Alkyl Hydroperoxide Reductase Subunit C (AhpC) Protects Bacterial and Human Cells against Reactive Nitrogen Intermediates

Molecular Cell, Vol. 1, 795–805, May, 1998, Copyright 1998 by Cell Press

Alkyl Hydroperoxide Reductase Subunit C (AhpC) Protects Bacterial and Human Cells against Reactive Nitrogen Intermediates Lei Chen, Qiao-wen Xie, and Carl Nathan* Seaver Laboratory Division of Hematology-Oncology Department of Medicine Cornell University Medical College New York, New York 10021

Summary In Salmonella typhimurium, ahpC encodes subunit C of alkyl hydroperoxide reductase, an enzyme that reduces organic peroxides. Here, we asked if ahpC could protect cells from reactive nitrogen intermediates (RNI). Salmonella disrupted in ahpC became hypersusceptible to RNI. ahpC from either Mycobacterium tuberculosis or S. typhimurium fully complemented the defect. Unlike protection against cumene hydroperoxide, protection afforded by ahpC against RNI was independent of the reducing flavoprotein, AhpF. Mycobacterial ahpC protected human cells from necrosis and apoptosis caused by RNI delivered exogenously or produced endogenously by transfected nitric oxide synthase. Resistance to RNI appears to be a physiologic function of ahpC. ahpC is the most widely distributed gene known that protects cells directly from RNI, and provides an enzymatic defense against an element of antitubercular immunity.

Introduction Aerobic metabolism leads to oxidative stress from generation of reactive oxygen intermediates (ROI). ROIs exert dose-dependent cellular effects ranging from (in)activation of kinases (Devary et al., 1992; Sundaresan et al., 1995; Konishi et al., 1997), phosphatases (Lander, 1997), ion channels (Taglialatela et al., 1997), and transcription factors (Abate et al., 1990; Schreck and Baeuerle, 1991) to mutagenesis (Demple and Linn, 1982; Storz et al., 1987), apoptosis (Hockenbery et al., 1993; Hug et al., 1997; Polyak et al., 1997), and necrosis (Nathan et al., 1979). Oxidative injury appears to play a critical role in many disease processes (Halliwell and Gutteridge, 1990), including ischemia-reperfusion (McCord, 1987), neurodegeneration (Beal, 1995), tumor promotion (Cerutti, 1985), and aging (Sohal and Weindruch, 1996). Plants, invertebrates, and mammals turn oxidative toxicity to their advantage by using ROI to signal other defenses (Levin et al., 1994), repel predators (Aneshansley et al., 1969), prevent polyspermy (Shapiro, 1991), or kill infectious agents (Klebanoff, 1998). Mammalian phagocytes, in particular, use a multisubunit flavocytochrome to generate large amounts of superoxide radical. Thus, it is not surprising that both pathogens (Miller and Britigan, 1997) and their hosts (Yu, 1994) * To whom correspondence should be addressed (e-mail: cnathan@ mail.med.cornell.edu).

have evolved multiple defenses against oxidative injury (Holmgren, 1989; Fahey and Sundquist, 1991; Farr and Kogoma, 1991; Chen and Helmann, 1995; Newton et al., 1996; Jin et al., 1997). Over the last decade, a fundamentally different source of oxidative and nitrosative stress has been identified in slime molds, insects, fish, birds, and mammals: reactive nitrogen intermediates (RNI) (Nathan and Xie, 1994). Like ROI, RNI can (in)activate enzymes, ion channels, and transcription factors, mutate DNA, and cause apoptosis or necrosis (Stamler, 1995; Keefer and Wink, 1996). As with ROI, the animal host not only suffers RNI, but also deploys them, equipping its cells with a multisubunit flavocytochrome, inducible nitric oxide synthase (NOS2; iNOS) that generates large amounts of RNI to combat infection (Fang, 1997; Nathan, 1997). For these reasons, it seems likely that microbes and mammals express genes that confer resistance not just to ROI, but also to RNI. We began a search for microbial RNI resistance genes in Mycobacterium tuberculosis. We considered the tubercle bacillus a likely source for two reasons. First, RNI generated by NOS2 are essential for the temporary control of tuberculosis in mice (Chan et al., 1995; MacMicking et al., 1997). Enzymatically active NOS2 is expressed in the tuberculous human lung within macrophages, the cells ultimately responsible for controlling the infection (Nicholson et al., 1996), and can prevent the replication of mycobacteria in human pulmonary macrophages in vitro (Nozaki et al., 1997). Second, M. tuberculosis is one of the most successful pathogens of the human species. The tubercle bacillus infects one person in three and kills 44% as many people each year as all types of cancer combined (Raviglione et al., 1995). Thus, RNI are likely to have exerted evolutionary pressure on M. tuberculosis, and M. tuberculosis is likely to have adapted. Alkyl hydroperoxide reductase was first cloned and purified from Salmonella typhimurium and Escherichia coli as proteins induced by oxidative stress under the positive control of the oxyR gene (Jacobson et al., 1989; Storz et al., 1989; Tartaglia et al., 1990). Hydroperoxides are mutagenic in bacteria (Farr and Kogoma, 1991). Overexpression of alkyl hydroperoxide reductase activity suppressed spontaneous mutagenesis associated with aerobic metabolism in DoxyR mutants of S. typhimurium and E. coli (Storz et al., 1987; Greenberg and Demple, 1988). The isolated enzyme uses NAD(P)H to reduce alkyl hydroperoxides to the corresponding alcohols. This activity is manifest by a tetramer comprised of two 57 kDa monomers of the NAD(P)H-oxidizing flavoprotein AhpF, and two 21 kDa monomers of its peroxidereducing partner, AhpC. Only a few homologs of AhpF have been identified (Chae et al., 1994b). In contrast, AhpC homologs are widely distributed among prokaryotes, and AhpC is z40% identical to thioredoxin peroxidase from yeast, rat, plants, amoebae, nematodes, rodents, and humans (Chae et al., 1994a, 1994b; Lim et al., 1994; Jin et al., 1997). Therefore, homologs of AhpC

