Stability of the Adenosine 5′-Triphosphate Pool in Coxiella burnetii : Influence of pH and Substrate

Abstract

The ability of Coxiella burnetii to couple oxidation of metabolic substrates to adenosine 5'-triphosphate (ATP) synthesis in axenic reaction buffers was examined. Pyruvate, succinate, and glutamate were catabolized and incorporated at the highest rates of 11 substrates tested. Glutamate oxidation, however, resulted in the greatest stability of the ATP pool and highest intracellular ATP levels over a 48-h period. At pH 4.5, the optimum for metabolism by C. burnetii, glutamate oxidation resulted in maintenance of the ATP pool at a concentration of approximately 0.7 nmol of ATP per mg of dry weight over a 96-h period. In the absence of substrate, ATP declined by 96 h to less than 0.01 nmol/mg of dry weight. When cells were maintained at pH 7.0 in the presence or absence of glutamate, ATP pools were considerably more stable, presumably due to the miniimal metabolic activity displayed by C. burnetii at pH 7. The stability of the ATP pool reflected viability as there was greater than an 8-log decrease in viable C. burnetii after incubation for 7 days at pH 4.5 in the absence of glutamate. Viability was retained in the presence of glutamate at pH 4.5 or 7.0 in the absence of any added substrate. The stability of the ATP pool was due to endogenous synthesis of ATP coupled to substrate oxidation as shown by depression of ATP levels in the presence of inhibitors of electron transport or oxidative phosphorylation. In addition, the adenylate energy charge increased from an initial value of 0.57 to 0.73 during glutamate oxidation with a concomitant rise in the total adenylate pool size. C. burnetii therefore appears able to regulate endogenous ATP levels in response to substrate availability and pH, thus effecting a conservation of metabolic energy in neutral or alkaline environments. Such a mechanism has been proposed to play a role in the resistance of C. burnetii to environmental conditions and subsequent activation upon entry into the phagolysosome in which this organism replicates.

Protein synthesis by intact Coxiella burnetii cells

“…This communication confirms earlier observations concerning the acidophilic nature of C. burnetii (6)(7)(8). Studies performed on rickettsiae derived from heavily infected baby hamster kidney (BHK-21) cells, released either naturally or through mechanical lysis, indicate that moderate rates of protein biosynthesis occur in this organism when incubated in an axenic medium at pH 4.5.…”

“…Further investigation is required to determine whether this effect is due to inhibition of DNA replication or repair. Previous studies of axenic protein synthesis by C. burnetii reported incorporation rates for glutamate (6,7), which is a preferred energy source for this organism (8) and is presumably a key intermediate in amino acid biosynthesis. To best approximate the protein synthesis rate, this study utilized leucine and methionine.…”

“…The acidophilic nature of this organism has been demonstrated through the stimulation of nutrient transport and catabolism in intact cells at pH 4.5 (6,7). Furthermore, prolonged maintenance of the adenylate energy charge of C. burnetii was possible at either pH 4.5 or 7.0 if the medium was supplemented with the appropriate substrate, notably glutamate or pyruvate (8). Biosynthetic pathways were also operative in those studies because radiolabeled glucose, glutamate, thymidine, uridine, and guanosine were incorporated into acid-insoluble material at pH 4.5 (6).…”

“…Maintenance of a high energy charge in isolated C. burnetii cells could provide a better opportunity to obtain axenic growth (8). Classic methods of propagation of C. burnetii in yolk sacs of embryonated hen eggs afford luxuriant growth of rickettsiae.…”

A Microbial Strategy to Multiply in Macrophages: The Pregnant Pause

“…Transport and catabolism of glutamate by freshly explanted extracellular C. burnetii is optimal at pH 3-5 and poor at neutral pH (60). These and other data indicate that intracellular C. burnetii exploit the proton gradient across its cytoplasmic membrane to drive critical biochemical reactions (45,61,62). How lysosomal Leishmania, C. burnetii, and L. pneumophila cope with the array of degradative enzymes remains a mys-tery.…”