Aconitase Functions as a Pleiotropic Posttranscriptional Regulator in Helicobacter pylori

Abstract

Posttranscriptional regulation in bacteria has increasingly become recognized as playing a major role in response to environmental stimuli. Aconitase is a bifunctional protein that not only acts enzymatically but also can be a posttranscriptional regulator. To investigate protein expression regulated by Helicobacter pylori AcnB in response to oxidative stress, a global proteomics study was conducted wherein the ⌬acnB strain was compared to the parent strain when both strains were O 2 stressed. Many proteins, including some involved in urease activity, in combating oxidative stress, and in motility, were expressed at a significantly lower level in the ⌬acnB strain. A bioinformatics prediction tool was used to identify putative targets for aconitase-mediated regulation, and electrophoretic mobility shift assays demonstrated that apo-AcnB is able to bind to RNA transcripts of hpn (encoding a nickel-sequestering protein), ahpC (encoding alkyl hydroperoxide reductase), and flgR (encoding flagellum response regulator). Compared to the wild type (WT), the ⌬acnB strain had decreased activities of the nickel-containing enzymes urease and hydrogenase, and this could be correlated with lower total nickel levels within ⌬acnB cells. Binding of apo-AcnB to the hpn 5= untranslated region (UTR) may inhibit the expression of Hpn. In agreement with the finding that AcnB regulates the expression of antioxidant proteins such as AhpC, ⌬acnB cells displayed oxidative-stress-sensitive phenotypes. The ⌬acnB strain has a lesser motility ability than the WT strain, which can likely be explained by the functions of AcnB on the FlgRS-RpoN-FlgE regulatory cascade. Collectively, our results suggest a global role for aconitase as a posttranscriptional regulator in this gastric pathogen. IMPORTANCEBacterial survival depends on the ability of the cell to sense and respond to a variety of environmental changes. For Helicobacter pylori, responding to environmental stimuli within the gastric niche is essential for persistence and host colonization. However, there is much to be learned about the regulatory mechanisms that H. pylori employs to orchestrate its response to different stimuli. In this study, we explore the role of aconitase, a bifunctional protein that has been found to act as a posttranscriptional regulator in several other bacteria. Our results shed light on the magnitude of aconitase-mediated regulation in H. pylori, and we propose that aconitase acts as a global regulator of key genes involved in virulence.

Oxidative and nitrosative stress defences ofHelicobacterandCampylobacterspecies that counteract mammalian immunity

“…In particular, the abundance of AhpC was significantly lower in the acnB strain compared to the wild-type (Austin, Wang and Maier 2015). The acnB mutant also displayed decreased survival during exposure to atmospheric O 2 as well as hypersensitivity towards cumene hydroperoxide (Austin, Wang and Maier 2015).…”

“…The acnB mutant also displayed decreased survival during exposure to atmospheric O 2 as well as hypersensitivity towards cumene hydroperoxide (Austin, Wang and Maier 2015). Binding of apo-AcnB to the 3 UTR of the ahpC transcript was confirmed by electrophoretic mobility shift assay (EMSA) (Austin, Wang and Maier 2015).…”

Protein Activity Sensing in Bacteria in Regulating Metabolism and Motility

“…In H. pylori, some of those are connected to sensing performed by TlpD (Behrens et al, 2016). The authors identified several novel intracellular protein interaction partners of TlpD by mass spectrometry, including the iron-sulfur cluster-containing enzyme aconitase (AcnB) (Austin et al, 2015;Behrens et al, 2016). Besides the metabolic enzyme AcnB and the chemotaxis histidine kinase CheAY2, catalase KatA was detected as direct physical interactor of TlpD.…”

“…Conversely, TlpD had an effect on transcriptional regulation, impacting the expression of numerous genes involved in redox homeostasis and metabolism (Behrens et al, 2016). Therefore, it has been hypothesized that TlpD could serve as a linker between the taxis response and metabolic regulatory responses, possibly via the bifunctional regulatory protein AcnB (Austin et al, 2015;Behrens et al, 2016) (see next chapter for AcnB regulatory function). This direct link might be required to cope with energy limitation conditions, thus providing an immediate mechanism to improve the intracellular state of the cell.…”

“…AcnB has been described as a moonlighting protein with dual functions in E. coli, H. pylori and other organisms including Homo sapiens (Tang et al, 2005;Austin and Maier, 2013;Jeffery, 2014). Several studies concordantly show that aconitases of various organisms can perform a biphasic switch between a metabolically active and a regulatory function by protein conformational changes (Kiley and Beinert, 2003;Tang et al, 2005;Austin and Maier, 2013;Lushchak et al, 2014;Austin et al, 2015). Under oxidative stress or iron depletion, which destroys the iron-sulfur clusters essential for its enzymatic activity, aconitase partially unfolds and can itself bind to particular mRNA sequences, resulting in either an increased or decreased mRNA stability and altered translation of the respective proteins (Lushchak et al, 2014).…”