Heme degrading protein HemS is involved in oxidative stress response of Bartonella henselae

Abstract

Bartonellae are hemotropic bacteria, agents of emerging zoonoses. These bacteria are heme auxotroph Alphaproteobacteria which must import heme for supporting their growth, as they cannot synthesize it. Therefore, Bartonella genome encodes for a complete heme uptake system allowing the transportation of this compound across the outer membrane, the periplasm and the inner membranes. Heme has been proposed to be used as an iron source for Bartonella since these bacteria do not synthesize a complete system required for iron Fe³⁺ uptake. Similarly to other bacteria which use heme as an iron source, Bartonellae must transport this compound into the cytoplasm and degrade it to allow the release of iron from the tetrapyrrole ring. For Bartonella, the gene cluster devoted to the synthesis of the complete heme uptake system also contains a gene encoding for a polypeptide that shares homologies with heme trafficking or degrading enzymes. Using complementation of an E. coli mutant strain impaired in heme degradation, we demonstrated that HemS from Bartonella henselae expressed in E. coli allows the release of iron from heme. Purified HemS from B. henselae binds heme and can degrade it in the presence of a suitable electron donor, ascorbate or NADPH-cytochrome P450 reductase. Knocking down the expression of HemS in B. henselae reduces its ability to face H₂O₂ induced oxidative stress.

Figure 1. HemS and heme degrading or trafficking enzymes sequence alignment.

ClustalW alignment of HemS from B. henselae, PhuS from Pseudomonas aeruginosa , ChuS from E. coli , ShuS from Shigella dysenteriae , HemS from Yersinia enterocolitica , HmuS from Yersinia pestis . This alignment was generated by Clustal W. Amino acids conserved in five or more polypeptides are highlighted in grey. Amino acids conserved in all protein are indicated with a star.

Figure 2. Functional complementation of the E. coli mutants impaired in iron release from heme.

E. coli strains FB8.27 pAM::hasR (pBAD24) (A), FB8.27 efeB::Kan yfeX::Cmp (pAM::hasR) (pBAD24) (B) and FB8.27 efeB::Kan yfeX::Cmp (pAM::hasR ), (pBAD24::hemS _his) (C) were tested for the use of heme as an iron source on iron depleted medium M63 (Gly 0.4%, Ara 0.2%, Dip 70 µM, Spc, Amp). Growth around the wells containing 1 µM, 5 µM, 10 µM, or 50 µM Hb were performed as described in “Materials and Methods”. Growth around the wells was assessed by visible turbidity in the agar. These pictures were taken after 48 hours of growth at 37°C. Experiment was repeated three times. A representative result is presented.

Figure 3. HemS heme blotting.

After SDS gel electrophoresis, one gel was stained with comassie brilliant blue R. Another gel was transferred to a nitrocellulose filter to do heme blotting and detected by ECL. (A) Coomassie blue staining: Line 1, 5 µg BSA; Line 2, 4 µg HemS; (B) Heme binding: Line 1, 5 µg BSA; Line 2, 4 µg HemS. Experiment was performed in triplicate and a single representative experiment is presented.

Figure 4. Binding of heme to HemS.

(A): Increasing amounts of heme (1 µM–20 µM final concentration) were added to HemS (10 µM) as decribed in “Materials and Methods” and the spectrum (300 nm–700 nm) was recorded after 5 min for each addition. The Soret band at 411 nm increases with each addition of heme as demonstrated by absorbance peak increases at 411 nm. (B): Absorbance at 411 nm was measured for each sample and plotted versus heme concentration. Experiments were performed in triplicate and a single representative experiment is presented.

Figure 5. HemS dependent degradation of heme.

(A): 10 mM final concentration of ascorbate was added to the HemS-heme complex (10 µM). The spectral changes from 300–700 nm were recorded every 1 min. (B): Cytochrome P450 reductase was added to 10 µM HemS-heme complex with a 0.3∶1 molar ratio and heme degradation was initiated by adding NADPH 10 µM increments to a final concentration of 100 µM. The spectra were recorded from 300–700 nm after each addition. All Experiments were performed in triplicate and a single representative experiment is presented.

Figure 6. Detection of HemS expression level in B. henselae pNS2Trc and B. henselae pNS2Trc::hemS_AS by immunoblotting.

20 µg samples of B. henselae pNS2Trc (1) and B. henselae pNS2Trc::hemS _AS (2), 20 ng sample of purified his-tagged HemS (3) were loaded on SDS-PAGE. After electrophoresis, one gel was stained with comassie brilliant blue R (A). Another gel was transferred to a nitrocellulose filter to do immune blotting as decribed in “Materials and Methods” (B). Measurement of HemS band intensity using Image J software gave the following results: B. henselae pNS2Trc: mean gray value: 24, integrated density: 3218; B. henselae pNS2Trc::hemS AS: mean gray value: 14, integrated density: 2075.

Figure 7. HemS knockdown decreases B. henselae ability to face exposure to H₂O₂.

B. henselae pNS2Trc and B. henselae pNS2Trc::hemS _AS were challenged with 10 mM H₂O₂ as described in “Materials and Methods”. Experiments were performed in triplicate and a single representative experiment is presented.