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Figure 1. Genomic Environment of ahpC in M. tuberculosis Contrasts with that in S. typhimurium Clone pMtb-ahpC contains 3934 bp of DNA from M. tuberculosis including the coding region of ahpC and four additional putative coding regions (I–IV) with the indicated number of codons (aa) in the orientation shown by the arrows (see also GenBank accession number Z81451). Gene III was earlier termed ahpD (GenBank accession number U44840), but bears no relationship to ahpC or ahpF. The latter comprise a bicistronic operon in S. typhimurium (GenBank accession number J05478).

define a large family of antioxidants present in organisms from all kingdoms. Herein, a new function is demonstrated for ahpC from M. tuberculosis, as well as the canonical ahpC of S. typhimurium: the ability to protect cells from RNI. Results Lack of a Bicistronic ahpCF Operon in M. tuberculosis From M. tuberculosis Erdman, we cloned and sequenced 3934 bp of DNA that included ahpCMtb flanked by four additional genes (Figure 1). Cloning of ahpCMtb was subsequently reported (Deretic et al., 1995; Sherman et al., 1995), and all five genes were contained in cosmid Y428 (GenBank accession number Z81451). Just 59 to ahpC lies a disrupted oxyR (Deretic et al., 1995; Sherman et al., 1995). At the protein level, AhpCMtb is 32% identical to the canonical AhpC from S. typhimurium (AhpC Sty) (Tartaglia et al., 1990), with conservation of the two Cys and surrounding residues (not shown). In contrast, ahpF, encoding the flavoprotein component of alkyl hydroperoxide reductase within a bicistronic operon in S. typhimurium (Figure 1), was lacking from the vicinity of ahpCMtb, nor has an ahpF homolog been reported elsewhere in the M. tuberculosis genome. Gene I is homologous to g-glutamyl phosphate reductase in E. coli, while genes II–IV are of unknown function. The lack of any known ahpF in M. tuberculosis suggested two possibilities for ahpCMtb: it might be vestigial, or it might subserve the same or related functions as in enterobacteria, but with a different mechanism for its own reduction. Expression of AhpC in M. tuberculosis That ahpC is vestigial in most clinical isolates of M. tuberculosis was suggested by negative immunoblots with cross-reactive anti-AhpC antibodies (Zhang et al., 1996), this being attributed to the disruption of oxyR (Deretic et al., 1995; Sherman et al., 1995). However, expression of AhpC was demonstrated by microsequencing in catalase-deficient isolates of M. tuberculosis in association with up-regulatory mutations in the

Figure 2. Expression of AhpCMtb in Mycobacteria (A) Purification of recombinant AhpCMtb. Lysates of E. coli M15 (pAhpC-3) treated with IPTG (lane 2) or not (lane 1) were loaded onto a 12% gel. Lanes 3–7 carry recombinant AhpCMtb eluted with 100 mM EDTA from Ni 1-NTA resin onto which the IPTG-treated M15 (pAhpC-3) lysate had been passed. Lane M, molecular markers. Arrow indicates AhpCMtb . Identity and purity of the AhpCMtb band were confirmed by amino acid sequencing. (B) Detection of AhpCMtb. Following SDS–PAGE on a 12% gel, immunoblot was performed with antiserum raised against antigen purified in (A) (1:2000 dilution). Upper panel: lysates of E. coli M15 (pAhpC3) (lane 1), M. tuberculosis H37Ra (lane 2, 25 mg), and M. smegmatis mc2155 (lane 3, 22 mg). Arrows indicate native AhpC and recombinant AhpC fusion protein (rec.). Lower panel: clinical isolates of M. tuberculosis characterized as isoniazid-sensitive (S) or -resistant (R) and as catalase-positive (1), -negative (2), or not determined (ND). Lane 8 represents H37Rv (ATCC 25618). Protein per lane varied depending on availability: lane 1, 20 mg; lane 2, 20 mg; lane 3, 9.6 mg; lane 4, 20 mg; lane 5, 20 mg; lane 6, 12 mg; lane 7, 12 mg; lane 8, 10 mg.

ahpC promoter, mutations that were presumably fixed because they afforded compensation for the lack of catalase (Sherman et al., 1996). Many isoniazid-resistant isolates are catalase-deficient, because catalase helps activate the pro-drug isoniazid to its active form (Zhang et al., 1992). Conversely, most isoniazid-sensitive isolates are catalase-replete (Stoeckle et al., 1993). Based on these studies, isoniazid-sensitive isolates of M. tuberculosis have been regarded as predominantly AhpCnegative. Since the foregoing impression was based in part on negative results with antibodies raised against AhpC from other species, we purified recombinant AhpCMtb (Figure 2A) and raised a specific antibody (Figure 2B). This antibody detected no polypeptides in E. coli M15 (not shown), but stained two bands in E. coli M15 (pAhpC-3)

