Proteome studies of bacterial antibiotic resistance mechanisms Iosif Vranakis, Ioannis Goniotakis, Anna Psaroulaki, Vassilios Sandalakis, Yannis Tselentis, Kris Gevaert, Georgios Tsiotis PII: DOI: Reference:

S1874-3919(13)00542-3 doi: 10.1016/j.jprot.2013.10.027 JPROT 1600

To appear in:

Journal of Proteomics

Received date: Accepted date:

20 December 2012 19 October 2013

Please cite this article as: Vranakis Iosif, Goniotakis Ioannis, Psaroulaki Anna, Sandalakis Vassilios, Tselentis Yannis, Gevaert Kris, Tsiotis Georgios, Proteome studies of bacterial antibiotic resistance mechanisms, Journal of Proteomics (2013), doi: 10.1016/j.jprot.2013.10.027

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ACCEPTED MANUSCRIPT Proteome studies of bacterial antibiotic resistance mechanisms

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Iosif Vranakis1, Ioannis Goniotakis2, Anna Psaroulaki1, 2, Vassilios Sandalakis1,

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Yannis Tselentis1, Kris Gevaert3,4 and Georgios Tsiotis5*

1. Regional Laboratory of Public Health of Crete, Heraklion 71110, Greece 2. Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical

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Medicine, Medical School, University of Crete GR-71110 Heraklion, Greece 3. Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium 4. Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium

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2208, GR-71003 Voutes, Greece

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5. Division of Biochemistry, Department of Chemistry, University of Crete, P.O. Box

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*Address correspondence to: Georgios Tsiotis

Division of Biochemistry

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Department of Chemistry University of Crete P.O. Box 2208

GR-71003 Voutes, Greece Email: [email protected] Tel:++302810545006

Fax:++302810545001

Keywords: Antibiotic resistance, MS-driven proteomics, Quantitative proteomics, COFRADIC

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ACCEPTED MANUSCRIPT Abstract Ever since antibiotics were used to help humanity battle infectious diseases,

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microorganisms straight away fought back. Antibiotic resistance mechanisms indeed

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provide microbes with possibilities to by-pass and survive the action of antibiotic

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drugs. Several methods have been employed to identify these microbial resistance mechanisms in an ongoing effort to reduce the steadily increasing number of treatment failures due to multi-drug-resistant microbes. Proteomics has evolved to an

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important tool for this area of research. Following rapid advances in whole genome

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sequencing, proteomic technologies have been widely used to investigate microbial gene expression. This review highlights the contribution of proteomics in identifying

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microbial drug resistance mechanisms. It summarizes different proteomic studies on

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bacteria resistant to different antibiotic drugs. The review further includes an overview of the methodologies used, as well as lists key proteins identified, thus

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providing the reader not only a summary of research already done, but also directions

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for future research.

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ACCEPTED MANUSCRIPT Introduction Infectious diseases have been estimated to be the second leading cause of death in the

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world [1]. The introduction of antibiotics provided the medical community the

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necessary tools for treating these diseases. Antibiotics were extensively used, amongst

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other drugs following their implementation in advanced therapeutic procedures such as transplantations or aggressive surgical practices, which would have been impossible without antibiotics [2]. However, diseases that were thought to be fully

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controlled by antibiotics are now returning in new forms resistant to antibiotic

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treatment [3]. Sexually transmitted diseases such as syphilis and gonorrhea were considered to be under control using common treatment schemes. Yet, the emergence

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of resistant strains poses a global public health threat [4]. This emergence of

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pathogenic organisms that are resistant to antibiotics is a serious concern as it limits the effectiveness of current drugs and causes treatment failure [5]. Further, reduced

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antibiotic susceptibility increases the risk of complications and the overall chances of fatal outcomes. Antibiotic resistance and the decreasing approval rate of new

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antibacterial compounds, turned infectious diseases into a major threat to human health [6].

Bacteria can get protected against chemical threats by an integrated network of different elements. A vast number of scientific reports indicate that there are five general ways for a bacterium to acquire antimicrobial resistance: (1) mutation in the target site; (2) enzymatic modification or degradation of the antibiotic; [7] active efflux of antibiotics from the cell; (4) resistance through reduced permeability to antibiotics, which restricts their access to target sites; and (5) acquisition of alternative metabolic pathways [8]. Recently, a link between bacterial metabolism and antibiotic resistance has also been suggested [9-12]. The elucidation of drug resistance

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ACCEPTED MANUSCRIPT mechanisms is a very active research area that bridges several disciplinary boundaries since understanding the mechanism(s) by which drug resistance develops, may lead to

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improvements in extending the efficacy of current antimicrobials [13-14]. The

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mechanisms steering bacterial antibiotic resistance can be linked to genetic variability

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and the evolutionary pressure that favors the fit, whereby the surviving organisms constitute a bigger threat to human health as our defense strategies against them are

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An overview of proteomics methods

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rendered useless [15].

Proteomics complements comparative genomics and transcriptome profiling for gene

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expression analysis by providing data on the nature of the final gene product, the

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protein. More in particular, proteomics provides information, not readily available by other methods, on the occurrence of posttranslational modifications, protein

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subcellular localization, and protein turnover rates, amongst others. Proteomic technologies have been widely used to investigate microbial gene expression. This

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was supported by the availability of whole genome sequences for a large number of bacteria, including many bacterial pathogens [16]. Proteome studies are based on gel-based and gel-free approaches. The first impact proteomics had on microbiology was the ability to generate proteome maps, and thus a detailed view on overall gene expression on given conditions, for bacteria. An obvious extension of both approaches is to establish and compare catalogs of proteins expressed by an organism under several conditions. Several proteomes of bacteria growing in vitro provided an opportunity to carry out comparative studies under highly controlled conditions and have been used to identify protein correlating to resistance. In this respect, mass spectrometry based quantitative proteomics is a

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ACCEPTED MANUSCRIPT powerful technology, capable of addressing such questions. The work horse of quantitative proteomics was for long time 2-DE followed by image analysis [17].

