Can't merge a non object mapping [body.sections.section.para.display.textbox.textbox-body.sections.section.section.section-title] with an object mapping [body.sections.section.para.display.textbox.textbox-body.sections.section.section.section-title]

{ "body": { "sections": { "section": [ { "@id": "sec1", "section-title": "Introduction", "para": [ { "@id": "p0010", "#text": [ "During early stages of infection the innate immune system is essential for limiting microbial replication and spread before an adaptive response is mounted. Accordingly, pathogens have evolved virulence strategies to antagonize innate immune function (", "). The interplay between host innate immune function and pathogen virulence mechanisms largely determines the outcome of most infections. Despite the logic of this conceptual framework, our understanding of the molecular interactions driving the emergence of virulence mechanisms remains relatively poor." ], "cross-refs": { "@refid": "bib28 bib47 bib70", "#text": "Hedrick, 2004; Rausher, 2001; Woolhouse et al., 2002" } }, { "@id": "p0015", "#text": [ "Innate immune receptors detect infection by recognizing conserved microbial features common to broad classes of microbes (", "). The Toll-like receptors (TLRs) target a range of microbial ligands, including lipopolysaccharide (TLR4), lipoproteins (TLR2), flagellin (TLR5), unmethylated CpG motifs in DNA (TLR9), double-stranded RNA (TLR3), and single-stranded RNA (TLR7 and TLR8) (", "). Expression of TLRs on innate immune cells links microbial recognition to induction of antimicrobial mechanisms, such as production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and expression of antimicrobial peptides (AMPs). In addition, TLR activation can promote adaptive immunity through control of dendritic cell (DC) maturation (", ")." ], "cross-refs": [ { "@refid": "bib31 bib39", "#text": "Janeway, 1989; Medzhitov, 2007" }, { "@refid": "bib1 bib32", "#text": "Akira et al., 2001; Kawai and Akira, 2005" } ], "cross-ref": { "@refid": "bib30", "#text": "Iwasaki and Medzhitov, 2004" } }, { "@id": "p0020", "#text": [ "To study the evolution of pathogen virulence and its relationship to innate immunity, we have focused on TLR-mediated recognition of", "serovar", ".", "is a gram-negative bacterium that can survive and replicate within host macrophages (", "). Survival within macrophages requires a set of genes, many of which are encoded within", "pathogenicity island 2 (SPI-2) (", "). SPI-2 encodes a type 3 secretion system (T3SS) that is expressed after the bacterium is phagocytosed (", "). Translocation of SPI-2 effectors into the host cell transforms the phagosome into a compartment that supports bacterial replication, the", "-containing vacuole (SCV) (", "). Multiple signals have been implicated in the transcriptional induction of SPI-2, including cation deprivation, phosphate starvation, and low pH (", "). Most of the studies implicating these signals have been performed on bacteria grown in vitro; whether the same signals are responsible for induction of SPI-2 genes within the phagosome remains unclear." ], "italic": [ "Salmonella enterica", "typhimurium", "S. typhimurium", "Salmonella", "Salmonella" ], "cross-ref": [ { "@refid": "bib12", "#text": "Coburn et al., 2007" }, { "@refid": "bib37", "#text": "Marcus et al., 2000" } ], "cross-refs": [ { "@refid": "bib22 bib50 bib64", "#text": "Galan, 2001; Shea et al., 1996; Waterman and Holden, 2003" }, { "@refid": "bib11 bib43 bib56", "#text": "Cirillo et al., 1998; Pfeifer et al., 1999; Valdivia and Falkow, 1997" }, { "@refid": "bib10 bib11 bib15 bib33 bib45", "#text": "Chakravortty et al., 2005; Cirillo et al., 1998; Deiwick et al., 1999; Kim and Falkow, 2004; Rappl et al., 2003" } ] }, { "@id": "p0025", "#text": [ "Recognition of", "is largely mediated by TLR2, TLR4, and TLR5 (", "). Consistent with a central role for these receptors,", "has evolved mechanisms to subvert this recognition or to avoid the consequences of TLR activation. For example, modification of lipid A by pagP reduces recognition by TLR4, although this modification is probably most relevant for resistance to AMPs (", "). Mice lacking TLRs, especially TLR4, are more susceptible to", "(", "). To circumvent the problem of redundancy, many studies have used mice lacking the common TLR adaptor MyD88 or lacking both MyD88 and another adaptor, TRIF (", "). Although these mice are very susceptible to", ", these studies suffer from the caveat that MyD88 is also required for signaling by members of the IL-1 receptor (IL-1R) family. Because mice deficient in IL-1R are more susceptible to infection, the phenotype of MyD88 knockout (KO) mice cannot be unequivocally attributed to TLRs (", ")." ], "italic": [ "S. typhimurium", "S. typhimurium", "S. typhimurium", "S. typhimurium" ], "cross-refs": [ { "@refid": "bib18 bib27 bib41 bib48 bib52 bib54 bib58", "#text": "Feuillet et al., 2006; Hapfelmeier et al., 2005; O'Brien et al., 1980; Royle et