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Metabolic events mediating early killing of host cells infected by Shigella flexneri
1Service des Entérobactéries, U.199 INSERM, Institut Pasteur, Paris, France.
Abstract:
J774, a continuous macrophage cell-line, was infected by M90T, an invasive isolate of Shigella flexneri serotype 5 and BS176, its non invasive derivative--which does not harbor the 220 kbase virulence plasmid pWR100. Killing of host cells by intracellular M90T, commenced one hour after infection and was completed by 4 hours. Intracellular BS176 did not kill cells during the same period. Cell protein biosynthesis was totally inhibited by both strains within 2 hours of infection thus indicating that shiga-like toxin 1 (SLT1) could not account for early killing. On the other hand a sharp decrease in intracellular ATP was observed after 1 hour in cells infected with M90T. No significant increase in ATPase activity could be detected. A sharp increase in pyruvate production starting immediately after infection indicated impairement in mitochondrial respiration, which accounts for most ATP produced intracellularly. In addition, fermentation appeared to be totally blocked thus leaving no chance of the infected cells regenerating NAD. Concurrent increase in cAMP concentration within the first hour of infection may contribute to the rapid and efficient cell killing. Cells infected by BS176 always showed an intermediate phenotype (i.e. ATP depletion, pyruvate increase, lactate decrease). Early lysis of the phagocytic vacuole by M90T may account for this difference by allowing toxic products of the bacteria to diffuse more efficiently within the cytosol.
Insights
Invasive Shigella flexneri M90T rapidly kills macrophages by disrupting cellular energy metabolism and increasing cAMP. Non-invasive strains cause less severe metabolic changes, highlighting the role of virulence factors in host cell death.
Area of Science:
- Microbiology
- Cell Biology
- Pathogenesis
Background:
- Shigella flexneri is an invasive bacterial pathogen responsible for bacillary dysentery.
- Virulence plasmids, such as pWR100, encode factors essential for Shigella pathogenesis.
- Macrophage cell lines are crucial models for studying host-pathogen interactions.
Purpose of the Study:
- To investigate the mechanisms of host cell killing by invasive Shigella flexneri M90T compared to its non-invasive derivative BS176.
- To elucidate the role of cellular energy metabolism and signaling molecules in Shigella-induced cytotoxicity.
- To determine if Shiga-like toxin 1 (SLT1) contributes to the early stages of host cell death.
Main Methods:
- Infection of J774 macrophage cell line with Shigella flexneri M90T and BS176.
- Monitoring of host cell viability, protein biosynthesis, intracellular ATP levels, pyruvate production, and cAMP concentration.
- Analysis of mitochondrial respiration and fermentation pathways.
Main Results:
- Invasive M90T caused rapid host cell lysis within 4 hours, while non-invasive BS176 did not.
- Both strains inhibited protein biosynthesis, suggesting SLT1 is not responsible for early killing.
- M90T infection led to a sharp decrease in intracellular ATP and an increase in pyruvate, indicating impaired mitochondrial respiration and blocked fermentation.
- Increased cAMP concentration was observed in M90T-infected cells within the first hour.
- BS176-infected cells exhibited intermediate metabolic changes, including ATP depletion and pyruvate increase.
Conclusions:
- Early lysis of the phagocytic vacuole by M90T facilitates the rapid dissemination of bacterial toxic products into the host cell cytosol.
- Disruption of cellular energy metabolism, particularly mitochondrial respiration and ATP production, is a key mechanism for M90T-induced rapid host cell killing.
- Increased intracellular cAMP levels may contribute to the efficient and rapid cytotoxicity observed during M90T infection.