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Total cellular proteomes from bacterial isolates that exhibit differences in antibiotic

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resistance can in this way be compared to identify proteins correlating with antibiotic

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resistance [18-19]. A breakthrough for such gel-based quantitative proteome analysis came with the Differential In-Gel Electrophoresis (DIGE) technique [20]. Significant progress in interfacing mass spectrometry with liquid chromatography

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(LC-MS/MS), overall instrument sensitivity and acquisition speed made gel-free

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approaches very popular [21]. In general, two main approaches can be distinguished. The first approach separates peptides over orthogonal chromatographic systems order

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to obtain optimal separation before MS/MS analysis [21]. In the second approach, the

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complexity of the peptide mixture is reduced by selecting a subset of peptides that is highly representative for the original proteins, and analysis is then restricted to this

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subset [22]. COmbined FRActional DIagonal Chromatography (COFRADIC) is an example of the latter approach [23-24].

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Mass spectrometry allows for direct quantitative comparison of different proteomes. In order to achieve this, a variety of techniques were introduced, such as isotopecoded affinity tags (ICAT) [25], stable isotope labeling by amino acids in cell culture (SILAC) [26], isobaric tags such as tandem mass tags (TMT) [27] and isobaric tags for relative and absolute quantitation (iTRAQ) [28]. In addition, other post-metabolic labeling strategies such as reductive dimethylation and acylation with different isotope-coded tags, have been used [29]. Recently, the labeling of the N- and Cterminus of peptides with

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C3-propionyl after digestion with endoproteinase Lys-C

was applied for quantitative proteome analysis [10-12, 30]. A variant of the different labeling by using

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C4-butyryl was also applied for comparative studies using N-

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ACCEPTED MANUSCRIPT terminomics [31]. It is clear that proteomics has become an established tool to study bacteria and in the

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following paragraphs we attempt to summarize several comparative proteomic studies

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of bacteria resistant, or under pressure, to different antibiotic drugs.

Cell wall-acting antibiotics

The cell wall is a crucial structure of the bacterial cell. It is responsible for the

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maintenance of the bacterial shape as well as other important functions such as

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reproductive division, prevention of cell lysis due to high osmotic pressure and extracellular presentation of several virulence factors. Peptidoglycan is the main

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structural component of the bacterial cell wall.

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Antibiotics, targeting the bacterial cell wall, use mechanisms to inhibit the activity of enzymes involved in the synthesis, maturation and layer formation of peptidoglycan.

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Several categories of antibiotics are used to target the cell wall such as β-lactams (alone or in combination with β-lactamase inhibitors), glycopeptides (vancomycin, lipoglycopeptides

(telavancin,

oritavancin),

cyclic

peptides

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teicoplanin),

(daptomycin), D-Cycloserine, fosfomycin and bacitracin [32-34].

β-lactams According to their chemical structure, β-lactams are mainly divided into penicillins, cephalosporins, carbapenems, monobactams and other minor categories. Bacteria use three main strategies to get protected against β-lactams: alteration in PenicillinBinding Proteins (PBPs) which reduces the affinity of β-lactams, efflux pumps which remove the antibiotic from the bacterial periplasmic space and production of β-

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ACCEPTED MANUSCRIPT lactamases i.e. enzymes which hydrolyze the ring of β-lactams [35-37]. The presence of β-lactamases is the leading cause of resistance in several Gram-negative bacteria.

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Resistance to cell wall-acting antibiotics, particularly β-lactams, is a matter of global

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discussion among all infectious diseases specialists. Many authors have pointed their

Staphylococcus

aureus,

Klebsiella

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concern upon the group of pathogens named as ESKAPE ( Enterococcus faecium, pneumoniae,

Acinetobacter

baumannii,

Pseudomonas aeruginosa and Enterobacter spp.) [38-40]. A recent review of the US

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Surveillance Network (TSN) data revealed continuous increase of ESKAPE

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organisms' resistance rates on β-lactam antibiotics suggesting the need of closer

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monitoring of multi-drug-resistant (MDR) strains and new drug targets [41].

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Soualhine and colleagues studied penicillin resistance in Streptococcus pneumoniae using a proteomic approach. The investigators studied susceptible and resistant

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isogenic strains. Resistance in penicillin was selected in vitro after subsequent cultures of the sensitive strains in increasing concentrations of the antibiotic. Two

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bacterial clones, which exhibited eightfold increase in penicillin resistance compared to the susceptible strain, were selected. Comparision of the proteome maps of the susceptible reference strain and both resistant strains revealed eight differentially expressed proteins. Among these proteins, a subunit of the phosphate ABC transporter, PstS, posed particular interest. Increased expression of this protein in both resistant strains of S.pneumoniae caused increased RNA expression of the entire ABC transporter-related operon. Inactivation of the PstS-related gene led to reduction in penicillin resistance thus suggesting a potential of this protein in resistance to penicillin [42].

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ACCEPTED MANUSCRIPT In another study, Chaussee and colleagues determined whether penicillin-induced killing of Streptococcus pyogenes is promoted by changes of a regulatory response to

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penicillin via the transcriptional regulator Rgg. 2D-PAGE was used to study proteins

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from wild-type and rgg mutant cultures with or without exposure to penicillin.

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Exposure to penicillin for one hour induced increased presence of 12 proteins associated with fatty acid biosynthesis, the pentose phosphate pathway, glycolysis and stress-related responses in the wild-type strains. Eight of these proteins presented

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greater abundance in the mutant strains even prior to penicillin exposure. The addition

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of cerulenin, a fatty acid biosynthesis inhibitor, promoted killing of the wild-type strain induced by penicillin. These results indicate the presence of tolerance to

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mediated by penicillin [43].

