https://orcid.org/0000-0001-8064-5615
Figures
Abstract
We have identified an acyl-carrier protein, Rv0100, that is up-regulated in a dormancy model. This protein plays a critical role in the fatty acid biosynthesis pathway, which is important for energy storage and cell wall synthesis in Mycobacterium tuberculosis (MTB). Knocking out the Rv0100 gene resulted in a significant reduction of growth compared to wild-type MTB in the Wayne model of non-replicating persistence. We have also shown that Rv0100 is essential for the growth and survival of this pathogen during infection in mice and a macrophage model. Furthermore, knocking out Rv0100 disrupted the synthesis of phthiocerol dimycocerosates, the virulence-enhancing lipids produced by MTB and Mycobacterium bovis. We hypothesize that this essential gene contributes to MTB virulence in the state of latent infection. Therefore, inhibitors targeting this gene could prove to be potent antibacterial agents against this pathogen.
Citation: Gutka HJ, Bondoc JMG, Patwell R, Khan S, Grzelak EM, Goswami R, et al. (2024) Rv0100: An essential acyl carrier protein from M. tuberculosis important in dormancy. PLoS ONE 19(6): e0304876. https://doi.org/10.1371/journal.pone.0304876
Editor: Selvakumar Subbian, Rutgers Biomedical and Health Sciences, UNITED STATES
Received: January 21, 2024; Accepted: May 20, 2024; Published: June 7, 2024
Copyright: © 2024 Gutka et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The raw data and statistics used to generate the figures in this manuscript are available in the figshare repository (accession number DOI: 10.6084/m9.figshare.25559904).
Funding: Farahnaz Movahedzadeh (FM) Grant No. RG-82534-N American Lung Association The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Mycobacterium tuberculosis (MTB), a causative agent of tuberculosis (TB), has infected more than a quarter of the human population and was the second leading cause of death by a single infectious agent in 2022 [1]. The infection usually develops into an asymptomatic state of latency which may last for many years. Changes in environmental conditions, such as compromised immunity of the host, may induce the dormant bacterium to begin proliferating and cause a shift from latent infection to pathogenesis. Furthermore, the increasing prevalence of drug resistance to the antibiotics that are part of the first-line treatment regimen has led to the proliferation of multiple drug-resistant (MDR) TB and extensively drug-resistant (XDR) TB [2, 3]. Consequently, there is an urgent need to identify new targets and more effective anti-mycobacterial agents to combat this disease.
Sequencing the MTB genome has revealed that over one third of its genome consists of unannotated genes; 15% with an unknown function and 23% associated with only hypothetical proteins. Subsequent proteomics and microarray analyses found many of these unannotated genes showed significant changes in expression under hypoxic conditions [4–9]. A basic understanding of the metabolic and regulatory pathways that allow MTB to maintain dormancy during a latent infection will be crucial to future therapeutic development. Yet, we still lack enough knowledge of these pathways to adequately describe the mechanisms of dormancy in MTB. Additionally, drug discovery efforts will be better served by focusing on proteins from MTB that do not share any functional homology with the human host, such as the unannotated protein Rv0100.
Rv0100 is a small, soluble protein that is composed of 78 amino acids with 2 cysteines and is likely located in the cytoplasm of MTB. Rv0100 was previously suggested to be essential for MTB survival in a mouse model of TB infection. This study by Sassetti and Rubin suggested that approximately 5% of the MTB genome was critical for growth, including Rv0100 [10].
