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A Microarray-Based Analysis of Differentially Expressed Genes in
Journal of Infectious Diseases & Preventive Medicine

Journal of Infectious Diseases & Preventive Medicine
Open Access

ISSN: 2329-8731

+44 1300 500008

Research Article - (2018) Volume 6, Issue 2

A Microarray-Based Analysis of Differentially Expressed Genes in Intracellular Brucella abortus 544 within Mdbk Cells

Huynh Tan Hop#, Alisha Wehdnesday Bernardo Reyes#, Lauren Togonon Arayan, Tran Xuan Ngoc Huy, Son Hai Vu, Wongi Min, Hu Jang Lee and Suk Kim*
Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea
#Contributed equally to this work
*Corresponding Author: Suk Kim, Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea, Tel: +82–55–772–2359, Fax: +82–55–772–2349 Email:

Abstract

Background: Modulation of gene expression is a fundamental requirement for adaptation of intracellular Brucella abortus. Since most of the current understanding mainly focus on professional phagocytes and renal involvement is uncommon in brucellosis, our aim was to identify and analyze changes in B. abortus gene expression in response to intracellular environment within a bovine kidney cell line.
Methodology: B. abortus RNA were isolated from Madin-Darby bovine kidney (MDBK) epithelial cells during replicative phase and the transcriptional profile of intracellular B. abortus was characterized using microarray analysis.
Results and interpretation: The microarray analysis revealed a total of 1,623 differentially expressed genes of ≥ 2-fold–788 (25.44%, 788/3098) upregulated and 835 (26.95%,835/3098) down-regulated genes as compared with free-living Brucella. Among these identified genes, 81 and 185 were upregulated and down-regulated at ≥ 7-fold, respectively, showing a marked induction of genes involved in transcription and distinct repression of genes involved in translation, ribosomal structure and biosynthesis.
Conclusion: The identified genes in this study may provide new insights into the molecular interactions between B. abortus and non-phagocytic bovine cell line, MDBK. In addition, several differentially highly expressed transcripts were hypothetical genes with unknown function and/or unclassified which require further characterization due to their potential contribution in the virulence and strategy of Brucella to survive and proliferate within the host.

 

Keywords: Brucella abortus; MDBK; Microarray; RNA

Introduction

Brucellosis, one of the most important zoonotic diseases worldwide, has managed to elude eradication and is readily transmissible to humans resulting to acute febrile illness and undulant fever that may progress to a more chronic form, virtually affecting all of the organs [1,2]. Brucella is classified in risk group III by the World Health Organization (WHO) laboratory biosafety manual and possesses important characteristics including no classic virulence factors such as exotoxins or endotoxins, its lipopolysaccharide (LPS) pathogenicity is not typical, and ability to invade and persist through inhibition of programmed cell death [2,3]. The progression of brucellosis into a chronic form is related to the pathogen’s ability to persist for prolonged periods within host cells, evading the host’s immune system throughout the infection [4,5]. After entering the host–most commonly in mucous membranes of the respiratory and digestive tracts, brucellae are taken up by local tissue lymphocytes, transmitted through regional lymph nodes into the circulation and subsequently seeded throughout the body with tropism for lymphoreticular and reproductive systems [3,4,6].

As a facultative intracellular pathogen, B. abortus is exposed to many different microenvironments during its lifecycle; hence regulation of gene expression is a fundamental requirement for its physiological adaptation [7]. Identification and analysis of these changes in Brucella gene expressions in response to intracellular environments for survival may hold the key to provide better understanding of its pathogenesis and bring new insights into the molecular interactions between Brucella and its host, particularly in non-phagocytic host cells. Among the techniques employed in identifying differences in global gene expressions is the microarray technology which provides a high-throughput screening method to simultaneously measure the expression levels of a large number of genes or to genotype multiple genomic regions, hence it is the most widely used method for profiling mRNA expression [8]. Microarray analysis was used to identify B. abortus genes necessary for its intracellular survival in RAW 264.7 cells and revealed that 7.82% (244/3334) and 5.4% (180/3334) of all B. abortus genes were up-regulated and down-regulated, respectively [8], and in our previous study, 25.12% (801/3190) and 16.16% (515/3190) of the total B. abortus genes were upregulated and down-regulated, respectively, at ≥ 2-fold within bone marrow-derived macrophages (BMDMs) [9].

