intracellular pathogen associated with epididymitis and abortion in sheep, being less studied than the zoonotic species of Brucella, although the infection produced by these bacteria represents an important economical problem in many countries.Survival and multiplication in host phagocytic cells are crucial in the pathogenesis of Brucella infections (Young, 2009;von Bargen et al., 2012).
In B. ovis, DnaK and GroEL have been identified as immunogenic proteins (Teixeira-Gomes et al., 1997), but the involvement of these proteins in the resistance of B. ovis cells in macrophages are not mentioned in the literature, as far as we know.
In this study, the chaperone proteins expression of B. ovis grown inside murine J774A.1 cells has been compared with that of bacteria grown in the different cell culture media under various stress conditions, such as acidic pH, high temperature and oxidative stress, which reproduce macrophage intracellular environment.We analyzed also the importance of DnaK and GroEL for B. ovis cells survival in the presence of high concentration of glucose.
A sensitive and powerful technique for quantification of nucleic acids, real time quantitative RT-PCR, was used for the estimation of the transcription of dnaK and groEL genes.In addition, the DnaK and GroEL expression was also analyzed at the translational level by SDS-PAGE and Western blot analysis.

Bacterial Strain and Culture Conditions
B. ovis 202 was obtained from the Bacteriology Department, IDAH (Institute for Diagnosis and Animal Health), Romania.This strain was confirmed as B. ovis by standard biotyping procedures described by Alton et al. (1988).B. ovis cells were grown in an atmosphere containing 5% CO2 for 72 to 89 h at 37°C in three different culture media: tryptic soy broth (TSB) (Oxoid, Cat.No. CM0129B); brain heart infusion (BHI) broth (Oxoid, Cat.No. CM1135B) and Columbia agar base (Oxoid, Cat.No. CM0331B) with 5% sterile defibrinated blood.For all the experiments, in parallel with untreated culture (normal conditions), mentioned above, the stress conditions were induced in the three culture media.For the heat shock response, the temperature was raised to 42°C (Lin et al., 1992).For the reduced pH (5.5), the protocol described by Rafie-Kolpin et al. (1996) was followed.The oxidative stress was induced by supplementing the medium with 50 mM H2O2 (Teixeira-Gomes et al., 2000).The response to osmotic stress was tested with B. ovis cultures in BHI broth by adding 5% glucose; B. ovis grown in TSB in normal conditions was used as positive control.

SDS-PAGE Analysis
Proteins were extracted with BioRad ReadyPrep Protein Extraction kit.Essentially, the wet cells (50 µl containing 50 µg of protein) were mixed with 1 ml of complete 2-D Rehydration/Sample Buffer 1, lysed with Greenbeads (Roche), and centrifuged.The supernatant was mixed with sample buffer and incubated for 5 min at 95ºC.Triplicate of identical protein concentration, as well as Broad Range molecular weight standard (Bio-Rad) were applied on polyacrylamide (PAA) gel.Gels were stained with 0.1% Coomassie Brilliant Blue R-250, destained by a solution containing methanol, acetic acid and water (4:1:5) and photographed dried.

Western Blotting (Immunoblotting)
After electrophoresis, proteins were transferred to a PVDF (polyvinylidene diflouride) membrane using Semi-Dry Transfer Cell (BioRad) according to the instructions recommended by the manufacturer.The mouse anti-GroEL monoclonal antibodies, and the mouse anti-DnaK monoclonal antibodies were purchased from StressGen Biotechnologies Corporation.Detection of stress proteins was done with Immun-Blot Assay kit (BioRad).

RNA Extraction and Reverse Transcription (RT)
RNA was stabilized in RNAprotect Bacteria Reagent (Qiagen).Extraction of total RNA from both the bacterial cultures and from the infection mixtures of macrophages and bacteria was performed with the Qiagen RNeasy Mini Protocol.Complementary DNA (cDNA) was generated from total RNA with M-MLV RT according to the manufacturer's instructions (Promega) using 2,5 µM of each reverse primer and 100 ng total RNA (40 µl final volume).

Controls for Semi-Quantitative and Quantitative RT-PCR
B. ovis 202 grown in TSB pH 7.0 was used as positive control.No-template controls were used for in vitro experiments and RNA of uninfected macrophage was isolated as a negative control.16S rRNA gene (a housekeeping gene) was chosen as internal control for semi-quantitative RT-PCR analysis for normalization of cDNA samples.The dilutions of cDNA from 10° to 10 -3 were analysed with primers for 16S rRNA gene and the cDNA samples were normalized according to quantified of 16S rRNA amplicons.For real time quantitative RT-PCR, the 16S rRNA gene, the expression of which is relatively constant in bacteria, was used as a reference.

Semi-Quantitative PCR
Selected genes were amplified from normalized cDNA samples with specific primers (0.6 µM of each primer).The PCR products were analysed on 1.5% agarose gel and visualized by ethidium bromide staining.