Rv0100 is predicted to be an acyl carrier protein (ACP) with a phosphopantetheine attachment site [19], suggesting it plays a critical role in the fatty acid biosynthesis pathway, which is one of the primary targets for anti-mycobacterial therapeutics. Fatty acid precursors are important for cell wall synthesis and the energy storage required to maintain dormancy. In particular, MTB uses triacylglycerol to accumulate lipid droplets to initiate a dormancy-like phenotype [11]. Rv0100 is contained within an operon (Fig 1) that includes the nrp gene (Rv0101), which encodes an adenosine monophosphate-dependent peptide synthase involved in either polyketide non-ribosomal peptide synthase or fatty acid degradation. The operon also encodes a protein predicted to be a fatty acid CoA ligase (FadD10, Rv0099). Both Rv0100 and Rv0101 have phosphopantetheine attachment sites. Fatty acid adenylating enzymes (FADD) and fatty acyl-AMP ligase (FAAL) are essential for mycobacteria, specifically for linking fatty acids and polyketide synthesis [12, 13]. FAAL has been shown to be responsible for transferring fatty acids to Rv0100 [14]. When MTB transitions into a hypoxic stage, the energy supply pathway may shift to utilize fatty acids as an energy source, and Rv0100 and surrounding genes in the operon may be involved in this process. The entire operon is conserved in the pathogens Mycobacterium bovis (M. bovis), Mycobacterium leprae and Mycobacterium avium. Based on bioinformatics methods to predict protein partners, Rv0100 is co-expressed with Rv0101, as the Rv0100 stop codon overlaps with the start codon of Rv0101, with the genes being 18 nucleotides apart on the genome. Rv0100 may also be a protein partner with FadD10, as these genes are only 5 nucleotides apart. Additionally, this highly conserved group of genes has been implicated in the biosynthesis of phthiocerol dimycocerosates (PDIMs), the virulence-enhancing lipids produced by MTB and M. bovis [15]. Indeed, Hotter et. al (2005) found that the mutation of the Mb0100 gene (equivalent to MTB Rv0097) disrupted the synthesis of PDIMs and caused a loss of virulence.
The genomic structure surrounding Rv0100 including all the genes contained in the same operon. This includes all genes contained within the same operon as our target of interest, Rv0100. Here, the nrp gene (encoding the Rv0101 protein) is shown to be only 18 nucleotides apart from Rv0100. The overlapping stop codon of Rv0100 and start codon of Rv0101 make them likely protein partners.
Genomic sequencing studies as well as protein analyses of a persistent MTB mutant have shown that the formation of the mutant persistence phenotype is related to several pathways involving lipid biosynthesis and other carbon pathways [10, 16]. In particular, under hypoxic conditions, lipid inclusion bodies play an important role in drug resistance [17]. Further, several studies have shown that statins (inhibitors of cholesterol biosynthesis) as well as other acyl protein metabolizing enzymes have anti-tubercular effects [18], which suggests that acyl carrying proteins or other key elements of the lipid metabolism pathway can serve as promising drug targets.
In addition to Rv0100, there are other ACPs identified in the MTB genome such as Rv2244, Rv0033, and Rv1344 [19]. These three proteins share an identity of 15–23% and a similarity of 26–39% with Rv0100 [20]. However, these proteins were not found to be up-regulated in the whole genome expression experiments mentioned above while, in contrast, Rv0100 has been shown to be upregulated in several other studies using a dormancy model under hypoxic conditions [6, 21, 22]. Therefore, the other three ACPs are less likely to be major contributors to survival during a state of latent infection.
Our lab has previously overexpressed and purified the Rv0100 protein [23, 24]. However, understanding the function of Rv0100 in a mouse model of TB infection and in a dormancy-like state will be necessary to determine its potential as a drug target. In this study, we generated Rv0100 knockout mutant (KO) and a complement strain to determine if Rv0100 is essential for the growth and survival of MTB in infected mice, the Wayne model of persistence and a macrophage model.
Materials and methods
Bacterial strains, plasmids and media
Bacterial strains and plasmids used are shown in Table 1. E. coli DH5α was used for all plasmid constructions. MTB H37Rv was cultured on Middlebrook 7H10 agar with 10% oleic acid-albumin-dextrose-catalase (OADC) supplement. Middlebrook 7H9 broth with 10% OADC supplement and 0.05% Tween 80, Sauton’s medium (0.025% Tyloxypol, 2% glycerol) [25] or Dubos Tween-albumin were used to grow liquid cultures. Hygromycin (100μg/ml), kanamycin (20μg/ml), gentamicin (10μg/ml) was added where appropriate.
PCR amplification and cloning of Rv0100
Cloning of Rv0100 was performed using standard techniques previously described in detail [27]. Briefly, MTB sequences of the target gene were retrieved from Genebank (NC_000962.2) and used to design primers for DNA amplification (Table 2). The primers, Rv0100 Flank-1 and Rv0100 Flank-2, were designed to amplify the target gene and 1kb of the upstream and downstream flanking region. The amplification reaction was performed with Platinum PCR Supermix High Fidelity (Invitrogen) according to the manufacturers protocol with an annealing temperature of 55°C. PCR products were then cloned into pUC19 generating the construct pFM214.