Research has focused on identifying virulence factor-encoded genes involved in the replication and survival of B. abortus mainly within professional phagocytes; however, the interaction between this pathogen and epithelial cells is also crucial in the outcome of infection for a deeper understanding of the pathogenesis of brucellosis. However, the current understanding on the molecular mechanisms and factors that Brucella employed within epithelial cells is limited [10]. A previous study investigated the relationship between the severity of apoptotic and autophagic cell death based on the distribution of Brucella antigens in the different tissues of aborted bovine fetuses due to natural infection and showed that the bacterial antigens were highly evident in the kidneys [11]. Interestingly, involvement of the renal parenchyma in the acute phase of brucellosis is very rare but generally manifested as acute interstitial nephritis, chronic interstitial nephritis or glomerulonephritis [12]. Although the involvement of kidney is not common, it has been reported that brucellosis is about ten times more prevalent in patients with renal failure and concluded that the disease can cause nephropathy [13]. Macrophages are the known primary target of B. abortus but has also been reported that the pathogen can invade a variety of other cell types including the Madin-Darby bovine kidney (MDBK) cells [14,15]. However, the pathogenic mechanisms of the pathogen within this cell line have not been elucidated at the cellular and molecular levels. To our knowledge, there have been no further studies done on the infection of Brucella in MDBK cells. Consequently, we focus on identifying critical genes involved in the interaction between Brucella and this epithelial host cell line MDBK cells.

Materials and Methods

Bacterial strain

The standard wild-type strains used were from B. abortus 544 (ATCC 23448) cultivated and maintained in Brucella broth or on agar (1.5%) (Becton Dickinson, USA). The bacterial culture used in the experiment was grown in broth at 37˚C with shaking until stationary phase was reached and serial dilutions on agar plates were performed in the assessment of the number of viable bacterial cells.

Cell culture

MDBK cell line (NBL-1) (ATCC CCL 22) was maintained in Dulbecco’s modified minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37˚C under 5% CO2. All the reagents were purchased from Gibco (USA).

Bacterial infection assay

MDBK epithelial cells were cultured at a concentration of 1 × 106 cells per well in 6-well plate overnight. The medium was changed into fresh medium and then infected with B. abortus at multiplicities of infection (MOIs) of 100. The cells were centrifuged at 150 xg for 10 min and incubated at 37˚C under 5% CO2 for 1 h. The cells were then washed with DMEM and incubated in fresh medium containing gentamicin (30 μg/ml) for 30 min to kill the remaining extracellular and/or adhered bacteria.

Brucella RNA extraction and purification

Isolation of intracellular B. abortus was done as previously described [9]. In brief, the infected cells were washed, lysed with distilled water and incubated at 37˚C for 5 min. The cells were then scraped and centrifuged at 14,000 xg for 2 min to collect the pellets. The pellets were resuspended in a 1 ml solution containing 890 μl distilled water, 100 μl RQ1 DNase Reaction Buffer (Promega, USA) and 10 μl RQ1 RNase-free DNase (1 μg/ml) (Promega, USA), and incubated at 37˚C for 30 min. The mixture was centrifuged at 8,000 xg for 2 min and the bacteria were pelleted at the bottom.

The bacterial pellets from free-living or intracellular B. abortus were incubated in 500 μl RNAse-free water at room temperature for 30 min. One ml of RNA Protect Bacteria Reagent (Qiagen, Germany) was added and then incubated further for 5 min. The mixture was centrifuged at 5,000 xg and the bacteria were dissolved in 1 ml solution containing 850 µl RNase-free water, 150 µl 1% or 10% sodium dodecyl sulfate (SDS) and 10 µl proteinase K (Promega, USA), and incubated at 37˚C for 1 h followed by the addition of 200 µl Chloroform (Sigma, USA). The total RNA extraction was performed using Qiagen RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Genomic DNA contamination was removed using RNase-Free DNase set kit (Qiagen, Germany).

The integrity of RNA was assessed by standard denaturing agarose gel electrophoresis, and the quantity and quality of RNA was evaluated using Optizen POP Nano Bio spectrophotometer (Mecasys Co., Ltd, Korea).

Brucella RNA sequencing and analysis

Sequencing and analysis of bacterial RNA were performed as previously described [9]. In brief, libraries for Illumina sequencing were made using TruSeq Stranded mRNA Sample Preparation Kit (Illumina, USA) following the manufacturer’s instructions. RNA sequencing was performed using HiSeq 2500 Sequencing System (Illumina, Korea) with single-end 50 bp sequencing. The sequence data for the reference genome retrieved from NCBI database was used to align with the quality-filtered reads using Bowtie 2. The genes were clustered into functionally related groups and metabolic pathway using eggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases, respectively. Mapping results and differentially expressed genes were visualized and analyzed, respectively, using CLRNASeq™ Program (ChunLab, Korea). The genetic alterations of intracellular Brucella were determined by comparison with that of the gene transcription levels of free-living Brucella. The microarray analysis was performed using three biological replicates from each infection and control time point using different preparations of cells and the fold changes of genes were calculated from three different samples. 

