Dissecting the Mycobacterium bovis BCG Response to Macrophage Infection to Help Prioritize Targets for Anti-Tuberculosis Drug and Vaccine Discovery

New strategies are required to reduce the worldwide burden of tuberculosis. Intracellular survival and replication of Mycobacterium tuberculosis after macrophage phagocytosis is a fundamental step in the complex host–pathogen interactions that lead to granuloma formation and disease. Greater understanding of how the bacterium survives and thrives in these environments will inform novel drug and vaccine discovery programs. Here, we use in-depth RNA sequencing of Mycobacterium bovis BCG from human THP-1 macrophages to describe the mycobacterial adaptations to the intracellular environment. We identify 329 significantly differentially regulated genes, highlighting cholesterol catabolism, the methylcitrate cycle and iron homeostasis as important for mycobacteria inside macrophages. Examination of multi-functional gene families revealed that 35 PE/PPE genes and five cytochrome P450 genes were upregulated 24 h after infection, highlighting pathways of potential significance. Comparison of the intracellular transcriptome to gene essentiality and immunogenicity studies identified 15 potential targets that are both required for intracellular survival and induced on infection, and eight upregulated genes that have been demonstrated to be immunogenic in TB patients. Further insight into these new and established targets will support drug and vaccine development efforts.


Introduction
Mycobacterium tuberculosis, the cause of tuberculosis (TB), is estimated to have killed 1.5 million people in 2020 and is the leading cause of death by a bacterial agent, despite being a treatable disease. The disease burden is not distributed evenly, as 86% of those who fall sick with TB are in 30 countries, and 47% of all cases face catastrophic costs (>10% household income) to access treatment [1]. Progress has been hindered by the SARS-CoV-2 pandemic, with reduced access to medical support causing a drop in newly identified cases, an increase in deaths, and a reduction in treatment provision [1].
TB vaccination programs use the Bacille Calmette-Guerin (BCG) vaccine created from an attenuated culture of Mycobacterium bovis approximately 100 years ago [2]. Meta-analysis indicates that BCG vaccination significantly reduces risk of TB by 50%, though protection against death, TB meningitis and disseminated disease is higher [3]. There is large interstudy variation from 0-80% efficacy, likely due to a combination of factors including pre-exposure to environmental mycobacteria [4]. In the search for new and more-effective

Macrophage Culture and Infection
Human monocyte THP-1 cells (ATCC:TIB-202) were maintained at 37 • C, 5% CO 2 in RPMI medium supplemented with 2 mM L-glutamine, 10% v/v fetal bovine serum and 1 mM sodium pyruvate without antibiotics. THP-1 cells were chosen as a well-characterized immortalized cell line frequently used for mycobacterial research, where sufficient cell numbers could be reproducibly generated in a macrophage of human genetic background. Monocytes (1.1 × 10 6 cells/mL) were differentiated into macrophage-like cells by 24 h stimulation with phorbol 12-myristate 13-acetate (20 nM final concentration). After washing twice with phosphate-buffered saline (PBS), cells were rested for 48 h in RPMI before infection. M. bovis BCG harvested from log phase culture was washed in PBS, resuspended in RPMI media, and syringed five times to generate a homogenous cell suspension before infecting macrophages at a multiplicity of infection of 10 bacilli: 1 macrophage for 4 h.
After infection, cells were washed with sterile PBS to remove extracellular bacteria before incubation with fresh RPMI for 20 h at 37 • C in a 5% CO 2 humidified incubator [16].

Mycobacterial RNA Extraction and RNA Sequencing
RNA was extracted from intracellular mycobacteria and from in vitro log phase M. bovis BCG using the GTC/TRIzol differential lysis method [11]. Mycobacterial RNA was DNasetreated and purified using the RNeasy spin-column system (Qiagen, Germantown, MD, USA). RNA quality and yield were evaluated using the Qubit Broad Range RNA assay (Thermo Scientific, Waltham, MA, USA), Nanodrop One (Thermo Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Three independent biological replicates of intracellular M. bovis BCG (2 µg/replicate) and 3 replicates of in vitro log phase M. bovis BCG RNA (2 µg/replicate) were depleted of mycobacterial rRNA using Ribo-Zero rRNA Removal (Bacteria) kit (Illumina, San Diego, CA, USA). Sequencing libraries were prepared using the NEBNext Ultra II kit (New England Biolabs, Ipswich, MA, USA) and paired-end sequenced using the Illumina NextSeq500 (75 × 2 paired end) instrument.

