Investigation of Rifampicin Resistance Mechanisms in Brucella

Article pubs.acs.org/jpr

Investigation of Rifampicin Resistance Mechanisms in Brucella abortus Using MS-Driven Comparative Proteomics Vassilios Sandalakis,†,‡ Anna Psaroulaki,†,‡ Pieter-Jan De Bock,§,∥ Athanasia Christidou,† Kris Gevaert,§,∥ Georgios Tsiotis,*,⊥ and Yiannis Tselentis†,‡ †

Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Medical School, University of Crete, GR-71110 Heraklion, Greece ‡ Regional Laboratory of Public Health-71110, Heraklion, Crete, Greece § Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium ∥ Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium ⊥ Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes, Greece S Supporting Information *

ABSTRACT: Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12 836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level. KEYWORDS: Brucella abortus, comparative, proteomics, rifampicin, in vitro, mutant, resistance, MIC

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INTRODUCTION Therapy of human brucellosis caused by the pathogenic to humans and mammals, Gram-negative coccobacilli Brucella spp. is mainly based on a combination of antibiotics. Monotherapy (doxycycline, azithromycin, aminoglycosides, rifampicin, or fluoroquinolones) has produced unsatisfying results.1 Currently, the preferred regiment for brucellosis is rifampicin (RIF) or streptomycin (STR) along with doxycycline (DOX) (100 mg DOX twice/day with 900 mg RIF for 45 days, or with STR 1 g/day for 14 days). In cases of pregnancy and for children under the age of eight, DOX and STR are avoided; thus, RIF is the alternative choice plus trimethoprim−sulfamethoxazole (SMZ/TMP).2 RIF’s mode of action relies on the inhibition of the DNAdependent RNA polymerase. The antibiotic acts selectively on bacterial RNA polymerase by binding to its β-subunit preventing transcription and consequently protein synthesis.3 Even though RIF can block mRNA transcription, it cannot stop elongation of mRNA once binding to the DNA template has occurred.4 © 2012 American Chemical Society

Resistance to RIF can develop easily, in a single-step fashion, and cross-resistance has been shown with other rifamycins.5 Mutations in the rpoB gene, coding for DNA-dependent RNA polymerase, have been shown to contribute to RIF resistance.6−8 Thus, bacteria exhibiting resistance to RIF produce RNA polymerases with slightly different β-subunits that disfavor binding of RIF. The types of mutations that confer resistance are not related to the “pressure” of RIF concentration to the bacteria.9 Until today, most studies in Brucella melitensis, Brucella abortus RB51, and Brucella suis are on the genetic bases of the rpoB gene identifying several positions that significantly influence rifampicin resistance (154, 526, 536, 539, 541, and 574 based on B. melitensis 16 M rpoB numbering [AE009516]).8,10 A different approach was performed with the bepC gene (TolC protein homologue) of B. suis which was cloned and shown to be involved in the efflux of several compounds and antibiotics.11 Isolation of RIF resistant strains has been reported12 and the first report was by De Rautlin in Received: November 10, 2011 Published: February 24, 2012 2374

dx.doi.org/10.1021/pr201122w | J. Proteome Res. 2012, 11, 2374−2385

Journal of Proteome Research

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1986,13 who isolated a B. melitensis rifampicin resistant strain from a relapsed patient that had been treated for 6 weeks with RIF−DOX. Even though there have been several proteomic based analyses on Brucella sp., in an attempt to analyze the bacterial metabolic procedures and proteomic composition,14−23 none has been performed for elucidating rifampicin resistance. The first attempt to understand the mechanisms of resistance to RIF on the proteome level has only been recently performed on Neisseria meningitidis.24 Most of the proteomic analyses on Brucella were performed using 2D-PAGE. As shown in previous studies, whole proteome investigation using gelbased methods is relatively laborious as multiple overlapping IPG strips with narrow pH ranges are required, for example, 3− 5, 4.5−5.5, 5−6, 4−7, 6−9, and 6−11.16 More efficient gel-free proteomics methods have also been applied on B. abortus.21,25,26 Such methods have been used for the comparison of global proteomes of intracellularly and extracellularly grown B. abortus from Lamontagne et al. in 2009.21 The same approach has been also performed with their subproteomes (cytosolic and membrane fractions).25 The objective of our research reported here was to further explore the rifampicin resistance mechanisms from a proteome viewpoint, and to investigate the effect to other groups of antibiotics used in the therapy of Brucellosis. In our study, the rpoB gene of an in vitro developed rifampicin resistant strain of B. melitensis biovar Abortus 2308 (BabRIFres) was sequenced revealing a previously characterized mutation, V154F, described in B. suis and B. melitensis RBM14.8,10 The resistant strain was tested against other groups of antibiotics and its global proteome was analyzed and compared to that of a RIF susceptible reference strain (BabRIFsus) using a gel-free, shotgun comparative proteomics approach.