Results

Analysis of RNA quality

Intracellular B. abortus total RNA from B. abortus-infected MDBK epithelial cells were successfully isolated using the present protocol. RNA integrity was assessed both on agarose gel and spectrophotometry. Electrophoresis on a denaturing agarose gel showed distinct bands of 23S, 16S and 5S rRNA indicating that the total RNA of B. abortus was intact and spectrophotometric analysis revealed an OD260/OD280 ratio of >1.8 indicating that the RNA samples were of superior quality suitable for microarray analysis.

Determination of bacterial gene regulation during the course of infection

Brucella was demonstrated to achieve an intensive replication from 24-48 h post infection after reaching endoplasmic reticulum (ER)-like compartment 14, hence the transcriptional profile of Brucella at the replicative phase (24 h post-infection) was investigated to better understand the pathogen’s mechanisms of infection within a non-phagocytic bovine kidney cell line, MDBK. The microarray analysis of the bacterial transcripts with at least 2-fold changes revealed that the response of Brucella during its infection within host cells was repression of most of its genes. These differentially expressed transcripts were consisted of 788 (25.44%, 788/3098) upregulated and 835 (26.95%, 835/3098) down-regulated genes as compared with free-living Brucella. The products of most highly expressed and repressed genes were N-formylglutamate amidohydrolase (B977_RS107740) with a 77.56-fold increase and uncharacterized protein pYV0051 (B977_RS118555) with a 540.66-fold reduction, respectively. The replicative phase of Brucella within MDBK cells suggested down-regulation of most bacterial transcripts as the pathogen’s strategy for its intracellular survival.

Functional analysis of bacterial transcripts

Among the 788 upregulated and 835 down-regulated genes analyzed by microarray assay, 81 upregulated and 185 down-regulated genes were differentially expressed by ≥ 7-fold. Functional analysis showed that these upregulated genes were mostly of unknown function followed by those that are involved in the transcription and in the transport and metabolism of amino acid, carbohydrate and inorganic ion (Figure 1 and Table 1).

ancient-diseases-preventive-remedies-expressed-transcripts

Figure 1: The differentially expressed transcripts of intracellular B. abortus at ≥ 7-fold within MDBK epithelial cells sorted by COG categories. Upregulated and downregulated genes of B. abortus at replicative phase were indicated as white and black bars, respectively. Data represent the average from at least three separated experiments.