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penicillin-mediated killing due to changes in the proteome of rgg mutant strains not

A year later, Hu and colleagues reported the decreased ceftriaxone resistance in a

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Salmonella enterica serovar Typhimurium strain attributed to a transposon insertion in yjeH gene, encoding for a possible transporter protein. Reduction of permeability

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and increased drug export are not commonly referred as mechanisms of resistance in Salmonella. The mutant strain, which was selected after subculturing the R200 ceftriaxone-resistant Salmonella strain in sub-inhibitory concentrations of ceftriaxone, exhibited altered expression of porin OmpD, putative Outer Membrane Proteins STM1530 and STM3031, heat shock protein MopA and a subunit of the protonpumping oxidoreductase NuoB. The results suggest the association of these proteins with ceftriaxone resistance and the influence of altered expression by the potential transporter gene yjeH [44]. Moving further, the aim of a study by dos Santos and colleagues was to identify changes in the proteome of a laboratory-derived piperacillin/tazobactam-resistant

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ACCEPTED MANUSCRIPT strain of Escherichia coli as compared with its susceptible wild-type strain using 2DDIGE followed by MALDI-TOF/TOF MS. The authors detected 12 proteins in the

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resistant strain with increased abundance. These proteins were related to bacterial

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virulence, antibiotic resistance and DNA protection during stress. On the other hand,

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14 proteins were found with decreased level, including proteins involved in glycolysis, protein biosynthesis, pentose-phosphate shunt, amino acid transport, cell division and oxidative stress response. Among the proteins with increased abundance

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in the resistant strain, the outer membrane protein TolC, which belongs to the

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multidrug efflux pump system, was identified [45]. Since TolC-increased abundance is consistent with reports that this protein combines with AcrAB, forming the major

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typical multidrug efflux pump in E. coli, a potential role of a multi-drug efflux system

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in E. coli resistance to piperacillin - tazobactam was suggested [46]. A more recent study by Monteiro and colleagues examined methicillin resistance of

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S. aureus from clinical origin. Using proteome mapping with 2-DE and MALDITOF/TOF, 227 proteins were identified in the methicillin resistant S. aureus (MRSA)

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strain. The authors presented data that suggest that alanine dehydrogenase 2 could be associated with antibiotic resistance by its involvement in peptidoglycan layer formation. The layer can contain L-alanine, which is associated with the mechanism of resistance that may reduce the antibiotic’s affinity for its target [47]. Multi-drug resistant bacterial strains are becoming a serious public health issue. In this context, Liu and colleagues studied the proteomic profile of a Stenotrophomonas maltophilia clinical isolate, which exhibited concurrent presence of blaNDM-1, blaL1 and blaL2 β-lactamase genes. Under imipenem-induced stress conditions, compared to the K279a control strain, the isolated strain DCPS-01 presented differential expression of several proteins. This protein repertoire included proteins with known

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ACCEPTED MANUSCRIPT imipenem resistance function (L1 MBL), key stress proteins (chaperone DnaK, heat shock chaperone ClpB etc), elongation-related proteins (elongation factor Tu, seryl-

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tRNA and cysteinyl-tRNA synthetases), metabolism-related proteins that may alter

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bacterial catabolic procedures and others. The different expression of L1 MBL

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precursor which is known to get implicated in imipenem resistance should be pointed out. The results suggest that the expression of genes related with imipenem resistance is induced by the presence of the antibiotic short after the beginning of clinical

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treatment [48].

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Using the same microorganism species and the same antibiotic, Oudenhove and colleagues studied the effect of imipenem in the microbe’s proteome. The scientists

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used a Stenotrophomonas maltophilia clinical isolate with MIC>32 μg/ml against

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imipenem. After exposure of the microorganism in sub-inhibitory imipenem conditions, the enriched cytoplasmic and membrane protein fractions were studied

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using iTRAQ differential labeling and 2-D liquid chromatographic separation (2DLC) MS/MS. The study showed differential expression of 73 proteins following

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imipenem exposure which are implicated in different metabolic procedures (protein folding, transcription, translation, nucleotide metabolism, energy metabolism, lipid metabolism, carbohydrate metabolism, amino acid metabolism) as well as in β-lactam resistance. Interestingly, increased production of β-lactamase L1 and L2 precursors and proteins related to their activation (LysR transcriptional factor AmpR) and was observed. The proteome analysis suggests mechanisms of cell adaptation in βlactamase production after exposure in β-lactamic antibiotics [49]. In the same context, Tiwari and colleagues studied the inner membrane fraction from carbapenem-resistant Acinetobacter baumannii clinical isolates (three strains with high, intermediate and low resistance profiles, respectively) and a reference strain

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ACCEPTED MANUSCRIPT using DIGE, followed by Decyder, Progenesis and LC-MS/MS analysis. They reported 23 differentially expressed proteins (19 over-expressed, 4 down-regulated) in

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resistant strains. The up-regulated proteins were directly or indirectly associated with

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carbapenem resistance. Such proteins were β-lactamases (AmpC, OXA-51), enzymes

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implicated in metabolism (ATP synthase, malate dehydrogenase, 2-oxoglutarate dehydrogenase), ribosomal proteins and elongation factor Tu. These enzymes seem to facilitate the enhanced metabolic needs for survival followed by enhanced production

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of proteins [50].