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MIC Determination

Determination of minimum inhibitory concentration (MIC) of RIF (Rifadine Lyophilizee, Sanofi-Aventis, France) was performed with the standard agar dilution method, on Müeller-Hinton agar with 5% sheep blood, that was prepared in doubling concentrations of RIF starting from 512 to 0.0625 mg/L. Inoculums were prepared as above. Cultures were allowed to grow at 35 °C and were interpreted 48 h postinoculation. Rifampicin MIC was also determined by the E-test (Biomerieux, France). The method was performed on Müeller-Hinton agar supplemented with 5% sheep blood. Potential cross-resistance in the BabRIFres strain was evaluated using just the E-test method for the following antibiotics; Norfloxacin (NX), Levofloxacin (LE), Moxifloxacin (MX), Ciprofloxacin (CI), Ofloxacin (OF), Tetracycline (TC), Rifampicin (RI), Streptomycin (SM), Gentamycin (GM), Trimethoprim/Sulfamethoxazole (TS), and Tigecycline (TGC). Escherichia coli ATCC 25922 was used as control. DNA Extraction and PCR Sequencing of the rpoB Gene

Bacterial DNA was extracted using the QIAampTissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. PCR amplifications were performed using MyCycler (Bio-Rad) DNA thermal cycler. The primers and cycling conditions used to amplify the rpoB gene as well as the sequencing primers are described by Marianelli et al.8 Positive PCR products were purified using the QIAquick PCR purification spin kit (Qiagen, Germany) and were directly sequenced on an Applied Biosystems 3500 sequencer (DNABiolab, Heraklion, Greece). Resulting sequences were processed using the Seqscanner v1.0 (Applied Biosystems) and DNAStar Lasergene Ver. 7.1 software package (Madison, WI). The process was performed for both BabRIFres and BabRIFsus. Harvesting of Bacteria

MATERIALS AND METHODS

Upon reaching the maximum antibiotic resistance for RIF, BabRIFres was cultured on Müeller-Hinton agar supplemented with 5% sheep blood with 120 mg/L RIF. The bacteria were allowed to grow under the above-described conditions. After 48 h, in order to harvest the bacteria, dH2O was poured in the cultures and they were gently shaken until the bacterial colonies were detached from the surface of the agar. Samples were centrifuged for 30 min at 6000g and the pellets were resuspended in 3.5 mL of dH20. Bacterial suspensions were then heated for 1 h at 65 °C and then three volumes of ice-cold acetone were added to each sample followed by vortexing. The suspensions were incubated for 18 h at 4 °C and centrifuged for 30 min at 6000g after which excess of acetone was removed and samples were freeze-dried. As a safety precaution measure, part of the final pellet was used to inoculate TSA and 5% sheepblood Mueller-Hinton agar. Cultures were observed for sterility for 7 days. Sterile samples were then used for proteome analysis.

Bacterial Culture and in Vitro Development of Rifampicin Resistance

The RIF resistant B. melitensis biovar Abortus strain 2308 (BabRIFres) was derived from the susceptible reference strain (MIC: 0.75 mg/L). The reference strain was cultured on Müeller-Hinton agar supplemented with 5% sheep blood, agar plates (Biomerieux, France). Parallel culture of the reference strain was performed with the addition of a 30 μg RIF antibiotic disc (Bio-Rad). Culture inoculums were prepared by suspending bacteria in 0.9% NaCl until turbidity was equivalent to 0.5 on the McFarland scale. Both cultures were incubated at 35 °C in a 5% CO2 incubator. Growth and inhibition zone diameter were observed and recorded 48 h postinoculation. Individual colonies growing closer to the disc were harvested and subcultured. This was repeated until there was no inhibition zone. Upon obtaining resistance, the BabRIFres strain was cultured five times sequentially on Müeller-Hinton agar sheep blood agar, in the absence of rifampicin, to reassure the insistence of mutations. All manipulations involving viable bacteria were performed in a Biosafety Level III laboratory as indicated by international safety protocols for potential bioterrorism agents. As an additional safety measure, upon completion of the experiments, all antibiotic resistant strains were inactivated by suspension in formaldehyde 8% for 48 h followed by autoclaving. Viability of the inactivated bacteria was tested by inoculation on 5% sheep blood Müeller-Hinton agar plates prior to discarding them.

Proteome Analysis

The deactivated bacterial pellets were used to perform a shotgun proteomics analysis for the comparison of the BabRIFres and BabRIFsus proteomes. Prior to digestion with endoproteinase Lys-C, the proteomes were reduced with TCEP and alkylated with iodoacetamide.27 After digestion, the peptides were labeled by N-propionylation.28 Here, the BabRIFres proteome digest was labeled with 12C3-propionate and the BabRIFsus proteome digest with 13C3-propionate. Each 2375



