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Published on: December 7, 2015
Response mechanisms to acid stress promote LF82 replication in macrophages
Ting Yao1,2, Yu Huang1,2, Zimeng Huai1,2
1TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin, China.
Background:
Adherent-invasive E. coli (AIEC) LF82 is capable of adhering to and invading intestinal epithelial cells, as well as replicating within macrophages without inducing host cell death.
Methods:
We compared the transcriptomics of LF82 at pH=7.5 and pH=5.8 by RNA-sequencing, and qRT-PCR verified differentially expressed genes (DEGs). The deletion mutants of DEGs in the treatment group (pH=5.8) compared to the control group (pH=7.5) were constructed by λ recombinant. The replication differences between the mutants and WT infected Raw 264.7 at 24 h.p.i were analyzed by combining LB solid plate count and confocal observation. NH4Cl and chloroquine diphosphate (CQ) were used for acid neutralization to study the effect of pH on the replication of LF82 in macrophages. Na2NO3 was added to RPMI 1640 to study the effect of nitrate on the replication of LF82 in macrophages. 0.3% solid LB was used for flagellar motility assay and Hela was used to study flagellar gene deletion mutants and WT adhesion and invasion ability.
Results:
In this study, we found that infection with LF82 results in acidification of macrophages. Subsequent experiments demonstrated that an intracellular acidic environment is necessary for LF82 replication. Transcriptome and phenotypic analysis showed that high expression of acid shock genes and acid fitness genes promotes LF82 replication in macrophages. Further, we found that the replication of LF82 in macrophages was increased under nitrate treatment, and nitrogen metabolism genes of LF82 were upregulated in acid treatment. The replication in macrophages of ΔnarK, ΔnarXL, ΔnarP, and Δhmp were decreased. In addition, we found that the expression of flagellar genes was downregulated in acidic pH and after LF82 invading macrophages. Motility assay shows that the movement of LF82 on an acidic semisolid agar plate was limited. Further results showed that ΔfliC and ΔfliD decreased in motility, adhesion ability, and invasion of host cells, but no significant effect on replication in macrophages was observed.
Conclusion:
In this study, we simulated the acidic environment in macrophages, combined with transcriptome technology, and explained from the genetic level that LF82 promotes replication by activating its acid shock and fitness system, enhancing nitrate utilization, and inhibiting flagellar function.
Insights
Adherent-invasive E. coli (AIEC) LF82 replicates in macrophages by activating acid shock genes and enhancing nitrate use. Acidic conditions and nitrate boost AIEC LF82 replication, while flagellar genes are downregulated, reducing motility and invasion.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Adherent-invasive Escherichia coli (AIEC) LF82 exhibits adherence, invasion, and intracellular replication in intestinal epithelial cells and macrophages.
- AIEC LF82 can replicate within macrophages without inducing host cell death, highlighting its survival mechanisms.
Purpose of the Study:
- To investigate the transcriptomic and genetic basis of AIEC LF82 replication within macrophages under acidic conditions.
- To elucidate the role of acid shock genes, nitrate metabolism, and flagellar function in AIEC LF82 pathogenesis.
Main Methods:
- RNA sequencing and qRT-PCR to analyze differentially expressed genes (DEGs) in LF82 at different pH levels.
- Construction of deletion mutants for DEGs and analysis of their replication, adhesion, and invasion capabilities.
- Assays for flagellar motility and macrophage replication under various conditions, including acid neutralization and nitrate supplementation.
Main Results:
- Intracellular acidification is crucial for AIEC LF82 replication within macrophages.
- Upregulation of acid shock and acid fitness genes promotes LF82 replication; nitrate treatment further enhances replication.
- Nitrogen metabolism gene deletions (ΔnarK, ΔnarXL, ΔnarP, Δhmp) decreased LF82 replication; flagellar gene downregulation (ΔfliC, ΔfliD) impaired motility, adhesion, and invasion but not replication.
Conclusions:
- AIEC LF82 promotes replication in macrophages by activating acid shock/fitness systems and enhancing nitrate utilization.
- Acidic environments and nitrate availability are key factors influencing LF82 intracellular survival and proliferation.
- Inhibition of flagellar function under acidic conditions contributes to reduced motility and host cell interaction.
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