Glycopeptides

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Vancomycin and teicoplanin, the two currently used glycopeptide antibiotics, have

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been extensively used against serious infections caused by multi-drug resistant bacterial strains such as MRSA, E. faecium and others. Vancomycin targets the D-

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Ala–D-Ala terminus of the UDP-N-acetylmuramyl-pentapeptide precursor of peptidoglycan, ultimately interfering with peptidoglycan crosslinking. Resistance

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arises as a result of the synthesis of abnormal pentapeptide precursors possessing altered termini (e.g. D-Ala–D-lactate or D-Ala–D-Ser) with lower affinity for vancomycin, catalysed by the products of the van genes [51]. Using subcellular fractionation and protein separation by 2DE-PAGE, Pieper and colleagues studied changes in the proteome of three isogenic S. aureus strains generated from a clinical vancomycin intermediate S. aureus (VISA) strain after suitable subculture conditions (VP32: MIC 32 μg/ml, HIP5827: MIC 8 μg/ml, P100: MIC 2 μg/ml). Proteomic analysis using MALDI TOF/TOF-MS and LC-MS/MS identified 65 proteins which exhibited differential expression pattern. Many enzymes implicated in purine ribonucleotide biosynthesis pathway showed increased

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ACCEPTED MANUSCRIPT abundance in strain VP32 compared to the other two strains. Changes in expression of other

proteins

such

as

LytM

(peptidoglycan

hydrolase),

SceD

(putative

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transglycosylase), PBP2 and D-Ala-D-Ala ligase might be responsible for the altered

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rate of cell wall turnover and might suggest an altered peptidoglycan structure in

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strain VP32 [52]. Trying to find a potential biomarker for the detection of VISA strains, Drummelsmith and colleagues used comparative proteomics (high resolution 2-DE, iTRAQ) to detect differentially expressed proteins in clinical MRSA and VISA

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strains of the same Multi Locus Sequence Typing (MLST). The scientists revealed the

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presence of 93 differentially expressed proteins implicated in different cellular functions (carbohydrate metabolism, cell surface structure metabolism, membrane

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and cell wall secreted proteins, co-factor biosynthesis, stress response regulation, fatty

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acid metabolism, nucleic acid metabolism, protein biosynthesis, proteolysis and others). The putative lytic transglycosylase SAV2095 was selected for its increased

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expression in all VISA strains compared to MRSA strains as well as in heterogenous VISA strains which appeared to be vancomycin-sensitive using standard methods.

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The molecular data presented in this study suggest that SAV2095 expression levels could be used as a molecular diagnostic biomarker for the rapid detection of VISA strains [53].

In another study, Wang and colleagues used proteomics to detect differences in protein expression in two vancomycin-resistant E. faecalis strains (reference strain V583 and clinical isolate V309) with and without vancomycin treatment. The scientists coupled their results with molecular techniques such as semi-quantitative RT-PCR to further study the induction of differential gene expression which was observed in the level of proteome. The combination of these techniques revealed 28(V583)/20(V309) up-regulated proteins, 8(V583)/6(V309) down-regulated proteins

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ACCEPTED MANUSCRIPT and a total of 6 proteins which exhibited post-translational modifications (mobility in 2-DE) upon treatment with vancomycin. These proteins included proteins with known

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implication in vancomycin resistance (VanA, VanB, VanX), virulence factors (gls24,

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endocarditis specific antigen EF2076, adhesion lipoprotein EF0577 and pheromone

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cAD1 precursor lipoprotein EF3256), proteins related to stress response and many proteins implicated in cell metabolism and cell growth, translation and conjugation. This study suggests the role of vancomycin presence not only in induction of

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resistance but also in stress-related modifications involved in survival [54].

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In the same context, Chen and colleagues coupled molecular techniques with proteomics to identify genes associated with glycopeptides resistance in clinical

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hVISA strains. The scientists studied two pairs of isogenic vancomycin susceptible S.

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aureus (VSSA) and hVISA strains isolated from two patients. Proteomic analysis revealed five differentially expressed proteins with up-regulated expression in hVISA

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strains (isaA, MsrA2, Asp23, GpmA, AhpC). In hVISA strains, the only statistically significant up-regulation appeared in the isaA gene, a gene encoding for a protein

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which acts as a probable transglycosylase precursor which acts on peptidoglycan cleavage pathway. This data suggest that isaA gene might be related to hVISA vancomycin resistance [55].

Lipopeptides Daptomycin (formerly LY146032) is a cyclic lipopeptide antibiotic exhibiting a novel mechanism of action. This agent acts on the cell wall membrane structure and synthesis by binding to the cell membrane via a calcium-dependent mechanism thus causing efflux of potassium ions off the bacterial cells. This series of events leads to bacterial cell death [56]. Daptomycin is active against Gram-positive bacteria and is

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ACCEPTED MANUSCRIPT clinically used to treat serious infections caused by these organisms (MRSA bacteremia, VRE infections, skin and soft tissue infections, endocarditis).

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Daptomycin-resistant strains are continuously referred in international literature [57-

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58].

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Studies that attempt to link alterations in drug targets as a mechanism of antibiotic resistance have also been done by proteomics techniques. Fischer and colleagues compared an isogenic daptomycin-susceptible (DAPS) and a daptomycin-resistant

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(DAPR) S. aureus strain isolated from a patient with relapsing endocarditis during

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daptomycin treatment. Daptomycin is classified as an inhibitor of membrane function by inducing membrane pore formation leading to bacterial death due to imbalance in

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transmembrane ion fluxes [59]. Combining IPG-IEF and LC-MS/MS 616 (DAPS) 701

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(DAPR) proteins were identified. This analysis revealed that several proteins belonging to various functional categories, including cell wall-associated targets, were

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differentially expressed [60].