Transcriptomic Analyses
Paired-end raw sequence reads were initially processed with Trimmomatic (v0.36) [17] to remove low quality reads. The cleaned reads were mapped to the M. bovis AF2122/97 genome and to the human genome GRCh38 using Hisat2 [18], yielding 2-5 million reads mapped to the M. bovis genome per biological replicate. Gene expression was quantified using FeatureCounts from the Subread package v1.5.2. Differentially expressed genes in intracellular M. bovis BCG compared to log phase M. bovis BCG were identified using DESeq2 R package v.3.6.0, RLE normalized, and false discovery rates reduced using the Wald test with Benjamini and Hochberg multiple testing correction [19]. Reads mapping to multiple locations with the same number of mis-matched bases were ignored, and only primary alignments were counted. Genes with a log2-fold change (L2FC) <−1 or >1 with a corrected p-value < 0.05 were considered to be significantly differentially expressed (Table S1). To compare to the published literature, M. bovis AF2122/97 gene IDs were converted to M. tuberculosis H37Rv identifiers [20]. Significant overlaps with previously published transcriptional signatures, gene essentiality datasets and functional categories were identified using the hypergeometric function, p-value < 0.05. Differentially expressed genes were also mapped to M. tuberculosis metabolic pathways using gene set enrichment analysis [21] and DAVID Functional Annotation tool [22]. Fully annotated RNAseq data have been deposited in ArrayExpress; accession number E-MTAB-11107.

Mycobacterial Transcriptional Adaptations to Macrophage Infection
To inform new treatment strategies for tuberculosis, we mapped the transcriptional adaptations of M. bovis BCG to the intracellular macrophage environment. Mycobacterial RNA was isolated from human THP-1 macrophages 24 h after infection. RNA, extracted using a differential lysis method ( Figure 1A), was bacterial rRNA depleted and sequenced without further selection or amplification to maintain as representative a transcriptional signature as possible. RNAseq generated 2 to 5 million reads that aligned to the M. bovis AF2122/97 genome from each independent biological replicate ( Figure 1B) [16]. A total of 329 genes were significantly differentially expressed (L2FC >1/<−1, corrected p-value < 0.05), of which 284 were induced in intracellular M. bovis BCG and 45 genes were repressed in comparison to in vitro log phase bacilli ( Figure 1C, Table S1). The two functional categories most represented in the upregulated genes (excluding 'conserved hypotheticals' and 'undefined') were 'lipid metabolism' and 'intermediary metabolism and respiration' ( Figure 1D). Gene set enrichment analysis (GSEA) of induced genes supported these findings with the top functional cluster (enrichment score 5.6) involving fatty acid beta-oxidation using acyl-CoA dehydrogenase (p = 3.4 × 10 −7 ), lipid metabolism (p = 4.5 × 10 −4 ), and electron carrier activity (p = 1.2 × 10 −7 ). The significance of altered carbon metabolism was further demonstrated by the second functional cluster (enrichment score 5.02) involving steroid metabolism (p = 1.1 × 10 −7 ) and cholesterol catabolism (p = 4.6 × 10 −7 ) ( Figure 2). The M. bovis BCG intracellular signature overlapped closely with previously defined M. tuberculosis intracellular transcriptional profiles with changes to respiratory and metabolic pathways exemplified by induction of KstR and DosR regulons, upregulated to metabolise cholesterol and on exposure to hypoxia/nitric oxide, respectively, and upregulation of the iron-scavenging siderophore mycobactin biosynthetic genes A total of 329 genes were significantly differentially expressed (L2FC >1/<−1, corrected p-value < 0.05), of which 284 were induced in intracellular M. bovis BCG and 45 genes were repressed in comparison to in vitro log phase bacilli ( Figure 1C, Table S1). The two functional categories most represented in the upregulated genes (excluding 'conserved hypotheticals' and 'undefined') were 'lipid metabolism' and 'intermediary metabolism and respiration' ( Figure 1D). Gene set enrichment analysis (GSEA) of induced genes supported these findings with the top functional cluster (enrichment score 5.6) involving fatty acid beta-oxidation using acyl-CoA dehydrogenase (p = 3.4 × 10 −7 ), lipid metabolism (p = 4.5 × 10 −4 ), and electron carrier activity (p = 1.2 × 10 −7 ). The significance of altered carbon metabolism was further demonstrated by the second functional cluster (enrichment score 5.02) involving steroid metabolism (p = 1.1 × 10 −7 ) and cholesterol catabolism (p = 4.6 × 10 −7 ) ( Figure 2). The M. bovis BCG intracellular signature overlapped closely with previously defined M. tuberculosis intracellular transcriptional profiles with changes to respiratory and metabolic pathways exemplified by induction of KstR and DosR regulons, upregulated to metabolise cholesterol and on exposure to hypoxia/nitric oxide, respectively, and upregulation of the iron-scavenging siderophore mycobactin biosynthetic genes (mbtA/B/C/D/E/F/G/I/J) ( Figure 2). Accounting for these findings, we focused on conserved mycobacterial metabolic pathways with implications for drug and vaccine discovery pipelines.
Vaccines 2022, 10, x FOR PEER REVIEW 5 of 14 (mbtA/B/C/D/E/F/G/I/J) ( Figure 2). Accounting for these findings, we focused on conserved mycobacterial metabolic pathways with implications for drug and vaccine discovery pipelines.

