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• W2034408518 abstract "•Individual Salmonella experience varying oxidative and nitrosative stresses in vivo•Neutrophils and monocytes kill Salmonella with bactericidal oxidative stress•Sublethal oxidative bursts in resident red pulp macrophages allow Salmonella to spread•Regional nitrosative stress triggers an effective Salmonella detoxification mechanism Reactive oxygen and nitrogen species function in host defense via mechanisms that remain controversial. Pathogens might encounter varying levels of these species, but bulk measurements cannot resolve such heterogeneity. We used single-cell approaches to determine the impact of oxidative and nitrosative stresses on individual Salmonella during early infection in mouse spleen. Salmonella encounter and respond to both stresses, but the levels and impact vary widely. Neutrophils and inflammatory monocytes kill Salmonella by generating overwhelming oxidative stress through NADPH oxidase and myeloperoxidase. This controls Salmonella within inflammatory lesions but does not prevent their spread to more permissive resident red pulp macrophages, which generate only sublethal oxidative bursts. Regional host expression of inducible nitric oxide synthase exposes some Salmonella to nitrosative stress, triggering effective local Salmonella detoxification through nitric oxide denitrosylase. Thus, reactive oxygen and nitrogen species influence dramatically different outcomes of disparate Salmonella-host cell encounters, which together determine overall disease progression. Reactive oxygen and nitrogen species function in host defense via mechanisms that remain controversial. Pathogens might encounter varying levels of these species, but bulk measurements cannot resolve such heterogeneity. We used single-cell approaches to determine the impact of oxidative and nitrosative stresses on individual Salmonella during early infection in mouse spleen. Salmonella encounter and respond to both stresses, but the levels and impact vary widely. Neutrophils and inflammatory monocytes kill Salmonella by generating overwhelming oxidative stress through NADPH oxidase and myeloperoxidase. This controls Salmonella within inflammatory lesions but does not prevent their spread to more permissive resident red pulp macrophages, which generate only sublethal oxidative bursts. Regional host expression of inducible nitric oxide synthase exposes some Salmonella to nitrosative stress, triggering effective local Salmonella detoxification through nitric oxide denitrosylase. Thus, reactive oxygen and nitrogen species influence dramatically different outcomes of disparate Salmonella-host cell encounters, which together determine overall disease progression. Host defense against pathogens depends on generation of reactive oxygen species (ROS), using NADPH oxidase, and reactive nitrogen species (RNS), using inducible nitric oxide synthase (iNOS) (Fang, 2004Fang F.C. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies.Nat. Rev. Microbiol. 2004; 2: 820-832Crossref PubMed Scopus (1228) Google Scholar, Nathan and Shiloh, 2000Nathan C. Shiloh M.U. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens.Proc. Natl. Acad. Sci. USA. 2000; 97: 8841-8848Crossref PubMed Scopus (1156) Google Scholar). ROS and RNS can inhibit or kill microbes, but it remains controversial if this is their main role in infection control (Fang, 2011Fang F.C. Antimicrobial actions of reactive oxygen species.MBio. 2011; 2 (00141–00111)Crossref PubMed Scopus (231) Google Scholar, Horta et al., 2012Horta M.F. Mendes B.P. Roma E.H. Noronha F.S. Macêdo J.P. Oliveira L.S. Duarte M.M. Vieira L.Q. Reactive oxygen species and nitric oxide in cutaneous leishmaniasis.J. Parasitol. Res. 2012; 2012: 203818Crossref PubMed Scopus (84) Google Scholar, Hurst, 2012Hurst J.K. What really happens in the neutrophil phagosome?.Free Radic. Biol. Med. 2012; 53: 508-520Crossref PubMed Scopus (91) Google Scholar, Liu and Modlin, 2008Liu P.T. Modlin R.L. Human macrophage host defense against Mycobacterium tuberculosis.Curr. Opin. Immunol. 