Category Accession No. Protein Fold
Energy production and conversion B977_RS116170 Sulfite reductase 7.07
B977_RS114775 Cytochrome c oxidase 10.24
B977_RS118655 Aldehyde dehydrogenase family 2 member B4 15.1
Amino acid transport and metabolism B977_RS106785 Pyruvate decarboxylase 7.79
B977_RS105275 Spermidine/Putrescine ABC transporter substrate-binding protein 7.99
B977_RS115580 NAD-dependent dihydropyrimidine dehydrogenase subunit PreT 8.19
B977_RS107745 Urocanate hydratase 58.76
B977_RS118750 usg family protein 72.86
B977_RS107740 N-formylglutamate amidohydrolase 77.56
Nucleotide transport and metabolism B977_RS107725 S-adenosylhomocysteine deaminase 23.13
Carbohydrate transport and metabolism B977_RS106925 Putative binding protein BruAb2_0484 7.83
B977_RS107770 Sugar ABC transporter substrate-binding protein 8.64
B977_RS105020 ABC transporter permease 9.28
B977_RS106780 5-dehydro-2-deoxygluconokinase 9.72
B977_RS115915 Sugar ABC transporter substrate-binding protein 10.32
Coenzyme transport and metabolism B977_RS116180 Siroheme synthase 1 8.61
Lipid metabolism B977_RS110990 Isobutyryl-CoA dehydrogenase 7.01
B977_RS116925 Methylcrotonoyl-CoA carboxylase beta chain 10
Transcription B977_RS105675 TetR family transcriptional regulator 7.17
B977_RS114710 UPF0301 protein BMEI1454 7.21
B977_RS106620 Transcriptional regulator 7.38
B977_RS114390 Transcription elongation factor GreA 7.48
B977_RS107655 GntR family transcriptional regulator 7.55
B977_RS106775 RpiR family transcriptional regulator 8.19
B977_RS105895 GntR family transcriptional regulator 8.19
B977_RS116080 Fis family transcriptional regulator 9.19
B977_RS105765 MarR family transcriptional regulator 9.56
B977_RS108350 GntR family transcriptional regulator 9.98
B977_RS112035 Hypothetical protein 10.14
B977_RS114380 AbrB family transcriptional regulator 10.8
B977_RS107710 Aldehyde dehydrogenase 11.4
B977_RS115215 Fur family transcriptional regulator 13.43
B977_RS107720 GntR family transcriptional regulator 17.97
Replication, recombination and repair B977_RS118540 Integrase 8.7
B977_RS116150 Methylated-DNA--protein-cysteine methyltransferase 15.41
B977_RS106070 Transposase 23.93
Posttranslational modification, protein turnover, chaperones B977_RS113485 Arginyl-tRNA-protein transferase 11.03
Inorganic ion transport and metabolism B977_RS116520 Sulfate-binding protein 7.63
B977_RS108615 Iron ABC transporter substrate-binding protein 7.91
B977_RS106815 Iron ABC transporter substrate-binding protein 7.99
B977_RS116165 Probable phosphoadenosine phosphosulfate reductase 10.46
B977_RS117800 ATPase 11.93
Secondary metabolites biosynthesis, transport and catabolism B977_RS107730 Histidine ammonia-lyase 47.66
B977_RS107735 Histidine ammonia-lyase 75.49
General functional prediction only (Typically, prediction of biochemical activity) B977_RS105045 ABC transporter D family member 1 7.65
B977_RS110255 Flagellar biosynthesis protein FlgM 7.85
B977_RS111530 Protein DJ-1 homolog D 9.16
B977_RS107210 ABC transporter substrate-binding protein 12.67
B977_RS111260 Inosine-5-monophosphate dehydrogenase 32.33
Signal transduction B977_RS112185 Universal stress protein 10.18
ftrB Transcriptional regulator 15.17
Defense mechanism B977_RS119425 MFS transporter 7.54
Function unknown B977_RS109985 Hypothetical protein 7.17
B977_RS113610 Hypothetical protein 7.26
B977_RS106970 Membrane protein 7.5
B977_RS08290 Hypothetical protein 7.55
B977_RS119015 Hypothetical protein 7.8
B977_RS118615 Ribosomal RNA large subunit methyltransferase H 8.66
B977_RS107110 Hypothetical protein 8.78
B977_RS107905 Nodulation protein NodN 9.45
B977_RS02240 Hypothetical protein 9.51
B977_RS110395 Hypothetical protein 10.57
B977_RS106975 Fusaric acid resistance protein 11.07
B977_RS118610 Ribosomal silencing factor RsfS 11.61
B977_RS112500 Hypothetical protein 11.83
B977_RS112495 Hypothetical protein 12.38
B977_RS118755 Fusaric acid resistance protein FusB 12.83
B977_RS111555 Hypothetical protein 13.27
B977_RS116250 Membrane protein 13.65
B977_RS107750 Hypothetical protein 54.73
Unclassified B977_RS109460 Hypothetical protein 7.22
B977_RS0103625 Hypothetical protein 7.45
B977_RS110120 DNA methyltransferase 7.55
B977_RS106065 Transposase 7.86
B977_RS115400 Hypothetical protein 8.06
B977_RS110910 Hypothetical protein 8.15
B977_RS0104155 Hypothetical protein 11.17
B977_RS11365 Hypothetical protein 11.96
B977_RS105565 Hypothetical protein 14.15
B977_RS02335 Hypothetical protein 19.83
B977_RS13980 Hypothetical protein 25.12

Table 1: B. abortus gene expression induced at 24 h post infection within MDBK cells.

Several of these genes were unclassified. On the other hand, the genes that appeared to repress were mostly involved in transcription, translation, energy production and conversion, and amino acid metabolism and transport (Figure 1 and Table 2). Similarly, several products of these highly repressed genes have unknown function and few were unclassified. Changed expression patterns of hypothetical genes were also observed which indicates that they may play important roles in the survival of Brucella within epithelial cells. Down-regulation of several ribosomal genes and RNA polymerase indicates amino acid starvation possibly due to poor nutritional intra-vacuolar microenvironment of Brucella. As a response, highly expressed genes that encode for transcriptional regulators are involved in regulation of virulence genes or bacterial responses to environmental stress. The products of the most highly expressed genes (B977_RS107745, B977_RS118750, B977_RS107735 and B977_RS107740) were involved in the metabolism and transport of amino acid suggesting the need for amino acids in the synthesis of proteins during the replicative phase; however, the products of the most highly repressed genes (B977_RS114825, B977_RS114830, rplT, B977_RS114840, B977_RS117320 and B977_RS113155) were involved in the translation, ribosomal structure and biosynthesis indicating that the need for amino acids could be primarily as sources for carbon, energy and/or nitrogen. These findings indicate the regulation of Brucella genes as a mechanism for metabolic adaptation in response to changes in the nutritional environment for optimum utilization of the nutrients available in infected epithelial host cells.

