Mycobacterium Tuberculosis Mce1 Protein Complex Initiates Rapid Induction of Transcription of Genes Involved in Substrate Trafﬁcking

ORIGINAL ARTICLE Mycobacterium tuberculosis Mce1 protein complex initiates rapid induction of transcription of genes involved in substrate trafﬁcking

R Stavrum1, H Valvatne1,8, A-K Stavrum2,3, LW Riley4, E Ulvestad5, I Jonassen3,6, TM Doherty7,9 and HMS Grewal1,5

The mammalian cell entry (Mce)1 protein complex has an important role during the initial phase of a Mycobacterium tuberculosis (M. tuberculosis) infection. Murine macrophages were infected with M. tuberculosis H37Rv or D-mce1 H37Rv, and total RNA was isolated from the host cells at 15, 30 and 60 min, and 4 and 10 h post-infection. With the aim of studying the role for the Mce1 protein complex on host gene expression, the RNA was hybridized onto 44 K whole-genome microarrays. Selected genes were verified by reverse-transcriptase quantitative PCR (RT-QPCR). ‘Transport’ was the most overrepresented biological process during the first hour post H37Rv infection. Five genes (Abca1 (21.0-fold), Slc16a10 (3.1-fold), Slc6a12 (17.9-fold), Slc6a8 (2.3-fold) and Nr1h3, (5.5-fold)) involved in substrate trafficking were verified by RT-QPCR to be upregulated by 42-fold 1 h post H37Rv infection. By 1 h post D-mce1 H37Rv infection, only Abca1 and Slc6a12 were upregulated by 42-fold. A number of other genes, which may be directly involved in substrate trafficking or share the same transcription, were found to have expression profiles similar to the genes involved in substrate trafficking. The Mce1 protein complex has a significant role in the transcriptional activation of genes involved in substrate trafficking during the initial phase of an M. tuberculosis infection.

Genes and Immunity (2012) 13, 496–502; doi:10.1038/gene.2012.24; published online 14 June 2012 Keywords: M. tuberculosis; Mce 1; host response; substrate trafﬁcking

INTRODUCTION has also recently been shown that the mce1 operon, in the The initial interactions between the cell wall of a pathogen and M. bovis BCG strain, is an important component in the switch from structures on the surface of macrophages may be critical in slow to fast growth, suggesting an involvement in the transition 7 determining the outcome of an infection. A Mycobacterium between acute and persistent infection. Previous studies have tuberculosis (M. tuberculosis) DNA fragment cloned into a non- shown that the M. tuberculosis Mce1 protein complex facilitates pathogenic Escherichia coli bacterium has been shown to confer the invasion of the bacterium into host cells, possibly through 3 ability to invade HeLa cells, augment macrophage phagocytosis cholesterol-dependent clathrin-coated pits, and that infection and survive for at least 24 h inside the human macrophage.1 This with an Mce1-deficient M. tuberculosis strain fail to induce a DNA fragment belonged to a gene termed mammalian cell entry protective pro-inflammatory response, causing a defective (mce) 1 and was shown, by genomic analyses, to be a member of granuloma formation with subsequent uncontrolled replication 6 an operon consisting of six mce1 genes (mce1A–mce1F) and two of the bacterium. However, more recent studies show that the 8 genes, yrbE1A and yrbE1B, coding for two integral membrane mce operons encode for lipid uptake transporters and that the proteins, preceding the Mce1 protein.2 All six Mce1 proteins mce1 operon is involved in fatty acid transport/metabolism; and 9 localize to the cell wall fraction of M. tuberculosis, and Mce1A is more specifically, mycolic acid recycling. exposed on the surface of M. tuberculosis.3 Further analyses of the In a previous study, we showed that a macrophage infection by M. tuberculosis genome showed that M. tuberculosis contains four a wild-type M. tuberculosis H37Rv strain resulted in the differential copies of the mce operon (mce1-4) organized in a similar manner transcription of a large number of genes, by the macrophage, as to mce1. The genes within the mce operons 1–4 have been shown early as 15 min post-infection, whereas the deletion of the Mce1 to be conserved in all members of the M. tuberculosis complex, the protein complex from the M. tuberculosis H37Rv strain, (M. Mycobacterium avium complex and Mycobacterium scrofolaceum, tuberculosis D-mce1 H37Rv) delayed the uptake of the bacteria except mce3, which is absent from Mycobacterium bovis, into the host macrophage, in addition to inducing an anti- Mycobacterium microti, Mycobacterium pinnipedii and a subgroup inflammatory response by 4 h post-infection.10 For the current of Mycobacterium africanum.4,5 The mce1 operon has been manuscript, the focus was shifted to genes showing sustained suggested to be involved in modulating the host inflammatory effects. Thus, only genes which showed altered transcription over response in such a way that the bacterium can enter a persistent all three time points during the first hour post-infection were state without being eliminated or causing disease in the host.6 It analyzed further. Although, by using this strategy, genes whose