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peptide thus had a label on its N-terminus and on its Cterminal lysine. So, there is a difference of 6 Da between light and heavy peptides. Equal amounts of both samples were mixed and analyzed by LC−MS/MS on an Ultimate 3000 HPLC system (Dionex) in-line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Electron). Instrument settings for LC−MS/MS analysis and generation of MS/MS peak lists were as described.29 MS/MS peak lists were searched with Mascot using the Mascot Daemon interface (version 2.3.0). The searches were performed in the B. abortus (strain 2308) database, downloaded from Uniprot on January 4, 2011 which contained 3022 entries from a total of 3494 detected ORFs (open reading frames). Lys-C/P was set as the used protease with one missed cleavage allowed, and the mass tolerance on the precursor ion was set to ±10 ppm and on fragment ions to ±0.5 Da. S-Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionine as a variable modification. In addition, Mascot’s C13 setting was set to 1. The light and heavy labels were defined in Mascot’s quantitation method. Peptide quantifications were carried out using the Mascot Distiller Quantitation Toolbox (version 2.2.1). The quantification method details were as follows: constrain search, yes; protein ratio type, average; report detail, yes; minimum peptides, 1; protocol, precursor; allow mass time match, yes; allow elution shift, no; all charge states, yes. Ratios for identified proteins were calculated by comparing the XIC peak areas of all matched light peptides with those of the heavy peptides, and the results were verified by visual inspection of MS spectra with the in-house developed Rover tool.30 All identified MS/MS spectra are publicly available in the PRIDE database (http:www.ebi.ac.uk/pride) under the experiment number 19733. Considering the low Mascot identification thresholds when using highly accurate peptide masses, peptides shorter than eight amino acids, with Mascot ion scores that were less than 10 units higher than the corresponding spectrum identity score, as well as falsely calculate peptide ratios (as indicated by the quantification module), were omitted from the results.

Table 1. MIC Levels for 10 Additional Antibiotics Used for Assessing Cross-Resistance of the BabRIFres Strain MIC (mg/L) E-test (Biomerieux abrev.) Rifampicin (RI) Streptomycin (SM) Gentamycin (GM) Tetracycline (TC) Tigecycline (TGC) Trimethoprim/ Sulfamethoxazole (TS) Norfloxacin (NX) Ciprofloxacin (CI) Ofloxacin (OF) Levofloxacin (LE) Moxifloxacin (MX) a

antibiotic group

BabRIFres

0.75 1.5 0.25 0.125 0.75 0.125/2.375

>32 Resa 1.5 0.38 0.125 0.75 1/19

2nd G+. Fluoroquinolones 2nd G+. Fluoroquinolones 2nd G+. Fluoroquinolones 3rd G+. Fluoroquinolones 4th G+. Fluoroquinolones

0.75

0.75

0.25

0.25

0.5

0.5

0.5

0.5

0.5

0.5

The MIC observed by the agar dilution method was 128 mg/L. Generation.

+

in Müeller-Hinton agar with 120 mg/L RIF. The susceptible reference strain was also cultivated in the same culture medium but in the absence of antibiotic. Resistance against RIF is known to develop quickly and in a one-step fashion.9,31 Still, it was of particular note that resistant strains were obtained in the first subculture as there were already colonies present within the initial inhibition zone and even next to the antibiotic disc. The mutants closest to the disc were subcultured, and 48 h postinoculation, full resistance was observed by absence of an inhibition zone. For the sequence analysis of the rpoB gene, DNA was extracted from both resistant and susceptible (reference strain) bacteria. Polymerase chain reaction was performed for both DNA extracts and produced a single band of 4134 bp in size. The full-length of the amplicons was sequenced and the products were compared to the NCBI published rpoB gene sequence (BAB1_1264) of the B. melitensis biovar Abortus 2308 chromosome I (AM040264). The reference strain, BabRIFsus, contained no mutations and the sequence was 100% identical to the published one.32 The BabRIFres rpoB gene sequence contained a single mutation, 460G>T, which corresponds to an amino acid alteration, V154F. The BabRIFsus and BabRIFres strains were used to perform the agar dilution MIC method and E-tests for RIF. For BabRIFres, the recorded MIC, based on the agar-dilution method, was 128 mg/L and, using the E-test, there was bacterial growth through the whole length of the strip, since the RIF concentration reaches a maximum of only 32 mg/L. We then tested cross-resistance to other antibiotics used in brucellosis treatment schemes and the results are summarized in Table 1. The RIF resistant strain altered only the MIC of SMZ/TMP. The MICs of the rest of the antibiotics remained unaffected (Table 1).

Bioinformatics

Analysis of the amino acid sequences of all identified proteins was carried out using several Web-based software tools, freely accessible from the “ExPASy Proteomics Server” of the Swiss Institute of Bioinformatics (SIB) (http://au.expasy.org/). Cellular role categories were assigned based on the Comprehensive Microbial Resource online database (http:// cmr.jcvi.org). Cellular localization of the resulting proteins was assigned based on the PSORTb v3.0 tool (http://psort.org/ psortb/index.html). Possible interactions between the identified proteins were tested using the STRING software (Search Tool for the Retrieval of Interacting Genes/Proteins) tool (http://string-db.org/). The prediction parameters used in STRING were neighborhood, gene fusion, co-occurrence, coexpression, experiments, databases, and text mining with medium confidence score (0.400).