Category Accession No. Protein Fold
Energy production and conversion B977_RS116810 Cytochrome o ubiquinol oxidase subunit III 8.35
B977_RS113220 NADH:ubiquinone oxidoreductase subunit M 9.14
B977_RS118220 Dihydrolipoamide succinyltransferase 9.22
B977_RS118200 Malate dehydrogenase 9.44
B977_RS105455 NADPH quinone oxidoreductase 10.61
B977_RS118810 ATP synthase epsilon chain 11.14
B977_RS113225 NADH:ubiquinone oxidoreductase subunit L 11.71
B977_RS118210 Succinyl-CoA synthetase subunit alpha 11.8
B977_RS118800 ATP synthase subunit gamma 16.57
sucA 2-oxoglutarate dehydrogenase E1 component 16.94
sucC Succinyl-CoA ligase subunit beta 17.43
B977_RS113060 Sorbitol dehydrogenase 18.35
B977_RS111520 Isocitrate dehydrogenase 18.51
B977_RS118795 ATP synthase subunit alpha 19.81
B977_RS117910 Inorganic pyrophosphatase 20.3
B977_RS118805 ATP synthase subunit beta 24.39
B977_RS118790 ATP synthase subunit delta 30.56
Amino acid transport and metabolism B977_RS105460 Leu/Ile/Val-binding protein homolog 8 7.08
B977_RS110215 Aspartate aminotransferase A 7.34
B977_RS109890 Anthranilate synthase 7.35
B977_RS113990 4-hydroxy-tetrahydrodipicolinate synthase 7.35
B977_RS116670 Argininosuccinate synthase 8.42
B977_RS116890 3-phosphoshikimate 1-carboxyvinyltransferase 10.03
B977_RS110705 Ketol-acid reductoisomerase 11.97
B977_RS108680 Ornithine decarboxylase 13.81
B977_RS115505 Acetylornithine aminotransferase 20.51
B977_RS117485 Imidazole-4-carboxamide isomerase 7.93
Nucleotide transport and metabolism B977_RS112190 Xanthine phosphoribosyltransferase 8.37
B977_RS113075 Adenylosuccinate lyase 9.93
B977_RS105375 Inosine-5'-monophosphate dehydrogenase 11.23
nrdI Protein NrdI 11.26
ndk Nucleoside diphosphate kinase 40.21
Carbohydrate transport and metabolism B977_RS115180 MFS transporter 7.84
gapA Glyceraldehyde-3-phosphate dehydrogenase B 9.78
B977_RS104685 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase 11.95
B977_RS108745 2-dehydro-3-deoxy-phosphogluconate aldolase 13.47
B977_RS119075 Transketolase 13.8
B977_RS119145 Inositol-1-monophosphatase 20.59
Coenzyme transport and metabolism B977_RS116010 Putative thiamine biosynthesis oxidoreductase ThiO 7.06
B977_RS116005 Phosphomethylpyrimidine kinase 7.28
B977_RS116025 Thiamine-phosphate synthase 7.315
B977_RS116015 Hypothetical protein 8.56
B977_RS117135 S-adenosylmethionine synthase 8.77
B977_RS113825 4-hydroxythreonine-4-phosphate dehydrogenase 9.19
B977_RS117430 Adenosylhomocysteinase 18.23
Lipid metabolism B977_RS105125 Hypothetical protein 7.12
B977_RS114805 3-oxoacyl-ACP synthase 7.31
Translation, ribosomal structure and biosynthesis B977_RS108305 Histidine--tRNA ligase 7.01
B977_RS113850 Cysteine--tRNA ligase 7.11
B977_RS110045 Peptidyl-tRNA hydrolase 7.32
gatB Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit B 7.37
B977_RS111420 30S ribosomal protein S14 7.62
B977_RS112905 Serine--tRNA ligase 7.73
B977_RS111465 30S ribosomal protein S13 7.88
B977_RS116355 Tryptophan--tRNA ligase 7.89
B977_RS111750 Glutamate--tRNA ligase 1 8.02
B977_RS111425 30S ribosomal protein S8 8.11
B977_RS112635 Valine--tRNA ligase 8.68
rpmE 50S ribosomal protein L31 9.39
B977_RS112425 Methionine--tRNA ligase 9.86
B977_RS111430 50S ribosomal protein L6 10.15
B977_RS111405 50S ribosomal protein L14 10.67
B977_RS111395 50S ribosomal protein L29 11.1
B977_RS110050 50S ribosomal protein L25 11.46
B977_RS118565 50S ribosomal protein L21 11.47
rpmH 50S ribosomal protein L34 11.83
B977_RS111435 50S ribosomal protein L18 11.86
B977_RS111410 50S ribosomal protein L24 13.71
B977_RS118900 50S ribosomal protein L32 14.04
B977_RS118570 50S ribosomal protein L27 14.08
B977_RS111470 30S ribosomal protein S11 14.47
B977_RS111440 30S ribosomal protein S5 14.51
B977_RS111445 50S ribosomal protein L30 14.8
B977_RS111450 50S ribosomal protein L15 14.84
B977_RS117005 30S ribosomal protein S20 15.01
B977_RS111415 50S ribosomal protein L5 15.52
B977_RS117330 Translation initiation factor IF-3 15.88
B977_RS111380 50S ribosomal protein L22 16.01
B977_RS111400 30S ribosomal protein S17 16.62
B977_RS106330 50S ribosomal protein L33 16.81
B977_RS111685 Elongation factor Ts 17.1
B977_RS104815 Ribonuclease P protein component 18.43
B977_RS111390 50S ribosomal protein L16 18.86
B977_RS118575 N-acetyltransferase GCN5 19.74
B977_RS111385 30S ribosomal protein S3 21.26
B977_RS111340 30S ribosomal protein S7 21.3
B977_RS111375 30S ribosomal protein S19 21.65
B977_RS111310 50S ribosomal protein L10 21.93
B977_RS111360 50S ribosomal protein L4 22.05
B977_RS117815 50S ribosomal protein L28 22.54
rplB 50S ribosomal protein L2 23.31
B977_RS111365 50S ribosomal protein L23 23.86
B977_RS111355 50S ribosomal protein L3 24.36
B977_RS111335 30S ribosomal protein S12 24.77
B977_RS118690 30S ribosomal protein S16 24.95
rpsJ 30S ribosomal protein S10 25.37
B977_RS119150 Elongation factor P 25.49
B977_RS111480 50S ribosomal protein L17 26.7
B977_RS111315 50S ribosomal protein L7/L12 27.93
tuf Elongation factor Tu 1 28.07
fusA Elongation factor G 28.2
B977_RS106125 Ribosomal protein S12 methylthiotransferase 28.59
B977_RS117090 Polyribonucleotide nucleotidyltransferase 34.5
B977_RS112765 Peptide chain release factor 2 39.36
B977_RS113155 30S ribosomal protein S4 43.72
B977_RS117320 50S ribosomal protein L35 44.78
B977_RS114840 50S ribosomal protein L9 53.78
rplT 50S ribosomal protein L20 64.9
B977_RS114830 30S ribosomal protein S18 103.4
B977_RS114825 30S ribosomal protein S6 206.57
Transcription B977_RS111325 DNA-dependent RNA polymerase subunit beta 8.5
B977_RS111320 DNA-directed RNA polymerase subunit beta 8.97
B977_RS118580 N-acetyltransferase GCN5 11.86
B977_RS117585 Transcription termination factor Rho 13.54
B977_RS110175 Transcription elongation factor GreA 14.46
B977_RS110225 Cold shock protein CspA 14.84
B977_RS119120 Probable transcriptional regulatory protein BQ11790 15.22
B977_RS105450 HTH-type transcriptional regulator McbR 17
B977_RS111475 DNA-directed RNA polymerase subunit alpha 24.74
B977_RS110400 ArsR family transcriptional regulator 7.78
Replication, recombination and repair B977_RS106085 Transposase 13.56
B977_RS112160 ATP-dependent RNA helicase-like protein DB10 32.45
B977_RS116440 DEAD-box ATP-dependent RNA helicase CshB 33.82
B977_RS119480 Transposase 41.19
B977_RS109550 Transposase 41.19
B977_RS118555 Integrase 540.69
Cell wall/membrane/envelope biogenesis B977_RS112080 D-alanyl-D-alanine carboxypeptidase DacF 7.51
B977_RS115700 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 9.31
B977_RS113065 Lauroyl acyltransferase 12.22
B977_RS115385 Antiholin-like protein LrgB 16.34
B977_RS110180 Lipopolysaccharide core biosynthesis mannosyltransferase LpcC 19.18
B977_RS117240 Hypothetical protein 110.41
B977_RS110440 Lytic transglycosylase 209.1
B977_RS107825 Hypothetical protein 76.47
Posttranslational modification, protein turnover, chaperones B977_RS105230 Glutaredoxin 8.56
B977_RS108265 Molecular chaperone GroES 11.06
groEL Molecular chaperone GroEL 11.23
B977_RS118135 Peptidylprolyl isomerase 12.87
B977_RS117325 Isoprenylcysteine carboxyl methyltransferase 19.18
B977_RS111980 Peptidyl-prolyl cis-trans isomerase 10.44
       