1Department of Microbiology and Immunology, The Gade Institute, University of Bergen, Bergen, Norway; 2Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Department of Clinical Medicine, Bergen, Norway; 3Department of Informatics, University of Bergen, Bergen, Norway; 4Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, CA, USA; 5Department of Microbiology, Haukeland University Hospital, Bergen, Norway; 6Computational Biology Unit, Uni Computing, Uni Research AS, Bergen, Norway and 7Department of Infectious Disease Immunology, Statens Serum Institute, Copenhagen, Denmark. Correspondence: Dr R Stavrum, Department of Microbiology and Immunology, The Gade Institute, University of Bergen, Bergen 5021, Norway. E-mail: [email protected] 8Current address: Intervet International B.V.,Wim de Ko¨rvestraat 35, PO Box 31 5830 AA Boxmeer, The Netherlands. 9Current address: GlaxoSmithKline, Brøndby, Copenhagen, Denmark. Received 22 February 2012; revised and accepted 24 April 2012; published online 14 June 2012 Host response to M. tuberculosis infection R Stavrum et al 497 transcription was highly (but transiently) modulated (upregulated differentiation’ (P ¼ o0.001) were the most overrepresented and/or downregulated) post-infection were lost, we were able to biological processes. ‘Immunity and defense’ (Po0.001) and identify genes that were stably transcribed for all three time- ‘Cytokine and chemokine-mediated signaling pathway’ points during the first hour and thus, determine the most (Po0.001) were the most overrepresented biological processes important biological processes during the very early stages of at 10 h post H37Rv infection. an M. tuberculosis H37Rv infection. As most proteins require a A total of 19 genes, belonging to the biological process network of other proteins to exert a biological function, ‘Transport’, were upregulated during the first hour post H37Rv information about the time of gene transcription and infection as compared with the uninfected controls. Of these 19 transcriptional co-regulation may generate knowledge on which genes, 63.2% (12 out of 19) were also upregulated at 4 h and/or proteins interact to coordinate a biological function.11 Thus, the 10 h post-infection, whereas 36.8% (7 out of 19) were only putative role for the Mce1 protein complex on the early biological upregulated during the first hour post-infection (data not shown). processes post-infection was determined, including information on the co-transcription of genes. RT-QPCR verification Relative quantification (reverse-transcriptase quantitative PCR RESULTS (RT-QPCR)) of gene transcription was performed on RNA isolated Microarray analysis from uninfected macrophages, and on RNA isolated from macro- To determine which genes were consistently upregulated, by the phages at the 15, 30 and 60 min, and 4 and 10 h time-points post macrophage, during the first hour post-infection by M. tuberculosis H37Rv infection, and D-mce1 H37Rv infection for the seven genes H37Rv, the rank product (RP) lists for the time-points 15, 30 and meeting the pre-defined criteria. This was done to verify that the 60 min were inspected, and only the genes which were present in transcriptional level of the seven genes belonging to the biological all the three lists were selected for further analysis. Functional process ‘Transport’ could indeed discriminate between the first hour classification of upregulated genes that were mapped by the post H37Rv infection and the two latter time points post H37Rv Panther Classification System 6.1 was undertaken for the three RP infection, 4 and 10 h, in addition to measuring the transcriptional lists comprising genes that were upregulated during the first hour, level of these seven genes post D-mce1 H37Rv infection. RT-QPCR and at 4 and 10 h post H37Rv infection. The biological processes analysis showed that five out of the seven (Abca1 (18.5-fold), that were overrepresented post H37Rv infection for each of the Slc16a10 (2.3-fold), Slc18a1 (1.7-fold), Slc23a2 (0.8-fold), Slc6a12 (18.0- lists are shown in Table 1a. fold), Slc6a8 (2.5-fold) and Nr1h3 (4.0-fold)) genes that were analyzed During the first hour post H37Rv infection, six biological were upregulated by two-fold or more at 15 min post H37Rv processes met the criteria for being classified as significantly infection as compared with the uninfected controls. By 15 min post overrepresented. These were Transport (P ¼ 0.002), Cell commu- D-mce1 H37Rv infection, only one gene, Slc6a12 was upregulated nication (P ¼ 0.009), Immunity and defense (P ¼ 0.009), Cell (2.4-fold; Table 1b). The most upregulated gene during the first hour adhesion (P ¼ 0.007), Cell motility (P ¼ 0.005) and Steroid hormone post H37Rv infection and post D-mce1 H37Rv infection, was the metabolism (P ¼ 0.004). The biological process ‘Transport’ was the gene encoding the cholesterol efflux pump Abca1,whichshowedan most overrepresented biological processes during the first hour average fold change of 21.0 and 3.6, respectively, as compared with post H37Rv infection comprising 11.7% (19 out of 162) of the the uninfected reference (Table 1b). number of genes in the search list. At 4 h post H37Rv infection Analyses showed that there was no significant mean difference ‘Cell communication’ (Po0.001) and ‘Cell proliferation and in level of transcription within the first three time points