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BabRIFsusc

Rifamycins Aminoglycosides Aminoglycosides Tetracyclines Glycylcyclines Benzylpyrimidines/ Sulfonamides

RESULTS

RIF Resistance and Cross-Resistance

The reference strain in our study presented an MIC of RIF at 0.75 mg/L. This susceptible strain was turned into RIF-resistant in vitro. Resistance occurred after the first subculture in RIFcontaining culture medium. The resistant strain reached an MIC of 128 mg/L and was then cultivated in 5% sheep blood

Differentially Regulated Proteins and Their Cellular Function

Here, we compared whole cell lysate proteomes of the RIF resistant strain to the susceptible strain. A total of 6753 2376



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Figure 1. Pie chart of the 456 proteins identified, categorized according to their cellular role categories.

receptor (Q2YKP4), which is involved in transport and binding functions (Table 2). Any direct correlation between these differentially expressed proteins (protein−protein interactions) was tested and visualized using the STRING software (Figure 5).

peptides were identified using 12 836 MS/MS spectra. As a result, 456 proteins were detected in both strains and these proteins were divided into distinct groups according to their cellular role (Supporting Information Table 1, Figure 1). The limits of the protein ratio interval out of which a protein was considered to be statistically significantly (p < 0.05) regulated were 0.06 and 13.7. Here, proteins with a protein ratio above 13.7 were considered to be up-regulated in the BabRIFres strain and proteins with protein ratio below 0.06 were considered to be up-regulated in the BabRIFsus strain. This wide interval is a result of the large differences between the two proteomes, a condition seen in another research investigating antibiotic resistance.33 From the total of 456 proteins identified, 38 were up-regulated in BabRifres and one was up-regulated in BabRifsus (Table 2). These differentially expressed proteins were further divided into groups according to the cellular role of the individual proteins (Figure 2). The majority (13) of the 38 overexpressed proteins in the BabRIFres strain are involved in energy metabolism, while the rest function in amino acid biosynthesis, transport and binding, central metabolic processes, nucleotide and nucleoside metabolism, cell envelope processes, fatty acid and phospholipid metabolism, detoxification, protein fate, protein processing, protein synthesis, and three up-regulated proteins were of unknown function (Table 2). Concerning the down-regulated proteins in the resistant strain, we identified only one at the end of the filtering process, the solute-binding protein/glutamate

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DISCUSSION Rifampicin is a very effective and broad-spectrum antibiotic against bacterial pathogens. Depending on its concentration, this antibiotic can have bacteriostatic or bactericidal effects.34 Rifampicin can have bactericidal activity against slow and irregularly growing Mycobacterium tuberculosis organisms and it also plays a significant role in the treatment of methicillinresistant Staphylococcus aureus (MRSA),35 Neisseria meningitidis,36 N. gonorrheae,37 Haemophilus inf luenza,38 Listeria species,39 Legionella pneumophila,40 and Brucella species.1 Mutations on the rpoB gene often occur under rifampicin stress. Such mutations have an affect not only on rifampicin resistance levels, but also on gene expression. In E. coli, certain Rif mutants, even in the absence of the stringent response, present a “stringent”-like behavior.41 In a similar way, Streptomyces coelicolor A33 and Streptomyces lividans rpoB mutants can suppress their antibiotic production deficiency.42 It is thus clear that rpoB mutations can have different effects on different organisms. In these former studies, rifampicin resistance could be attributed to the rpoB mutations only. Consequently, one could argue that all the differentially 2377


2378

apt tig hslV tsf nhaA

Adenine phosphoribosyltransferase

Trigger factor ATP-dependent protease subunit HslV Elongation factor Ts Na(+)/H(+) antiporter nhaA

Protein kinase:General substrate transporter:Sugar transporter superfamily:Major facilitator superfamily (MFS) PTS system fructose subfamily IIA component Periplasmic binding protein ATP/GTP-binding site motif A (P-loop):Fumarylacetoacetate (FAA) hydrolase

