A study on some aspects of Brucella central carbon metabolismThe entner-doudoroff, gluconeogenesis and erythritol pathways

Resumen

The brucellae causes brucellosis, a zoonosis causing abortions and sterility in livestock, symptomatology related to their intense proliferation in placenta and genitals. Although this strongly suggests a pathogenicity-metabolism link, little is known about Brucella metabolism. All brucellae lack the phosphofructokinase gene (pfk) and glucose catabolism through the Embden-Meyerhof-Parnas pathway has been experimentally ruled out. However, whereas genomic data suggest a full Entner-Doudoroff (ED) pathway, biochemical studies in B. abortus, B. melitensis and B. suis biovar 1 are inconsistent with its functionality. In Chapter 1, we have investigated whether these apparently contradictory data could mean that ED is an ancestral pathway whose functionality has been lost in some brucellae. To this end, we used B. suis 513 because of its phylogenomic position and ability to grow in vitro with glucose as the sole C source. We have proved that Edd (putative Entner-Doudoroff dehydratase) plays a role in glucose catabolism, and that it was necessary to simultaneously mutate Edd, PpdK (phosphoenolpyruvate dikinase) and Pyk (pyruvate kinase) to abrogate growth on glucose, xylose or ribose. These results support an active ED route in B. suis 513 that, with the Pentose Phosphate pathway, sustains growth on glucose or pentoses in vitro. Remarkably, the triple mutant in Edd-PpdK-Pyk was not attenuated in mice. It has been previously shown that B. abortus 2308W and B. suis 513 Fbp-GlpX mutants unexpectedly grow on glutamate-lactate-glycerol, which implies active gluconeogenesis. In Chapter 2, we show that the brucellae carry an undescribed gluconeogenic broad substrate bisphosphatase (Bbp) active not only on F1,6bP but also on sedoheptulose-1,7-bisphosphate (S1,7bP) and several bisphosphate polyols. We also show that Brucella gluconeogenesis requires the activity of the fructose-bisphosphate aldolase (Fba). Bbp and Fbp can link the Fba-dependent dihydroxyacetone-phosphate + glyceraldehyde 3-phosphate ⇌ F1,6bP reaction with fructose-6-phosphate generation. Similarly, they can link the Fba-dependent dihydroxyacetone-phosphate + erythrose 4-phosphate ⇌ S1,7bP reaction with sedoheptulose-7-phosphate for fructose-6-phosphate synthesis through the Pentose Phosphate shunt. On glutamate-lactate-glycerol, erythrose-4-phosphate can result from the transketolase-catalyzed fructose-6-phosphate + glyceraldehyde-3-phosphate ⇌ xylulose-5-phosphate + erythrose-4-phosphate interconversion, where fructose-6-phosphate results from the Bbp and Fbp F1,6bP bisphosphatase activity. Remarkably, dysfunction of all gluconeogenic enzymes did not result in attenuation in mice. These results suggest that the brucellae thrive intracellularly by using 3 and 4 C substrates with a limited supply of 6 and 5 C sugars. Erythritol is a preferred Brucella substrate present in the placenta and genitals. Since erythritol is catabolized into erythrose-4-phosphate, we re-examined in Chapter 3 the current model of erythritol catabolism through the Pentose Phosphate cycle. We have found that growth on erythritol proceeds in both a B. suis 513 transaldolase mutant and a double Bbp-Fbp mutant, which rules out a single entry of erythrose-4-P in the Pentose Phosphate cycle. Moreover, consistent with a two-entry model, growth on erythritol was abrogated in a double Fba-Tal mutant.