Inverse PCR reaction
To create a deletion in Rv0100, inverse PCR was first performed on pFM214 [27] with the primers Rv0100 Inverse-1 and Rv0100 Inverse-2 (Table 2) using Phusion High-Fidelity DNA Polymerase (NEB) following the manufacturers protocol. These primers are designed in such a way that the flanking regions and plasmid sequence of pFM214 are amplified leaving out most of the target gene’s coding sequence. Next, the amplified template was digested with DpnI for 1h at 37°C followed by blunt end religation to restore the circular form of the plasmid. The new plasmid, pFM216, contains the deletion in Rv0100. The deletion in the resulting plasmid was confirmed with sequencing.
Cloning in p2NIL and cloning of the marker gene cassette
The SmaI restriction site present in pUC19 was used to digest the deleted versions of the target gene from the pFM216 plasmid and clone it into p2NIL. The pFM216 plasmid were digested with KpnI and BamHI and cloned into the KpnI and BamHI sites of p2NIL, generating the plasmid pFM218. A marker gene cassette derived from pGOAL19 was cloned into the PacI site of p218, generating the suicide delivery plasmid pFM220. The marker gene cassette contains the hygromycin resistance gene, the lacZ gene for blue/white selection, and the sacB gene that confers sucrose sensitivity. This set of genes is used for the selection of a single cross-over and ultimately a double cross-over.
In order to complement the Rv0100 mutant, this gene was cloned into pFM209 [29] under the Ag85 promoter, to produce pFM225. Then the HindIII cassette of pUC-Gm-int, which has the integration part and gentamicin gene, was cloned into the HindIII site of pFM225 to produce pFM227.
In vitro growth under hypoxic conditions using the Wayne model
The effect of Rv0100 deletion on growth under hypoxic conditions was performed using the commonly used protocol originally established by Wayne and Hayes [30]. Briefly, cultures of WT (H37Rv) MTB, along with the Rv0100 KO mutant and the Rv0100 complement strains were grown in Dubos-Tween-Albumin (Becton Dickinson) to an OD600 of 0.4 to 1. For hypoxic cultures, 16x100mm glass culture tubes containing 9ml DTA were inoculated to an OD580 of 0.04 and incubated at 37°C with 12mm magnetic stirrer bars stirring at 120rpm for up to two weeks. Screw-capped tubes were tightened to ensure an air-tight seal. Methylene blue 1.5ug/ml (final concentration) was added to one culture per strain as an indicator of oxygen depletion, which occurred completely by the 8th day. Two tubes were sampled at each time point of 4, 7, and 14 days in addition to the day 0 control. Colony forming units (CFU) were counted by serial dilution and plated on Middlebrook 7H11 medium containing OADC after 4 weeks incubation at 37°C. CFU measurements were obtained by averaging two technical replicates each from two biological replicates per strain at days 7 and 14. Only one biological replicate and two technical replicates were prepared for day 0 readings.
Detection and analysis of PDIMs
Given the involvement of Rv0100 in the fatty acid biosynthesis pathway, we wanted to know if its deletion would affect the synthesis of PDIMs, which are lipid components of the MTB cell wall that are known to be essential for its survival and virulence [31]. It is worth noting that while we observe no attenuation of growth compared to WT MTB when the Rv0100 KO mutant is cultured in 7H9 + OADC media, our initial attempts to culture this mutant in Sauton’s media failed. However, we were able to achieve marginal growth by lowering the concentration of glycerol from 6% to 2% (S1 Fig) which allowed us to collect the pellicles necessary for the PDIM analysis described below.
Bacterial pellicles were collected from 200ml cultures after 20 days by pouring off the excess media. The bacteria were inactivated by heating at 95°C for 2h and then transferred to a solution of CHCl3/CH3OH (2:1, v/v) for incubation at room temperature overnight. The lipids were extracted twice with CHCl3/CH3OH (1:1, v/v), concentrated under vacuum, then washed three times with water and dried. This crude lipid extract contains phosphoglucoisomerase (PGI) and various members of the DIM family such as dimycocerosates of phthiocerol A (DIM A) and dimycocerosates of phthiodiolone (DIM B) [32]. Thin-layer chromatography (TLC) was performed as previously described [33]. Briefly, TLC was conducted on silica gel G60 plates (20x3x20cm; Merck) using petroleum ether/diethyl ether (9:1 v/v) as the eluent. Lipid compounds were visualized by spraying the plates with 10% phosphomolybdic acid in ethanol and heating. Two biological and two technical replicates were run for each gel across two experimental runs. It should be noted that heat inactivation of the bacterial cultures may have led to some loss of PDIMs however, the reported qualitative differences are relative to the WT MTB strain, which was treated identically to the other strains.
