Concomitant Loss of the Glyoxalase System and Glycolysis Makes the Uncultured Pathogen "Candidatus Liberibacter asiaticus" an Energy Scavenger

Abstract

Methylglyoxal (MG) is a cytotoxic, nonenzymatic by-product of glycolysis that readily glycates proteins and DNA, resulting in carbonyl stress. Glyoxalase I and II (GloA and GloB) sequentially convert MG into d-lactic acid using glutathione (GSH) as a cofactor. The glyoxalase system is essential for the mitigation of MG-induced carbonyl stress, preventing subsequent cell death, and recycling GSH for maintenance of cellular redox poise. All pathogenic liberibacters identified to date are uncultured, including "Candidatus Liberibacter asiaticus," a psyllid endosymbiont and causal agent of the severely damaging citrus disease "huanglongbing." In silico analysis revealed the absence of gloA in "Ca Liberibacter asiaticus" and all other pathogenic liberibacters. Both gloA and gloB are present in Liberibacter crescens, the only liberibacter that has been cultured. L. crescens GloA was functional in a heterologous host. Marker interruption of gloA in L. crescens appeared to be lethal. Key glycolytic enzymes were either missing or significantly downregulated in "Ca Liberibacter asiaticus" compared to (cultured) L. crescens Marker interruption of sut, a sucrose transporter gene in L. crescens, decreased its ability to take up exogenously supplied sucrose in culture. "Ca Liberibacter asiaticus" lacks a homologous sugar transporter but has a functional ATP/ADP translocase, enabling it to thrive both in psyllids and in the sugar-rich citrus phloem by (i) avoiding sucrose uptake, (ii) avoiding MG generation via glycolysis, and (iii) directly importing ATP from the host cell. MG detoxification enzymes appear to be predictive of "Candidatus" status for many uncultured pathogenic and environmental bacteria.IMPORTANCE Discovered more than 100 years ago, the glyoxalase system is thought to be present across all domains of life and fundamental to cellular growth and viability. The glyoxalase system protects against carbonyl stress caused by methylglyoxal (MG), a highly reactive, mutagenic and cytotoxic compound that is nonenzymatically formed as a by-product of glycolysis. The uncultured alphaproteobacterium "Ca Liberibacter asiaticus" is a well-adapted endosymbiont of the Asian citrus psyllid, which transmits the severely damaging citrus disease "huanglongbing." "Ca Liberibacter asiaticus" lacks a functional glyoxalase pathway. We report here that the bacterium is able to thrive both in psyllids and in the sugar-rich citrus phloem by (i) avoiding sucrose uptake, (ii) avoiding (significant) MG generation via glycolysis, and (iii) directly importing ATP from the host cell. We hypothesize that failure to culture "Ca Liberibacter asiaticus" is at least partly due to its dependence on host cells for both ATP and MG detoxification.

Keywords: ATP/ADP translocase; carbonyl stress; citrus greening; culturability; glycolysis; glyoxalase; huanglongbing; liberibacter; methylglyoxal; sugar transporter.

FIG 1

Functional validation of L. crescens gloA in the heterologous host X. albilineans. (A) X. albilineans cells carrying L. crescens gloA (pMJ052), X. albilineans gloA (pMJ054), or empty vector (pUFR071) were grown for 4 days in modified Wilbrink's medium supplemented with 0 to 25 mM methylglyoxal. (B) Extractable glyoxalase I enzyme activity (S-lactoylglutathione, min⁻¹ mg of protein⁻¹) in X. albilineans cells carrying L. crescens gloA (pMJ052), X. albilineans gloA (pMJ054), or empty vector (pUFR071). The bars represent averages ± the standard errors (A, n = 8; B, n = 4). Asterisks represent significant differences at P < 0.05.

FIG 2

Relative expression of glyoxalase and key glycolysis pathway genes in L. crescens grown in culture and “Ca. Liberibacter asiaticus” in both its citrus and psyllid hosts. The transcript abundance of each gene was normalized against the expression levels of the chromosomal reference gene gyrB within each sample. Bars represent averages ± the standard errors of the means (n = 3). Asterisks represent significant differences (P < 0.05) in the transcript abundance with respect to prfA within each set.

FIG 3

Functional characterization of L. crescens sucrose transporter sut (B488_RS00965). Five-day-old wild-type BT-1 and interruption mutant Sut-1 cells (sut::pCR2.1-TOPO) were incubated in the presence of [¹⁴C]sucrose at room temperature. At the indicated time points, 500-μl culture aliquots were withdrawn, washed twice to remove external [¹⁴C]sucrose, and resuspended in PBS and scintillation fluid. The amount of radioactive sucrose taken up by the cells was expressed as counts per minute (cpm). Bars represent average data ± standard errors of at least three independent experiments.

FIG 4

(A) Relative expression of “Ca. Liberibacter asiaticus” ATP/ADP translocase (nttA [CLIBASIA_RS01005]) in both its citrus and psyllid hosts. Transcript abundances of nttA and prfA were normalized against expression levels of chromosomal reference gene gyrB within each host. Bars represent averages ± the standard errors of the means (n = 3). Asterisks represent significant differences (P < 0.05) in the transcript abundance with respect to prfA within each host. (B) Time- and dose-dependent [α-³²P]ATP uptake into L. crescens cells expressing “Ca. Liberibacter asiaticus” nttA. Five-day-old wild-type BT-1 cells and transformed BT-1/pMJ056 cells expressing nttA were incubated in the presence of [α-³²P]ATP at room temperature. At the indicated time points, 500-μl culture aliquots were withdrawn and washed twice to remove external [α-³²P]ATP. The cells were resuspended in PBS and scintillation fluid. The amount of radioactive ATP taken up by the cells was expressed as counts per minute (cpm). Bars represent average data ± the standard errors of at least three independent experiments.