The heparin-binding hemagglutinin protein of Mycobacterium tuberculosis is a nucleoid-associated protein

Abstract

Nucleoid-associated proteins (NAPs) regulate multiple cellular processes such as gene expression, virulence, and dormancy throughout bacterial species. NAPs help in the survival and adaptation of Mycobacterium tuberculosis (Mtb) within the host. Fourteen NAPs have been identified in Escherichia coli; however, only seven NAPs are documented in Mtb. Given its complex lifestyle, it is reasonable to assume that Mtb would encode for more NAPs. Using bioinformatics tools and biochemical experiments, we have identified the heparin-binding hemagglutinin (HbhA) protein of Mtb as a novel sequence-independent DNA-binding protein which has previously been characterized as an adhesion molecule required for extrapulmonary dissemination. Deleting the carboxy-terminal domain of HbhA resulted in a complete loss of its DNA-binding activity. Atomic force microscopy showed HbhA-mediated architectural modulations in the DNA, which may play a regulatory role in transcription and genome organization. Our results showed that HbhA colocalizes with the nucleoid region of Mtb. Transcriptomics analyses of a hbhA KO strain revealed that it regulates the expression of ∼36% of total and ∼29% of essential genes. Deletion of hbhA resulted in the upregulation of ∼73% of all differentially expressed genes, belonging to multiple pathways suggesting it to be a global repressor. The results show that HbhA is a nonessential NAP regulating gene expression globally and acting as a plausible transcriptional repressor.

Keywords: DNA damage; DNA topology; DNA transcription; DNA-protein interaction; Mycobacterium tuberculosis; genome organization; nucleoid-associated proteins (NAPs).

Figure 1

In-silico identification of HbhA from Mtb as a putative NAP.A, list of motifs that govern the DNA-binding ability of Histone H1 isoforms. B, presence of specific DNA-binding motifs in the amino acid sequence of different proteins encoded in the genome of Mtb and the number of repeats of DNA-binding motifs. C and D, the amino acid sequence of HbhA (C) and RplV (D) from Mtb shows the position of AKKA motifs. AKKA motifs are highlighted in red, and their positions are written in plum. Green highlighted portion of the amino acid sequence in (C) represents the C-terminal domain of HbhA. E, sequence- and structure-independent DNA-binding activity of RplV. A constant amount of supercoiled and linear forms of pUC19 DNA were incubated with increasing molar ratios (1:0, 1:50, 1:250, and 1:500) of RplV and Lsr2. Lsr2 was used as a positive control. GST at a DNA::protein molecular ratio of 1:500 was used as a negative control. GST, glutathione S-transferase; HbhA, heparin-binding hemagglutinin; Mtb, Mycobacterium tuberculosis.

Figure 2

HbhA from Mtb binds to DNA in a sequence- and structure-independent manner, and its C-terminal domain is DNA-binding domain.A and B, binding of HbhA with supercoiled and linear forms of pUC19 DNA, respectively. Constant amount of supercoiled and linear forms of pUC19 DNA were incubated with increasing molar ratios (1:0, 1:100, 1:250, 1:500, and 1:1000) of HbhA (lanes 1–5) and Lsr2 (lanes 6–10) with respect to pUC19. Lsr2 was used as a positive control and GST (lane 11) at a DNA::protein molar ratio of 1:1000 was used as a negative control. C, an illustration representing the generation of a C-terminal deleted mutant (HbhAΔC) and two N-terminal domain deleted mutants (HbhA_151-199, and HbhA_161-199) of HbhA. NTD and CTD represents N-terminal and C-terminal domains, respectively. D, constant amount of supercoiled pUC19 DNA were incubated with increasing DNA::protein molar ratios (1:0, 1:100, and 1:1000) of HbhA (lanes 1–3), HbhAΔC (lanes 4–6), HbhA_151-199 (lanes 7–9), and HbhA_161-199 (lanes 10–12) with respect to pUC19. Native DNA and DNA-protein complexes were resolved on 0.5% agarose gel and visualized using EtBr staining. E, CD spectra of HbhA and HbhAΔC. CTD, C-terminal domain; EtBr, ethidium bromide; GST, glutathione S-transferase; HbhA, heparin-binding hemagglutinin; NTD, N-terminal domain.

Figure 3

HbhA possesses characteristic features of a NAP.A, agarose gel image showing HbhA mediated protection of DNA from DNaseI enzymatic activity. Increasing DNA::HbhA molecular ratios enhanced DNA protection from DNaseI activity. +, ++, and ++++ denotes 1:250, 1:500, and 1:1000 DNA::HbhA molar ratios, respectively. B, agarose gel image showing inhibitory effect of HbhA on in vitro transcription reaction mediated by T7 RNA polymerase. Expected sizes of transcripts 1 and 2 are ∼2368 nucleotides and ∼1042 nucleotides, respectively. + and - denotes the presence and absence of specific components in the reaction mixture. C, agarose gel image showing the effect of HbhA on topoisomerase I-mediated relaxation of pUC19 DNA. + and - denotes the presence and absence of specific components in the reaction mixture. C and SC denote the covalently closed circular and supercoiled forms of pUC19 DNA, respectively. D and E, AFM images showing HbhA-mediated architectural modulations in supercoiled and linear forms of pUC19 DNA, respectively. 1:250 DNA::HbhA molar ratio was used to visualize the architectural effect of HbhA on pUC19 DNA. Upper panel in (D and E) denotes supercoiled and linear forms of pUC19 DNA alone, respectively, in 1× EMSA buffer. Blue arrows denote beads on a string like structure, lavender arrows denote bending of DNA molecule, cyan arrow denotes looping of DNA molecules. Scale bars are denoted in respective panels. AFM, atomic force microscopy; HbhA, heparin-binding hemagglutinin; NAP, nucleoid-associated proteins.