Inorganic ion transport and metabolism B977_RS116030 ABC transporter ATP-binding protein 8.6
B977_RS113140 Bcr/CflA family drug resistance efflux transporter 8.62
B977_RS107495 Magnesium transporter MgtE 29.63
General functional prediction only (Typically, prediction of biochemical activity) B977_RS106165 Invasion protein B 7.87
B977_RS117130 tRNA (guanine-N(7)-)-methyltransferase 8.11
B977_RS119285 Glutamine amidotransferase 10.19
B977_RS111585 NADPH-dependent 7-cyano-7-deazaguanine reductase 12.44
B977_RS115390 Murein hydrolase transporter LrgA 20.08
Signal transduction B977_RS107490 GTP-binding protein TypA 14.79
Intracellular trafficking and secretion B977_RS104810 Membrane protein insertase YidC 7.35
B977_RS111795 Preprotein translocase subunit SecG 10.69
B977_RS118130 Protein translocase subunit SecA 10.92
Defense mechanisms B977_RS116055 Hypothetical protein 25.6
B977_RS116050 ABC transporter 28.18
B977_RS116060 ABC transporter 35.29
Function unknown B977_RS117915 Uncharacterized protein Atu26591 7.57
B977_RS108245 Hypothetical protein 7.77
B977_RS113300 Hypothetical protein 8.13
B977_RS106190 Hypothetical protein 8.16
B977_RS110265 Aspartyl-tRNA amidotransferase subunit B 8.26
B977_RS115170 Polysaccharide deacetylase 8.3
B977_RS119280 Membrane protein 9.18
B977_RS119040 Hypothetical protein 9.28
B977_RS118225 Membrane protein 9.52
B977_RS107895 Hypothetical protein 9.57
B977_RS110130 Hypothetical protein 9.67
B977_RS109645 Hypothetical protein 9.88
B977_RS110160 Membrane protein 10.04
B977_RS112355 Hypothetical protein 10.32
B977_RS114835 Hypothetical protein 10.35
B977_RS113120 Hypothetical protein 10.87
B977_RS114075 AP endonuclease 10.98
B977_RS119390 Hypothetical protein 11.05
B977_RS118480 Hypothetical protein 11.26
B977_RS119290 Capsular polysaccharide biosynthesis protein J 12.98
B977_RS117125 Ribosome maturation factor RimP 16.24
B977_RS116645 Hypothetical protein 17.34
B977_RS107330 Hypothetical protein 21.32
B977_RS117310 Serine/threonine protein kinase 34.75
B977_RS111680 30S ribosomal protein S2 9.04
Unclassified B977_RS111105 Hypothetical protein 9.11
B977_RS01000000119490 Hypothetical protein 16.27
B977_RS116650 Hypothetical protein 23.58