Table 1a. Overrepresented biological processes among genes upregulated in the J774A.1 Mj during the ﬁrst, fourth and tenth hour following infection with Mycobacterium tuberculosis H37Rv

Biological process Reference (n ¼ 18950) First hour (n ¼ 162) 4 h (n ¼ 241) 10 h (n ¼ 249)

%%P-value % P-value % P-value

Biological process unclassiﬁed 34.9 25.9 0.009 29.0 0.032 26.5 0.003 Transport 6.1 11.7 0.002 8.7 0.065 7.2 0.264 Cell communication 5.6 10.5 0.009 11.6 o0.001 11.2 o0.001 Ligand-mediated signaling 1.8 3.7 0.080 3.3 0.080 4.8 0.003 Amino acid transport 0.2 0.6 0.320 1.2 0.020 1.6 0.003

Immunity and defense 6.5 11.7 0.009 11.2 0.004 16.1 o0.001 Cytokine/chemokine mediated immunity 0.5 2.5 0.010 2.1 0.009 2.0 0.010 B-cell- and antibody-mediated immunity 0.5 1.2 0.183 1.7 0.030 2.4 0.001 Granulocyte-mediated immunity 0.4 1.9 0.021 2.1 0.002 2.0 0.002 MHCII-mediated immunity 0.1 0.0 0.836 0.4 0.234 1.2 0.003 Macrophage-mediated immunity 0.6 1.9 0.081 2.1 0.018 3.2 o0.001 Detoxiﬁcation 0.4 1.2 0.133 1.2 0.069 2.0 0.003 Blood clotting 0.4 1.2 0.144 1.2 0.079 2.0 0.004

Signal transduction 18.4 21.6 0.168 23.2 0.034 24.5 0.010 Cytokine and chemokine mediated signaling pathway 1.1 3.7 0.011 3.3 0.007 4.8 o0.001 Cell adhesion 2.8 6.8 0.007 6.6 0.002 6.4 0.002 Cell proliferation and differentiation 4.3 8.6 0.010 9.5 o0.001 6.8 0.040 Cell structure and motility 4.9 9.3 0.014 9.5 0.002 8.8 0.006 Proteolysis 4.7 7.4 0.080 8.3 0.010 10.0 o0.001 Cell motility 1.6 4.9 0.005 4.6 0.002 5.2 o0.001 Steroid hormone metabolism 0.2 1.9 0.004 0.4 0.384 0.0 0.607

& 2012 Macmillan Publishers Limited Genes and Immunity (2012) 496 – 502 Host response to M. tuberculosis infection R Stavrum et al 498 Table 1b. Genes belonging to the most signiﬁcant biological process ‘Transport’ induced by the infection of J774A.1 Mj with M. tuberculosis H37Rv during the ﬁrst hour post-infection

Agilent probe ID Gene Molecular function Post H37Rv infection Post D-mce1 H37Rv infection name 15 min 30 min 60 min 4 h 10 h 15 min 30 min 60 min 4 h 10 h

A_52_P665675 Abca1 Cholesterol efflux 18.5 19.9 24.7 8.4 4.0 1.8 3.4 5.7 5.3 8.5 activity A_52_P527924 Slc16a10 Na( þ )-independent 2.3 3.3 3.7 1.3 1.4 0.5 1.1 1.3 0.3 0.2 transport of aromatic amino acids across the plasma membrane A_52_P212388 Slc18a1 Vesicular monoamine 1.7 3.3 2.1 0.3 0.4 0.3 0.6 0.5 1.3 3.1 transporter A_52_P286520 Slc23a2 Sodium-dependent 0.8 0.9 0.4 0.7 0.3 0.0 0.4 0.7 0.8 0.9 vitamin C transporter-2 A_51_P323812 Slc6a12 Cation transmember 18.0 17.9 17.8 0.0 0.0 2.4 2.2 1.7 0.0 2.4 transporter A_51_P450829 Slc6a8 Creatine transporter 1 2.5 2.1 2.2 1.6 2.9 0.3 0.7 1.8 1.0 2.2 A_52_P546421 Nr1h3 Oxysterols receptor LXR- 4.0 7.9 4.5 7.5 4.5 0.6 0.2 6.2 2.4 8.5 alpha involved in cholesterol homeostasis Genes highlighted in bold were significantly upregulated during the first hour post M. tuberculosis H37Rv infection compared with 4 and 10 h post infection. The relative quantification reflects the fold-change in gene transcription for each time point post-infection compared with the uninfected samples, as measured by reverse-transcriptase quantitative PCR.