Real-Time Quantitative PCR (qPCR)
Samples were amplified in a 20 µl reaction mixture containing 0.5 µM of each oligonucleotide primer, 0.5 µM fluorescent labelled probe for each gene (dnaK, groEL and 16S rRNA) and 5 µl normalized cDNA.Amplification program (iQ5 Real Time PCR System-BioRad): 3 min at 95°C, then 40 cycles of 30 s at 95°, 30 s at 56°C and 30 s at 72°C.The final extension was performed for 3 min at 72°C.Relative transcriptional level was determined by the method of 2 −∆∆ Ct (Livak and Schmittgen, 2001).

Statistics
Three replicates were used for all experiments.The relative transcription of the genes and the quantification of the proteins were calculated as the mean ± the standard deviation (S.D.)

Macrophage Infection Assay
Macrophage cell infection was assayed as described earlier (Buchmeier and Heffron, 1990).B. ovis 202 cells grown on tryptose agar plates for 89 h were harvested by centrifugation at 13.000 rpm.Then, bacteria were opsonized with polyclonal rabbit anti-Brucella ovis antibodies for 30 min at 37°C and washed once in PBS.The plates were infected with Brucella ovis at a density of 50 bacteria per macrophage and incubated at 37°C in 5% CO2 and a humidified atmosphere for 60 min.The cells were then washed three times with PBS to remove extracellular bacteria.The cells were incubated for 1 h in DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) (Life Technologies, Cat. No. 11320-074) supplemented with 100 µg/ml ampicillin plus 50 µg/ml kanamycin to kill the extracellular still present bacteria.To ensure that macrophage protein synthesis was inhibited, 50 µg/ml cycloheximide was added to uninfected macrophages.At various time points post infection, the macrophages were lysed with 0,2% (vol/vol) ice-cold Triton X-

Results and Discussion
Survival and replication of Brucella in host phagocytes are the key components of their virulence (Enright, 1990;Smith and Ficht, 1990).In order to understand any possible contribution of the molecular chaperones DnaK and GroEL for Brucella ovis stress survival and intracellular persistance, the expression in different culture media under different stress conditions were analysed at the transcriptional and translational levels and compared with that of bacteria grown inside murine J774A.1 cells.A proteomic approach was performed by other authors with B. suis, B. abortus and B. melitensis (Lin and Ficht, 1995;Kohler et al., 1996;Teixeira-Gomes et al., 2000) and the proteins implicated in intracellular survival were identified.In the proteomic analysis of Brucella melitensis, Teixeira Gomes et al. (2000) found 62 protein spots that showed either increased (DnaK, GroEL, AapJ, RRF, Fe Mn SOD, Cu Zn SOD) or decreased (Alpha-ETF, ClpP, bacterioferritin, BvrR, IalB, and pyruvate dehydrogenase E1 component beta subunit) levels depending of the stress.
The transcription level of the dnaK and groEL were determined by the combination of semiquantitative RT-PCR and quantitative real time RT-PCR enabled the sensitive quantification of the gene expression.The selected genes were amplified from normalized cDNA samples (Figure 1).Three replicate were used for each dnaK and groEL real time qRT-PCR experiment and the relative transcription (R) was calculated.The transcription of these two genes was modified under different stress conditions depending on media content ( The results of the real time quantitative PCR of dnaK and groEL genes were consistent with the observation that the induction of DnaK and GroEL depends on the transcription (Cardoso et al., 2010).The semi-quantitative PCR of the genes has been a less sensitive method that showed the different levels of the transcription, being a partial confirmation of the other two methods results (Figure 2).The quantification of the bands intensity for DnaK and GroEL proteins (Figure 3) and the determination of the apparent molecular weight from Western blots were done with UN-SCAN-IT gel™ software.The DnaK and GroEL proteins were identified by the Western blot analysis where the proteins with the same molecular mass as DnaK and GroEL cross-reacted with the antibodies specific for these proteins with the strong reactions.These protein spots were accepted as the DnaK (Figure 4) and GroEL (Figure 5).The results showed an increase of protein level corresponding to DnaK in TSB medium, as a response of B. ovis to the heat (Figure 3A).In addition, the DnaK level was increased in BHI medium with 5% glucose after exposure to the increased temperature (Figure 3B).In contrast, the DnaK expression of B. ovis on solid medium with peptone (Columbia agar) was unchanged (Figure 3C).The increased induction of groEL gene by heat shock was observed regardless of the media composition.The effect of the reduced pH on the chaperone synthesis was estimated by the level of DnaK and GroEL proteins extracted from B. ovis cells grown in the TSB, BHI with 5% glucose and Columbia agar, respectively, at pH 5.5 compared with the same media at pH 7.0 (Figure 3).A major increase in the level of these two molecular chaperones was observed at acid-stressed organisms cultured in TSB (Figure 3A) and BHI with 5% glucose (Figure 3B).The presence of peptone in the solid medium in the absence of glucose (Columbia agar) caused no change in the DnaK expression as comparing with normal pH (7.0).Under the same conditions, the level of GroEL expression was increased (Figure 3C).