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Figure 3. S. typhimurium ahpC::Tn10 Strain (TA4190) Is More Susceptible to RNI than Parental Wild-Type Strain (LT2) (A) Confirmation of canonical phenotype: disruption of ahpC (with associated decrease in aphF expression) causes hypersensitivity to cumene hydroperoxide in S. typhimurium. Discs were placed on agar containing LT2 or TA4190 and impregnated with 15 ml of 5% cumene hydroperoxide. Zones of inhibition were photographed after 18 hr at 378C. (B) Demonstration of new phenotype with respect to RNI—studies with GSNO. S. typhimurium LT2 (closed squares) or TA4190 (closed circles) was exposed to indicated concentration of GSNO at pH 5 for 7 hr at 378C in LB (a), or to 5 mM GSNO at pH 5 for the indicated times (b). Surviving organisms were determined as colony-forming units on agar (closed symbols; [a], [b]), while cell growth was measured by OD600 in (b) (open squares, LT2; closed squares, TA4190). Results are means 6 SE of triplicates or quadruplicates in 3–4 representative experiments. (C) Demonstration of new phenotype with respect to RNI—studies with acidified nitrite. Survival of S. typhimurium LT2 (closed squares) and TA4190 (closed circles) exposed to indicated concentrations of nitrite at pH 5 for 14 hr at 378C in LB (a), or to 3 mM nitrite in LB at pH 5 for the indicated times (b). (c): controls with nitrate at pH 5 (open diamonds, LT2; open triangles, TA4190) and nitrite at pH 7 (closed diamonds, LT2; closed triangles, TA4190) as indicated by cell growth measured by OD600. Surviving organisms were determined as colony-forming units on agar (closed symbols; [a], [b]), while cell growth was measured by OD600 (open squares, LT2; open circles, TA4190) in (b). Results are means 6 SE of triplicates or quadruplicates in 4 representative experiments. In (B) and (C), most error bars fall within the symbols.

(Figure 2B, upper panel). The band with apparent Mr z21.5 kDa most likely corresponded to AhpCMtb translated from the authentic start codon of the ahpCMtb ORF, while the higher Mr species represented the recombinant protein with its 2.8 kDa N-terminal tag. This antibody immunoblotted a single polypeptide with the apparent Mr of native AhpCMtb in Mycobacterium smegmatis and M. tuberculosis H37Ra, as well as in clinical isolates of M. tuberculosis characterized as isoniazid-sensitive or -resistant and as catalase-positive or -negative (lower panel). Thus, expression of ahpCMtb is not precluded by disruption of oxyR, expression of catalase, or sensitivity to isoniazid, and strains that express AhpCMtb do not appear to be rare. Inactivation of ahpC in S. typhimurium Causes Hypersensitivity to RNI Disruption of ahpC would be the most direct means to test its function. In M. tuberculosis, targeted gene disruption has met with limited success (Pelicic et al., 1997) and has not yet been accomplished for ahpCMtb. In contrast, ahpC has been inactivated in S. typhimurium strain LT2 by Tn10 insertion, generating strain TA4190 (Storz et al., 1989). The transposon disrupts the 33rd

codon of the ahpC ORF (not shown). Therefore, we used this organism to explore the function of ahpC. First, we confirmed the reported phenotype of TA4190, namely, its increased sensitivity to cumene hydroperoxide compared to LT2 (Figure 3A). We next asked whether disruption of ahpC rendered salmonella more sensitive to RNI. For this, we used two forms of RNI, both of which are physiologic products of the activated macrophage: S-nitrosoglutathione (GSNO) and mildly acidified nitrite. GSNO can be bactericidal at neutrality (e.g., Ehrt et al., 1997) but is more stable under mildly acidic conditions (Feelisch and Stamler, 1996). Because the bactericidal activity of GSNO requires its uptake (De Groote et al., 1996), enhanced stability is accompanied by enhanced potency. Hence, we used GSNO at pH 5. Disruption of ahpC in TA4190 led to marked hypersensitivity to GSNO compared to LT2 (Figure 3B). Mildly acidified nitrite, often used to preserve foods, has long been recognized as bactericidal. At the pH encountered in phagolysosomes, a small proportion of nitrite is protonated. The protonated form dismutates to generate NO, NO2, N2O3, and N2O4 (reviewed in Ehrt et al., 1997). When both LT2 and TA4190 were exposed to sodium nitrite (4 mM, pH 5) for 14 hr, z106-fold fewer

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Figure 4. Complementation of aphCF Deficiency in S. typhimurium ahpC::Tn10 Strain TA4190 by ahpC Mtb with and without ahpF Sty Lane numbers and X-axis designations correspond to the key at the upper right. (A) Expression of native and recombinant proteins in S. typhimurium. Parental wild-type strain LT2 (lane 1) and untransformed TA4190 (ahpC::Tn10) (lane 2) as controls were compared to TA4190 transformed with recombinant plasmids: pSty-C (lane 5), pSty-CF (lane 6), pMtb-C (lane 7), and pMtb-CF (lane 8). 100 mg of lysate protein (upper and middle rows) and 150 mg (lower row) from each strain were subjected to SDS–PAGE and immunoblotted with antibodies specific for AhpCSty (1:4000 dilution), AhpCMtb (1:2000), or AhpFSty (1:1000). (B) AhpCMtb partially restores resistance to cumene hydroperoxide. S. typhimurium LT2 (pRB3-273C), TA4190 (pRB3-273C), TA4190 (pSty-C), TA4190 (pMtb-C), and TA4190 (pMtbCF) were treated with 0 mM (black bars), 100 mM (open bars), or 150 mM (hatched bars) cumene hydroperoxide in LB for 14 hr at 378C (a) or with 100 mM cumene hydroperoxide in LB for 8 hr at 378C [(b); symbols: closed squares, LT2 (pRB3-273C); closed circles, TA4190 (pRB3-273C); closed triangles, TA4190 (pSty-C); closed diamonds, TA4190 (pMtb-C); open squares, TA4190 (pMtb-CF)]. (C) AhpCMtb restores resistance to RNI. S. typhimurium LT2 (pRB3-273C), TA4190 (pRB3273C), TA4190 (pSty-C), TA4190 (pSty-CF), TA4190 (pMtb-C), and TA4190 (pMtb-CF) were treated without (black bars) or with 2 mM NaNO 2 (hatched bars) in LB (pH 5) for 24 hr (a), and without (black bars) or with 4 (open bars) or 5 mM (hatched bars) GSNO in LB (pH 5) for 27 hr (b) at 378C. In (B) and (C), surviving organisms were determined as colony-forming units on LB agar containing ampicillin. Results are means 6 SE of triplicates or quadruplicates.

