ISSN: 2329-8731
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Research Article - (2018) Volume 6, Issue 2
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
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.
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.
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).
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.
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.
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.
The authors have no conflict of interest to declare.
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).