Fatty Acid Metabolism and Cholesterol Catabolism
M. tuberculosis inside macrophages metabolises fatty acids as a carbon source [25,26], and this is reflected in the induction of fatty acid metabolism genes intracellularly [9,12].
The key indicator gene icl1 (Mb0476; Rv0467), involved in the glyoxylate cycle to convert acetyl-CoA molecules derived from the β-oxidation of fatty acids into oxaloacetate, was induced (L2FC 2.67) intracellularly by M. bovis BCG. In addition, β-oxidation of odd-chain fatty acids leads to the formation of propionyl-CoA, which cannot be used in the glyoxylate cycle. Instead, propionyl-CoA is converted to pyruvate via the methylcitrate cycle, a process requiring methylcitrate synthase and methylcitrate dehydratase, encoded by prpC and prpD, respectively (Mb1162, Mb1161; Rv1131, Rv1130). These were the most highly upregulated genes intracellularly (L2FC 9.24 and 10.42), further highlighting the significance of changing carbon metabolism in intracellular mycobacteria. Unsurprisingly, as identified by GSEA analysis, genes involved in cholesterol catabolism, many of which are regulated by KstR, were also induced (chsE1, chsE2, chsH2, chsH1, hsaA, hsaD) ( Figure 3).

Fatty Acid Metabolism and Cholesterol Catabolism
M. tuberculosis inside macrophages metabolises fatty acids as a carbon source [25,26], and this is reflected in the induction of fatty acid metabolism genes intracellularly [9,12].
The key indicator gene icl1 (Mb0476; Rv0467), involved in the glyoxylate cycle to convert acetyl-CoA molecules derived from the β-oxidation of fatty acids into oxaloacetate, was induced (L2FC 2.67) intracellularly by M. bovis BCG. In addition, β-oxidation of oddchain fatty acids leads to the formation of propionyl-CoA, which cannot be used in the glyoxylate cycle. Instead, propionyl-CoA is converted to pyruvate via the methylcitrate cycle, a process requiring methylcitrate synthase and methylcitrate dehydratase, encoded by prpC and prpD, respectively (Mb1162, Mb1161; Rv1131, Rv1130). These were the most highly upregulated genes intracellularly (L2FC 9.24 and 10.42), further highlighting the significance of changing carbon metabolism in intracellular mycobacteria. Unsurprisingly, as identified by GSEA analysis, genes involved in cholesterol catabolism, many of which are regulated by KstR, were also induced (chsE1, chsE2, chsH2, chsH1, hsaA, hsaD) ( Figure 3).