(15, 30 and 60 min) and the last two time points (4 and 10 h) post significant mean difference (Po0.0001) in the transcriptional level H37Rv infection and D-mce1 H37Rv infection, suggesting that for all the genes analyzed, except Slc23a2 (P ¼ 0.4; Table 2). From measurements within the first hour (15,30 and 60 min) and the last the comparison between the ‘late’ H37Rv infection and the ‘late’ two time points (4 and 10 h) could be pooled, reducing the D-mce1 H37Rv infection, only Slc16a10 (1.4- vs 0.3-fold (P ¼ 0.008)) potential for minor variation between different time points. Thus, showed a significant mean difference between the two groups for the statistical analyses, the RT-QPCR results for the first three (Table 2). time points (15,30 and 60 min) were combined into one group As previously reported,10 the deletion of the mce1 operon caused termed ‘early’ for each of the infections (post H37Rv infection and a delay in the uptake of the M. tuberculosis D-mce1 H37Rv strain by post D-mce1 H37Rv infection), and the results for the last two time the macrophage. Thus, to see if this delay could be seen at the points (4 and 10 h) were combined into one group termed ‘late’ transcriptional level, the RT-QPCR results from the ‘late’ D-mce1 for each of the infections (post H37Rv infection and post D-mce1 H37Rv-infection group were compared with the RT-QPCR results H37Rv infection). from the ‘early’ H37Rv-infection group. The results from this For the purpose of identifying genes that could discriminate comparison showed that the Abca1 (21.0- vs 6.9-fold (Po0.0001)), between different stages post-infection (uninfected, ‘early’ and Slc16a10 (3.1- vs 0.3-fold (Po0.0001)), and Slc6a12 (17.9- vs 1.2-fold ‘late’), the RT-QPCR results from the uninfected macrophages were (Po0.0001)) genes showed significant mean difference at the compared with the RT-QPCR results of the ‘early’ and ‘late’ infection transcriptional level between these two groups, indicating that the groups for both H37Rv and D-mce1 H37Rv infection. RT-QPCR differences were not merely a consequence of the time delay. results showed that for all seven genes (Abca1 (21.0-fold), Slc16a10 (3.1-fold), Slc18a1 (2.7-fold), Slc23a2 (0.7-fold), Slc6a12 (17.9-fold), Slc6a8 (2.3-fold) and Nr1h3 (5.5-fold)) there was a significant mean Expression profile similarity analysis difference (Po0.0001) in the level of transcription between the A gene expression profile similarity search was performed, in uninfected macrophages and the macrophages during the ‘early’ which the transcriptional profile for each of the seven genes that time points post H37Rv infection. Only Abca1 (6.0-fold (P ¼ 0.005)), had been selected for verification by RT-QPCR was matched Nr1h3 (6.0-fold (Po0.0001)) and Slc6a8 (2.3-fold (Po0.0001)) against the gene expression profiles of all the genes that were showed a significant mean difference in the level of transcription expressed at all time points post H37Rv infection and D-mce1 between the uninfected macrophages and the macrophages H37Rv infection, as measured by microarray. during the ‘late’ time points post H37Rv infection (Table 2). Figures 1 and 2 demonstrate that by using a tolerance level of Post D-mce1 H37Rv infection, the genes Abca1 and Slc6a12 0.05%, the similarity search returned 14 gene sets (7 for the H37Rv showed significant mean differences in the level of transcription infection and 7 for D-mce1 H37Rv infection), each comprising 13 between the uninfected macrophages and the macrophages genes (including the index gene). The 14 clusters were divided during the ‘early’ time points post D-mce1 H37Rv infection (3.6- into two groups based on the results from the statistical analyses fold (Po0.0001)) and (2.1-fold (Po0.0001)), respectively. For the of the 7 index genes. Figure 1 comprises the first group clusters ‘late’ time points post D-mce1 H37Rv infection, the genes Abca1 where the index genes (Abca1, Slc6a12 and Slc16a10) showed a (6.9-fold (P ¼ 0.001), Slc6a12 (1.2-fold (P ¼ 0.002), Nr1h3 (5.5-fold significant mean difference (Po0.0001) in the transcriptional level (Po0.0001)) and Slc18a1 (2.2-fold (P ¼ 0.004)) showed significant between the ‘early’ H37Rv infection and the ‘late’ D-mce1 H37Rv mean difference in the level of transcription (Table 2). infection (Table 2). The second group is shown in Figure 2 and Next, we wanted to see whether any of these seven genes could comprises the gene expression profiles of the cluster of genes in discriminate between the two infection types (H37Rv and D-mce1 which the index genes (Nr1h3, Slc6a8 and Slc18a1) showed a H37Rv infection) during either the ‘early’ or ‘late’ stages post significant mean difference in the level of gene transcription infection. When comparing the ‘early’ post H37Rv-infection group between the ‘early’ H37Rv infection and the ‘early’ D-mce1 H37Rv with the ‘early’ post D-mce1 H37Rv-infection group, there was a infection in addition to Slc23a2, which did not differ significantly