In vivo growth and survival in mice
BALB/c female mice (n = 36) approximately 7–8 weeks old with an average weight of 20g were used for evaluating the effect of Rv0100 gene knockout on MTB infection in vivo. The mice were randomly divided into three groups (n = 18/group) and infected via aerosol using a Glas-Col inhalation exposure system with 2x107 CFU/ml of either WT, Rv0100 KO or the Rv0100 complement strain. Six mice from each group were sacrificed at post-infection days 1, 29 and 99. Carbon dioxide from a bottled source was used for asphyxiation followed by cervical dislocation. Whole lungs from individual mice were aseptically removed and homogenized in 3 ml of HBSS buffer. A hundred microliters of lung homogenate were used to make the serial dilutions that were then cultured on 7H11 media for CFU analysis. CFU measurements were obtained by averaging two technical replicates each from six biological replicates per strain per timepoint. The CFU limit of detection was 60 CFU/mouse. This study was approved by the ethics committee of the University of Illinois IACUC (protocol ACC # 09–100).
Macrophage assay
Inhibition of growth of the Rv0100 KO strain in comparison with its complement strain and WT was assessed as in a macrophage cell culture of J774A.1 cells as previously described [34, 35]. Briefly, J774A.1 cells were grown in DMEM medium supplemented with 10% FBS. Cells were detached and centrifuged, and the pellet was suspended to a final concentration of 1x105 to 3x105 cells/ml. One-milliliter aliquots of cell suspension were distributed into 24-well plates and incubated at 37°C in a 5% CO2 incubator for 16h. WT, Rv0100 KO, and Rv0100 complement strain cultures were diluted to a final concentration of 4x105 CFU/ml with DMEM, and 500μl of the dilution was dispensed to each well at a 1:1 multiplicity of infection (MOI) and incubated at 37°C for 2 hours to allow for phagocytosis. Cells were washed gently with HBSS to remove the extracellular bacteria, and fresh DMEM medium was added followed by incubation at 37°C under 5% CO2. Samples were collected at 0h (for pre-infection controls) and after 1 and 7 days of incubation. Macrophage cells were lysed with 0.25% sodium dodecyl sulfate, and CFU counts were performed by plating on solid agar. The CFU detection limit was 20 CFU/ml. Colonies were counted after 3 weeks of incubation at 37°C. Two biological replicates for CFU counts were prepared for each of three strains at the Pre and day 1 timepoints and 3 biological replicates for the day 7 timepoint. Each biological replicate was plated into two technical replicates for CFU analysis. More biological replicates were planned for the day 7 timepoint in anticipation of potential issues with cell clumping limiting our ability to accurately measure CFUs.
RNA isolation and microarray.
RNA isolation and microarray were performed as reported previously [36, 37]. Briefly, Cultures of MTB WT H37Rv and the Rv0100 KO mutant were grown stirring in Dubos-Tween-Albumin (Becton Dickinson) to an OD 600 of 0.15 in vented flasks prior to centrifugation for RNA isolation. RNA isolation was performed by bead agitation in Trizol reagent (Gibco-BRL) to disrupt bacterial cells followed by RNA purification with RNAeasy kits (Qiagen). Labeled cDNA was prepared using Superscript II (Invitrogen) Cy3 or Cy5-dUTP. Labeled cDNA was hybridized overnight to 70-mer oligonucleotide-based microarrays using the tuberculosis oligonucleotide set (Qiagen). Microarrays were scanned using a GenePix 4000A (Axon Instruments). Microarray-determined expression ratios were calculated from three biological replicates of H37Rv versus the Rv0100 KO and normalized using mean values of the middle 90% of all gene-specific spots on the microarray. Significance analysis of microarrays (SAM) was used to determine statistically significant regulated genes [38]. Only genes regulated at least 2-fold and had a SAM corresponding false discovery q value of zero were included in Table 3, column 1.