Cytochrome P450 Family
CYP450 enzymes are a superfamily of heme containing enzymes involved in a range of intermediary metabolism and respiration pathways. There are 20 CYP450s identified in M. tuberculosis H37Rv (and M. bovis BCG) that likely function in diverse metabolic processes. Of these, cyp51, cyp123, cyp125, cyp142a and cyp142b were upregulated in macrophages (Figure 4b). In line with cholesterol catabolism genes that were upregulated, the products of cyp125 and cyp142 have been shown to oxidise cholesterol side chains as mycobacteria adapt carbon metabolism in the intracellular environment [33].

Cytochrome P450 Family
CYP450 enzymes are a superfamily of heme containing enzymes involved in a range of intermediary metabolism and respiration pathways. There are 20 CYP450s identified in M. tuberculosis H37Rv (and M. bovis BCG) that likely function in diverse metabolic processes. Of these, cyp51, cyp123, cyp125, cyp142a and cyp142b were upregulated in macrophages (Figure 4b). In line with cholesterol catabolism genes that were upregulated, the products of cyp125 and cyp142 have been shown to oxidise cholesterol side chains as mycobacteria adapt carbon metabolism in the intracellular environment [33].

Overlap with Gene Essentiality Datasets
To highlight pathways for drug discovery that are both induced in the intracellular macrophage environment and essential for growth, we compared the M. bovis BCG intracellular transcriptional signature to genome-wide gene essentiality datasets. The most significant overlaps were with studies determining genes essential for cholesterol catabolism [15] (p-value = 1.3 × 10 −15 ) and genes essential for growth in murine bone marrow derived macrophages [14] (p-value = 0.018) (Figure 2). Focusing on these two studies, we found that, from 324 genes (mapped to M. tuberculosis H37Rv) defining the transcriptional adaptations to macrophage phagocytosis, 28 genes were essential for growth and cholesterol catabolism [15], 10 genes for survival in the macrophage [14], and eight genes for both cholesterol and macrophage environments ( Figure 5). As might be expected, these eight genes were all involved in cholesterol degradation (Figure 3). ChsE2 and ChsH2 have been linked to side chain degradation. KstD, HsaA and HsaD play roles in degradation of A and B rings, and FadE30 homologues in Actinomyces spp. have been shown to do the same. IpdA homologues in Actinomyces spp. drive the final stages of degradation, and FadE32 has been suggested to do the same [27].

Overlap with Gene Essentiality Datasets
To highlight pathways for drug discovery that are both induced in the intracellular macrophage environment and essential for growth, we compared the M. bovis BCG intracellular transcriptional signature to genome-wide gene essentiality datasets. The most significant overlaps were with studies determining genes essential for cholesterol catabolism [15] (p-value = 1.3 × 10 −15 ) and genes essential for growth in murine bone marrow derived macrophages [14] (p-value = 0.018) (Figure 2). Focusing on these two studies, we found that, from 324 genes (mapped to M. tuberculosis H37Rv) defining the transcriptional adaptations to macrophage phagocytosis, 28 genes were essential for growth and cholesterol catabolism [15], 10 genes for survival in the macrophage [14], and eight genes for both

Cytochrome P450 Family
CYP450 enzymes are a superfamily of heme containing enzymes involved in a range of intermediary metabolism and respiration pathways. There are 20 CYP450s identified in M. tuberculosis H37Rv (and M. bovis BCG) that likely function in diverse metabolic processes. Of these, cyp51, cyp123, cyp125, cyp142a and cyp142b were upregulated in macrophages (Figure 4b). In line with cholesterol catabolism genes that were upregulated, the products of cyp125 and cyp142 have been shown to oxidise cholesterol side chains as mycobacteria adapt carbon metabolism in the intracellular environment [33].

