Treponema pallidum 3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete

Abstract

In the causative agent of syphilis, Treponema pallidum, the gene encoding 3-phosphoglycerate mutase, gpm, is part of a six-gene operon (tro operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP in T. pallidum and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this, T. pallidum gpm was cloned, Gpm was purified from Escherichia coli, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective T. pallidum. Enzyme assays indicated that Gpm did not require Mn 2؉ while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25°C, retained only 50% activity after incubation for 20 min at 34°C or 10 min at 37°C, and was completely inactive after 10 min at 42°C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented E. coli strain PL225 (gpm) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of E. coli PL225 harboring pSLB2 from 34 to 42°C resulted in a 7-to 11-h period in which no growth occurred (compared to wild-type E. coli). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of T. pallidum.Syphilis, a sexually transmitted disease caused by the spirochete Treponema pallidum, remains a major public health problem in the world. T. pallidum cannot be cultivated in vitro, making it difficult to assess the role of genes in physiology, survival in the host, and pathogenesis. One approach to studying the functions of T. pallidum genes is to clone and overexpress these genes in Escherichia coli and then characterize the recombinant proteins in vitro. This approach was taken recently to characterize the TroR regulatory protein from T. pallidum (28). In the presence of Mn 2ϩ , TroR binds the operator of the transport-related operon (tro) and represses transcription. The tro operon contains six genes (14). The first four genes encode a putative ABC metal transport system (troA to -D), the fifth gene encodes TroR (troR), and the last gene encodes a glycolytic enzyme, 3-phosphoglycerate mutase (gpm, referred to as pgm in the T. pallidum genome database), which converts 3-phosphoglycerate (3-PGA) to 2-phosphoglycerate (2-PGA) (8, 11). Since T. pallidum can only generate ATP via glycolysis, 3-phosphoglycerate mutase is a key enzyme for the spirochete.Bacterial phosphoglycerate mutases are divided into two classes, based on their requirement for the cofactor 2,3-diphosphoglycerate (DPG) (10). Phosphoglycerate mutases from spore-forming Bacillus species, such as Bacillus megaterium, Bacillus subtilis, and Bacillus stearothermophilus, are DPG independent b...

The Treponema pallidum tro Operon Encodes a Multiple Metal Transporter, a Zinc-dependent Transcriptional Repressor, and a Semi-autonomously Expressed Phosphoglycerate Mutase

“…Gherardini and co-workers (16,20) further suggested that TroRmediated repression of Gpm within the central nervous system would decrease treponemal metabolism and thereby contribute to the latency of syphilitic infection. Because T. pallidum can only generate ATP by glycolysis, such a genetic arrangement seems inherently problematic because TroR-mediated repression of Gpm would hinder generation of ATP and thus the ability of the bacterium to escape a potentially metal-toxic environment.…”

“…In addition to encoding an ATPase (TroB), two cytoplasmic membrane permeases (TroC and TroD), and a DtxR-like metalloregulator (TroR), the operon also encodes the glycolytic enzyme phosphoglyceromutase (Gpm) (13). Because the T. pallidum Gpm has no requirement for metals (16), the physiological benefit of transcriptionally linking the spirochete's sole copy of this essential enzyme to the Tro transporter genes is unclear.…”

Efficient preparation and metal specificity of the regulatory protein TroR from the human pathogen Treponema pallidum

“…The protein TroR [22][23][24][25][26] in this study is from Treponema pallidum, a sexually transmitted human pathogen that usually disseminates from the genital area to diverse organs. The understanding of T. pallidum's biology and physiology remains limited largely because of the inability to culture it in vitro.…”

Biological Basis for Syphilis

“…The typical heat shock response regulated by 32 is lacking (105,296), possibly reflecting the sensitivity of the organism to growth temperature (95). At least one T. pallidum enzyme is unstable at normal body temperature (22), suggesting that the heat lability of enzymes may also contribute to the slow growth of the organism. Heat therapy for late neurosyphilis was introduced in 1918 by the Viennese psychiatrist Julius Wagner von Jauregg (326), a discovery for which he later won the Nobel Prize in Medicine.…”