pyrC-1

BAB2_0348 BAB1_2096 BAB2_0483 BAB1_0247

Q2YQY0 Q2YL07 Q2YP89

BAB1_0917 BAB1_2081 BAB1_1183 BAB1_0425

BAB1_1556

BAB1_0688

BAB1_1813 BAB1_0869 BAB1_2185

BAB2_0305 BAB1_2173 BAB1_0242 BAB2_0337 BAB1_1155 BAB2_0928 BAB1_1150

BAB2_0848 BAB2_0303 BAB1_0204 BAB1_1149 BAB1_1741

BAB2_0289 BAB1_0666 BAB1_1023 BAB2_1012 BAB2_0572 BAB1_0191 BAB2_0457 BAB1_1156 BAB1_1279 BAB2_0943

BAB1_1942

ordered locus name

Q2YL56

Q2YNL3 Q2YQZ3 Q2YRP5 Q2YMB3

Q2YM57

Q2YN31

Q2YLI1 Q2YNI8 Q2YQP7

tal rpe

rocF eno nosZ aceF

Q2YIL6 Q2YQQ9 Q2YP94 Q2YIG5 Q2YPV0 Q2YJW2 Q2YPV5

Q2YK32 Q2YIL8 Q2YP66 Q2YPV6 Q2YRE0

Q2YLB2 Q2YMY1 Q2YQ67 Q2YJN7 Q2YKS6 Q2YP48 Q2YL33 Q2YPU9 Q2YQG1 Q2YJU8

Q2YLU4

protein accession number

hutH fabB

lpdA-1 gap

katA hutU

folD kdsA

dapA glnA dapB

argJ

gene name

Dihydroorotase

Pyruvate decarboxylase Dihydrodipicolinate synthetase:Dihydrodipicolinate synthase subfamily Glutamine synthetase Dihydrodipicolinate reductase Aminotransferase, class IV Aminotransferase class-III:Maltose binding protein Bifunctional protein FolD 2-dehydro-3-deoxyphosphooctonate aldolase Bacterial transferase hexapeptide repeat Multicopper oxidase, type 1:Copper-containing nitrite reductase:Twin-arginine translocation pathway signal Catalase Urocanate hydratase Zinc-containing alcohol dehydrogenase superfamily:Zinc-containing alcohol dehydrogenase Dihydrolipoyl dehydrogenase Glyceraldehyde 3-phosphate dehydrogenase:TrkA potassium uptake protein: Glyceraldehyde-3-phosphate dehydrogenase, type I Histidine ammonia-lyase Beta-ketoacyl synthase Mandelate racemase/muconate lactonizing enzyme Arginase:Arginase/agmatinase/formiminoglutamase Enolase Twin-arginine translocation pathway signal Biotin/lipoyl attachment:Antifreeze protein, type I:Catalytic domain of components of various dehydrogenase complexes:2-oxo a Probable transaldolase Ribulose-phosphate 3-epimerase Enoyl-CoA hydratase/isomerase

Arginine biosynthesis protein ArgJ

protein name

Table 2. List of Differentially Expressed Proteinsa

14395.5 37504.9 29895

67375.6

52937.4 19838.8 31490.7 78587.4

19613.8

48838.4

23359.1 23853.3 28070.3

53253.6 43310.8 40774.8 33182 45261 71071.1 46748.7

56446.3 61228.5 36246.5 51726.9 36237.2

60717.2 31612.3 52159.3 27604.5 38747.1 49593.7 31231.2 29532 18592.3 40515

43244.4

MW (t)

5.01 5.85 5.08

8.8

4.85 6.15 5.02 9.17

6.17

6.24

5.47 5.34 5.48

6.1 5.21 5.67 5.7 5.03 5.49 5.89

6.52 6.04 5.76 6.87 6

6.14 6.26 5.43 6.36 7.03 5.99 6.18 6.12 6.05 5.64

6.27

pI (t)

Up Up Up

Up

Up Up Up Up

Up

Up

Up Up Up

Up Up Up Up Up Up Up

Up Up Up Up Up

Up Up Up Up Up Up Up Up Up Up

Up

regulationb Psort localization

Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Membrane Cytoplasmic Membrane Cytoplasmic Periplasmic Cytoplasmic

Cytoplasmic

Cytoplasmic

Cytoplasmic Cytoplasmic Cytoplasmic

Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Periplasmic Cytoplasmic

Periplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic

Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic Periplasmic

Cytoplasmic

cellular role category

metabolism metabolism metabolism metabolism metabolism metabolism metabolism

transport and binding transport and binding unknown function

transport and binding

energy metabolism energy metabolism fatty acid and phospholipid metabolism nucleotide and nucleoside metabolism nucleotide and nucleoside metabolism protein processing protein fate protein synthesis transport and binding

energy energy energy energy energy energy energy

Detoxification energy metabolism energy metabolism energy metabolism energy metabolism

fatty acid and phospholipid metabolism amino acid biosynthesis amino acid biosynthesis amino acid biosynthesis amino acid biosynthesis amino acid biosynthesis central metabolism processes central metabolism processes cell envelope processes cell envelope processes central metabolism processes

Journal of Proteome Research Article


Gene name, protein accession number, and the Ordered Locus Name were retrieved from the Uniprot database (http://www.uniprot.org/). Calculation of the molecular weight (MW) and isoelectric point (pI) was performed in http://au.expasy.org/tools/protparam.html and http://au.expasy.org/tools/pi_tool.html, respectively. bRegulation is characterized as Up or Down only in the BabRIFres in comparison to BabRIFsus.