Figure 4

HbhA colocalizes with nucleoid of Mtb and is nonessential for invitro growth of Mtb.A, nucleoids were isolated using 12 to 60% sucrose density gradient centrifugation. DNA concentration (ng/μl) in each fractions of 500 μl were plotted against fraction number in a XY-graph. Fractions with highest DNA content represent the nucleoid fractions. B, representative immunoblot of three independent experiments showing the presence of HbhA protein in nucleoid fractions. rHbhA represents His-HbhA recombinant purified protein. The presence of HbhA was detected using antibodies generated in mice against His-HbhA at 1:5000. C, a schematic representation of MT103, MT103ΔhbhA, and MT103ΔhbhA::hbhA strains. D and E, stationary phase cultures of MT103, MT103ΔhbhA, and MT103ΔhbhA::hbhA strains were inoculated at an initial A₆₀₀ of 0.05 in 7H9 (D) and Sauton's (E) medium. Growth kinetics were measured by enumerating CFUs at indicated time points. Data represented shows mean log₁₀ CFU/ml ± SD of independent biological triplicates. CFU, colony-forming units; HbhA, heparin-binding hemagglutinin; Mtb, Mycobacterium tuberculosis.

Figure 5

HbhA acts as a global transcriptional regulator.A, schematic representation of RNA-sequencing experiment. B, volcano plot showing the differentially expressed genes (DEGs) in MT103ΔhbhA in comparison to MT103. Red and blue dots denote significantly downregulated and upregulated genes upon applying a cutoff of log₂ fold change > 1.0 and adjusted p-value < 0.05. C, heatmaps showing the normalized read counts of all DEGs from four biological replicates of MT103 and MT103ΔhbhA. Red and blue color intensities indicate relative downregulation and upregulation, respectively. D, bar graph showing the upregulated (blue) and downregulated (red) DEGs belonging to various functional categories. E, the pie chart depicting percentage of DEGs that are operonic and nonoperonic. F, the pie chart depicting the percentage of DEGs that are significantly upregulated and downregulated. G, bar graph depicting the number of operonic and nonoperonic DEGs belonging to essential (ES), essential domain (ESD), growth advantage (GA), growth defect (GD), nonessential (NE), and uncertain (U) categories. H, bubble plot depicting significantly enriched biological process categories of upregulated DEGs. I, bar graph depicting the number of significantly upregulated (blue) and downregulated (red) DEGs belonging to different biotypes. DEGs, differentially expressed genes; HbhA, heparin-binding hemagglutinin.

Figure S1

Multiple sequence alignment (MSA) and phylogenetic analysis of HbhA from different actinobacterial species.A, MSA was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). Percent coverage (cov) and percent identity (pid) matrix are shown. B, a phylogenetic tree representing evolutionary relationships of HbhA from different actinobacterial species was generated using Neighbor-Joining analysis conducted in MEGA 11. The tree is drawn to scale with bootstrap values. Mtb, Mycobacterium tuberculosis; Mbo, Mycobacterium bovis; Mlep, Mycobacterium leprae; Mmar, Mycobacterium marinum; Msm, Mycobacterium smegmatis; Mglv, Mycobacterium gilvum; Mabs, Mycobacterium abscessus; Mav, Mycobacterium avium; Mvan, Mycobacterium vanbaalenii; Mcan, Mycobacterium canetti; Nfar, Nocardia farcinica; Ropa, Rhodococcus opacus.

Figure S2

Effect of HbhAΔC and Lsr2 on the architectural features of linear pUC19 DNA. Effect of (A) HbhAΔC and (B) Lsr2 on the architectural features of linear pUC19 DNA. Lsr2 was used as positive control for AFM imaging. Scale bars are denoted in respective panels.

Figure S3

Evaluation of the presence of GroEL2 with nucleoids of Mtb. Immunoblot showing the absence of GroEL2 protein in nucleoid fractions of Mtb. rHbhA represents His-HbhA recombinant purified protein. The presence of GroEL2 was detected using antibodies generated in mice against His-GroEL2 at 1:10,000.

Figure S4

Comparative analyses of HbhA, mIHF, and Lsr2 regulons.A, venn-diagram showing the percentage of overlap and uniqueness of HbhA, mIHF, and Lsr2 regulons upon applying a cut-off of log₂ fold change > 1.0 and adjusted p-value < 0.05. B, the pie-chart showing the percentage of HbhA regulon that overlaps with mIHF, Lsr2, both mIHF and Lsr2, and unique to HbhA. C–F, bar graph showing the upregulated (blue) and downregulated (red) DEGs of (C) HbhA specific regulon, (D) HbhA and mIHF overlapping regulon, (E) HbhA and Lsr2 overlapping regulon, and (F) HbhA, mIHF, and Lsr2 overlapping regulon belonging to various functional categories.