In a first experiment reported ( 2 ), suspensions of the T. whipplei
strain Twist were applied to a human squamous carcinoma cell line (HeLa cells; first established in 1952). Within 4 hours of incubation, some of the bacteria were internalized by the epithelial cells, whereby
the uptake of live bacteria was better than of heat-killed (100oC for 1 minute) bacteria. Intraepithelial bacteria were fluorescence-labelled with either polyclonal or monoclonal antibodies. During 3 days of
observation, more than 80% of intraepithelial bacteria stained positive with a nucleic acid dye (SYTO 9), indicating that bacterial DNA was not completely degraded within 3 days. SYTO9 staining was judged as
intracellular survival of bacteria, and dot-like fluorescence signals were counted semiquantitatively in software-generated virtual sections of 1 um thickness. Next, the pH within intracellular phagosomes was measured
indirectly by ratiometric analysis of fluorescence intensitites (DM-NERF). In HeLa cells infected with live bacteria, as well as in HeLa cells which ingested heat-killed bacteria, the phagosomal pH was estimated near
4.7 . Treatment of HeLa cells for 48 hours with pH-neutralizing drugs (ammonium chloride, bafilomycin, chloroquin, ethylmaleimide) resulted in increases of phagosomal pH near 6.4 , with only minor effects on HeLa cell
viability, but with reduced SYTO9 positivity of intracellular bacteria, consistent with partial degradation of bacterial DNA. It was concluded that survival of T.whipplei
strain Twist requires phagosome acidification, and manipulating pH may provide a new approach for the treatment.
Comment: The viability of intracellular Whipple-bacteria is a matter of debate, and experimental studies are clearly hampered by the very slow growth of bacteria. Until now, metabolically active bacteria
are observed (by FISH) in the intestinal lamina propria, where phagocytosis of extracellular bacteria is performed by macrophages. Ultrastructural studies argue against the hypothesis of survival of bacteria in
macrophages. On this background, interpretation of this short-term experimental study should be cautious.
1. Raoult D, Birg ML, La Scola B, Fournier PE, Enea M, Lepidi H, Roux V, Piette JC,
Vandenesch F, Vital-Durand D, Marrie TJ (2000). Cultivation of the bacillus of Whipple´s disease. N Engl J Med 342: 620-625
2. Ghigo E, Capo C, Aurouze M, Tung CH, Gorvel JP, Raoult D, Mege JL (2002). Survival of Tropheryma whipplei, the agent of Whipple´s disease,
requires phagosome acidification. Infect Immun 70: 1501-1506