Article

Figure 2. Pie chart classifying the differentially expressed proteins (upregulated and down-regulated proteins in BabRIFres) according to their cellular role.

expressed proteins picked in our and other studies are just a random result caused by the inability of the RNA polymerase to function properly, and by luck these proteins assist the cell to survive antibiotic stress. This statement is however weakened by a study performed in M. tuberculosis, where it was shown that 5% of rifampicin resistant strains carry no mutations on the rpoB gene.43 The vaccine strain RB51 of B. abortus 2308 is known for its high levels of rifampicin resistance. This strain was not chosen for analysis in the present study as it was derived by many successive cultures on medium containing high concentrations of rifampicin and penicillin.44 We preferred the use of a B. abortus 2308 strain that acquired resistance under minimal stress of rifampicin only, as we considered this to be closer to an in vivo condition where the concentration of the antibiotic within cells is low as it depends on the cells’ uptake and cytotoxicity capabilities. Investigation of the V154F Mutation

The genetic bases of the rpoB gene involved in RIF resistance have already been studied and six mutations were described that are considered responsible.8,10 The type of mutation detected herein, V154F, has been described in B. suis and B. melitensis RBM14 before, while H536Y was considered as the predominant mutation in B. abortus.8,10 The absence of other described mutations could be due to the minimum stress that was exerted on the bacteria as it was not intended to create high-level of resistance but a strain with resistance above the CLSI (Clinical and Laboratory Standards Institute) breakpoint (2 mg/L). Nevertheless, this single mutation contributes to a 170-fold increase in the MIC of RIF (128 mg/L), which is well above the CLSI breakpoint. Cross-Resistance Testing

Cross-resistance testing provided information of both clinical and basic research interest. As already mentioned, the suggested first line antibiotic regimens include the combination of DOX with RIF or STR. Thus, it is encouraging that RIF resistance leaves the rest of the first line antibiotics susceptibilities unaffected. Additionally, the susceptibility levels of gentamycin, fluoroquionolones, occasionally used in treatment,45,46 and tigecycline, recently introduced to the brucellosis treatment47 did not change.

a

cellular role category Psort localization

Up Up Down 5.58 5.22 4.78 BAB2_0644 BAB1_0837 BAB2_0612 Q2YKL5 Q2YNE8 Q2YKP4

25124.3 61272.1 27218.8

regulationb ordered locus name

UPF0173 metal-dependent hydrolase BAB2_0644 Beta-lactamase-like Solute-binding protein/glutamate receptor

Table 2. continued

protein name

gene name

protein accession number

MW (t)

pI (t)

Undefined Cytoplasmic Periplasmic

unknown function unknown function transport and binding


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Figure 3. Distribution of the log2 peptide ratio denoting the difference of the compared proteomic profiles. The x-axis indicates the number of peptide identifications (6753) and the y-axis represents the log2 of the peptide ratio.

that are mistranslation products.52 Even though these proteins are associated with aminoglycoside resistance, the BabRIFres strain did not exhibit increased MIC levels against streptomycin or gentamycin, suggesting a possible alternative mechanism of action.

The 8-fold decrease of susceptibility against SMZ/TMP is a quite disturbing finding as this second line antibiotic is used for cases of brucellosis in children under the age of eight and pregnant women.48 Some research groups also suggest RIF and cotrimoxazole for these sensitive groups.49 Resistance against SMZ and TMP involves mutations on the target genes and/or plasmid acquisition.50 Since in the current research the BabRIFres strain was not stressed with these antibiotics, it could be assumed that mutations on the target genes are unlikely to have occurred. Interestingly, we identified a protein involved in folate biosynthesis, FolD (Q2YL33), which might be linked to the 8-fold increase in the SMZ/TMP MIC levels. The mode of action of sulfamethoxazole is to act as a falsesubstrate inhibitor of dihydropteroate synthetase and subsequently inhibit the production of dihydropteroic acid which is necessary for the folate biosynthesis. Trimethoprim is blocking the action of dihydrofolate reductase, thus, inhibiting synthesis of tetrahydrofolic acid.51 The detection of the up-regulated FolD protein and the co-occurrence of the increased SXT MIC could be connected as they are all related to folate biosynthesis. Therefore, it becomes obvious that in cases of brucellosis, by a RIF resistant strain, the use of SMZ/TMP should be questioned before administration. Still, further research is needed as this is only an in vitro model and more strains of the genus need to be tested. The effects of rifampicin on the process of transcription and the production of hydroxyl radicals could be the cause for misfunctional proteins. The up-regulated hslV protein is a protease subunit of the HslU/HslV proteasome-like degradation complex. It has already been associated to the resistance against aminoglycosides.52 It is assumed that, along with other proteases, this protease provides protection through the detection and elimination of membrane-disruptive proteins