Overlap with Gene Essentiality Datasets
To highlight pathways for drug discovery that are both induced in the intracellular macrophage environment and essential for growth, we compared the M. bovis BCG intracellular transcriptional signature to genome-wide gene essentiality datasets. The most significant overlaps were with studies determining genes essential for cholesterol catabolism [15] (p-value = 1.3 × 10 −15 ) and genes essential for growth in murine bone marrow derived macrophages [14] (p-value = 0.018) (Figure 2). Focusing on these two studies, we found that, from 324 genes (mapped to M. tuberculosis H37Rv) defining the transcriptional adaptations to macrophage phagocytosis, 28 genes were essential for growth and cholesterol catabolism [15], 10 genes for survival in the macrophage [14], and eight genes for both cholesterol and macrophage environments ( Figure 5). As might be expected, these eight genes were all involved in cholesterol degradation (Figure 3). ChsE2 and ChsH2 have been linked to side chain degradation. KstD, HsaA and HsaD play roles in degradation of A and B rings, and FadE30 homologues in Actinomyces spp. have been shown to do the same. IpdA homologues in Actinomyces spp. drive the final stages of degradation, and FadE32 has been suggested to do the same [27]. cholesterol and macrophage environments ( Figure 5). As might be expected, these eight genes were all involved in cholesterol degradation (Figure 3). ChsE2 and ChsH2 have been linked to side chain degradation. KstD, HsaA and HsaD play roles in degradation of A and B rings, and FadE30 homologues in Actinomyces spp. have been shown to do the same. IpdA homologues in Actinomyces spp. drive the final stages of degradation, and FadE32 has been suggested to do the same [27].

Figure 5.
Overlap between transcriptional adaptations to macrophage infection and gene essentiality. Blue indicates the significantly differentially expressed genes after M. bovis BCG macrophage infection; green, the essential genes for growth on cholesterol [15]; red, the essential genes for macrophage infection [14]. Number of genes denoted in each section. Gene name and M. tuberculosis H37Rv identifiers marked for intersects; '+' upregulated in macrophage, '-' downregulated.
Away from cholesterol metabolism, seven M. bovis BCG genes (Table 1) were induced on macrophage infection that were also essential for survival in murine macrophages [14], but not essential for cholesterol catabolism in vitro [15] (Figure 5). Upregulated Rv0082 and Rv3552, encoding a probable oxidoreductase and possible CoA-transferase, respectively, are likely intermediate respiration and metabolism enzymes. Rv0195 encodes a LuxR family regulator linked to mycobacterial growth recovery and survival of hypoxic and reductive stress [34]. Of the remaining genes, two are associated with lipid catabolism (Rv3541c, Rv3556), one is a member of the PPE family (Rv0096), and one encodes a conserved hypothetical protein of unknown function (Rv0372c). These genes are of interest in identifying potentially druggable pathways (separate from cholesterol catabolism) that are essential for macrophage survival and induced on infection. The functions of these genes are not fully elucidated and, therefore, warrant further investigation as potential therapeutic targets. Blue indicates the significantly differentially expressed genes after M. bovis BCG macrophage infection; green, the essential genes for growth on cholesterol [15]; red, the essential genes for macrophage infection [14]. Number of genes denoted in each section. Gene name and M. tuberculosis H37Rv identifiers marked for intersects; '+' upregulated in macrophage, '-' downregulated.
Away from cholesterol metabolism, seven M. bovis BCG genes (Table 1) were induced on macrophage infection that were also essential for survival in murine macrophages [14], but not essential for cholesterol catabolism in vitro [15] (Figure 5). Upregulated Rv0082 and Rv3552, encoding a probable oxidoreductase and possible CoA-transferase, respectively, are likely intermediate respiration and metabolism enzymes. Rv0195 encodes a LuxR family regulator linked to mycobacterial growth recovery and survival of hypoxic and reductive stress [34]. Of the remaining genes, two are associated with lipid catabolism (Rv3541c, Rv3556), one is a member of the PPE family (Rv0096), and one encodes a conserved hypothetical protein of unknown function (Rv0372c). These genes are of interest in identifying potentially druggable pathways (separate from cholesterol catabolism) that are essential for macrophage survival and induced on infection. The functions of these genes are not fully elucidated and, therefore, warrant further investigation as potential therapeutic targets. Table 1. Genes induced by M. bovis BCG after macrophage infection that are also essential for growth in macrophages [14], but not essential for growth and cholesterol catabolism in vitro [15]. Catalyses the formation of 4-methyl-5-oxo-octanedioyl-CoA in steroid catabolic pathway