Table 2: B. abortus gene expression repressed at 24 h post infection within MDBK cells.

Discussion

Intracellular bacterial pathogens are equipped with specific virulence genes regulated at the transcriptional level that aid in their ability to survive and replicate within host cells [17]. The pathogenicity of Brucella is due to its ability to adapt to its environmental conditions encountered, hence understanding the bacterial response and the molecular characterization of the pathogen’s intracellular survival process would provide guidance for subsequent development of new therapeutic agents against brucellosis since infectious diseases are a major health threat representing the main cause of mortality worldwide [17,18]. However, most of the published studies primarily described the host response to infection. Consequently, a powerful and promising approach to identify the bacterial response at the transcriptional level is through microarray techniques.

Bacterial regulation of gene expression is a fundamental requirement for physiological adaptation; hence, the response of Brucella in its intracellular replicative niche at 24 h post infection at the transcriptional level was investigated and showed that the pathogen transcriptional profile with at least 2-fold changes was slightly downregulated. These findings were different from the response of B. abortus within professional phagocytes–both in RAW 264.7 cells and a primary cell line BMDM cells where most of the differentially expressed genes were upregulated revealing a unique mechanism of this pathogen to survive and proliferate in a different non-phagocytic host cell line. Furthermore, the present study showed higher rate of both upregulated and downregulated genes as compared to previous studies on phagocytes. Interestingly, few of these downregulated genes were previously reported as potential vaccine candidate against brucellosis which include nucleoside diphosphate kinase (Ndk), 50s ribosomal protein L7/L12 (RpIL) and phosphoglycerate kinase (Pgk) [19-21]. Many of these differentially regulated genes with ≥ 7-fold change are known to respond to stress such as amino acid starvation as a consequence of poor nutritional intra-vacuolar microenvironment of the pathogen. The most highly expressed genes found in the present study were associated with the transport and metabolism of amino acid suggesting the need for amino acids during the replicative phase of Brucella within MDBK epithelia cells. A wide range of microbial species utilize histidine as both a carbon and nitrogen source, and the N-formylglutamate amidohydrolase is known to catalyze the terminal reaction in the five-step pathway for histidine utilization in Pseudomonas putida [22]. The usg family protein may function as a subunit or stabilizer of phosphoribosylanthranilate isomerase (trpF) that catalyzes the fourth step of tryptophan biosynthesis which is important precursor to a large number of complex microbial natural products [23,24]. Polyamines, particularly spermidine and putrescine, have been associated with bacterial virulence and pathogenicity in human pathogens and in B. ovis, the defective uptake of spermidine and putrescine was contributed to the possible lack of bacterial virulence in humans [25]. Taken together, this suggests that Brucella is actively using the host amino acid for virulence and survival possibly as sources of carbon and/or nitrogen.