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Antibiotics acting on cell membrane Antimicrobial peptides Antimicrobial peptides (AMPs) are polypeptides which are produced endogenously in order to protect the host from microbial invasion and are active against a broad spectrum of microorganisms including MDR bacteria. New AMPs are being continuously developed targeting new structural sites. AMPs have a distinguished mechanism of action. This fact means that resistance to AMPs is not easily possessed [61]. In order to elucidate the AMPs mode of action Chiu and colleagues compared expression patterns of the outer membrane, inner membrane and cytoplasmic proteins (OMPs, IMPs and CPs) from Vibrio parahaemolyticus with or without exposure to

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ACCEPTED MANUSCRIPT cationic antimicrobial peptides (AMPs). Here, 2-DE coupled with LC-ESI-Q-TOF MS⁄MS was used to identify three OMPs (maltoporin, flagellin and OmpV), two

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IMPs (ATP synthase F1, alpha subunit; and OmpV) and three CPs (pyruvate

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dehydrogenase subunit E1, glyceraldehyde-3-phosphate dehydrogenase and inositol-

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5-monophosphate dehydrogenase) with differential expression levels [62]. The authors concluded that the bacterium may directly respond to AMPs through upregulation of the efflux channel, increased yield of energy, effective repair of

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damaged membranes and down-regulation of carbohydrate and nucleotide

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metabolism for energy preservation. In this study, RT-qPCR was used to determine mRNA expression levels of the corresponding genes and these correlated well with

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the proteomics data [62].

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Fehri and colleagues studied Mycoplasma pulmonis, a murine respiratory pathogen. Their results indicated that the activation of stress response (followed by mutations in

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the hrcA gene) might enhance the development of resistance to AMPs such as melittin or gramicidin D. In parallel, 2-DE analyses showed up-regulation of enzymes

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implicated in energy metabolism of M. pulmonis as a possible result of the increased energy demand of the resistant strains [63]. Using membrane subproteome analysis, Shen and colleagues tried to identify proteins which are implicated in Vibrio parahaemolyticus AMP resistance. Resistant strains were obtained after subculture of V.parahaemolyticus strains in the presence of four different AMPs. The analysis revealed two OMPs (TolC, flagellin) and five IMPs (transcription termination factor NusA, EF-Tu, ATP synthase α subunit, dihydrolipoamide dehydrogenase, long-chain FA transport protein, FadL) with significantly altered expression between the WT and AMP-resistant strain. The researchers suggested a possible mechanism of AMP resistance mediated by

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ACCEPTED MANUSCRIPT overexpression of the MDR efflux pump, TolC. Upregulation of the TolC pump is also described as a possible resistance mechanism described in other studies with

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different antibiotics [48, 64-65]. As a concluding statement, AMP resistance is

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possibly mediated via up-regulation of the energy-dependent MDR efflux transporter

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(TolC and F1-ATPa), effective repair of damaged membranes (DLD) and prevention of cellular penetration of AMPs (down-regulation of FadL) [66]. Similarly, the magainin (another AMP) resistance process of Escherichia coli was

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studied. These data indicated that resistance results in commitment of the resistant

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strains to several metabolic pathways such as energy metabolism, nitrogen

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metabolism, amino acid metabolism and others [67].

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Polypeptides

Polymyxins (obtained from Bacillus polymyxa) and colistins (obtained from

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Aerobacillus colistinus) are a collection of cyclic polypeptides which act by altering the permeability of the cytoplasmic membrane [68]. Resistance to polypeptides is

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currently referred especially as part of a MDR microbial profile in strains with resistance to multiple clinically used antibiotics. It is based upon chromosomallyinduced decrease in outer cell membrane permeability. Using 2-D DIGE, Fernandez-Reyes and colleagues tried to identify differences in protein expression among colistin-resistant and susceptible Acinetobacter baumannii strains. Their analysis revealed 35 differentially expressed proteins including OMPs, chaperones, enzymes implicated in metabolism (14 in total) and protein biosynthesis factors. Colistin-susceptible and resistant strains of A. baumannii were tested for growth on media containing a variety of individual carbon sources. Interestingly, the A. baumannii resistant strain presented enhanced growth on citrate as a single carbon

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ACCEPTED MANUSCRIPT compared to the colistin-susceptible strain. Isocitrate dehydrogenase was found upregulated in the resistant strain, thus the TCA cycle seems to play a role in

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providing metabolic requirements of colistin-resistant A. baumannii. This theory is

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further supported by changes in the expression of metabolic enzymes implicated

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directly or indirectly in the TCA cycle which result in loss of biological fitness of the resistant strains [69].

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Protein synthesis inhibitors

Tetracyclines

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Tetracyclines are broad-spectrum agents, exhibiting activity against a wide range of

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microorganisms. The favorable antimicrobial properties of these agents and the absence of major adverse side effects have led to their extensive use for the therapy of

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infected humans and animals.

The mode of action of tetracyclines is the inhibition of bacterial protein synthesis by

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preventing the association of aminoacyl-tRNA with the bacterial ribosome [70]. The binding of tetracyclines sterically blocks aminoacyl-tRNA binding and as a result inhibits protein synthesis [71]. Resistance to tetracyclines can occur mainly through five mechanisms. These are the production of ribosomal protection proteins (RPPs), the active efflux of tetracycline from the cell, the decreased drug permeability, the mutation of the antibiotic target, and the enzymatic degradation of the administered antibiotics [72]. Zhang and colleagues characterized the outer membrane proteome of E. coli in response to tetracycline. Using 2-DE and matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) they identified FimD, Tsx,

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ACCEPTED MANUSCRIPT OmpW, OmpC and TolC in upregulation and LamB in downregulation. Even though they confirmed using genetically modified strains with gene deletion or

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complementation that LamB, OmpC and TolC are important OM proteins for

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tetracycline resistance in E. coli additional functional validation is required to

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investigate whether the altered proteins obtained from two-dimensional gel electrophoresis play a direct or indirect role in phenotypic changes [73]. One year later Chao and co-workers proteomically compared doxycycline sensitive

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versus insensitive isolates of Orientia tsutsugamushi. Using 2D-PAGE and LC-

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MS/MS the authors tried to identify protein factors that may be partially responsible for differential drug sensitivity of isolates of Orientia. Their comparison of 2D-PAGE