Comparative Proteomic Analysis

Besides mutations on the rpoB gene, several other mechanisms have been described in bacteria for RIF resistance. Examples are integron-mediated rifampicin resistance in Pseudomonas aeruginosa, RIF efflux pumps in Pseudomonas f luorescence, RIF ribosylation in Mycobacterium smegmatis, glycosylation in Nocardia brasiliensis, phosphorylation in N. otitidiscavarium, and decomposition in Bacillus species.53 Bacterial adaptation and growth to antibiotic-containing environment is an ability that requires the adjustment of several metabolic processes, which in turn, result in changes of the bacterial physiology.33,54 A characteristic example are the metabolic alterations induced in a RIF resistant Bacillus subtilis strain, where additional metabolic capabilities were activated by the RIF-induced rpoB gene mutations.55 The BabRIFres strain presented a quite different proteomic profile compared to the susceptible strain, as evident from the wide ratio distribution (Figure 3). This restricted detection of proteins involved in RIF-resistance which, although differentially regulated, were excluded at 95% confidence settings. To include possibly missed, but interesting proteins, the confidence settings should be lowered. Unfortunately, this would mean that the risk of considering a protein unrelated to RIF-resistance as significant would increase. Therefore, we preferred to “miss” some proteins rather than making wrong assumptions. Equivalent conditions have also been observed in proteomic studies on antibiotic resistant strains of Coxiella burnetii56(I. Vranakis, 2380



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analysis of resistance mechanisms emerging through various metabolic pathways. The correlation between the proteins was also depicted by the hypothetical protein interaction network constructed by STRING, where 24 of the 39 differentially expressed proteins in the BabRIFres were associated with each other (Figure 5). A vast majority of the up-regulated proteins are involved in metabolic processes (TCA cycle, glycolysis/gluconeogenesis, fatty acid synthesis and metabolism, arginine biosynthesis, tyrosine catabolism, detoxification processes, salvaging purines/ pyrimidines. etc.). A crossroad molecule in all cells is pyruvate, as it can be involved in many metabolic processes. Proteins of the pentose phosphate pathway, ribulose-phosphate 3-epimerase, a transaldolase, and the GAPDH are forcing the production of fructose 6-phosphate. This product enters the third step of the glycolytic pathway, which, enforced by the up-regulated phosphopyruvate dehydratase, leads to the production of pyruvate that would be directed to the TCA cycle in the form of acetyl-CoA. Proteins catalyzing this final reaction were also overexpressed (dihydrolipoyl dehydrogenase and biotin/ lipoyl attachment of dehydrogenase complex). Acetyl coenzyme A is also important for fatty acid metabolism and arginine biosynthesis, procedures in which some of the up-regulated proteins belong (β-ketoacyl-ACP synthase I, bifunctional protein ArgJ, Enoyl-coA hydratase). These findings agree with other published proteomics-based analyses for rifampicin resistance.24

personal communication). Such dissimilarities in the proteomes (resistant and susceptible) come in support of the above hypothesis by Martinez et al.,33 and in this research, it becomes apparent by the role of the differentially expressed proteins detected. In compliance to the above, most of the differentially regulated proteins are cytosolic (Figure 4) directing toward the

Figure 4. Grouping of the differentially expressed proteins as determined by their predicted localization using PSORTb v3.0.

Figure 5. Hypothetical interaction network between the differentially expressed proteins. 2381



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formation.62 This process is also mediated by the TCA cycle and temporary depletion of NADH. Brucella abortus produces two siderophores (2,3-dihydrobenzoic acid and brucebactin) whose role could also be protective against the formation of the hydroxyl radicals,63−65 thereby contributing to the survival of the cell. In vivo, rifampicin stimulates the zymosan-induced production of H2O2.66 In vitro, it is unknown if there is an increase the production of H2O2 as a result of the antibiotic stress. Such a condition can only be hypothesized by the up-regulation of the periplasmic catalase (Q2YK32). This protein is involved in detoxification processes, and based on previous data, it has a complementary role in protecting the organism from H2O2 exposure.67 Consistency of the outer cell barriers (cell wall and cell membrane) is of a great importance for cell survival. This is the main idea behind the up-regulation of dihydrodipicolinate synthetase (Q2YMY1) and dihydrodipicolinate reductase (Q2YJN7) which are involved in the synthesis of diaminopimelate, an essential component of the cell wall peptidoglycan in both Gram-positive and Gram-negative bacteria.68 Further, the overexpressed 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase (Q2YPU9) is a key enzyme in the lipopolysaccharide biosynthesis of Gram-negative bacteria.69