Genes and Immunity (2012) 496 – 502 & 2012 Macmillan Publishers Limited Host response to M. tuberculosis infection R Stavrum et al 499 Table 2. Comparison of transcriptional level of selected genes (as measured by reverse-transcriptase quantitative PCR) between the different groups (uninfected, ‘early’, and ‘late’) post-infection with M. tuberculosis H37Rv and D-mce1 H37Rv

Gene name Post H37Rv (wt) infection Post D-mce1 H37Rv Comparison between combinations of groups (del) infection

Uninf. vs Uninf. vs Uninf. vs Uninf. vs wt ‘early’ wt ‘early’ wt ‘late’ wt ‘late’vs ‘early’ ‘late’ ‘early ‘late’ vs del ‘early’ vs del ‘late’ vs del ‘early del ‘late’

Abca1 xxxxx x –– Nr1h3 xx–xx – x– Slc16a10 x–––x x –x Slc18a1 x––xx – –– Slc23a2 x–––– – –– Slc6a12 x–xxx x x– Slc6a8 xxx–x – x–

Abbreviations: del: D-mce1 H37Rv; Uninf: uninfected; wt: H37Rv. Here, ‘x’ indicates a Bonferroni level of o0.0083 representing a signiﬁcance level of Po0.05 and ‘–’ indicates a Bonferroni level of 40.0083 representing a signiﬁcance level of P40.05. The ‘early’–group comprises the time–points 15, 30 and 60 min. The ‘late’-groups comprises the time–points 4 and 10 h.

3.5 3.5 3.0 a 3.0 b 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 -0.5 -0.5 -1.0 -1.0 -1.5 -1.5 -2.0 -2.0

3.5 3.5 3.0 c 3.0 d 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 -0.5 -0.5 -1.0 -1.0 Relative fold-change -1.5 -1.5 -2.0 -2.0

3.5 3.5 3.0 e 3.0 f 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 -0.5 -0.5 -1.0 -1.0 -1.5 -1.5 -2.0 -2.0 1234 5 6 1234 5 6 Time-points Figure 1. Expression profiles of co-transcribed genes. A gene expression profile similarity search was performed using three genes (Abca1, Slc6a12 and Slc16a10) showing a significant mean difference (Po0.0001) in the transcriptional level between ‘early’ H37Rv infection and ‘late’ D-mce1 H37Rv infection as index genes. A tolerance level of 0.05% returned 14 gene sets (7 for the H37Rv infection and 7 for the D-mce1 H37Rv infection). The values on the y axis represent the relative fold change between the different time points and the pooled common reference. The different time points are marked along the x axis: (1) Uninfected, (2) 15 min, (3) 30 min, (4) 60 min, (5) 4 h and (6) 10 h post- infection. Panels a, c and e represent results from the H37Rv-infection study, whereas panels b, d and f represent the D-mce1 H37Rv-infection study. Each gene is represented by a color on the graph and the gene names and position on the mouse chromosome is given in the two columns to the right. NA: not annotated. for any of the comparisons made between the H37Rv and D-mce1 immune response. Originally thought to be simply involved in H37Rv infections (Table 2). gaining entry to host cells,3 more recent work9 suggests that the Mce1 protein complex is also involved in the recycling of extracellular mycolic acids for use as a carbon source. Of DISCUSSION particular interest, we show that the genes that were This study compares transcriptional responses in monocytic cells significantly upregulated by the macrophages in the period infected with M. tuberculosis H37Rv or D-mce1 H37Rv, as a way of immediately after infection by M. tuberculosis H37Rv, but not assessing possible functions for the mce1 operon on the early host D-mce1 H37Rv, were primarily involved in substrate trafficking.12