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protein profiles of drug sensitive strain versus (vs.) insensitive isolates has led to the

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identification of 14 differentially expressed or localized proteins, including elongation factor Ts and Tu, DNA-directed RNA polymerase α-subunit, ATP synthase β-subunit,

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and several hypothetical proteins confirming the tremendous proteomic diversity of isolates of Orientia and suggesting that drug insensitivity in this species may arise

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from multiple mechanisms [74]. Yun and colleagues analyzed the surface proteome and secretome of Acinetobacter baumannii under tetracycline stress conditions [75]. 2DE-PAGE was used to separate membrane proteins and ESI-Q-TOF MS for protein identification, while secreted proteins were fractionated by SDS-PAGE followed by LC-MS/MS for protein identification. It was found that membrane expression of major outer membrane proteins [27] was significantly decreased in response to tetracycline however, their transcript levels were not significantly changed [75]. Analysis of the secreted proteins indicated increased secretion of several Omps under tetracycline stress. These results clearly indicate that A. baumannii actively regulates membrane expression and

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ACCEPTED MANUSCRIPT secretion of Omps in order to overcome tetracycline induced stress. Here, an interesting observation is that the increased secretion of particular Omps was not due

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to transcriptional but rather to translational regulation, reflecting the necessity of

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using proteomics tools.

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Peng and colleagues used 2-DE and MALDI-TOF/MS to study the membrane proteome of P. aeruginosa showing enhanced resistance to ampicillin, kanamycin and tetracycline. Apart from previously identified porins associated with antibiotic

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resistance, the authors detected five novel porins in the insoluble sarcosine fraction

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[76] and concluded that P. aeruginosa showed significant diversity, concerning membrane protein expression, against these three antibiotics. Their proteomics data

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provided valuable information for elucidating antibiotic-resistant differences between

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different species of bacteria, while the detected proteins shared by different bacteria or a bacterium against different antibiotics could provide candidate targets for the

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development of new drugs that control antibiotic-resistant bacteria. In another study, the proteome of C. burnetii under tetracycline stress using

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COFRADIC was studied and 531 proteins were identified [12]. These proteins were classified according to their predicted cellular function. Interestingly, many proteins were found to be involved in cell metabolism (amino acid biosynthesis 3%, DNA metabolism 6%, energy metabolism 9%, fatty acid and phospholipid metabolism 3%, multi-biosynthetic pathways 4% and others). Five proteins were up-regulated under tetracycline stress conditions, and these were involved in energy metabolism and in the pentose phosphate pathway (Q83DM4), biosynthesis of pantothenate and coenzyme A (Q83EA2), aspartate biosynthesis (Q83A62) and others. Nineteen proteins were down-regulated under tetracycline stress conditions. Down-regulation of the phosphoenolpyruvate-protein phosphotransferase (Q83BF9) under tetracycline

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ACCEPTED MANUSCRIPT stress is interesting because of previous findings that this protein is only present in a model of C. burnetii acute infection compared with a persistent model of C.burnetii

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infection [30]. Further proteome studies on the Q fever causing agent could reveal

that energy is a key function that

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To conclude, it is intriguing to hypothesize

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new mechanisms of resistance reducing treatment failures.

guarantees cell survival. Resistant strains seem to change their metabolism trying to achieve a basic level of energy production which will guarantee survival when under

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stress by a harmful antibiotic agent. This hypothesis should be further studied in the

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future using proteomics. Aminoglycosides

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Aminoglycosides comprise an important group of antibiotics for the battle against

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serious infections. Streptomycin, which has been discovered and used since 1944, was the first member of this family but now it is only used in certain infections such as

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tuberculosis because of its toxicity and narrow spectrum of action [77]. Aminoglycosides inhibit protein synthesis by binding to the 30S bacterial subunit of

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the bacterial ribosomes. Resistance mechanisms have been described in aminoglycosides and they include drug-modifying enzymes, active efflux and membrane impermeabilization and post-transcriptional methylations of 16S rRNA [78-79]. Resistance to aminoglycosides such as streptomycin can also arise from target site (i.e. ribosomal) mutations [80]. Sharma and colleagues compared the proteome profiles of streptomycin [81] susceptible and resistant M. tuberculosis clinical isolates using 2-DE and MALDITOF-MS. Over-expression of nine proteins in SM-resistant strains were found. Malate dehydrogenase, which converts malate to oxaloacetate, was found to be overexpressed. Its role in M. tuberculosis drug resistance remains unknown. However,

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ACCEPTED MANUSCRIPT streptomycin interacts with amino acids of malate dehydrogenease conserved active site, which suggests the potential effect of the drug in the activity of this enzyme.

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Over-expression of another protein (oxidoreductase), probably involved in cellular

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metabolism, was observed. Results from previous studies suggested this protein as a

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candidate antigen for development of a new vaccine against M. tuberculosis. Moreover, oxidoreductase was observed to be differentially expressed in isoniazidresistant and susceptible strains. Over-expression of an electron transfer flavoprotein

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alpha subunit (transferring electrons to the respiratory chain and taking part in fatty

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acids oxidation) was also found. Finally, it was postulated that the identified proteins (some of which are implicated in metabolic procedures) are expected to contribute in

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conferring a resistant phenotype to the strains [82].

Macrolides

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Macrolides comprise a class of antibiotics which act by binding to the 50S subunit of the bacterial ribosomes thus inhibiting protein synthesis. Erythromycin complex was

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the first antibiotic of this category to be described and used in clinical medicine. The most common mechanism of resistance to macrolides involves modification of their target site on the ribosome, specifically methylation of an adenine residue in domain V of the 23S rRNA [46,47] Cash et al. investigated erythromycin resistance in Streptococcus pneumonia which is associated with two distinct phenotypes; the M and MLS phenotypes. Using 2DEPAGE, increased synthesis of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) in erythromycin-resistant isolates possessing the M phenotype was revealed. No such alteration was observed in isolates possessing the MLS phenotype and erythromycinsusceptible strains. The erythromycin resistance M phenotype is mediated by an

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ACCEPTED MANUSCRIPT active efflux mechanism. GAPDH up-regulation might provide energy, via NADH, for this process according to the authors’ hypothesis. However, the role of GAPDH in

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M phenotype S. pneumoniae remains to be further investigated [83].