TA4190 survived than LT2 (Figure 3Ca). Similarly, sodium nitrite (3 mM, pH 5) killed z105-fold of TA4190 cells within 24 hr but was not bactericidal to LT2. Neither pH 5 alone (not shown), sodium nitrate at pH 5 (Figure 3Cb), nor sodium nitrite at pH 7 (Figure 3Cc) affected the growth curves of LT2 and TA4190, which were indistinguishable. ahpC from M. tuberculosis Complements ahpCF Deficiency in S. typhimurium for Resistance to RNI We then asked whether ahpCMtb could functionally replace ahpCSty. The ORFs of ahpCMtb, ahpCSty, and the ahpCFSty operon were individually subcloned into pRB3273C (Berggren et al., 1995) under the ahpCFSty promoter to yield plasmids pMtb-C, pSty-C, and pSty-CF. In TA4190, ahpF expression is thought to be eliminated by polarity as a result of Tn10 insertion (Storz et al., 1989). To investigate the role of AhpFSty, ahpFSty was also placed downstream of ahpCMtb to form a chimeric bicistronic operon in pMtb-CF. Expression of AhpCSty, AhpCMtb , and AhpFSty after transformation of TA4190 was confirmed by immunoblotting (Figure 4A). Anti-AhpCSty antibody did not immunoblot TA4190, whose ahpC has been disrupted by

Tn10 insertion, but did detect a single polypeptide species in the wild-type strain LT2 and in TA4190 carrying either pSty-C or pSty-CF. Immunoblots with anti-AhpCMtb antibody were negative with LT2, TA4190, and TA4190 (pSty-C), indicating that this antibody does not crossreact with AhpCSty. When TA4190 was transformed with pMtb-C or pMtb-CF, anti-AhpCMtb antibody immunoblotted AhpCMtb. Immunoblots confirmed expression of AhpFSty (z57 kDa) from the chromosome in LT2 and from pSty-CF and pMtb-CF in transformed TA4190. AntiAhpFSty also reacted faintly with TA4190, suggesting that Tn10 insertion into ahpCSty greatly reduced but did not totally abolish the transcription of ahpF (Figure 4A). Next, LT2 and TA4190, transformed with the vector to comprise positive and negative controls, respectively, were compared to TA4190 transformed with pSty-C, pMtb-C, and pMtb-CF for their resistance to cumene hydroperoxide. TA4190 was the most susceptible, succumbing to micromolar cumene hydroperoxide, while LT2 was not sensitive to the same concentrations (Figure 4B). Surprisingly, transformation of TA4190 with pSty-C alone restored resistance to cumene hydroperoxide to the same level displayed by wild-type control LT2. Given that purified AhpCSty requires AhpF to reduce

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with cumene hydroperoxide (Figure 4B). When concentrations of either nitrite or GSNO were increased to the point that complementation was only partial, coexpression of ahpF still did not enhance the resistance against RNI conferred by ahpFSty or ahpCMtb (not shown).

Figure 5. Expression of AhpCMtb in Stably Transfected Human Cells (A) Immunoblot. Lysates of human 293, 293/neor, 293/AhpC-1, and 293/AhpC-2 cells (100 mg each) were subjected to SDS–PAGE in a 12% gel and immunoblotted with anti-AhpCMtb antiserum (1:4000). (B) Immunocytochemistry. 293/neo r cells ([a] and [b]), 293/AhpC-1 cells ([c] and [d]) and 293/AhpC-2 cells ([e] and [f]) were stained with preimmune serum ([a], [c], and [e]) or anti-AhpCMtb antiserum ([b], [d], and [f]). Original magnification 31000.

cumene hydroperoxide (Jacobson et al., 1989), this suggests that the residual level of AhpF in TA4190 was sufficient to cooperate with AhpCSty to constitute a functional alkyl hydroperoxide reductase. ahpCMtb, expressed from pMtb-C, was less efficient than ahpCSty in affording partial protection against cumene hydroperoxide. ahpF expression in trans in conjunction with expression of ahpCMtb from pMtb-CF provided TA4190 with additional protection (Figure 4B). The impaired efficiency of AhpCMtb operating in anti-ROI mode with salmonella AhpF presumably reflected the phylogenetic distance between the donor and recombinant host. Viable vector-transformed TA4190 were reduced by 4–5 orders of magnitude by nitrite (2 mM, pH 5) or GSNO (5 mM, pH 5), while LT2 was resistant (Figure 4C). The deficiency in TA4190’s ability to survive in RNI was fully complemented in trans not only by expression of ahpCSty but also by ahpCMtb. Complementation conferred by either species’ ahpC for resistance to RNI did not require the help of ahpF Sty (Figure 4C), in contrast to the situation

