The multifunctional histone-like protein Lsr2 protects mycobacteria against reactive oxygen intermediates - PubMed (original) (raw)

. 2009 Mar 17;106(11):4414-8.

doi: 10.1073/pnas.0810126106. Epub 2009 Feb 23.

A Haq, V L Arcus, E Summers, R S Magliozzo, A McBride, A K Mitra, M Radjainia, A Khajo, W R Jacobs Jr, P Salgame, D Alland

Affiliations

The multifunctional histone-like protein Lsr2 protects mycobacteria against reactive oxygen intermediates

R Colangeli et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Mycobacterium tuberculosis has evolved a number of strategies to survive within the hostile environment of host phagocytes. Reactive nitrogen and oxygen intermediates (RNI and ROI) are among the most effective antimycobacterial molecules generated by the host during infection. Lsr2 is a M. tuberculosis protein with histone-like features, including the ability to regulate a variety of transcriptional responses in mycobacteria. Here we demonstrate that Lsr2 protects mycobacteria against ROI in vitro and during macrophage infection. Furthermore, using macrophages derived from NOS(-/-) and Phox(-/-) mice, we demonstrate that Lsr2 is important in protecting against ROI but not RNI. The protection provided by Lsr2 protein is not the result of its ability to either bind iron or scavenge hydroxyl radicals. Instead, electron microscopy and DNA-binding studies suggest that Lsr2 shields DNA from reactive intermediates by binding bacterial DNA and physically protecting it. Thus, Lsr2 appears to be a unique protein with both histone-like properties and protective features that may be central to M. tuberculosis pathogenesis. In addition, evidence indicates that lsr2 is an essential gene in M. tuberculosis. Because of its essentiality, Lsr2 may represent an excellent candidate as a drug target.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

DNA protection studies. (A) Linear DNA (≈0.2 μM) was used alone or after incubation with either 2 μM of purified Lsr2 or BSA. Both proteins were suspended in identical imidazole buffers. Samples were treated for 5 min with either FeSO4 (20 mM) plus H2O2 (5 mM) or left untreated as indicated. The samples were analyzed on an ethidium bromide stained 1% agarose gel. Lane 1, linear DNA; lane 2, linear DNA + Lsr2; lane 3, linear DNA + Lsr2 + FeSO4/H2O2; lane 4, linear DNA + BSA; lane 5, linear DNA + BSA + FeSO4/H2O2. (B) Supercoiled φhX174 plasmid (≈0.1 μM) was used alone or incubated with Lsr2 in imidazole buffer. The Lsr2 was used without or with prior extensive dialysis in buffer containing 800 mM NaCl. Samples were treated for 5 min with either FeSO4 (20 mM) plus H2O2 (5 mM) or left untreated as indicated and then analyzed on an ethidium bromide stained 1% agarose gel. Lane 1, φhX174 plasmid; lane 2, φhX174 plasmid + Lsr2; lane 3, φhX174 plasmid in buffer containing 800 mM NaCl; lane 4, φhX174 plasmid in buffer containing 800 mM NaCl + dialyzed Lsr2; lane 5, φhX174 plasmid + Lsr2 + FeSO4/H2O2; lane 6, φhX174 plasmid in buffer containing 800 mM NaCl + dialyzed Lsr2 + FeSO4/H2O2; lane 7, φhX174 plasmid + FeSO4/H2O2; lane 8, φhX174 plasmid in buffer containing 800 mM NaCl + FeSo4/H2O2.

Fig. 2.

Fig. 2.

Electron microscopic structure of Lsr2/DNA complexes. (A) Micrograph of purified, recombinant Lsr2 embedded in uranyl-acetate (UA, negative stain) displaying a filamentous structure. (B) Five representative class averages of Lsr2 following reference-free classification of ≈700 boxed (244 × 244 Å) segments and isolated from the image shown in A and 2 other images. The fibrillar structure with helical winding is apparent. The diameter of the fibrils is ≈90 Å. (C) A cryoelectron micrograph revealing protofibrils of the Lsr2–DNA complex. Arrows indicate the protofibrils and lines have been placed to the immediate left of the protofibrils to further clarify their position. The diameter of the protofibrils is ≈45Å.

Fig. 3.

Fig. 3.

Sensitivity of M. smegmatis and M. tuberculosis mutants to H2O2 and to NO. Five mM of H2O2, 10 mM of sodium nitrite, or 50 mM of sodium nitrite were added to cultures of each strain. Culture aliquots were removed at different time points and plated on solid media. Results are expressed as colony forming units (cfu) per milliliter of culture. (A) M. smegmatis strains treated with H2O2: (open diamonds) mc2 155 (control); (filled squares) NJS22 (lsr2 knockout); and (filled triangles) NJS22.1 (lsr2 knockout strain overexpressing lsr2 on an expression plasmid). (B) M. smegmatis strains treated with sodium nitrite. Red indicates treatment with 10 mM sodium nitrate: (red open diamonds) mc2 155; (red squares) NJS22; and (red triangles) NJS22.1. Black indicates treatment with 50 mM sodium nitrate: (open diamonds) mc2 155; (filled squares) NJS22; and (filled triangles) NJS22.1. (C) M. tuberculosis strains treated with H2O2: (open diamonds) H37Rv(pMV261) (control) (filled triangles); NJS18 (lsr2 overexpression strain). Error bars indicate plus and minus 1 standard deviation of a minimum of 3 experiments. The _P_-value at all time points (panel A) and at 3 and 4 h (panel B) was <0.05 in a Tukey's Studentize range.

Fig. 4.

Fig. 4.

Survival of M. smegmatis mutants within macrophages. Bone marrow derived macrophages (BMM) from (A) wild-type C57BL/6 mice; (B) NOS−/− mice; (C) Phox−/− mice; and (D) wild-type C57BL/6 mice were infected with (open diamonds) M. smegmatis mc2 155 (control) (red squares); NJS22 (lsr2 knockout); and (blue triangles) NJS22.1 (lsr2 knockout strain overexpressing lsr2 on an expression plasmid). In panel D, the BMM were treated with 500 μM of Fenbufen 1 h before infection and again 48 h after infection. For each strain, the cfu at each time point are expressed relative to the cfu at time 0. Results from a minimum of 3 replicates are shown for each time point. Error bars indicate plus and minus 1 standard deviation. The _P_-values between wild-type and lsr2 knockout strains for time points 48 and 72 h (panel A); and time points 24, 48, and 72 h (panel B) were <0.05 in a Turkey's Studentize range. The _P_-values between wild-type and lsr2 knockout strains were not significantly different in panels C and D at all time points tested.

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