The differentially expressed proteins did not point directly to mechanisms of resistance related to drug/multidrug efflux pumps or transporter families like MFS, resistance nodulation division (RND), drug-metabolite transporters (MDR, SMR), and multidrug and toxic compound exporters (MATE). Nevertheless, there are data suggesting their possible involvement and contribution to antibiotic resistance. Interestingly, overexpression of a Na + /H + antiporter, nhaA (Q2YMB3), was detected in BabRIFres. Antiporters of this type are used to establish a sodium-motive force (SMF).57 The SMF and the proton motive force (PMF) are providing the necessary functioning energy to drug/multidrug efflux pumps, MFS, RND, MDR, SMR, or MATE.57 A possible hypothesis here could be that overexpression of a secretion system is not needed at this point, as long as the powering force of these systems, the Na+/H+ antiporter, is able to provide sufficient energy as to render excretion systems more efficient under antibiotic stress. In support of this hypothesis is the underexpressed protein Q2YKP4, a solute-binding protein/ glutamate receptor. Glutamate receptors are ligand-gated ion channels (LGICs) and build up a major class of ion channels. These proteins are nonselectively permeable to Na+, K+, and Ca+ ions.58 It seems that down-regulating this protein reinforces the antiporters’ attempt to establish the sodium motive force at higher rates. Under this conception, overexpression of the energy providing mechanisms seems convenient. This finding could also be associated with Q2YL56. The latter is a general substrate transporter belonging to the sugar transporter superfamily. These transporters are included in the Major Facilitator Superfamily (MFS). This protein kinase was analyzed using the TMHMM Server v. 2.0 web based tool, which predicted that the protein contains 11 transmembrane helices. Transporters involved in antibiotic transportation typically possess 12−14 transmembrane helices.59 Those proteins show a great sequence similarity to sugar transporters that could be considered to also be involved in similar processes.59 It is apparent that there is lack of direct evidence proving the relation of this transporter with antibiotic resistance and the overall scenario described. Nevertheless, the differentially expressed proteins coupled with the increased expression of the Na+/H+ antiporter could suggest a correlation between them and an alternative path to antibiotic or toxic substances transportation. Another protein that was up-regulated was Q2YL07. This is a periplasmic binding protein with iron ion transmembrane transporter activity. Iron acquisition systems retrieve iron which is essential for bacterial growth. It has been shown that the iron transfer system is capable of transferring antibiotics containing iron, like sideromycins. An exception to this was a semisynthetic rifamycin derivative, rifamycin CGP 4832, which, although it shared no similarity to sideromycins or ferrichrome, could be transported inside the cell.60,61 This is not the case though for rifamycin that could not enter through this route. Rifampicin is also a semisynthetic rifamycin, but we assume that it cannot pass either through that transportation pathway as, in such case, the most possible outcome would be the decrease and not an increase in the MIC levels. Thus, we believe that upregulation of this protein is mostly related to iron acquisition. Perhaps this increased iron uptake is related with the Fenton reactions. In these reactions, Fe2+ reacts with hydrogen peroxide forming hydroxyl radicals. In a recent study, it was suggested that bactericidal drugs utilize the iron from iron− sulfur clusters and promote Fenton-mediated hydroxyl radical

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CONCLUSION

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ASSOCIATED CONTENT

In this study, we attempted to approach bacterial resistance from multiple angles, proteomic, genomic, and microbiological. The aim was to investigate the proteomic profile differences between a susceptible and a RIF-resistant strain of Brucella. This research provides insight in the metabolic processes and responses induced by RIF in a confirmed in vitro developed RIF resistant Brucella strain. As shown for other bacteria, like N. meningitidis, resistance against RIF is not the result of just a single mutated protein,24 on the contrary, resistance is the end result of many underlying cellular processes. Our findings indicate a more active metabolic profile in BabRIFres compared to BabRIFsusc. The cell needs to act fast against stress conditions, to correct translational mistakes, to produce components of the cell wall and membrane, and to avoid toxic metabolic byproducts. The quick building up of the SMF could perhaps be just enough to excrete rifampicin from the internal compartment since the antibiotics effectiveness has been reduced due to the mutation on its target. Consequently, RIF resistant strains have to struggle for their survival and successfully cope with the high biological cost. Of particular clinical interest was the parallel increase of the SMZ/TMP MIC level, a finding that was in part backed up by the proteomic approach results. This finding is adequate for questioning the use of SMZ/TMP in the cases where RIF resistance is detected. Future research with deletion mutants of the proteins of interest and in vivo models might help to unravel their potential participation in rifampicin resistance mechanisms of Brucella.

S Supporting Information *

Table of the identified proteins in both Brucella strains. This material is available free of charge via the Internet at http:// pubs.acs.org. 2382



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AUTHOR INFORMATION

Corresponding Author

*Institution: Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box 2208, GR-71003 Voutes, Greece. E-mail: [email protected]. Tel: 00302810545006. Fax: 00302810545001. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Special thanks to the institution of University of Crete and the Regional Laboratory of Public Health of Crete for allowing the use of several instruments and to Dr. Iosif Vranakis and Dr. Dimosthenis Chochlakis for their valuable comments on the manuscript. Finally, many thanks to Nikolaos Lianeris for the creation of a data-mining software.

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ABBREVIATIONS RIF, rifampicin; DOX, doxycycline; STR, streptomycin; rpoB, DNA-dependent RNA polymerase gene; SMZ/TMP, trimethoprim−sulfamethoxazole; MIC, minimum inhibitory concentration; bepC, TolC protein homologue gene; IPG, immobilized pH gradient; BabRIFres, rifampicin resistant B. abortus 2308; BabRIFsus, rifampicin susceptible B. abortus 2308

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