2008; 20: 371-376Crossref PubMed Scopus (152) Google Scholar, Slauch, 2011Slauch J.M. How does the oxidative burst of macrophages kill bacteria? Still an open question.Mol. Microbiol. 2011; 80: 580-583Crossref PubMed Scopus (267) Google Scholar). Various pathogens are highly resistant to ROS and RNS stress due to protective mechanisms that directly interfere with NADPH oxidase or iNOS activities, detoxify ROS and RNS before these compounds can damage the pathogen, and/or repair or replace damaged pathogen components. Moreover, ROS and RNS have additional important functions as host signaling molecules that regulate a wide variety of innate immune mechanisms, including chemotaxis, signaling, cell activation, vasculature tension, etc., all of which could contribute to infection control. Oxidative and nitrosative stresses have been extensively studied in various Salmonella infection models. In cell culture models, infected macrophages kill most Salmonella in the first few hours after uptake in a NADPH oxidase-dependent manner, whereas iNOS inhibits growth of surviving Salmonella from 5 hr after infection (Vazquez-Torres et al., 2000aVazquez-Torres A. Jones-Carson J. Mastroeni P. Ischiropoulos H. Fang F.C. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro.J. Exp. Med. 2000; 192: 227-236Crossref PubMed Scopus (446) Google Scholar). On the other hand, Salmonella can inhibit assembly of NADPH oxidase and intracellular targeting of iNOS, using its SPI-2 type III secretion system (Chakravortty et al., 2002Chakravortty D. Hansen-Wester I. Hensel M. Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates.J. Exp. Med. 2002; 195: 1155-1166Crossref PubMed Scopus (267) Google Scholar, Vazquez-Torres et al., 2000bVazquez-Torres A. Xu Y. Jones-Carson J. Holden D.W. Lucia S.M. Dinauer M.C. Mastroeni P. Fang F.C. Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase.Science. 2000; 287: 1655-1658Crossref PubMed Scopus (451) Google Scholar). In mouse models, NADPH oxidase is crucial for infection control similar to cell cultures (Mastroeni et al., 2000Mastroeni P. Vazquez-Torres A. Fang F.C. Xu Y. Khan S. Hormaeche C.E. Dougan G. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo.J. Exp. Med. 2000; 192: 237-248Crossref PubMed Scopus (319) Google Scholar), but it is unclear if this is due to a direct bactericidal effect of ROS (Fang, 2011Fang F.C. Antimicrobial actions of reactive oxygen species.MBio. 2011; 2 (00141–00111)Crossref PubMed Scopus (231) Google Scholar, Slauch, 2011Slauch J.M. How does the oxidative burst of macrophages kill bacteria? Still an open question.Mol. Microbiol. 2011; 80: 580-583Crossref PubMed Scopus (267) Google Scholar). NADPH oxidase remains crucial for infection control over many days. However, it is unclear if Salmonella killing continues after the first few hours of infection (Grant et al., 2008Grant A.J. Restif O. McKinley T.J. Sheppard M. Maskell D.J. Mastroeni P. Modelling within-host spatiotemporal dynamics of invasive bacterial disease.PLoS Biol. 2008; 6: e74Crossref PubMed Scopus (155) Google Scholar). A recent report even suggested that ROS levels in vivo are generally too low to have a significant direct impact on wild-type Salmonella (Aussel et al., 2011Aussel L. Zhao W. Hébrard M. Guilhon A.A. Viala J.P. Henri S. Chasson L. Gorvel J.P. Barras F. Méresse S. Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst.Mol. Microbiol. 2011; 80: 628-640Crossref PubMed Scopus (80) Google Scholar). iNOS is dispensable for Salmonella control throughout the first 7 days of infection (Mastroeni et al., 2000Mastroeni P. Vazquez-Torres A. Fang F.C. Xu Y. Khan S. Hormaeche C.E. Dougan G. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo.J. Exp. Med. 2000; 192: 237-248Crossref PubMed Scopus (319) Google Scholar, White et al., 2005White J.K. Mastroeni P. Popoff J.F. Evans C.A. Blackwell J.M. Slc11a1-mediated resistance to Salmonella enterica serovar Typhimurium and Leishmania donovani infections does not require functional inducible nitric oxide synthase or phagocyte oxidase activity.J. Leukoc. Biol. 2005; 77: 311-320Crossref PubMed Scopus (46) Google Scholar) in spite of the substantial bacteriostatic effect of iNOS within a few hours after infection in cell culture infection models. Most of these studies relied on in vitro cell culture infections or bulk analyses of infected tissues, but such approaches ignore the remarkable diversity of host cell types and microenvironments that are encountered by Salmonella during infection. It is possible that, in these complex host environments, Salmonella subsets are exposed to widely varying ROS and RNS levels that have differential impacts. Common bulk average measurements would miss this heterogeneity and thus might be difficult to interpret. Here, we developed single-cell approaches to determine the impact of ROS and RNS on individual Salmonella in a mouse typhoid fever model. We focused on the first few days of acute infection. Our goal was to clarify controversial issues, including the extent of Salmonella killing by host defenses, the impact of ROS and RNS on Salmonella properties and fates, and the potential role of diverse Salmonella-host encounters on overall disease progression. To determine in which tissue microenvironments Salmonella reside during infection, we analyzed fixed spleen cryosections using immunohistochemistry. At day 4 after infection, Salmonella colonized spleen red pulp, but rarely the white pulp (Figures 1A and 1B ), consistent with previous observations (Nix et al., 2007Nix R.N. Altschuler S.E. Henson P.M. Detweiler C.S. Hemophagocytic macrophages harbor Salmonella enterica during persistent infection.PLoS Pathog. 2007; 3: e193Crossref PubMed Scopus (76) Google Scholar). Neutrophils and inflammatory monocytes accumulated in inflammatory lesions in infected regions, as expected (Richter-Dahlfors et al., 1997Richter-Dahlfors A. Buchan A.M. Finlay B.B. Murine salmonellosis studied by confocal microscopy: Salmonella typhimurium resides intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes in vivo.J. Exp. Med. 1997; 186: 569-580Crossref PubMed Scopus (416) Google Scholar, Rydström and Wick, 2007Rydström A. Wick M.J. Monocyte recruitment, activation, and function in the gut-associated lymphoid tissue during oral Salmonella infection.J. Immunol. 2007; 178: 5789-5801PubMed Google Scholar). Salmonella resided in neutrophils and monocytes within lesions and primarily in resident red pulp macrophages outside of these lesions (Figures 1C–1H). An antibody to Salmonella lipopolysaccharide (LPS) stains both live and dead Salmonella, but intracellular retention of fluorescent proteins discriminates live from dead Salmonella (Barat et al., 2012Barat S. Willer Y. Rizos K. Claudi B. Mazé A. Schemmer A.K. Kirchhoff D. Schmidt A. Burton N. Bumann D. Immunity to intracellular Salmonella depends on surface-associated antigens.PLoS Pathog. 2012; 8: e1002966Crossref PubMed Scopus (61) Google Scholar). Using Salmonella expressing the red fluorescent protein mCherry (RFP), we determined that most Salmonella within neutrophils and inflammatory monocytes in inflammatory lesions were dead (LPS+ RFP−; Figures 1D, 1E, and 1I). Large lesions contained little detectable LPS, suggesting successful Salmonella clearance. In comparison, red pulp macrophages outside of inflammatory lesions contained lower proportions of dead Salmonella (LPS+ RFP+; Figures 1F, 1G, and 1I). Salmonella killing in macrophages was almost abolished in mice treated with a neutralizing antibody to interferon gamma (IFNγ; Figure 1J), consistent with the crucial role of IFNγ in early Salmonella control (Gulig et al., 1997Gulig P.A. Doyle T.J. Clare-Salzler M.J. Maiese R.L. Matsui H. Systemic infection of mice by wild-type but not Spv- Salmonella typhimurium is enhanced by neutralization of gamma interferon and tumor necrosis factor alpha.Infect. Immun. 1997; 65: 5191-5197Crossref PubMed Google Scholar, Muotiala, 1992Muotiala A. Anti-iFN-gamma-treated mice—a model for testing safety of live Salmonella vaccines.Vaccine. 1992; 10: 243-246Crossref PubMed Scopus (13) Google Scholar, VanCott et al., 1998VanCott J.L. Chatfield S.N. Roberts M. Hone D.M. Hohmann E.L. Pascual D.W. Yamamoto M. Kiyono H. McGhee J.R. Regulation of host immune responses by modification of Salmonella virulence genes.Nat. Med. 1998; 4: 1247-1252Crossref PubMed Scopus (111) Google Scholar) and activation of macrophage bactericidal activity (Vazquez-Torres et al., 2000aVazquez-Torres A. Jones-Carson J. Mastroeni P. Ischiropoulos H. Fang F.C. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro.J. Exp. Med. 2000; 192: 227-236Crossref PubMed Scopus (446) Google Scholar). Cybb−/− mice deficient for cytochrome b-245 heavy chain, an essential subunit of NADPH oxidase, are hypersusceptible to Salmonella infection (Mastroeni et al., 2000Mastroeni P. Vazquez-Torres A. Fang F.C. Xu Y. Khan S. Hormaeche C.E. Dougan G. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo.J. Exp. Med. 2000; 192: 237-248Crossref PubMed Scopus (319) Google Scholar). The high spleen loads in such mice (Figure 2A) correlated with less Salmonella killing in neutrophils and inflammatory monocytes, whereas Salmonella live/dead ratios in resident macrophages remained unaltered (Figure 2B). As a consequence, higher proportions of live Salmonella resided in neutrophils and inflammatory monocytes in Cybb−/− mice (Figure 2C). These data indicated that neutrophils and inflammatory monocytes effectively killed Salmonella using NADPH oxidase, while resident macrophages used less effective, largely NADPH oxidase-independent Salmonella killing mechanisms. NADPH oxidase generates superoxide O2•−, which spontaneously dismutates to hydrogen peroxide H2O2 and molecular oxygen. Neutrophils and inflammatory monocytes, but not resident macrophages, express myeloperoxidase (MPO), which converts almost all O2•− or H2O2 into highly bactericidal hypohalites: hypochlorite OCl− (bleach), hypobromite, and/or hypoiodite (Klebanoff et al., 2013Klebanoff S.J. Kettle A.J. Rosen H. Winterbourn C.C. Nauseef W.M. Myeloperoxidase: a front-line defender against phagocytosed microorganisms.J. Leukoc. Biol. 2013; 93: 185-198Crossref PubMed Scopus (459) Google Scholar, Swirski et al., 2010Swirski F.K. Wildgruber M. Ueno T. Figueiredo J.L. Panizzi P. Iwamoto Y. Zhang E. Stone J.R. Rodriguez E. Chen J.W. et al.Myeloperoxidase-rich Ly-6C+ myeloid cells infiltrate allografts and contribute to an imaging signature of organ rejection in mice.J. Clin. Invest. 2010; 120: 2627-2634Crossref PubMed Scopus (86) Google Scholar). Myeloperoxidase preferentially colocalized with dead Salmonella (Figures 2D and 2E), and MPO−/− mice deficient for myeloperoxidase had slightly elevated Salmonella loads (Figure 2F). Together, these data suggest a contribution of hypochlorite (and/or related species) in Salmonella killing. Nevertheless, myeloperoxidase was largely dispensable for Salmonella control, indicating alternative NADPH oxidase-mediated killing mechanisms. In the absence of myeloperoxidase, neutrophils accumulate O2•− and H2O2 (Winterbourn et al., 2006Winterbourn C.C. Hampton M.B. Livesey J.H. Kettle A.J. Modeling the reactions of superoxide and myeloperoxidase in the neutrophil phagosome: implications for microbial killing.J. Biol. Chem. 2006; 281: 39860-39869Crossref PubMed Scopus (497) Google Scholar). To explore their potential impact on Salmonella, we combined a published computational model for oxidative bursts in neutrophil phagosomes (Winterbourn et al., 2006Winterbourn C.C. Hampton M.B. Livesey J.H. Kettle A.J. Modeling the reactions of superoxide and myeloperoxidase in the neutrophil phagosome: implications for microbial killing.J. Biol. Chem. 2006; 281: 39860-39869Crossref PubMed Scopus (497) Google Scholar) with in vivo expression data for Salmonella ROS defense enzymes (Steeb et al., 2013Steeb B. Claudi B. Burton N.A. Tienz P. Schmidt A. Farhan H. Mazé A. Bumann D. Parallel exploitation of diverse host nutrients enhances Salmonella virulence.PLoS Pathog. 2013; 9: e1003301Crossref PubMed Scopus (117) Google Scholar). This in silico model predicted superoxide and hydrogen peroxide accumulation in the phagosomal lumen in the absence of myeloperoxidase (Figure 3), as expected (Winterbourn et al., 2006Winterbourn C.C. Hampton M.B. Livesey J.H. Kettle A.J. Modeling the reactions of superoxide and myeloperoxidase in the neutrophil phagosome: implications for microbial killing.J. Biol. Chem. 2006; 281: 39860-39869Crossref PubMed Scopus (497) Google Scholar). According to the model, superoxide was largely present in the deprotonated form of O2•− that poorly penetrates into bacteria (Korshunov and Imlay, 2002Korshunov S.S. Imlay J.A. A potential role for periplasmic superoxide dismutase in blocking the penetration of external superoxide into the cytosol of Gram-negative bacteria.Mol. Microbiol. 