Comparison to the TB Vaccination Pipeline
Successful vaccination strategies target antigens that are expressed in vivo; therefore, we asked whether mapping the M. bovis BCG genes induced in macrophages might inform vaccine discovery efforts. There was no overlap between upregulated genes and antigens in vaccinations currently in clinical trials [35]. This is not surprising, as major vaccine candidate antigens such as ESAT-6 and CFP-10 are not present in M. bovis BCG. Intracellular growth had no significant impact on the expression of the other commonly included vaccine antigen, Ag85A.
The pre-clinical recombinant vaccine CMV-6Ag [36] includes the antigens Rv1733c, Rv2626c and Rv2389c. All three of the genes encoding these target proteins were significantly upregulated on macrophage infection (L2FC 1.46, 1.88, 2.19, respectively). The gene hspX (Rv2031c) was also induced (L2FC 4.28), the product of which is known to be highly immunogenic and an inducer of a strong Th1 immune response in mice [37]. Further comparison to 23 M. tuberculosis antigens, shown to be immunogenic through ELISA testing of TB patient blood IFN-γ responses [38], found that eight genes (five of which are regulated by DosR) were significantly induced (Table 2), and one gene (Rv0440) repressed by intracellular M. bovis BCG. The three most upregulated genes encoding immunogenic antigens from the Kassa et al. study were Rv0079 (encoding a dormancy associated translation inhibitor), rpfD (coding for a resuscitation-promoting factor) and fdxA (involved in electron transfer). The remaining five genes encoded a transcriptional regulator of the response to hypoxia (Rv0081), a transmembrane protein (Rv1733c), a universal stress-associated protein (Rv2028c), and two conserved hypotheticals (Rv1734c, Rv2627). In addition, 14 mycobacterial genes induced intracellularly were also listed in the top 45 predicted vaccine candidate antigens selected by Zvi et al. [39] from comprehensive literature and in silico analyses ( Table 2). These genes, the products of which have been determined to be immunogenic in TB patients or are predicted to be, and that show enhanced gene expression in the intracellular environment of a macrophage, should be prioritised for further examination. Table 2. Genes induced by M. bovis BCG after macrophage infection relative to in vitro growth that were also demonstrated to be immunogenic in patients with active pulmonary tuberculosis [38] and/or are listed in the top 45 vaccine candidate antigens by Zvi et al. [39]. No asterisk = Kassa et al. [38] only; * = Zvi et al. [39] only; ** = identified in both studies.