Statistical analysis
All statistical comparisons were conducted in Prism (version 9, Graphpad, La Jolla, CA). Log10 transformed CFUs/ml were used as the primary outcome measure in the Wayne, macrophage, and mouse models. Samples were obtained from different strains at various time points and compared using either two-way ANOVA or, in the case of missing data points, a mixed-effects model analysis. Tukey’s test was used for post-hoc follow up testing in the presence of a significant interaction of main effects. It should be noted that cell clumping is an inherent limitation to using CFU as a readout as it makes it difficult to discern individual colonies, particularly with regards to the later time points where cell density is high. To minimize this limitation, all CFU counts were averaged across 2–3 technical replicate plates, as described in detail in the methods section for each experiment. Klett units were used as the primary outcome measure for the qualitative analysis of growth in Sauton’s media experiment. A Klett unit is a measurement of optical density used by a Klett-Summerson Photoelectric Colorimeter (Scienceware, www.belart.com), where 20 Klett units is approximately equal to an OD of 0.1 at 540 nm. This instrument is part of a proprietary system including special culture flasks that allowed us to measure pathogenic anaerobic cultures in a Bio-Safety Level 3 environment.
Results
Growth of the Rv0100 KO strain in the Wayne model
Given that our hypothesis suggests Rv0100 could play an essential role during a latent infection, we monitored and compared the growth of our Rv0100 KO and complement strains to WT in the Non-replicating Persistence model (Wayne model) [30] at several different time points (Fig 2). A mixed model repeated measures analysis uncovered significant main effects of time [F(1.706,3.413) = 142.6, p = 0.0005] and strain [F(2,3) = 122.2, p = 0.0013] as well as a significant time by strain interaction [F(6,6) = 77.08, p<0.0001]. Tukey’s post hoc comparisons test was then used to explore the details of the significant interaction. There was significantly fewer CFUs in the Rv0100 KO strain compared to the complement strain at day 7 (p = 0.0005) but not the WT strain (p >0.05), likely due to high variability in the samples from this time point. Similarly, there were significantly fewer CFUs in the KO strain at day14 compared to both the WT (p = 0.0170) and the complement strain (p = 0.0281). Overall, these data suggest that knocking out Rv0100 can lead to significant attenuation of growth following exposure to anaerobic conditions.
The Rv0100 KO strain showed significantly attenuated growth following exposure to anaerobic conditions for 7 days compared to the complement and WT strains. This effect was further pronounced after 14 days of exposure where the difference in growth reached statistical significance compared to both complement and WT strains. n = 1(day0)-2 biological replicates. Data represented as mean ± SEM CFU/ml across 2 technical replicates per timepoint. Repeated measures mixed-effect ANOVA followed by Tukey’s test. *p<0.05:Rv0100 KO vs. WT and complement; †p<0.05:Rv0100 KO vs. complement. CFU = colony forming unit; KO = knock out; WT = wild type.
PDIM analysis
A qualitative comparison of the bands corresponding to DIM A and DIM B revealed disrupted synthesis of DIM B in the Rv0100 KO strain but not the WT (Fig 3). PGI was also greatly reduced in the ko strain. No differences in DIM A abundance were observed between the strains. We also examined PDIM content in the Rv0100 KO complement strain using TLC and found that PGI synthesis was restored (S2 Fig).
Qualitative analysis of lipid extracts from MTB WT and Rv0100 KO reveals that disruption of the Rv0100 gene is associated with disrupted synthesis PGI and the virulence enhancing PDIM, DIM B, but not DIM A. In a separate repeat experiment, we also compared the complement strain to WT and found that PGI was restored (S2 Fig). Representative image shown from two biological and two technical replicates. MTB = Mycobacterium tuberculosis; WT = wild type; KO = knock out; PDIM = phthiocerol dimycocerosates; PGI = phosphoglucoisomerase: Dim B = dimycocerosates of phthiodiolone; Dim A = dimycocerosates of phthiocerol A.