2002; 43: 95-106Crossref PubMed Scopus (124) Google Scholar), whereas H2O2 reached levels around 15 μM within Salmonella, far above the lethality threshold for Salmonella (∼2 μM; Seaver and Imlay, 2001Seaver L.C. Imlay J.A. Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli.J. Bacteriol. 2001; 183: 7182-7189Crossref PubMed Scopus (357) Google Scholar). This was the consequence of phagosomal H2O2 (17 μM) readily diffusing through the Salmonella envelope (Seaver and Imlay, 2001Seaver L.C. Imlay J.A. Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli.J. Bacteriol. 2001; 183: 7182-7189Crossref PubMed Scopus (357) Google Scholar) at rates matching the Salmonella detoxification rate (0.15 × 106 molecules/s). Salmonella killing by moderate, but stable, levels of luminal H2O2 was consistent with previous data for high lethality of continuous H2O2 exposure (Park et al., 2005Park S. You X. Imlay J.A. Substantial DNA damage from submicromolar intracellular hydrogen peroxide detected in Hpx- mutants of Escherichia coli.Proc. Natl. Acad. Sci. USA. 2005; 102: 9317-9322Crossref PubMed Scopus (273) Google Scholar). Interestingly, increasing Salmonella detoxification by 0.15 × 106 molecules/s (thus doubling its rate) would marginally affect predicted phagosomal H2O2 (16.3 μM versus 17 μM), due to buffering by rapid H2O2 diffusion from the phagosome to the host cell cytosol (3.8 × 106 molecules/s; Figure 3). This diffusion is, by definition, proportional to the concentration gradient between phagosome and cytosol, and a slight decrease of phagosomal H2O2 from 17 μM to 16.3 μM would lower its rate by 0.15 × 106 s-1. As Salmonella detoxification increased, less H2O2 would thus be lost to the host cell cytosol, and this compensated for the increase in Salmonella detoxification, resulting in almost unaltered phagosomal and Salmonella concentrations. Together, these data suggested NADPH oxidase-dependent oxidative killing of Salmonella in neutrophils (and inflammatory monocytes) either by hypohalites or by overwhelming hydrogen peroxide if myeloperoxidase was absent. In addition to such direct bactericidal ROS effects, synergism with other bactericidal mechanisms, including antimicrobial peptides and hydrolases, might contribute to Salmonella killing. While Salmonella in neutrophils and monocytes were largely killed through NADPH oxidase-dependent mechanisms, most live Salmonella resided in macrophages with apparently little impact of NADPH oxidase (Figure 2B). To determine if such live Salmonella experienced any oxidative stress, we used Salmonella carrying an episomal katGp-gfpOVA fusion as a ROS biosensor (Figure 4A). The katGp promoter is activated when the transcription factor OxyR reacts with H2O2 (Dubbs and Mongkolsuk, 2012Dubbs J.M. Mongkolsuk S. Peroxide-sensing transcriptional regulators in bacteria.J. Bacteriol. 2012; 194: 5495-5503Crossref PubMed Scopus (131) Google Scholar). This promoter has low baseline activity and a large dynamic range compared to previously used ahpCp (Aussel et al., 2011Aussel L. Zhao W. Hébrard M. Guilhon A.A. Viala J.P. Henri S. Chasson L. Gorvel J.P. Barras F. Méresse S. Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst.Mol. Microbiol. 2011; 80: 628-640Crossref PubMed Scopus (80) Google Scholar) (Figure 4B). We used the unstable GFP variant GFP_OVA (Rollenhagen et al., 2004Rollenhagen C. Sörensen M. Rizos K. Hurvitz R. Bumann D. Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen.Proc. Natl. Acad. Sci. USA. 2004; 101: 8739-8744Crossref PubMed Scopus (72) Google Scholar) to measure current promoter activities instead of integrating over many hours with stable GFP. We coexpressed RFP from the sifBp promoter with constitutive in vivo expression (Rollenhagen et al., 2004Rollenhagen C. Sörensen M. Rizos K. Hurvitz R. Bumann D. Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen.Proc. Natl. Acad. Sci. USA. 2004; 101: 8739-8744Crossref PubMed Scopus (72) Google Scholar) to distinguish autofluorescent host cell fragments and dead RFP− Salmonella from live RFP+ Salmonella regardless of their GFP content (Figure 4C). Biosensor Salmonella showed normal virulence in infected mice and stably maintained the episomal katGp-gfpOVA fusion (>99% plasmid maintenance at day 5 after infection). Proteome analysis of ex vivo purified biosensor Salmonella revealed unaltered expression of OxyR regulon members compared to Salmonella without episomal fusion (Figure S2A), indicating negligible OxyR titration by multicopy katGp. Live RFP+ biosensors had heterogeneous green fluorescence distributions, with large GFPdim and small, but highly reproducible, GFPbright subpopulations (Figure 4D). This reflected heterogeneous katGp activities, as gfpOVA fusions to unrelated promoters had unimodal GFP distributions (Figure S2B). GFPbright Salmonella resided in various host cell types (Figure S2C) but were absent in Cybb−/− mice, indicating specific responses to ROS generated by host NADPH oxidase (Figure 4D). In contrast, myeloperoxidase-deficient MPO−/− mice contained a larger fraction of GFPbright Salmonella, consistent with enhanced H2O2 levels and leakage in these mice (see above). GFPdim biosensors had green fluorescence levels close to those of control Salmonella without GFP but maintained active katGp-gfpOVA fusions as demonstrated by ex vivo sorting followed by in vitro stimulation or reinjection into mice (Figure 4E). This suggested that their low in vivo GFP content reflected limited ROS exposure instead of plasmid loss or mutation. Together, these data indicated heterogeneous oxidative stress levels in live Salmonella. Heterogeneous ROS exposure could reflect temporal dynamics of host cell oxidative bursts, with peak ROS generation early after bacterial contact followed by extended periods with little ROS generation (VanderVen et al., 2009VanderVen B.C. Yates R.M. Russell D.G. Intraphagosomal measurement of the magnitude and duration of the oxidative burst.Traffic. 2009; 10: 372-378Crossref PubMed Scopus (75) Google Scholar). To test this hypothesis, we injected ex vivo sorted RFP+ GFPdim biosensor Salmonella into mice preinfected with nonfluorescent Salmonella (to ensure ongoing tissue inflammation). A large majority of biosensor Salmonella activated katGp within 1 hr but became less active at 3 hr after injection (Figure 4E). At 20 hr after injection, we again observed the typical distribution with a small tail of GFPbright Salmonella. We also constructed a modified katGp-gfp biosensor expressing stable GFP instead of unstable GFP_OVA. This modified biosensor showed larger proportions of NADPH oxidase-dependent GFPbright Salmonella (Figure 4F) compared to the unstable GFP-biosensor, as expected for prolonged GFP retention after transient expression. Together, these data were consistent with ROS exposures during transient host cell oxidative bursts. The small steady-state number of ROS biosensors with high katGp activities at later time points might reflect ongoing exposure of some Salmonella after spreading to new host cells. To determine Salmonella responses to this transient ROS stress, we purified GFPdim and GFPbright subpopulations of katGp-gfpOVA biosensor Salmonella ex vivo (Figure 4G) and compared their proteomes. Abundance data for 966 different proteins revealed upregulation of several proteins involved in Salmonella oxidative stress defense, including catalase G and YaaA in GFPbright biosensor Salmonella (Figure 4H, Table S1), supporting enhanced oxidative stress in this subpopulation. The protein profiles were otherwise highly similar, suggesting no major physiological differences between the two subpopulations. Interestingly, several Salmonella ROS defense proteins had very high abundance even in GFPdim Salmonella (e.g., SodCI, 52,000 ± 2,000 copies per Salmonella cell; TsaA, 22,000 ± 2,000 copies; AhpC, 16,000 ± 2,000 copies). This could reflect residual low-level ROS exposure in this subset. Alternatively, Salmonella might stay prepared to cope with rapid onsets and short durations of host oxidative bursts (both within a few minutes), which cannot be efficiently countered by comparatively slow de novo protein synthesis. Why were these oxidative bursts sublethal in resident red pulp macrophages? In part, this could reflect generally low NADPH oxidase activities in resident red pulp macrophages (Imlay, 2009Imlay J.A. Oxidative Stress.in: Curtiss J.B. Squires C.L. Karp P.D. Neidhardt F.C. Slauch J.M. EcoSal, R.I.K. ASM Press, Washington, DC2009Google Scholar, Nusrat et al., 1988Nusrat A.R. Wright S.D. Aderem A.A. Steinman R.M. Cohn Z.A. Properties of isolated red pulp macrophages from mouse spleen.J. Exp. Med. 1988; 168: 1505-1510Crossref PubMed Scopus (15) Google Scholar). To explore this issue, we built a computational model of Salmonella oxidative stress in macrophage phagosomes based on our model for neutrophils (see above), but incorporating lower oxidative burst activities and acidic phagosomal pH (Figure 3). This in silico model predicted effective Salmonella ROS detoxification to sublethal concentrations in macrophages, in agreement with previous semiquantitative estimates (Imlay, 2009Imlay J.A. Oxidative Stress.in: Curtiss J.B. Squires C.L. Karp P.D. Neidhardt F.C. Slauch J.M. EcoSal, R.I.K. ASM Press, Washington, DC2009Google Scholar, Slauch, 2011Slauch J.M. How does the oxidative burst of macrophages kill bacteria? Still an open question.Mol. Microbiol. 2011; 80: 580-583Crossref PubMed Scopus (267) Google Scholar). Interestingly, periplasmic SodCI was the only individual Salmonella defense enzyme with predicted critical impact on any ROS level. In the absence of SodCI, predicted HO2• concentration in the periplasm increased some 12,000-fold from 0.38 nM to 4.7 μM (Figure 3). Such high levels are likely to damage periplasmic biomolecules (Gort and Imlay, 1998Gort A.S. Imlay J.A. Balance between endogenous superoxide stress and antioxidant defenses.J. Bacteriol. 1998; 180: 1402-1410Crossref PubMed Google Scholar). SodCI deficiency was also predicted to increase cytosolic HO2•, but the resulting level (0.3 nM) was likely sublethal, given that external amino acids are available in vivo (Gort and Imlay, 1998Gort A.S. Imlay J.A. Balance between endogenous superoxide stress and antioxidant defenses.J. Bacteriol. 1998; 180: 1402-1410Crossref PubMed Google Scholar, Steeb et al., 2013Steeb B. Claudi B. Burton N.A. Tienz P. Schmidt A. Farhan H. Mazé A. Bumann D. Parallel exploitation of diverse host nutrients enhances Salmonella virulence.PLoS Pathog. 2013; 9: e1003301Crossref PubMed Scopus (117) Google Scholar). These results are fully consistent with previous experimental data on the role of various Salmonella defense proteins (Craig and Slauch, 2009Craig M. Slauch J.M. Phagocytic superoxide specifically damages an extracytoplasmic target to inhibit or kill Salmonella.PLoS ONE. 2009; 4: e4975Crossref PubMed Scopus (64) Google Scholar, De Groote et al., 1997De Groote M.A. Ochsner U.A. Shiloh M.U. Nathan C. McCord J.M. Dinauer M.C. Libby S.J. Vazquez-Torres A. Xu Y. Fang F.C. Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase.Proc. Natl. Acad. Sci. USA. 1997; 94: 13997-14001Crossref PubMed Scopus (312) Google Scholar, Uzzau et al., 2002Uzzau S. Bossi L. Figueroa-Bossi N. Differential accumulation of Salmonella[Cu, Zn] superoxide dismutases SodCI and SodCII in intracellular bacteria: correlation with their relative contribution to pathogenicity.Mol. Microbiol. 2002; 46: 147-156Crossref PubMed Scopus (62) Google Scholar). Together, these data supported the hypothesis that NADPH oxidase activities in macrophages during early infection might be insufficient to overwhelm the potent and redundant Salmonella antioxidative defense. Our simplif" @default.
• W2034408518 created "2016-06-24" @default.
• W2034408518 creator A5025005632 @default.
• W2034408518 creator A5026093971 @default.
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• W2034408518 date "2014-01-01" @default.
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• W2034408518 title "Disparate Impact of Oxidative Host Defenses Determines the Fate of Salmonella during Systemic Infection in Mice" @default.
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