Brucella has been found to be able to survive and replicate within membrane-bound compartments of non-professional phagocytes, and few Brucella genes have already been identified such as the type IV secretion system (T4SS), which is encoded by the virB operon that has been found to be involved in the maturation process of Brucella-containing vacuoles (BCVs) constituting a major determinant of Brucella virulence in HeLa cells [26]. Similarly, we observed upregulation of virB genes (virB1 to virB11; data not shown) although virB1 to virB7 were observed to increase at ≥ 2-fold indicating that they play an important role in the intracellular survival of the pathogen in MDBK epithelial cells. The expression of the T4SS-encoding virB operon is regulated by multiple transcription factors belonging to different families including a MarR-Type regulator [27] which is similar to our study where a high expression of MarR family transcriptional regulator (10.8-fold) was observed. Bacterial survival in environment exposed to variations in nutrient availability and presence of toxic molecules requires a wide range of rapid, adaptive responses triggered by regulatory proteins [28]. Many of the upregulated genes in this study were involved in the transcription. Particularly, the transcriptional regulators belonging to MarR family of transcription factors aid in the regulation of bacterial responses to environmental stress [27]. Several transcriptional regulators belonging to TetR, GntR, RpiR, Fis, AbrB, Fur families were observed to increase at ≥ 7-fold. Proteins of the TetR family are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes and pathogenicity [28,29]. The transcriptional regulator gene, gntR, has been shown to play an important role in the control of B. melitensis virulence, and the GntR regulators to play important roles in maintenance of fatty acids concentrations, amino acid catabolism, organic acids, regulation of carbon catabolism and degradation of complex organics, as well as reported to affect expression of T4SS and quorum sensing system (QSS) in Brucella during infection in macrophages [29]. Attenuated six mutants that belong to the GntR family were identified and confirmed to replicate at lower rates in murine BALB/c model, while only one was slightly attenuated in HeLa cells indicating their role in Brucella virulence and proposed that GntR4 was a direct or indirect activator of virB transcription [30]. A conserved transcriptional regulator Fis was reported to be involved in the virulence of Dickeya zeae which is a causal agent of rice foot rot disease [31]. Binding of metal ions to DNA is typically required by Fur family proteins to exert their transcriptional regulatory activities such as utilizing iron as a co-repressor and represses siderophore synthesis in pathogens that directly or indirectly controls expression of enzymes that protect against toxic reactive oxygen species (ROS) damage [32,33].

The interaction between the pathogen and host includes uptake and secretion of substances facilitated by a family of transporters. The ATP-binding cassette (ABC) transporters use a variety of substrates such as amino acids, sugars, inorganic ions, polysaccharides, peptides and proteins [34] and several ABC transporters have been associated to the full virulence and survival of Brucella in vitro and in vivo, hence the increase in the transcription of genes that encode for these proteins could help in the virulence and survival of Brucella. MFS transporter was also observed to increase which functions as a secondary carrier that transports small solutes in response to chemiosmotic ion gradients and its upregulation may be important for the adaptation of Brucella in the ionic intracellular environment [12]. Furthermore, several transcripts for hypothetical protein were observed to increase suggesting their important roles in the survival of B. abortus in epithelial host cells, thus impose special attention to be studied further.  

Conclusion

In summary, the present microarray analysis identified 81 and 185 genes that were upregulated and down-regulated at ≥ 7-fold, respectively in B. abortus within MDBK epithelial cells at 24 post-infection as compared with free-living Brucella, showing a marked induction of genes with unknown function followed by those involved in transcription while a drastic repression of genes observed were involved in translation, ribosomal structure and biosynthesis followed by those whose products were of unknown function. Since renal involvement during Brucella infection is uncommon, the identified genes in the present study could provide additional insights on the strategy of B. abortus in their virulence and survival within non-phagocytic cells. Furthermore, genes with unknown function that represent 22% of the differentially highly expressed genes at >7-fold deserve some special attention as this group might contain some potential unknown virulence factors utilized by Brucella in their survival within the host.

Conflict of Interest

The authors have no conflict of interest to declare.

Acknowledgement

This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Korea (HI16C2130).

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Citation: Hop TH, Reyes AWB, Arayan LT, Ngoc Huy TX, Hai Vu S, et al. (2018) A Microarray-Based Analysis of Differentially Expressed Genes in Intracellular Brucella abortus 544 within Mdbk Cells. J Infect Dis Preve Med 6: 181.

Copyright: © 2018 Hop HT, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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