& 2012 Macmillan Publishers Limited Genes and Immunity (2012) 496 – 502 Host response to M. tuberculosis infection R Stavrum et al 500

Figure 2. Expression profiles of co-transcribed genes. A gene expression profile similarity search was performed using four genes (Nr1h3, Slc6a8 and Slc18a1) showing a significant mean difference (Po0.0001) in the transcriptional level between ‘early’ H37Rv infection and ‘early’ D- mce1 H37Rv infection, but not between the ‘early’ H37Rv infection and the ‘late’ D-mce1 H37Rv infection as index genes, and Slc23a2, which did not differ significantly for any of the comparisons made between the H37Rv and D-mce1 H37Rv infections. A tolerance level of 0.05% returned 14 gene sets (7 for the H37Rv infection and 7 for the D-mce1 H37Rv infection). The values on the y axis represents the relative fold change between the different time points and the pooled common reference. The different time points are marked along the x axis: (1) Uninfected, (2) 15 min, (3) 30 min, (4) 60 min, (5) 4 h and (6) 10 h post-infection. Panels a, c, e and g represent results from the H37Rv-infection study, whereas panels b, d, f and h represent the D-mce1 H37Rv-infection study. Each gene is represented by a color on the graph and the gene names and position on the mouse chromosome is given in the two columns to the right. NA: not annotated.

Our study shows that the most transcriptionally upregulated manipulation of cholesterol efflux during the initial stages of gene post H37Rv infection was the ATP-binding cassette infection. In a previous study, it was shown that mRNA synthesis of transporter 1 (Abca1), which is directly involved in the regulation mce1A was repressed by 4 h post H37Rv infection.16 The of cholesterol efflux from the host cell and can be present on both decreased transcription of Abca1 by 4 and 10 h in comparison the cell surface, and in early and late endosomes.13 Besides being with 60 min post H37Rv infection support a role for the Mce1 used as an energy source, cholesterol is essential for receptor- protein complex on transcription of Abca1. However, a steady mediated internalization through clathrin-coated pits.14 Previous increase in the transcription of the Abca1 gene was also observed studies have suggested that the active domain of the Mce1 from the 30 min time point to 10 h post D-mce1 H37Rv, suggesting protein complex for gaining entry into the host cell is a 58 amino a time-dependent role for the Mce1 protein complex on the Abca1 acid long domain located between position 105 and 163 of the gene transcription (Table 1b). To ensure sufficient availability of Mce1; the most hydrophilic part of the protein,3,15 and that this cholesterol, the mycobacteria rely on lipid import by the host peptide induces a membrane invagintion with structures macrophage17 and M. tuberculosis has been demonstrated to resembling clathrin-coated pits.3 For this study, the upregulation stimulate the accumulation of cholesterol in the cell wall.18 The of the gene Abca1 by 15 min was only observed post H37Rv storage of cholesterol in the cell wall of the bacterium has been infection (18.5-fold) and not post D-mce1 H37Rv (1.8-fold), shown to result in altered cell wall permeability and decreased suggesting that the Mce1 protein complex is involved in the uptake of rifampin.18 The transcription of Abca1 is tightly