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Chloramphenicol

Chloramphenicol was discovered in 1947 as a physical product of the actinomycete Streptomyces venezuelae (initially named as chloromycetin) [84]. Chloramphenicol

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acts by binding to the 50S bacterial ribosomal subunit inhibiting protein synthesis.

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Resistance to chloramphenicol has been described as part of the presence of the chloramphenicol acetyltransferase (CAT), an enzyme which inactivates the drug.

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Using 2-DE-IEF (Isoelectric focusing) and MALDI-TOF-MS, Li and co-workers

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identified six outer membrane proteins and one protein of unknown location that were responsible for chloramphenicol (CAP)-resistant Escherichia coli and for survival in

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medium with suddenly strong CAP treatment [64]. Using Western blotting and gene mutants the authors concluded that 4 out of the 7 proteins, namely TolC, OmpT,

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OmpC, and OmpW, were critically altered and could be considered as potential targets for designing new drugs against CAP-resistant E. coli. Antibodies against the identified OM proteins were then used to demonstrate antibody-combating bacterial growth. It was observed that anti-TolC showed a very significant inhibition on bacterial growth in medium with CAP, which points to a potential new way for treating infection by antibiotic-resistant bacteria. SDS-PAGE coupled with LC nano electrospray MS/MS was used to study antibiotic resistance mechanisms in Burkholderia thailandensis [85]. The chloramphenicolinduced resistant strain was also resistant against structurally unrelated antibiotics including quinolones and tetracyclines. It was demonstrated that the multidrug

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ACCEPTED MANUSCRIPT resistance phenotype, identified in chloramphenicol-resistant variants, was associated with over-expression of two different efflux pumps capable of expelling antibiotics

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from several families, including chloramphenicol, quinolones, tetracyclines,

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trimethoprim and some β-lactams.

DNA synthesis/metabolism inhibitors Fluoroquinolones

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Quinolones are inhibitors of the essential bacterial enzymes DNA gyrase and DNA

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topoisomerase IV [86].

Resistance to quinolones has been a problem ever since nalidixic acid was introduced

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into clinical medicine more than 40 years ago. Generally, three mechanisms of

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resistance to quinolones are currently recognized: mutations that alter the drug targets, mutations that reduce drug accumulation and plasmids that protect cells from the

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lethal effects of quinolones [87].

Identification and functional characterization of the outer membrane proteome of

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genetically modified to resist inhibition of nalidixic acid E. coli strains was reported by Lin and colleagues [88]. Using 2-DE and MALDI-TOF/MS the authors report that the OM proteins TolC, OmpT, OmpC and OmpW were found to be up-regulated, and FadL was down-regulated in the nalidixic acid-resistant E. coli strains. They further investigated the possible roles these altered proteins played in regulation of nalidixic acid resistance using genetically modified strains with the deletion of these genes. They concluded that TolC and OmpC may play more important roles in the control of nalidixic acid resistance than the other identified outer membrane proteins. They went a step further by showing that the pathway responsible for the regulation of OmpC

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ACCEPTED MANUSCRIPT expression in response to nalidixic acid resistance, namely the two-component system EnvZ/OmpR, was also found to be involved in the process of resistance.

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A study of Leptospira interrogans by quantitative proteomic analysis of cells

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inoculated with ciprofloxacin highlighted a set of 26 protein complexes of L.

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interrogans after analyzing an extensive proteome list (2221) generated by quantitative mass spectrometry. Among the alterations ATP-synthase, ClpB and Hsp15were found to be increased in L. interrogans after treatment with ciprofloxacin

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[89]

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In veterinary medicine, S. uberis is an important pathogen causing mastitis. 2-D DIGE has been used to assess the impact of ciprofloxacin on the S. uberis proteome. Differential expression of 24 proteins was revealed in strains after treatment with

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ciprofloxacin (1.0 μg/mL). These proteins were implicated in various cell functions including carbohydrate metabolism, nucleotide/amino acid synthesis and stress-

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related responses. The data indicated that ciprofloxacin-induced down-regulation of metabolic pathways producing NADH and imbalanced synthesis of dNTPs could

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stimulate mutagenesis and promote development of resistance [90] Using 2D-PAGE and 2D-HPLC-MS for examining protein expression levels Coldham and colleagues identified 43 proteins with increased expression in Salmonella enterica serovar Typhimurium strains when a fluoroquinolone was added in the bacterial culture [91]. Most of these proteins were merely a physiological response to fluoroquinolone, however the identified over-expressed AcrAB/TolC efflux pump could be associated with resistance [91]. Our lab has extensively studied changes in the proteome of Coxiella burnetii and tried to identify proteins involved in levofloxacin resistance. Using COFRADIC, we compared the proteomes of in vitro developed levofloxacin-resistant (NMres) and

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ACCEPTED MANUSCRIPT susceptible strains (NMsus). Fifteen proteins presented differential expression among the 381 proteins which were identified in both strains. In the NM C. burnetii

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susceptible strain, up-regulated expression of proteins involved in DNA metabolism

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(topoisomerase IV subunit B), purine biosynthesis (adenylosuccinate lyase),

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biosynthesis of cofactors (erythronate-4-phosphate dehydrogenase) and others. A protein involved in phospholipid and fatty acid metabolism (3-hydroxymyristoyldehydratase) was found to be over-expressed in the resistant strain [11].