Discussion
M. tuberculosis is able to survive and replicate in macrophages, and the initial hostpathogen interplay shapes subsequent pathogenesis [8]. The identification of metabolic pathways or immunogenic proteins important at this key stage of infection may offer new targets, and substantiate existing targets, for drug and vaccine discovery research. Here, we used differential RNA extraction methods alongside RNAseq to define the global transcriptional adaptations of M. bovis BCG, the TB vaccine, to human THP-1 macrophage infection. Aside from bacterial ribosomal RNA depletion and RNAseq library preparation, the RNA was not manipulated to retain a representative intracellular transcriptional signature. Good quality bacterial RNA, indicated by intact 16s and 23s ribosomal peaks, was isolated from three independent macrophage infections ( Figure 1A). Degraded human macrophage RNA in the intracellular samples, denoted by minor 18s and 28s ribosomal peaks and spread of low/high molecular weight species, mapped to the human genome as expected. Two to five million reads per sample were mapped with high stringency to the M. bovis genome. This enabled the differential expression of highly repetitive gene sequences to be mapped with greater confidence, such as members of the PE/PPE gene family that are often ignored in genome-wide comparisons due to the repetitive nature of their sequences.
We used the vaccine strain M. bovis BCG Montreal containing a GFP plasmid in this study. M. bovis BCG shares 99% genome sequence similarity with virulent M. tuberculosis, there are also several important genomic modifications responsible for M. bovis BCG attenuation [40]. This includes Region of Difference 1 (RD1) encoding an ESX-1 system responsible for the secretion of CFP-10/ESAT-6 virulence factors [41]. Thus, macrophage infection models using M. bovis BCG are missing key pathogenic mechanisms of M. tuberculosis but capture the broad metabolic processes necessary for intracellular survival. Correspondingly, the 329 genes differentially expressed by M. bovis BCG intracellularly in this study overlap significantly with previously defined M. tuberculosis transcriptional adaptations to macrophage infection [9][10][11][12]42], featuring induction of glyoxylate shunt, methylcitrate cycle, cholesterol catabolism, and iron homeostasis signatures.
To highlight pathways for drug discovery that are important for mycobacterial survival in vivo and to avoid targets that become non-essential during infection [43], we compared the M. bovis BCG intracellular transcriptional response to genome-wide gene essentiality studies [14,15]. We found an overlap of 36 genes that were essential for growth with cholesterol and significantly induced after macrophage infection. This analysis underlines the importance of cholesterol catabolic pathways intracellularly, targeting drug discovery towards in vivo lifecycle stages of M. tuberculosis. This comparison also identified seven genes ( Table 1) that were upregulated on macrophage infection and essential to survival in macrophages, but not linked to cholesterol gene essentiality. Many of these gene functions are unknown, and further investigation may highlight important roles in the adaptation of mycobacteria to the intracellular environment.
An alternative strategy to the above unsupervised approaches to identify potentially druggable pathways active in vivo is to explore the expression of gene families that likely have distinct roles across a diverse range of cellular processes. Such analyses for the highly repetitive PE/PPE family (pe, ppe, pe_pgrs gene families) and cytochrome P450s (cyp gene family) show induction of subsets of these gene families 24 h after macrophage infection (Figure 4), revealing novel pathways to target. Although the functions of many of these gene products are not well-understood, genes linked to survival under stress (pe34), iron acquisition (ppe62) and perturbation of the host response (ppe27, ppe37, pe-pgrs41) were highlighted as potentially important in vivo. Of the cyp genes induced intracellularly, enzymes encoded by cyp125 and cyp142 are involved in cholesterol catabolism and are suggested therapeutic targets, as inhibition of either of these enzymes leads to M. tuberculosis growth inhibition through cholest-4-en-3-one accumulation [44]. The sterol demethlyase encoded by cyp51 is the target of azole antifungal drugs, such as econazole that is also effective against M. tuberculosis. The CYP450 enzymes, or the pathways that they operate in, identified by their upregulation after macrophage infection might make promising drug targets in M. tuberculosis [45].
To help define novel targets that are expressed during disease for vaccination strategies, we compared the genes upregulated by M. bovis BCG after macrophage infection with antigens used in subunit vaccines or found to be immunogenic in patients with TB [38] or predicted to be [39]. Three gene products (Rv1733c, Rv2626c and Rv2389c) overlapped with antigens in the preclinical CMV-6Ag subunit vaccine [36]. The products of 18 genes upregulated in macrophages were also demonstrated to be immunogenic in TB patients or predicted to be highly immunogenic ( Table 2), suggesting that these may be potential targets for the development of future subunit vaccines.
In summary, we have characterized the M. bovis BCG response to human macrophage infection, generating RNAseq datasets for future investigations. The importance of β-oxidation of fatty acids, glyoxylate shunt, methylcitrate cycle, cholesterol catabolism and iron acquisition were reflected in the transcriptional adaptations to the intracellular environment. Overlaps with gene essentiality and antigen discovery studies highlight targets with potential for future anti-TB drug and vaccination strategies.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/vaccines10010113/s1, Supplementary Table S1: The M. bovis BCG response to THP-1 macrophage infection at 24 h. Differentially expressed genes in intracellular M. bovis BCG compared to log phase M. bovis BCG were identified using DESeq2 R package v.3.6.0, RLE normalized, and false discovery rates reduced using the Wald test with Benjamini and Hochberg multiple testing correction. Genes with a log2-fold change <−1 or >1 with an adjusted p-value < 0.05 were considered to be significantly differentially expressed. The table lists 329 differentially expressed genes, 284 induced and 45 repressed after macrophage infection relative to log phase in vitro growth. Table ordered