Genes and Immunity (2012) 496 – 502 & 2012 Macmillan Publishers Limited Host response to M. tuberculosis infection R Stavrum et al 501 regulated by the amount of cholesterol in the cell and is phase H37Rv or D-mce1 H37Rv bacteria at a multiplicity of infection of 10. stimulated by the liver X receptor (LXR; encoded by the Nr1h3 Three biological replicates were included for each time point for each gene).19 This gene also has a role in the survival of macrophages strain. After 15, 30 and 60 min, and 4 and 10 h following infection, the infected by intracellular pathogens.20 Thus, the combined medium was removed and total RNA was harvested from each flask using upregulation of the Abca1 and Nr1h3 genes during the the RNAeasy Kit (Qiagen Sciences Inc, Germantown, MD, USA). RNA concentration was measured using a NanoDrop scanning spectro- immediate phase of infection by the wild-type M. tuberculosis photometer (NanoDrop Technologies, Wilmington, DE, USA) and the H37Rv strain, suggest a role for the Mce1 protein complex in the quality was measured using the Eukaryote Total RNA Nano 6000 assay bacteria’s survival mechanism, possibly by accumulation of (Agilent RNA 6000 Nano LabChip Kit; Agilent Technologies, Santa Clara, CA, USA). cholesterol after mycobacterial entry into the host macrophage during M. tuberculosis H37Rv infection. Hybridization The cell wall of M. tuberculosis is dominated by covalently linked mycolic acids that protect the organism from environmental stress A pooled common reference was prepared by mixing 10 ml of total RNA isolated from each biological replicate of uninfected MF. Cy-5-labeled and contributes to disease persistence and the resistance of 21 cRNA (825 ng) from M. tuberculosis H37Rv- or H37Rv-D-mce1-infected M. tuberculosis to many antibiotics. Furthermore, mycolic acids J774A.1 MF was randomly hybridized against 825 ng Cy-3-labeled pooled have been shown to strongly interfere with lipid metabolism of common reference cRNA onto 4 Â 44 K 60-mer oligo whole-mouse macrophages, triggering intracellular accumulation of cholesterol, genome—micro-arrays (Agilent Technologies). Following hybridization, increased cell size and multiple vacuole formation.17 Thus, the the arrays were washed according to the manufacturer’s description decreased expression of Abca1 post D-mce1 H37Rv infection as (Agilent no. 5188-5325, 5188-5326). Acetonitrile (Sigma-Aldrich, St Louis, compared with post H37Rv infection may be the result of an anti- MO, USA) and Agilent stabilization and drying solution (Agilent inflammatory response initiated through the accumulation of free Technologies) were included in the two final steps in the washing mycolic acids on the mycobacterial cell envelope caused by a procedure. The arrays were scanned using the Agilent’s dual laser DNA microarray scanner (part number G2505B). The scans were converted to disruption of the fatty acid recycling mechanism coded for by the data files with Agilent’s Feature Extraction software (v. 9.1.3.1). mce1 operon. All microarray data are MIAME compliant and fully annotated raw Interestingly, the other genes differentially expressed are also microarray data has been deposited in ArrayExpress (accession number: responsible for transport functions being members of a family of E-TABM-1170). membrane transporters called solute carriers (Slcs). Membrane transporters are involved in the maintenance of cellular home- Data analysis ostasis, but also in the absorption, distribution and excretion of Microarray data analysis. Preprocessing and analysis was undertaken various drugs and metabolic reagents. Other Slcs that have been using the software J-Express Pro 2.9.25 Control spots, as well as all spots shown to be important for the control of tuberculosis in humans that were flagged by Feature Extraction (v. 9.1.3.1) (Agilent Technologies) 22,23 are SLC11A1 (alias NRAMP1) and SLC6A3. The importance of or where the signal was saturated in both channels were removed from the solute carriers during a tuberculosis infection has been further the analysis. Log 2 ratios were calculated between the Cy 5 and Cy 3 demonstrated by the increased mRNA expression of a range of signals from the remaining spots. Processed Signal values, which by the Slcs in various tissues during murine pulmonary tuberculosis.24 Feature Extraction default settings had been background-corrected and Those shown to be differentially affected in this study are normalized with respect to dye effects, were chosen to represent the signal transporters of compounds, such as lactate, pyruvate and intensity values. The data was divided into two data subsets, one for the aromatic amino acids, all of which are important metabolites. A time series following H37Rv infection and one for time series following the D-mce1 H37Rv infection. Genes differentially expressed post H37Rv gene expression similarity search identified genes with a similar infection, as compared with uninfected controls, for each set of time transcriptional profile to Slc6a12, of which two genes, Ela1 and series were identified using RP.26 Serpina3g, have been shown to be involved in host defense against infection by intracellular Salmonella typhimurium. The Functional classification. Functional classification of RP lists of upregu- decline in the transcription of genes in the gene set represented lated genes at various time points post H37Rv infection was performed by the Slc6a12 gene at 4 and 10 h post-infection could be the using the Panther Classification System 6.1.27 To detect genes that were result of secreted mycobacterial effector proteins, or a part of the consistently upregulated during the first hour, the gene lists for the three cellular defense mechanisms employed by the macrophage, or it first time points (15, 30 and 60 min) were compared. A new list comprising may simply indicate that Mce1 complex acts as a facilitator for the genes present in all three RP lists (15, 30 and 60 min) were created and compounds that ultimately effect these changes in gene termed 1 h. The 1, 4 and 10 h lists post H37Rv infection (using a false expression, allowing M. tuberculosis to more rapidly affect host discovery rate of 10%) were compared with the entire list of genes with detectable expression in at least one of the 21 samples from the post functions. Although, such direct links between the genes H37Rv-infection study (n ¼ 18950) on the 4 Â 44 K 60-mer oligo whole- belonging to the other gene sets could not be determined, it is mouse genome—microarrays (Agilent Technologies). Statistically over- and possible that some of the genes within the other gene sets also underrepresented annotated biological processes were determined by work in concert in order to exert a protective response to an binominal statistics, using the observed number of genes versus the intracellular infection. numbers expected by chance within a certain annotation group. The findings from this study indicate that M. tuberculosis H37Rv Categories meeting the threshold of P-value below 0.001 were exported with an intact Mce1 protein complex initiates an immediate host for further analyses. transcriptional response, which may facilitate the survival of the bacilli by promoting an anti-inflammatory, nutrient-rich and RT-QPCR verification sustainable milieu inside the host macrophage. Genes that were shown to belong to the most overrepresented biological process by the Panther Classification System 6.127 and that were only present in the RP lists from the first three time points (15, 30 and 60 min MATERIALS AND METHODS (false discovery rate 10%)) and not in the RP lists from the 4 and 10 h (false Infection discovery rate 10%) post infection with M. tuberculosis H37Rv were As the use of macrophages in different states of development/maturation selected for verification by RT-QPCR. The RT-QPCR mixture for genes and terminal specificity may generate ‘background noise’ that may meeting the predefined criteria and for two genes (Hmbs and Ubc) interfere with an ability to clearly discern the role of the Mce1 protein included as endogenous controls was prepared as described previously.10 complex, and as most of the published microarray work on in vitro RT-QPCR amplification and analysis were performed using the ABI 7500 infection of monocytic cells has been done on cell lines, we decided to use instrument with software version 2.0.3 (Applied Biosystems, Carlsbad, CA, murine J774A.1 macrophages for this study. As described previously,10 USA), and the relative amount of gene expression was calculated using a B107 murine J774A.1 macrophages (MF) were infected with mid-log pooled common reference as reference sample.