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The identification of one of the targets of fluoroquinolones, topoisomerase IV subunit

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B (Q83F84), over expresed in NMsus is of particular interest. Although mutations at the binding sites of the particular enzyme (QRDR) as a fluoroquinolone resistance

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mechanism in pathogens have been described [92], this is not the case with C.

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burnetii where recent work has shown that nucleotide mutations within QRDRs of C. burnetii are observed in all the subunits of gyrase and topoisomerase apart from the

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subunit B of topoisomerase IV [93]. Thus, under-expression of this particular enzyme in the NMres strain indicates a reduction in DNA replication due to the

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fluoroquinolone action.

The results of this analysis support the participation of detoxification processes indicated by the alteration of the amount of unsaturated lipids in the membranes as a probable mechanism of quinolone resistance. Additionally, the decrease of the expression of developmental stage-specific proteins in the NMres indicates for the first time possible metabolic changes of the resistant strain compared to the NMsus. A study that added to our understanding of antibiotic-resistant mechanisms in content of metabolic regulation is that of Li and colleagues. The authors, using 2DE and MALDI-TOF-MS, investigated differential proteins of Vibrio alginolyticus in resistance to balofloxacin [94]. Ten proteins were altered, and following western

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ACCEPTED MANUSCRIPT blotting confirmation they indicated that downregulation of Na(+)–NQR complex is

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essential for V. alginolyticus resistance to balofloxacin.

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ACCEPTED MANUSCRIPT RNA synthesis inhibitors Rifampicin

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Rifampicin is a member of rifamycins family. It has been broadly used as an anti-

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tuberculosis agent. It acts by inhibiting a DNA-dependent bacterial RNA polymerase,

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blocking mRNA synthesis and inhibiting protein synthesis. Rifampicin resistance is mainly connected with mutations in the rpoB gene, which encodes for the β subunit of the RNA polymerase. Neri and colleagues analyzed the proteomes of rifampicin-

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susceptible and resistant strains of Neisseria meningitidis using 2-DE and MS.

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Twenty three proteins showed differential expression, some of which are involved in major metabolic pathways such as catabolism of pyruvate and the TCA cycle [95].

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Proteins involved in the metabolism of carbohydrates and enzymes involved in the

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TCA cycle were over-expressed in resistant strains (phosphoenolpyruvate synthase, isocitrate dehydrogenase, glutamate dehydrogenase and others). Proteins involved in

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ATP production were down-regulated. Further analysis revealed that changes in protein expression were genetically encoded rather than induced by exposure to

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rifampicin. Bacteria were expected to over-express stress-related proteins because of exposure to rifampicin (bacterial shock). Additionally, rifampicin-resistant strains over-expressed positive transcription regulators to assist the process of transcription under the presence of rifampicin. Changes in meningococcal metabolism might result in loss of biological fitness, metabolic disadvantages of the resistant strains and decreased invasion capacity. All of this explains the relatively low rate of rifampicinresistant Neisseria meningitidis in the population . In a more recent study, Sandalakis and colleagues conducted a promising project combining proteomic, genomic and microbiological research methods to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella

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ACCEPTED MANUSCRIPT abortus 2308. The resistant strain presented the described mutation V154F, in the rpoB gene. Among 456 proteins which were identified using MS/MS, the resistant

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strain possessed 39 differentially expressed proteins involved in various metabolic

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pathways such as fatty acid and phospholipid metabolism, amino acid biosynthesis,

Interestingly,

the

resistant

strain

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energy metabolism, protein processing and protein fate, transport and binding etc. possessed

a

parallel

increase

of

the

trimethoprim/sulfamethoxazole MIC, a finding consistent with the different proteomic

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profile of the resistant microbe. The authors conclude that resistance is the end point

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of a complex cellular processes network rather than the result of changes in single

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proteins [10].

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Conclusion

The vast majority of proteomic-based researches that have been performed until today

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do not just point out a few proteins that are directly related to an antibiotic resistance mechanism. On the contrary, the revealed proteomic profiles expose to researchers a

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large number of differentially regulated proteins involved in various metabolic procedures, most of which have already been mentioned in this review, regardless of the antibiotic used [10-11, 69, 73-74, 82-83, 90, 94-95]. One must also consider that scientists are investigating a few parameters at given times and conditions, when the proteomic response, as well as the life-saving mutations are habitat-related (intracellular, extracellular, nutrient availability etc.) and life-cycle related (dormant cells, infection stage, replication stage, and growing stage etc.). The majority of the differentially expressed proteins are not just a collateral response of a stressed cell. A resistant phenotype is an innate controlled reprogramming of genome expression of bacteria adapting to a new environment, rather than a simple over-expression of a

28

ACCEPTED MANUSCRIPT pump. Thus, most metabolic alterations observed in resistant strains do not constitute a metabolic malfunction of the bacteria [9]. Although complex, all of the response

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mechanisms of bacteria aim on a single target, survival. The simplest example is the

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prompting of genomic mutations through the mechanism of SOS response,

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specialized polymerases (e.g. SOS-dependent and RecA-dependent DNA pol V) and controlling of the ROS production. The sooner a life-saving mutation is incorporated

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to the genome, the greater are the chances of survival [9, 96].

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Antibiotics speed up the natural process of evolution and this makes imperative the simultaneous re-adjustment of various metabolic procedures, impacting on cell

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physiology. Proteomics along with other high-throughput methodologies can

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contribute in the understanding of metabolic networks and their effect on antibiotic resistance. Hopefully the next step will be improved therapeutic strategies and,

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intimately, novel drug targets.

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Figure 1: Simplified diagram summarizing the major mechanisms of antibiotic

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Graphical Abstract

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Proteomics studies to identify microorganism antibiotic resistance mechanisms

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Used proteomics methods to identify key proteins

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Bacteria resistant to different antibiotic drugs

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Future research directions

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