Leptospira interrogans Aer2: an Unusual Membrane-Bound PAS-Heme Oxygen Sensor

Abstract

In this study, we provide the first characterization of a chemoreceptor from Leptospira interrogans, the cause of leptospirosis. This receptor is related to the Aer2 receptors that have been studied in other bacteria. In those organisms, Aer2 is a soluble receptor with one or two PAS-heme domains and signals in response to O₂ binding. In contrast, L. interrogans Aer2 (LiAer2) is an unusual membrane-bound Aer2 with a periplasmic domain and three cytoplasmic PAS-heme domains. Each of the three PAS domains bound b-type heme via conserved Eη-His residues. They also bound O₂ and CO with similar affinities to each other and other PAS-heme domains. However, all three PAS domains were uniquely hexacoordinate in the deoxy-heme state, whereas other Aer2-PAS domains are pentacoordinate. Similar to other Aer2 receptors, LiAer2 could hijack the E. coli chemotaxis pathway but only when it was expressed with an E. coli high-abundance chemoreceptor. Unexpectedly, the response was inverted relative to classic Aer2 receptors. That is, LiAer2 caused E. coli to tumble (it was signal-on) in the absence of O₂ and to stop tumbling in its presence. Thus, an endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2, and its displacement for gas-binding turned signaling off. This response also occurred in a soluble version of LiAer2 lacking the periplasmic domain, transmembrane (TM) region, and first two PAS domains, meaning that PAS3 alone was sufficient for O₂-mediated control. Future studies are needed to understand the unique signaling mechanisms of this unusual Aer2 receptor. IMPORTANCE Leptospira interrogans, the cause of the zoonotic infection leptospirosis, is found in soil and water contaminated with animal urine. L. interrogans survives in complex environments with the aid of 12 chemoreceptors, none of which has been explicitly studied. In this study, we characterized the first L. interrogans chemoreceptor, LiAer2, and reported its unique characteristics. LiAer2 is membrane-bound, has three cytoplasmic PAS-heme domains that each bound hexacoordinate b-type heme and O₂ turned LiAer2 signaling off. An endogenous ligand in the deoxy-heme state was correlated with signal-on LiAer2 and its displacement for O₂-binding turned signaling off. Our study corroborated previous findings that Aer2 receptors are O₂ sensors, but also demonstrated that they do not all function the same way.

Keywords: Aer2; Leptospira interrogans; PAS domain; chemoreceptor; heme; oxygen sensing.

FIG 1

The L. interrogans che2 operon and LiAer2 structural models. (A) Organization of the che2 operon from L. interrogans serovar Pomona strain Pomona. Che2 encodes a chemoreceptor (Aer2), two histidine kinases (HK and CheA2), a coupling protein (CheW3), two response regulators (RR and CheY2), two adaptation proteins (CheD1 and CheB3), and an anti-sigma factor antagonist (σ*). (B) The structure of a LiAer2 dimer as predicted by AlphaFold. LiAer2 contains a potential periplasmic ligand-binding domain (LBD), followed by a transmembrane (TM) region, three cytoplasmic PAS domains, a di-HAMP unit, and a kinase control module with 34 heptad repeats (34H). (C) LiAer2 PAS3 dimer model based on the unliganded dimer structure of P. aeruginosa Aer2 PAS (PaPAS) with heme (PDB 4HI4) (16). PaPAS residues involved in heme- and O₂-binding (Eη-His and Iβ-Trp) are shown as sticks. (D) Proposed LiAer2 interactions and phosphotransfer reactions based on known interactions and reactions in the P. aeruginosa Che2 system (9) (see text for details). By analogy to the methylation sites of E. coli Tsr and Tar, LiAer2 has three predicted methylation sites (residues 729, 736, and 904), and one putative methylation site (residue 911, one heptad C-terminal to residue 904), all of which are surrounded by predicted CheD binding sites.

FIG 2

LiAer2-directed behavior in E. coli BT3312. (A) The average percentage of E. coli BT3312 cells tumbling over a 1 s period, 30 s after switching to air or N₂. Results are shown for BT3312 expressing full-length LiAer2_1-976, truncated LiAer2 receptors, or cells containing the empty vector pProEXHTa (pProEX). Error bars represent the standard deviation from three to six independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. There was no significant difference in percent tumbling for LiAer2_597-976 or pProEX in air or N₂ (P > 0.05). (B) E. coli BT3312 expressing LiAer2_1-976, LiAer2_203-976, or containing pProEX, in tryptone soft agar with 0, 50, or 100 μM IPTG. Plates were incubated at 30°C for 16 h.

FIG 3

LiAer2-directed behavior in E. coli UU2610 (CheRB⁻) and UU2612 (CheRB⁺) in the absence and presence of EcTar. LiAer2_1-976 and EcTar were expressed from compatible plasmids in isogenic, chemoreceptorless E. coli strains in the absence and presence of the adaptation enzymes CheR and CheB. Graphs represent the average percentage of E. coli cells tumbling over a 1 s period, 30 s after switching to air or N₂. Error bars represent the standard deviation from two to four independent experiments. ****, P < 0.0001.

FIG 4

LiPAS sequence alignment and structural elements. (A) An alignment of the Aer2 PAS domain sequences from L. interrogans serovar Pomona strain Pomona and P. aeruginosa PAO1 as generated by Clustal Omega. Stars indicate conserved residues, colons indicate similar amino acids, and periods indicate amino acids with weakly similar properties. The conserved Eη-His that coordinates heme in PaPAS, and the Iβ-Trp that stabilizes O₂-binding in PaPAS, are boxed and colored blue and orange, respectively. Residues that line the heme cleft of PaPAS are highlighted gray. The C-terminal “DxT” motifs that conformationally couple PAS to a C-terminal domain are boxed and colored red. Secondary structure elements are based on the solved structures of PaPAS (16, 17). (B) LiPAS3 cartoon from Fig. 1C (showing the right-side protomer rotated by −10° on the y-axis) with secondary structure elements labeled.

FIG 5

PAS1-3 heme spectra and dissociation constants. Absorption spectra of 5 to 10 μM purified LiAer2 PAS domains in the reduced (deoxy), oxygen-bound (oxy), and carbon monoxide-bound (carbonmonoxy) states. The wavelengths of each absorbance maximum (the Soret and β and α peaks) are indicated. Inserts show an expanded view of the β and α peaks between 500 and 600 nm. The O₂ and CO affinities for each PAS domain are also shown.

FIG 6

Analysis of PAS Eη-His and Iβ-Trp mutants. (A) Average heme content of PAS domains containing Eη-His or Iβ-Trp replacements, given as a percentage of WT PAS heme content and corrected for protein concentration. Error bars represent the standard deviation from three independent experiments. (B) Representative O₂ titration for 10 μM purified PAS1-W329L showing O₂ concentrations of interest. The “?” indicates that the heme is bound to an unidentified ligand.