Sample
SDS-PAGE profiles and Western blot analysis of proteins synthesized in B. ovis incubated in TSB medium (Figure 3A) and Columbia agar (Figure 3C) under oxidative conditions show no change in the synthesis of DnaK protein.
The high concentration of glucose in medium induced a decrease in DnaK protein synthesis in B. ovis exposed to 50 mM H2O2 (Figure 3B).The GroEL expression under oxidative conditions was influenced by the media composition.The high concentration of glucose in medium induced an increase in the GroEL protein synthesis under oxidative conditions (Figure 3B) and a small increase of this molecular chaperone level was noticed in the absence of glucose in the solid medium with peptone (Figure 3C).In contrast, the level of GroEL protein was decreased in TSB medium with 50 mM H2O2 (Figure 3A).
GroEL protein is up-regulated when B. ovis cultures were grown on media with peptone regardless of glucose under all stress conditions.It seems that too much peptone (relative to glucose) present in the medium increases the pH by the overproduction of ammonium stimulating only the GroEL expression.Under the similar alkaline conditions, the DnaK expression was unchanged, suggesting the response may be shifted by the medium.B. ovis is a fastidious organism with a low growth and metabolic activity (Tsolis et al., 2009;Al Dahouk et al., 2010).Meyer (1969) showed that B. ovis is defective in oxidative metabolism of glucose and other sugars, this finding being confirmed by the recent studies that show the inability of B. ovis to use glucose as a primary carbon source, suggesting a correlation between this metabolic deficiency and the narrowing of the tissue tropism and host range of B. ovis (Tsolis et al., 2009;Al Dahouk et al 2010).However, McAlpin and Slanetz (1928) showed that all species of Brucella except B. abortus are able partly to use the available glucose in the medium, the degree of acid production varied with the different strains (Zobell and Meyer, 1930).In this context, the high levels of dnaK and groEL transcripts in BHI medium with 5% glucose could be explain by the acidification of the medium, probably by the use of glucose in the fermentation process.As a consequence, these two proteins in B. ovis might be involved in the adaptive acid tolerance response.

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In this study, using various media for B. ovis culture under stress conditions we have shown that the B. ovis stress response was significantly influenced by the media composition.dnaK and groEL transcript levels are higher under reduced pH and heat shock in rich medium (BHI broth with 5% glucose) than in minimal medium (TSB); similar data were reported by Lin et al. (1995b) and Foster et al. (1991)  Our results demonstrate that heat, the acid pH and intramacrophagic cell infection induce the expression of DnaK and GroEL in B. ovis, confirming the previously described data on other Brucella species involved in human pathology, as mentioned above.The pH reductions are mainly responsible for the induction of these genes inside phagocytic cells providing that the temperature is not changes.dnaK and groEL genes may play possibly an important role in the resistance against the bactericidal activity of macrophages and against the pathogenicity of B. ovis.
The expression of molecular chaperones genes in bacteria is controlled at transcriptional level by both positive and negative mechanisms.In Escherichia coli, the induction of the majority of up-regulated cytoplasmic heat shock proteins occurs through the sigma factor (σ 32 ) located upstream of heat-shock genes (Chuang et al., 1993).The identification of putative heatshock promotor -35 and -10 consensus sequence for B. melitensis, B. abortus and B. ovis suggest that σ 32 could be responsible for the increased cellular levels of DnaK and GroEL expression in Brucella (Cellier et al., 1992;Lin et al., 1992;Teixeira-Gomes et al., 2000).Delory et al. (2006)  In addition, the negative heat shock regulation appears to be more prominent in bacteria than positive regulation by σ factors.The identification of any putative CIRCE element upstream of the groES operon, but not upstream the dnaKJ operon (Narberhaus, 1999) could explain the differential regulation observed between these two operons in B. ovis.
This study suggests that the molecular chaperones, DnaK and GroEL, are regulated at the transcriptional and translational levels by various stress conditions and that this regulation may help improving the resistance of B. ovis cells to environmental stress, including the macrophage microbicidal mechanisms.Our data represent the first transcription analysis of the molecular chaperones in a non-zoonotic Brucella species, regarding the resistance of B. ovis cells in macrophages and to various environmental conditions.
identified a RpoHlike sigma factor of the RNA polymerase (σ H2 ) with multiple roles in the adaptation of B. melitensis to heat, cold and oxidative stress by the control of dnaKJ and groESL operons.These findings could be the potential explanations for the increased cellular levels of DnaK and GroEL in B. ovis inside macrophages and under various stress conditions.International Journal of Veterinary Medicine: Research & Reports _______________ Luminita Monica Vanghele, Maria Ionescu, Handan Coste and Elena Ganea (2013), International Journal of Veterinary Medicine: Research & Reports, DOI: 10.5171/2013.729541

Table 1
); the results of Western blot analysis under all the stress conditions used in vitro are a confirmation of this.Real Time RT-PCR showed that DnaK and GroEL were induced during macrophage cell infection (Table 1).International Journal of Veterinary Medicine: Research & Reports _______________ Luminita Monica Vanghele, Maria Ionescu, Handan Coste and Elena Ganea (2013), International Journal of Veterinary Medicine: Research & Reports, DOI: 10.5171/2013.729541