Expression of ahpCMtb in Stably Transfected Human Cells and Protection from GSNO Macrophages can control the replication of M. tuberculosis through expression of NOS2 (Chan et al., 1995; MacMicking et al., 1997). However, activated macrophages also produce large amounts of ROI (Nathan and Root, 1977), making them unsuitable as a test cell in which to assess the effect of AhpC selectively against NOS2-derived RNI. As a substitute, we used a transfectable human epithelial cell line, 293, that expresses neither the respiratory burst oxidase nor NOS2. Transient transfection of NOS2 or vector alone into 293 cells permitted us to test the effects of physiologically relevant (macrophage-like) amounts of RNI in the presence of no more than basal levels of ROI. To this end, we cloned two independent lines of 293 cells that stably expressed transfected ahpCMtb, termed 293/AhpC-1 and 293/AhpC-2. Immunoblot demonstrated full-length AhpCMtb in both cell lines (Figure 5A); immunocytochemistry showed that nearly every cell in each line was positive (Figure 5B). Parental 293 cells and 293 cells stably transfected with vector alone (293/neor ) were nonreactive by both assays. Addition of GSNO to the medium killed 293 cells in a dose-dependent fashion. In 24 hr, 4 mM GSNO reduced the proportion of 293/neo r cells excluding trypan blue to 15% of that of untreated cells; expression of ahpCMtb increased the proportion of cells excluding trypan blue by 2.5-fold (not shown). After 48 hr in the presence of 4 mM GSNO, tetrazolium reduction in 293/neor was reduced by 95% compared to control. However, the concentration of GSNO required to kill 50% of 293/AhpC-1 by the tetrazolium assay was z5-fold higher than needed to kill 50% of 293/neor (Figure 6). AhpCMtb Protects Human Cells from Cytotoxicity and Apoptosis Induced by NOS2 The independent 293 cell lines stably expressing AhpCMtb (293/AhpC-1 and 293/AhpC-2), along with 293/neor as control, were transiently transfected with either control vector (pcDNAI/Amp) or NOS2 on this vector (pL8Amp). After z40 hr, cells excluding trypan blue were counted. 293/AhpC-1 and 293/AhpC-2 survived 35%–60% better than 293/neor (Figure 7A). Improved viability was not explained by differential expression of NOS2 protein (Figure 7B) or product (nitrite) (not shown). Inspection of the cultures revealed more striking differences. Figure 7C shows representative fields. Without NOS2, vector-transformed 293/neo r cells remained adherent and nearly confluent (panel [a]). After transfection with NOS2, many 293/neor cells seemed to disappear. Most of the remainder detached from the plate and rounded up (panel [b]). In contrast, expression of NOS2 had little impact on the morphology of 293/AhpC-1 or 293/AhpC-2 (panels [c]–[f]). The greater destruction caused by NOS2 as evaluated

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Figure 6. Expression of AhpCMtb Increases Resistance of Human Cells to GSNO 293/neor (open circles) or 293/AhpC-1 (closed circles) cells were exposed to the indicated concentrations of GSNO in DMEM medium with 10% FBS for 48 hr. Viability was assayed by reduction of a tetrazolium salt with the value for untreated cells set to 100%. Results are means 6 SE for triplicates and are representative of three experiments.

morphologically compared to that evident with trypan blue was consistent with the possibility that many of the cells were undergoing apoptosis, since apoptotic cells can exclude trypan blue (e.g., Lazebnik et al., 1993). RNI generated by NOS2 can induce apoptosis (Xie et al., 1995; Sandau et al., 1997). DNA isolated from cultures like those in Figure 7C revealed internucleosomal cleavage, a marker of apoptosis (Enari et al., 1998), only in NOS2-transfected 293/neo r cells (Figure 7D). DNA from NOS2-transfected 293/AhpC-1 and 293/AhpC-2 remained intact. Hence, expression of ahpCMtb prevented 293 cells from undergoing apoptotic cell death induced by expression of NOS2. Discussion ahpC protected both bacterial and human cells against RNI. Protection was effective against RNI generated chemically (by GSNO or nitrite) or biochemically (by NOS2), and was evident against levels of injury ranging from stasis to lysis for bacteria and from apoptosis to necrosis for human cells. ahpCs as diverse as those from S. typhimurium (a purple bacterium) and M. tuberculosis (an actinomycete) had anti-RNI functions. The degree of protection against RNI conferred on salmonella by its own ahpC was comparable to if not greater than the protection conferred against alkyl hydroperoxides. Considering the ubiquity of RNI as products of soil commensals (Conrad, 1996) as well as animal hosts (Nathan and Xie, 1994), the physiologic function of ahpC may consist in resisting RNI as much as in reducing alkyl hydroperoxides. These findings cast ahpC in a new light, as the most widely distributed RNI resistance gene known. Other RNI Resistance Genes Several other microbial RNI resistance genes have recently been identified, one from M. tuberculosis and

the others from enterics. noxr1 (for nitrogen oxides and oxygen intermediates resistance-1) appears confined to the M. tuberculosis complex (Ehrt et al., 1997). In E. coli, NO shares the ability of intracellularly generated (but not exogenous) O2.- to induce the soxRS regulon (Nunoshiba et al., 1993). In E. coli but not S. typhimurium (Fang et al., 1997), the soxRS system confers modest resistance to NO (Nunoshiba et al., 1993, 1995) via induction of Mn-superoxide dismutase (sodA), glucose 6-phosphate dehydrogenase (zwf), endonuclease IV (nfo), and micF, a suppressor of OmpF porin. SOD may confer resistance to RNI by diverting O2.- from reaction with NO and thus forestalling the production of peroxynitrite (De Groote et al., 1997), a mechanism distinct from the protection against preformed RNI seen with AhpC. The mechanisms by which other genes in the soxRS regulon contribute to RNI resistance are unknown. In S. typhimurium, mutations in genes encoding enzymes involved in the repair of DNA (De Groote et al., 1995) or the synthesis of homocysteine (metL) (De Groote et al., 1996) enhance susceptibility to S-nitrosothiols. Finally, in E. coli, both H2O2 (Farr and Kogoma, 1991) and S-nitrosothiols (Hausladen et al., 1996) induce oxyR, which controls a regulon of 9 genes, including those encoding catalase, glutathione reductase, and AhpCF. When oxidized or S-nitrosylated, the transcription factor OxyR induces the regulon, whose products confer resistance to H2O2, alkyl hydroperoxides, and S-nitrosothiols. Which gene(s) in the oxyR regulon are responsible for resistance to S-nitrosothiols was unknown. The present findings suggest that ahpC is (one of) the responsible gene(s).