& 2012 Macmillan Publishers Limited Genes and Immunity (2012) 496 – 502 Host response to M. tuberculosis infection R Stavrum et al 502 Similarity search. Genes meeting the criteria for verification by RT-QPCR 10 Stavrum R, Stavrum A-K, Valvatne H, Riley LW, Ulvestad E, Jonassen I et al. were selected as index genes in the search for other genes with a similar Modulation of transcriptional and inflammatory responses in murine macro- gene expression profile. Euclidean distance was used as the distance phages by the Mycobacterium tuberculosis mammalian cell entry (Mce) 1 Complex. measure, and the tolerance level was set at 0.05% (that is, 5% of the genes PLoS ONE 2011; 6: e26295. in the entire data set that were most similar were reported back for each 11 Fessele S, Maier H, Zischek C, Nelson PJ, Werner T. Regulatory context is a crucial search). part of gene function. Trends Genet 2002; 18: 60–63. 12 Hediger MA, Romero MF, Peng J-B, Rolfs A, Takanaga H, Bruford EA. The ABCs of RT-QPCR data analysis. Statistical analyses were undertaken to detect solute carriers: physiological, pathological and therapeutic implications of human differences between groups of measurements. The t-test was applied membrane transport proteinsIntroduction. Pflu¨gers Archiv Eur J Physiol 2004; 447: when comparing groups of two, whereas analysis of variance was applied 465–468. when comparing more than two groups. Multiple testing effects were 13 Knight BL. ATP-binding cassette transporter A1: regulation of cholesterol efflux. taken into account, thus adjusting the significance level by the Bonferroni Biochem Soc Trans 2004; 32(Part 1): 124–127. rule using a significance level of 0.05/6 ¼ 0.00833. The computations were 14 Subtil A, Gaidarov I, Kobylarz K, Lampson MA, Keen JH, McGraw TE. Acute done using SPSS 17. cholesterol depletion inhibits clathrin-coated pit budding. Proc Natl Acad Sci USA 1999; 96: 6775. 15 Casali N, Konieczny M, Schmidt MA, Riley LW. Invasion activity of a Mycobacterium ACKNOWLEDGEMENTS tuberculosis peptide presented by the Escherichia coli AIDA Autotransporter. Infect Immun 2002; 70: 6846–6852. We thank Karl-Henning Kalland and Kari Rostad for constructive advice, Kristi Øvreås 16 Casali N, White AM, Riley LW. Regulation of the Mycobacterium tuberculosis mce1 and Kjell Petersen for technical assistance, Haukeland University Hospital for operon. J Bacteriol 2006; 188: 441–449. P3-laboratory facilities, the Norwegian Microarray Consortium, Bergen, for microarray 17 Korf H, Vander Beken S, Romano M, Steffensen KR, Stijlemans B, Gustafsson J-A facilities, and the FUGE bioinformatics platform for valuable bioinformatics support. et al. Liver X receptors contribute to the protective immune response against This study was funded by the Research Council of Norway project no. 179342 and Mycobacterium tuberculosis in mice. 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