Abstract
Chlamydia trachomatis is an obligate intracellular bacterium that alternates between two metabolically different developmental forms. We performed fluorescence lifetime imaging (FLIM) of the metabolic coenzymes, reduced nicotinamide adenine dinucleotides [NAD(P)H], by two-photon microscopy for separate analysis of host and pathogen metabolism during intracellular chlamydial infections. NAD(P)H autofluorescence was detected inside the chlamydial inclusion and showed enhanced signal intensity on the inclusion membrane as demonstrated by the co-localization with the 14-3-3β host cell protein. An increase of the fluorescence lifetime of protein-bound NAD(P)H [τ2-NAD(P)H] inside the chlamydial inclusion strongly correlated with enhanced metabolic activity of chlamydial reticulate bodies during the mid-phase of infection. Inhibition of host cell metabolism that resulted in aberrant intracellular chlamydial inclusion morphology completely abrogated the τ2-NAD(P)H increase inside the chlamydial inclusion. τ2-NAD(P)H also decreased inside chlamydial inclusions when the cells were treated with IFNγ reflecting the reduced metabolism of persistent chlamydiae. Furthermore, a significant increase in τ2-NAD(P)H and a decrease in the relative amount of free NAD(P)H inside the host cell nucleus indicated cellular starvation during intracellular chlamydial infection. Using FLIM analysis by two-photon microscopy we could visualize for the first time metabolic pathogen-host interactions during intracellular Chlamydia trachomatis infections with high spatial and temporal resolution in living cells. Our findings suggest that intracellular chlamydial metabolism is directly linked to cellular NAD(P)H signaling pathways that are involved in host cell survival and longevity.
Highlights
The obligate intracellular bacterium Chlamydia trachomatis (C. trachomatis) has two metabolically different developmental forms, which ensure its infectivity and replication
It seems obvious that a system to import NAD(P) from the host cell must exist to that of environmental chlamydiae, no NAD(P) transporter has yet been identified in C. trachomatis according to sequence homology searches [7]
A more detailed understanding about the metabolic activity and needs of C. trachomatis and its specific interactions with the host cell would be the basis for the development of novel treatment strategies
Summary
The obligate intracellular bacterium Chlamydia trachomatis (C. trachomatis) has two metabolically different developmental forms, which ensure its infectivity and replication. The infectious form, the elementary body, enters the host cell and differentiates into a metabolically active form, the reticulate body. Within 24 hours post infection (hpi), numbers and size of reticulate bodies are maximized and chlamydiae start to re-differentiate to infectious elementary bodies. An ADP/ATP transporter has been found [2,3], the genome sequence unraveled the existence of several glucose metabolizing enzymes [4]. It seems obvious that a system to import NAD(P) from the host cell must exist to that of environmental chlamydiae, no NAD(P) transporter has yet been identified in C. trachomatis according to sequence homology searches [7]. It is reasonable to assume that intracellular chlamydial development strongly depends on host cell NAD availability
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