Mechanistic Considerations RNI can inactivate glutathione peroxidase (Asahi et al., 1995), an important component of mammalian cell defenses against ROI (Nathan et al., 1981). Cells less able to redox-cycle glutathione might be more sensitive to endogenous ROI, against which AhpC might protect them, giving a misleading appearance that AhpC protects directly from exogenous RNI. Such a scenario is exceedingly unlikely. First, the foregoing postulate could not explain why the protection afforded to S. typhimurium by AhpC was independent of AhpF in the case of RNI, but dependent on AhpF in the case of ROI. Second, to our knowledge, depletion of any element of an ROI resistance path has never been found to lead to rapid, extensive cell death from endogenous oxidants; such depletion only serves to sensitize cells to exogenous oxidants. A germane illustration is provided by S. typhimurium strain TA4190, which is completely deficient in AhpC and extensively deficient in AhpF. TA4190 grows normally in the absence of an exogenous oxidant stress, even though it is highly sensitive when such a stress is supplied. Thus, in contrast to the situation with SOD (De Groote et al., 1997), the anti-RNI effect of AhpC is distinct from its anti-ROI effect. In short, AhpC appears to be bifunctional. AhpCF can act as a lipid hydroperoxide reductase. Glutathione peroxidase serves as a precedent for a mammalian lipid hydroperoxide reductase that can metabolize RNI. While glutathione peroxidase normally uses glutathione to reduce lipid hydroperoxides to the

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Figure 7. Expression of AhpCMtb Increases Resistance of Human Cells to Cytotoxicity and Apoptosis Caused by Expression of NOS2 (A) Viability. 293/neor cells (open bars), 293/AhpC-1 (closed bars), or 293/AhpC-2 (hatched bars) were transiently transfected with NOS2 on pL8Amp (for brevity, NOS2) or with the vector pcDNAI/Amp (for brevity, vector). At indicated times, cell viability was determined by trypan blue exclusion. Viability in NOS2-transfected cultures was expressed as a percentage of viable vector-transfected cultures derived from the same starting population. Results were summarized from 6 independent experiments. (B) Expression of NOS2 and AhpCMtb. Immunoblots were performed with antisera specific for mouse NOS2 (upper row, 200 mg/sample after SDS–PAGE on a 7.5% gel) or AhpCMtb (lower row, 100 mg/sample, 12% gel). Lane 1, 293/neor transfected with vector; lane 2, 293/neor transfected with NOS2; lane 3, 293/AhpC-1 transfected with vector; lane 4, 293/AhpC-1 transfected with NOS2. Expression of NOS2 in transfected 293/AhpC-2 was confirmed in a separate experiment. (C) Morphology of cultures prepared as in (A). (a), 293/neor transfected with vector; (b), 293/neor transfected with NOS2; (c), 293/AhpC-1 transfected with vector; (d), 293/AhpC-1 transfected with NOS2; (e), 293/AhpC-2 transfected with vector; (f), 293/AhpC-2 transfected with NOS2. (D) DNA fragmentation. Genomic DNA was prepared from harvested cells as in (A), and equal amounts were subjected to agarose gel electrophoresis. Lane 1, 293/neor transfected with vector; lane 2, 293/neor transfected with NOS2; lane 3, 293/AhpC-1 transfected with vector; lane 4, 293/AhpC-1 transfected with NOS2; lane 5, 293/AhpC-2 transfected with vector; lane 6, 293/AhpC-2 transfected with NOS2.

corresponding alcohols or hydrogen peroxide to water, it can use GSNO in place of glutathione, releasing an unidentified form of RNI (Freedman et al., 1995). However, because the product of this reaction is more potent than GSNO as an inhibitor of platelet aggregation (Freedman et al., 1995), the reaction does not appear to constitute a detoxification. Alternatively, glutathione peroxidase can use glutathione to catabolize peroxynitrite to nitrite, a reaction dependent on the enzyme’s distinctive selenocysteine residue (Sies et al., 1997). Neither of the foregoing mechanisms is likely to explain the anti-RNI function of AhpC: AhpC alone lacks activity as a lipid hydroperoxide reductase; AhpC is not known to contain selenium; mycobacteria do not contain glutathione (Newton et al., 1996); and nitrite itself is one of the RNI against which ahpC conferred protection. Given the relative inefficiency of AhpCMtb operating against ROI in salmonella, it was striking how proficiently AhpCMtb protected both bacterial and human cells against RNI. Because a small number of molecules of AhpC can protect cells against a vast molar excess of RNI, the action of AhpC is probably catalytic. AhpC’s catalytic mechanism against RNI may involve another protein that serves as a reducing cofactor. AhpC in S. typhimurium

requires AhpF to detoxify alkyl hydroperoxides. Defense against RNI does not involve AhpF, and M. tuberculosis appears to lack AhpF (Wilson and Collins, 1996; and this work), so that for anti-RNI defense, another protein may shuttle electrons from NAD(P)H to AhpC in place of AhpF. A candidate for such an AhpF equivalent is thioredoxin reductase. The thioredoxin reductase cloned from M. tuberculosis (GenBank accession number X95798) is 32% identical to the C-terminal half of AhpF from S. typhimurium (L. C., unpublished data). Thioredoxin, also identified in M. tuberculosis (Wieles et al., 1995), accelerates decomposition of S-nitrosothiols in vitro (Nikitovic and Holmgren, 1996). Thioredoxin may be part of the RNI resistance mechanism that operates through AhpC. Implications Isoniazid is the mainstay of antituberculous therapy. It is for isoniazid-resistant tuberculosis that new therapeutic approaches are most sorely needed. Many isoniazidresistant isolates are catalase-deficient (Zhang et al., 1992). In such organisms, inhibition of AhpC might cripple a major backup path for ROI resistance, while also rendering the bacteria susceptible to RNI. Mycobacteria whose AhpC has been inhibited may be more readily