Growth of the Rv0100 KO strain in a mouse model of MTB infection
The effect of Rv0100 gene knockout on growth in lung tissue compared to both the control WT and the Rv0100 complement strain was evaluated in vivo using an aerosol infection mouse model (Fig 4). A two-way repeated measures ANOVA revealed significant main effects of both time [F(1.690,25.35) = 791.8, p<0.0001] and strain [F(2,15) = 119.9, p<0.0001] as well as a significant time by strain interaction [F(4,30) = 28.89, p<0.0001]. To further explore this interaction, we used Tukey’s test to compare the CFUs of the KO and complement strains to the control WT strain at each of time points. There were significantly fewer CFUs in the KO strain compared to both WT and complement at day 29 (both p<0.0001) and day 99 (pWT = 0.002, pCOM = 0.0004). In contrast, there were no significant difference between the complement strain and WT at either of these time points (p>0.05). There was also a small, but statistically significant increase in CFUs observed in the complement strain compared to WT at day 1 (mean log10(CFU) ± SD, complement: 0.3095±0.0961; WT: 0.1173±0.0909; p = 0.0132), however the cause of this difference is not clear. Together, these results indicate that knocking out Rv0100 attenuates the growth of this mutant MTB strain compared to WT MTB in the mouse lung and that restoring the Rv0100 gene through complementation can rescue this deficit.
The growth of MTB WT was compared with mutant Rv0100 KO and complement strains in vivo using a murine aerosol lung infection model. The Rv0100 KO strain exhibited significantly reduced growth compared to WT at post-infection days 29 and 99. In contrast, restoring Rv0100 through complementation resulted in a growth profile that was indistinguishable from WT at the same time points. There was also a small, but significant increase in CFUs found in the complement strain versus WT at post-infection day 1. Data represented as mean ± SEM CFU/ml lung tissue homogenate, n = 6 biological replicates per group for each time point. Two-way repeated measures ANOVA followed by Tukey’s test. *p<0.05:Rv0100 KO vs. WT and complement; #p<0.05:Rv0100 complement vs. WT. MTB = Mycobacterium tuberculosis; CFU = colony forming unit; KO = knock out; WT = wild type.
Growth of the Rv0100 KO strain in a macrophage model
We next sought to determine the effects of Rv0100 KO on the survivability of MTB in J774A.1 macrophages (Fig 5). Macrophages were infected with 4x105 CFU/ml of either WT, Rv0100 KO, or the Rv0100 complement strain and allowed to incubate for 0h (Pre), 1 day or 7 days before collecting the lysate and culturing the surviving MTB from each condition. All cultures were visually inspected under the microscope before lysis at each time point. No differences between the MTB strain conditions were noted neither in appearance following the initial phagocytosis, nor in macrophage viability. A mixed effects ANOVA of the resulting CFUs found a significant main effect of time [F(1.275,3.187) = 87.38, p = 0.0019] and a significant time by strain interaction [F(4,5) = 31.65, p = 0.0010]. A follow up Tukey’s test revealed a significant attenuation of growth in the Rv0100 complement strain compared to WT (p = 0.0083) at the 7 day post-infection time point. While the growth of the Rv0100 KO strain also appeared attenuated compared to WT at this time point, the effect did not reach statistical significance (p = 0.0636). It should be noted that there were two biological replicates planned for each of three strains at the Pre and day 1 timepoints and 3 biological replicates for the day 7 timepoint. However, one of the two biological replicates for WT failed at the day 1 timepoint and was not measurable. Overall, these data suggest that Rv0100 may play an important role in the survivability of MTB after phagocytosis by macrophages, however due to high statistical variability, this conclusion is limited to being based on qualitative observations of the differences in growth profile between the strains.
The ability of the mutant Rv0100 KO strain to survive phagocytosis by macrophages was assessed by comparing its growth with MTB WT and the Rv0100 complement strains at three time points. The results showed a significant reduction of CFUs in Rv0100 KO strain compared to complement at post-infection day 7. The WT strain also exhibited attenuation at this time point but the effect did not reach statistical significance. n = 1(day1;WT)-3 biological replicates per strain for each time point. Data represented as mean ± SEM CFU/ml across n = 1(day1;WT)-3 technical replicates per group for each time point. Repeated measures mixed-effect ANOVA followed by Tukey’s test. #p<0.05:Rv0100 complement vs. WT. MTB = Mycobacterium tuberculosis; CFU = colony forming unit; KO = knock out; WT = wild type.
