Host induced microRNA hsa-miR-643 promotes Mycobacterium tuberculosis survival inside macrophages by downregulating Gins2

Technical Summary: Host-induced microRNA hsa-miR-643 promotes Mycobacterium tuberculosis survival inside macrophages by downregulating Gins2

Problem Statement
Tuberculosis (TB) remains a global health crisis, exacerbated by the rise of multi-drug resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis (Mtb). A critical factor in Mtb pathogenesis is its ability to survive within host macrophages by subverting host defenses, specifically by arresting phagosome maturation and evading apoptosis. While bacterial genes facilitating this survival are well-documented, the role of host non-coding RNAs, particularly microRNAs (miRNAs), in regulating this host-pathogen interplay remains poorly understood. Previous work by the authors identified hsa-miR-643 as significantly upregulated in Mtb-infected human macrophages (THP-1 cells), but its functional impact and specific targets were unknown.

Methodology
The study employed a multi-faceted approach to characterize the role of hsa-miR-643:

• Cellular Models: Differentiated THP-1 human macrophages were infected with pathogenic Mtb (H37Rv) and non-pathogenic Mtb (H37Ra) strains.
• Transcriptome Analysis: Comparative whole-genome RNA sequencing (RNA-seq) was performed on THP-1 cells treated with Mtb infection versus those transfected with hsa-miR-643 mimics. Differentially expressed genes were identified using iDEP.96 software with strict statistical cutoffs (FDR 0.05, log2 fold-change >1.8 or <-1.8).
• Target Validation:
  • qRT-PCR and Western Blot: Used to quantify mRNA and protein levels of candidate genes (specifically Gins2) in response to Mtb infection, hsa-miR-643 mimic transfection, and hsa-miR-643 inhibitor treatment.
  • Luciferase Reporter Assay: The 1.9 kb 3'-UTR of the human Gins2 gene was cloned into a pmirGLO vector. THP-1 cells were co-transfected with this construct and hsa-miR-643 mimics to assess direct binding. A mutant construct lacking the seed sequence (pmirGLO:Gins2 3'-UTR∆SS) served as a control.
• Functional Assays:
  • Apoptosis Analysis: Flow cytometry using Annexin V/Propidium Iodide staining was used to measure apoptosis rates in cells treated with hsa-miR-643 inhibitors and infected with Mtb.
  • Bacterial Survival: Colony Forming Unit (CFU) assays were conducted to enumerate intracellular Mtb loads in cells treated with hsa-miR-643 inhibitors or Gins2 siRNA.

Key Results

1. Identification of Gins2 as a Target: Transcriptome analysis revealed that hsa-miR-643 mimics consistently downregulated a specific set of genes. Among these, Gins2 (GINS complex subunit 2) was selected for further study.
2. Direct Regulation: Experimental validation confirmed that hsa-miR-643 directly targets the 3'-UTR of Gins2. Luciferase assays showed a significant reduction in activity when hsa-miR-643 mimics were present, which was restored in the mutant construct lacking the binding site. Consequently, Gins2 mRNA and protein levels were significantly decreased in Mtb-infected cells and mimic-transfected cells, while inhibitor treatment restored Gins2 expression.
3. Pathogenic Specificity: The downregulation of Gins2 was observed with the pathogenic H37Rv strain but not the non-pathogenic H37Ra strain, linking the phenomenon specifically to virulent Mtb infection.
4. Impact on Apoptosis and Bacterial Survival:
   • Inhibition of hsa-miR-643 led to a marked increase in apoptosis in infected THP-1 cells (rising to ~20% compared to ~5.5% in infected controls).
   • This increase in apoptosis correlated with a significant reduction in intracellular Mtb survival (lower CFU counts).
   • Conversely, silencing Gins2 via siRNA increased the bacterial burden, confirming that Gins2 levels are inversely related to Mtb survival.

Significance and Claims
The paper claims to establish a novel regulatory axis where Mtb manipulates host cell signaling to ensure intracellular survival. Specifically, the study demonstrates that:

• Mtb infection induces hsa-miR-643 in host macrophages.
• Elevated hsa-miR-643 directly suppresses Gins2 expression.
• The suppression of Gins2 attenuates host cell apoptosis, thereby creating a protected niche for Mtb replication.
• This mechanism is distinct from the response to avirulent strains, highlighting a specific virulence strategy.

The authors propose that hsa-miR-643 represents a promising therapeutic target for host-directed therapy (HDT). By inhibiting hsa-miR-643, the host's natural apoptotic defense could be restored to clear intracellular bacteria, potentially offering a strategy to combat drug-resistant TB without inducing bacterial antibiotic resistance. The paper also notes the potential for repurposing oncology drugs targeting Gins2 (an established oncogene) for infectious disease management, though it acknowledges that hsa-miR-643 likely targets other genes contributing to mycobacterial progression, warranting further investigation.