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killed when exposed to RNI or ROI that are delivered by the host’s immune system, RNI delivered by inhalation (Frostell and Zapol, 1995), or NO-donating organochemicals (Hanson et al., 1995). Conversely, disruption of ahpC in M. tuberculosis or inhibition of its protein product might lead to overproduction of catalase, as observed in B. subtilis (Bsat et al., 1996), which might restore sensitivity to isoniazid. Although we have focused on microbial RNI resistance, these results have implications for eukaryotic cells. In the absence of ahpCMtb, epithelial cells underwent apoptosis and necrosis after they were transfected to express NOS2 at levels typical of activated macrophages. When transfected with ahpCMtb, the epithelial cells were substantially protected. Macrophages themselves can tolerate similar levels of NOS2 without evident autotoxicity (Vodovotz et al., 1994). The divergent fates of various NOS2-expressing mammalian cells may reflect differential expression of endogenous anti-RNI genes homologous to ahpC. Indeed, the antiapoptotic function of a human thioredoxin peroxidase has very recently been demonstrated (Zhang et al., 1997). Oxidant-induced apoptosis is a mechanism shared by such diverse processes as p53-dependent tumor suppression, ischemia-reperfusion, radiation therapy, and antineoplastic chemotherapy (Hug et al., 1997; Polyak et al., 1997). Inhibition or induction of such enzymes may facilitate therapeutic regulation of the extent of oxidantinduced apoptosis. Experimental Procedures Materials Chemicals and enzymes were obtained as follows: G418, X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside), and trypsin from GIBCO-BRL; IPTG from Boehringer Mannheim; restriction endonucleases, DNA polymerase I Klenow fragment, and calf intestinal alkaline phosphatase from New England Biolabs; Pfu DNA polymerase from Stratagene; AmpliTaq DNA polymerase, MuLV reverse transcriptase, dNTPs, and reagents for PCR and RT–PCR from Perkin Elmer; oligonucleotides from Oligo Etc.; and others from Sigma.

Cloning of ahpC from M. tuberculosis Portions of the ahpC coding region conserved in Mycobacterium avium (GenBank accession number U18263) and Mycobacterium leprae (GenBank accession number L01095) were used to design the primer pair ACCAGTTCCCCGCCTAC (sense) and ACCCTTGCG CCAGTTGCA (antisense). With M. tuberculosis H37Rv genomic DNA as template (gift of Lee Riley, Cornell University Medical College), PCR amplification yielded a product with the expected size (521 bp) whose sequence was homologous to that of M. avium and M. leprae. This was used to prepare a probe with which we screened a lgt11 library of M. tuberculosis Erdman DNA (gift of Richard Young, Whitehead Institute). Twenty clones were selected from z5 3 10 4 phage plaques. Two overlapping clones were combined and subcloned into the pT7-Blue vector (Novagen) to yield pMtb-ahpC, which was sequenced.

Bacterial Strains S. typhimurium TA4190 (ahpC::Tn10) and its congenic wild-type strain LT2 were from the Salmonella Genetic Stock Center (University of Calgary). E. coli strain DH5a (GIBCO-BRL) and XL1-Blue (Stratagene) were used for general genetic manipulation. Cultures were in LB broth or agar (Sambrook et al., 1989) with ampicillin (100 mg/ml), tetracycline (15 mg/ml), or kanamycin (25 mg/ml). Lysates of M. smegmatis mc2155 and M. tuberculosis H37Ra were kindly

provided by S. Ehrt (Cornell University Medical College). M. tuberculosis H37Rv (ATCC 25618) was from P. Bifani (Public Health Research Institute, New York, NY). J. T. Belisle (Colorado State University) supplied g-irradiated clinical isolates of M. tuberculosis with defined isoniazid sensitivity and catalase status. Strains were deemed isoniazid-sensitive if #1 mg/ml isoniazid suppressed CFU 100%. Plasmids The ahpCF operon was cloned from S. typhimurium LT2 by PCR with forward (59-GGCGGCCTTTTTACTTTAGATG-39) and reverse (59-AGGCCCGAATAGCTTACACTA-39) primers designed according to GenBank sequence J05478. The amplified 2.6 kb fragment was cloned into pT7-Blue (Novagen), resulting in pStahpCF. Plasmid pRB3-273C, a gift from F. Fang (University of Colorado), served to introduce ahpCMtb, ahpC Sty, and ahpF Sty into S. typhimurium TA4190. A SmaI fragment from pStahpCF containing ahpC Sty was cloned into pRB3-273C in the opposite orientation to the vector’s lacZ promoter, resulting in pSty-C. Plasmid pSty-CF was generated by subcloning an XbaI–NheI fragment carrying ahpFSty into the XbaI site in pStyC, and ahpFSty was placed downstream of ahpCSty to allow both genes to be expressed from the upstream promoter of ahpCFSty (Ps). To achieve a level of ahpCMtb expression similar to that of ahpCSty on pSty-C, the entire promoter of ahpCFSty (Ps) was amplified by PCR using the forward primer above and a reverse primer (59-GAATT CCATATGTACTTCCTCCGTGTTTTC-39) that engineered an NdeI site around the ATG start codon. The amplified P s-containing fragment was cloned into pT7-Blue, generating pPs-T7. To PCR-amplify the ORF of ahpCMtb from pMtb-ahpC, the forward primer (59-GAGGAGAC ATATGCCACTGCTAACCATTG-39) contained an NdeI site without changing codons of ahpC Mtb ORF, while the reverse primer (59-CCTC TAGATTATGCCGAAGCCTTGG-39) incorporated an XbaI site after the ahpC Mtb ORF. The amplified product was digested with NdeI and XbaI and cloned into pPs-T7 digested by the same enzymes, resulting in pPs-MtahpC. A SacI–XbaI fragment from pPs-MtahpC that contained both the P s element and the downstream ahpCMtb was cloned into the compatible sites on pRB3-273C, resulting in pMtb-C. To construct a chimeric bicistronic ahpC Mtb-ahpFSty operon whose transcription was directed by P s, an ahpFSty -containing XbaI– NheI fragment was subcloned into XbaI-digested pMtb-C to generate pMtb-CF. To express ahpCMtb in mammalian cells, a 612 bp HifI fragment containing the ORF of ahpCMtb was end-filled and cloned into pcDNAI/Amp (Invitrogen) such that ahpCMtb was expressed from the vector-borne CMV promoter, resulting in pAhpC-mtb1. Production of Antibody and Western Blot A BamHI–XhoI fragment (679 bp) from pAhpC-mtb1 was subcloned in-frame downstream of an IPTG-inducible promoter in pQE-30 (QIAGEN) and the resulting plasmid used to transform E. coli M15 (pREP4) (QIAGEN). The hexahistidine fusion protein overexpressed upon induction with IPTG was purified on Ni1-NTA resin and SDS– PAGE and transferred to a PVDF membrane (Millipore) for N-terminal sequencing (NYS Center for Advanced Technology, Cornell University). New Zealand rabbits were immunized seven times at 2–4 week intervals with 100 mg of purified recombinant AhpCMtb in Freund’s adjuvant (complete, first injection; incomplete thereafter). Clinical isolates of M. tuberculosis were suspended in sonication buffer (50 mM Na-phosphate [pH 7.8], 300 mM NaCl) and agitated with fine glass beads. Supernatant was concentrated (Microcon10, Millipore) and protein measured by the Bradford method (BIORAD). Overnight cultures of S. typhimurium were inoculated (1:100) into 100 ml of LB and shaken at 378C for 4 hr. Cells were resuspended in 5 ml of sonication buffer, frozen at 2808C, thawed, and sonicated. Supernatant proteins were separated by SDS–PAGE and electroblotted onto a 0.2 mm pore nitrocellulose membrane (Schleicher & Shuell). The membrane was blocked in 5% nonfat milk in TBST (25 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Tween 20) and probed with the indicated antiserum, washed with TBST, and incubated with horseradish peroxidase–conjugated donkey anti-rabbit IgG (Amersham) for detection by enhanced chemiluminescence (NENE Life Science Products). Disk Inhibition Salmonella grown in LB with antibiotics at 378C for 8 hr were diluted 10-fold. Aliquots (z107 cells) were plated in 2 ml of M9 soft agar.