Global gene expression profiling of the Rv0100 KO strain
To determine the regulatory scope of Rv0100, we performed global gene expression profiling using RNA microarrays comparing the Rv0100 KO strain with the H37Rv WT strain (S1 Table). While comparing Rv0100 KO vs. WT, we found several genes to be significantly upregulated using the SAM statistical analysis technique [38]. Amongst them, Rv0467 (aceA, isocitrate lyase) showed the most significant difference at 5.3-fold. Other notable genes displayed modest induction: AhpC (Alkyl hydroperoxidase), 3.8-fold; AhpD (member of AhpC/TSA family), 3.2-fold; RubA (rubredoxin A), 3.4-fold; RubB (rubredoxin B), 3.1-fold; Rv3249C (transcriptional regulator, TetR/AcrR family), 2.8-fold; Rv3269 (probable heat shock protein), 2.5-fold; and FadE23 (acyl coA dehydrogenase), 2-fold. In contrast, we also found several down regulated genes including Rv1361c (member of PPE family), 10.7-fold; LipF (a probable esterase), 3.5-fold; Rv0171 (part of the mce operon) 3.3-fold; Rv0173 (part of mce operon), 2.7-fold; Rv2295, 2.5-fold; Rv3723, 2.5-fold; and Rv2840C, 2-fold. We then compared our list of up-regulated genes with normalized expression data of other microarray studies evaluated in a large-scale meta-analysis [22]. We focused our comparisons on the expression pattern changes found in WT following exposure to hypoxic conditions for genes regulated at least 2-fold with a SAM corresponding false discovery q value of zero (Table 3). We found that knocking out Rv0100 produced significant changes in gene expression patterns that partially overlap patterns exhibited by WT strains under hypoxic conditions. Overall, these results support the role of Rv0100 as a potential mediator of the dormancy phenotype exhibited by MTB under hypoxic conditions.
Discussion
MTB has a unique ability to enter a state of dormancy during latent TB infection. The dormancy phenotype is a critical aspect of the bacterium’s life cycle and has significant implications for the spread and treatment of TB. During latent infection, MTB can persist in a non-replicating state within the host for years or even decades without causing symptoms. This state of dormancy allows the bacterium to evade the host’s immune response and survive in an environment that is hostile to its growth and replication. The immune system of the host keeps the bacteria under control but cannot eradicate it completely, leading to a delicate balance between the host and the pathogen. In this study have demonstrated that Rv0100 is not only essential for MTB growth in the Wayne model of non-replicating persistence, but also crucial for the pathogen’s survival in a macrophage assay as well as in a mouse model of TB infection.
A key challenge in tuberculosis research is interpreting genomic data within the context of host-pathogen interactions and leveraging this knowledge to develop a new generation of drugs and vaccines. The complete genome sequences of the MTB pathogen and the human host have been available since 2001, thanks to The Genome International Consortium. Analyzing these genomes enables us to identify potential gene products unique to the pathogen and absent in the host. This significantly narrows down the search for viable drug targets from a vast array to a select few. Most known antibacterial compounds function by inhibiting specific bacterial enzymes or proteins. Enzymes in MTB pathways that are dissimilar to any human protein are particularly promising as drug targets. In this study, we focused on the ACP, Rv0100, which is one of several proteins that play a role in the bacterial fatty acid biosynthesis pathway (FasII pathway) of MTB [39, 40], in contrast to the human FasI pathway, which utilizes a single multi-enzyme complex. Since the MTB FasII pathway is not similar to its human counterpart, Rv0100 represents a potentially attractive drug target as it would be likely to have minimal off-target effects.
Fatty acid metabolism is one of the primary targets for first-line antibacterial therapeutics. One ubiquitous example of a drug that targets bacterial fatty acid synthesis is the widely used Fabl inhibitor, Triclosan, which is usually directed against pathogenic Staphylococcus and E. coli infections. Fatty acid precursors are likely important for energy storage and cell wall synthesis that is required for maintenance of bacterial dormancy. In particular, MTB uses triacylglycerol to accumulate lipid droplets to acquire a dormancy-like phenotype [11]. Among the first-line therapeutics against MTB, isoniazid stands out for its targeted action on FasII system, crucial for the synthesis of mycolic acids, key components of the mycobacterial cell wall. This mechanism underscores the drug’s efficacy in disrupting the bacterial cell envelope, a vital barrier and structural component of MTB. Furthermore, the seminal report by Cole et. al (1998) predicted several MTB ACPs that would likely be required for the synthesis of mycolic acids [19]. These additional ACPs include Rv2244, Rv0033, and Rv1344. However, while these proteins share an identity of 15–23% and a similarity of 26–39% with Rv0100, our microarray analysis and comparisons with other dormancy models found no changes in the expression pattern of these ACPs. These results support our previous hypothesis that Rv0100 has more potential as a drug target than these other ACPs. Finally, several drugs are entering phase II trials to inhibit cell wall synthesis in MTB [41]. Taken together, these developments are highly suggestive of the promise held by inhibitors of fatty acid metabolism in combating MTB.
