Aconitase-Mediated Posttranscriptional Regulation of Helicobacter pylori Peptidoglycan Deacetylase

Abstract

Some bacterial aconitases are bifunctional proteins that function in the citric acid cycle and act as posttranscriptional regulators in response to iron levels and oxidative stress. We explore the role of aconitase (AcnB) in Helicobacter pylori as a posttranscriptional regulator of the cell wall-modifying enzyme peptidoglycan deacetylase, PgdA. Under oxidative stress, PgdA is highly expressed and confers resistance to lysozyme in wild-type cells. PgdA protein expression as well as transcript abundance is significantly decreased in an acnB mutant. In the wild type, pgdA mRNA half-life was 13 min, whereas the half-life for the acnB strain was 7 min. Based on electrophoretic mobility shift assays and RNA footprinting, the H. pylori apo-AcnB binds to the 3=-untranslated region of the pgdA RNA transcript. Some of the protected bases (from footprinting) were localized in proposed stem-loop structures. AcnB-pgdA transcript binding was abolished by the addition of iron. The acnB strain is more susceptible to lysozyme-mediated killing and was attenuated in its ability to colonize mice. The results support a model whereby apo-AcnB directly interacts with the pgdA transcript to enhance stability and increase deacetylase enzyme expression, which impacts in vivo survival.

A Novel DNA-Binding Protein Plays an Important Role in Helicobacter pylori Stress Tolerance and Survival in the Host

“…Particularly, several important antioxidant proteins, such as SodB, Tpx, TrxR, and NapA, were less expressed in the ⌬hp119 mutant than in the WT strain. Strikingly, oxidative stress induces a peptidoglycan modification enzyme (PgdA) in WT H. pylori (52), and the aconitase (AcnB) was shown to be involved in this regulation at the posttranscriptional level (53). The results in Table 4 show that the expression of PgdA was greatly decreased, although another isoform of PgdA was slightly increased, in the ⌬hp119 mutant compared to the WT, suggesting that HP119 may also be involved in the regulation of oxidative stress induction of PgdA.…”

Aconitase Functions as a Pleiotropic Posttranscriptional Regulator in Helicobacter pylori

“…Purified RNA targets (50 nM) were each incubated separately with purified apo-AcnB (1.5 M) (for AcnB protein purification, see reference 5) in a solution containing 10 mM Tris (pH 8.3), 20 mM KCl, and 10% glycerol with an excess of yeast tRNA for 15 min at room temperature. The experimental conditions described here were previously optimized for use with apo-AcnB and putative IRE-like targets (5). Reaction products were resolved on a 6% nondenaturing polyacrylamide gel.…”

“…PgdA confers lysozyme resistance and immune evasion; its expression is increased when H. pylori cells are exposed to oxidative stress or are in contact with host immune cells (19)(20)(21)(22). AcnB plays an important role in the posttranscriptional regulation of PgdA (5). As H. pylori lacks many of the oxidative stress response regulators found in other Gram-negative bacteria, we hypothesize that AcnB serves as a posttranscriptional regulator for many other genes.…”

“…H. pylori possesses one copy of aconitase encoded by acnB. Recently, our laboratory demonstrated that H. pylori apo-AcnB binds to the 3= UTR of the peptidoglycan deacetylase (pgdA) transcript, resulting in transcript stability and increased PgdA expression (5). PgdA confers lysozyme resistance and immune evasion; its expression is increased when H. pylori cells are exposed to oxidative stress or are in contact with host immune cells (19)(20)(21)(22).…”

“…The regulatory role of aconitase has been studied in several different bacteria, and these bacterial aconitases have been shown to bind not only to the eukaryotic consensus IRE but also to several IRE-like sequences (4)(5)(6). In Escherichia coli, AcnA and AcnB have been shown to bind to and subsequently regulate the expression of the acnA and acnB transcripts, respectively (7).…”