Alkyl Hydroperoxide Reductase and Resistance to NO 803

Cumene hydroperoxide (15 ml, 5%) was applied to a 0.25 inch paper disc (BBL Microbiology Systems) centered on the surface. Mammalian Cells 293 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (Sigma) with 10% heat-inactivated fetal bovine serum (HyClone), 200 U/ml penicillin, and 200 mg/ml streptomycin (complete medium) at 378C in 5% CO2. Cells were detached with 0.05% trypsin/ 0.02% EDTA and cotransfected with pcDNA3 (Invitrogen) containing neor and pAhpC-mtb1 carrying ahpC Mtb in the presence of calcium phosphate (Ruan et al., 1996). After 14 hr, cells were washed; complete medium containing G418 (500 mg/ml) was added and replaced every 2 days 3 5. Surviving cells were serially diluted with G418 (600 mg/ml). Individual colonies were isolated, expanded, verified by RT–PCR and anti-AhpCMtb immunoblot, and maintained in complete medium with G418 (500 mg/ml). Immunocytochemistry 293 cells (z2 3 104 ) were sedimented on a glass slide, fixed with 1% paraformaldehyde, 75 mM cacodylic acid, 0.72% sucrose [pH 7.4] followed by 3.7% formaldehyde in PBS, each for 10 min at RT, permeabilized with 0.05% Triton X-100, stained with preimmune serum or anti-AhpCMtb (1:300), and developed as described (Nicholson et al., 1996). Viability Assays 293 cells stably transfected with ahpC Mtb or the vector (neor) were transiently transfected in the presence of calcium phosphate with the vector pcDNAI/Amp or mouse NOS2 cDNA on this vector in pL8Amp (Ruan et al., 1996). z40 hr later, cultures were trypsinized and trypan blue–excluding cells counted in a hemocytometer. For the CellTiter 96 AQueous assay (Promega), in which viable cells convert a tetrazolium salt into a water-soluble formazan, 293/neor and 293/AhpC-1 cells were cultured for 48 hr in triplicate in a 96well plate (1 3 104 cells, 100 ml per well) in complete medium with G418 (500 mg/ml) and GSNO. After 1 hr with tetrazolium at 378C, OD490 was recorded (MR5000 microplate reader, Dynatech). Values for medium controls were deducted. Viability was determined as a percentage of dye reduction by untreated cells. DNA collected from transfected 293 cells (2 3 10 6) by the method of Liu et al. (1996) was electrophoresed on a 2% agarose gel at 50 V for 2 hr in 0.5 3 TBE buffer (Sambrook et al., 1989), stained with 2 mg/ml ethidium bromide, and visualized under UV light. Acknowledgments

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This work was supported by NIH grant HL51967 and by a fellowship to L. Chen from Merck and Co. We thank S. Ehrt, F. Fang, M. Fuortes, J.D. Helmann, L. Riley, and I. Smith for discussions; R. Young for the M. tuberculosis DNA library; G. Storz for antibodies to salmonella AhpC and AhpF; S. Ehrt, L. Riley, J. Belisle, and P. Bifani for lysates or cultures of M. tuberculosis; F. Fang for plasmids; O. Dussurget, P. Bifani, and B. Kreiswirth for access to equipment and help in preparation of lysates; and M. Fuortes for help with figure preparation. Samples from J. Belisle were provided under NIAID contract NO1-AI25147.

Deretic, V., Philipp, W., Dhandayuthapani, S., Mudd, M.H., Curcic, R., Garbe, T., Heym, B., Via, L.E., and Cole, S.T. (1995). Mycobacterium tuberculosis is a natural mutant with an inactivated oxidative-stress regulatory gene: implications for sensitivity to isoniazid. Mol. Microbiol. 17, 889–900.

Received January 15, 1998; revised March 13, 1998.

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