As previously mentioned, FADDs and FAAL are essential for linking fatty acids and polyketide synthesis in Mycobacterium [12, 13] and FAAL is responsible for transferring fatty acids to Rv0100 [14]. Additionally, there are multiple acyl-AMP ligases that are required for biosynthesis of PDIMs, which are important virulence lipids in the cell wall [42–45]. Interestingly, the disruption of PDIM synthesis observed in the Rv0100 KO strain in our study is similar to those found in M. bovis by Hotter et al. (2005) following the mutation of MB0100 (Rv0097 equivalent). Together, these findings may be indicative of a protein partner relationship between Rv0100 and Rv0097. Another study found that disruption of the ppe1-nrp operon harboring Rv0100 resulted in a disruption of PDIM synthesis but found no direct link to Rv0100 itself [46]. While outside the scope of the current study, it will be important for future work to investigate the specific role of Rv0100 in this context in order to further clarify the mechanisms of PDIM synthesis and its impact on virulence.
A comprehensive meta-analysis has shown that, across multiple sets of data, there is upregulation of Rv0100 in models of MTB dormancy [22]. This study normalizes experimental data gathered from microarray DNA gene expression ratios, accounting for maximum time end point from the source materials. Here, we compared our microarray results with the source data sets from the meta-analysis that used hypoxic conditions [6, 8, 21] and found a substantial overlap of genes, including Rv0100, that are upregulated (Table 3) [22]. In the microarray studies with starvation under 50% dissolved O2 [21], and microarray studies mimicking the Wayne model [6], Rv0100 was upregulated 3-fold and 4-fold, respectively. However, no changes were seen for Rv0100 in one of the comparative studies [8]. Others have also shown that the expression of Rv0100 was induced 2.2-fold by diamide, a chemical which triggers an oxidative stress reaction [47].
Collectively, the results from our work as well as others strongly implicate that Rv0100 is upregulated in response to oxidative stress and likely plays a key role in its survival during latent infections, potentially by mediating its dormancy state. However, future work needs to be performed to expand the scope of the current study in way that can firmly establish the essentiality of Rv0100 in the dormancy state of MTB and any potential role it may have on the synthesis of PDIMs. Only through increasing our understanding of how MTB survives during latent infection can we effectively develop desperately needed drugs to combat this ancient and deadly bacterium.
Supporting information
S1 Fig. Growth profile of MTB WT and Rv0100 KO in Sauton’s media in the presence of 2% or 6% glycerol.
The Rv0100 KO strain showed severely attenuated growth in the presence of 6% glycerol. Growth was achieved in the presence of 2% glycerol; however, it was much slower than WT in comparable media. n = 1 technical replicate per condition. KO = knock out; WT = wild type.
https://doi.org/10.1371/journal.pone.0304876.s001
(TIF)
S2 Fig. Lipid analysis of MTB WT versus the Rv0100 KO strain and the complement strain.
The various strains of MTB were grown in Sauton’s medium and bacterial cells were harvested in chloroform/methanol and lipids were subjected to thin layer chromatography. After resolving lipids on a thin layer plate, they were scanned using the ultra-violet scanner to visualize the bands. n = 3 biological replicates. Representative figure shown from three technical replicates. The blue arrows indicate PDI. The numbers represent Wild type (WT), Rv0100 KO (220) and Complement (227).
https://doi.org/10.1371/journal.pone.0304876.s002
(TIF)
S1 Table. Global gene expression profiling.
We performed global gene expression profiling using RNA microarrays to compare the Rv0100 KO strain with WT.
https://doi.org/10.1371/journal.pone.0304876.s003
(PDF)
S1 Raw images. Original image of Fig 3 TLC gel.
https://doi.org/10.1371/journal.pone.0304876.s004
(PDF)
Acknowledgments
The authors would like to thank Professor Scott Franzblau for the support of this project from the Institute of Tuberculosis Research and Dr Yuehong Wang for her help in animal preparation.
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