al., 2003; Smith et al., 2003; Uematsu et al., 2006; Vazquez-Torres et al., 2004" }, { "@refid": "bib3 bib16 bib25 bib26", "#text": "Bader et al., 2005; Detweiler et al., 2003; Guo et al., 1997, 1998" }, { "@refid": "bib27 bib65", "#text": "Hapfelmeier et al., 2005; Weiss et al., 2004" }, { "@refid": "bib38 bib46", "#text": "Mayer-Barber et al., 2010; Raupach et al., 2006" } ], "cross-ref": { "@refid": "bib65", "#text": "Weiss et al., 2004" } }, { "@id": "p0030", "#text": [ "In the studies described here, we sought to eliminate TLR-based recognition of", "and examine the effect on pathogen virulence, while avoiding the caveats associated with MyD88-KO mice. In addition, we were concerned that the extreme susceptibility of C57Bl/6 mice (the genetic background on which most studies with TLR-KO mice have been performed) to", "infection might mask any relationships between TLRs and bacterial virulence strategies. Many inbred mouse strains, including C57Bl/6, possess a nonfunctional allele of the", "gene.", "encodes a multipass transmembrane protein that localizes to lysosomes and functions as a transporter of divalent cations, and mice with the nonfunctional allele are extremely susceptible to a number of intracellular pathogens (", ")." ], "italic": [ "S. typhimurium", "S. typhimurium", "nramp-1", "nramp-1" ], "cross-refs": { "@refid": "bib20 bib23 bib59 bib60 bib61", "#text": "Forbes and Gros, 2001; Govoni et al., 1996; Vidal et al., 1993, 1995, 1996" } }, { "@id": "p0035", "#text": [ "To avoid the caveats associated with nonfunctional Nramp-1 and TLR-independent functions of MyD88, we generated mice with a functional allele of", "that lack individual or multiple TLRs. Studies in these mice led to a striking finding. Whereas mice lacking a subset of the TLRs involved in", "recognition showed increased susceptibility to infection, a lack of additional TLRs resulted in reduced susceptibility. The loss of virulence correlated with an inability of bacteria to survive and replicate within macrophages. We show that TLR signaling leads to rapid acidification of the SCV, and this signal is required for regulation of virulence gene expression. In the absence of this contextual cue,", "is unable to survive and replicate intracellularly. Altogether, this work describes the molecular interactions underlying a bacterial pathogen's dependence on the innate immune system for virulence." ], "italic": [ "nramp1", "S. typhimurium", "S. typhimurium" ] } ] }, { "@id": "sec2", "section-title": "Results", "section": [ { "@id": "sec2.1", "section-title": { "#text": "Multiple TLRs Are Involved in Recognition of", "italic": "S. typhimurium" }, "para": [ { "@id": "p0040", "#text": [ "To identify which TLRs are relevant for innate recognition of", ", we utilized HEK293 reporter cell lines expressing an NF-κB-luciferase reporter construct. Stimulation of these cells with heat-killed bacteria resulted in robust induction of NF-κB, which we attributed to endogenous TLR5 expressed by these cells (", "A", "). This response was abrogated when cells were stimulated with bacteria lacking flagellin. To measure activation of other TLR family members, HEK293 reporter cells stably expressing individual TLRs were stimulated with bacteria lacking flagellin (to eliminate the contribution of endogenous TLR5). Using this approach, we observed activation of TLR2 and TLR4 by", "(", "A). Furthermore,", "genomic DNA was capable of activating a surface-localized version of TLR9 (", "A)." ], "italic": [ "S. typhimurium", "S. typhimurium", "S. typhimurium" ], "cross-ref": [ { "@refid": "fig1", "#text": "Figure 1" }, { "@refid": "fig1", "#text": "Figure 1" }, { "@refid": "fig1", "#text": "Figure 1" } ], "float-anchor": { "@refid": "fig1" } }, { "@id": "p0045", "#text": [ "Although these results confirmed that TLR2, TLR4, TLR5, and TLR9 may play a role in recognition of", ", they did not address the relative importance of each TLR during infection. To this end, we infected bone marrow-derived macrophages (BMMs) lacking combinations of TLRs and measured production of nitric oxide (NO). In agreement with previously published studies, BMMs lacking both TLR2 and TLR4 (TLR2×4-KO) produced much less NO than wild-type BMMs (", "B). The remaining response was partially dependent on TLR9, as BMMs lacking TLR2, TLR4, and TLR9 (TLR2×4×9-KO) produced even less NO. Similar results were observed when tumor necrosis factor alpha (TNF) production was measured (", "C). Importantly, all genotypes of BMMs responded equivalently to the TLR7 ligand R848, indicating that the cells were otherwise equivalent (", "D and", "available online). The small amount of TNF and NO produced in TLR2×4×9-KO BMMs was dependent on other TLRs, as BMMs lacking both MyD88 and TRIF (and therefore all TLR-dependent signaling) did not respond to", "(", "B and 1C). As TLR5 is not expressed in murine BMMs, we reasoned that the residual TNF and NO produced by TLR2×4×9-KO BMMs was most likely due to TLR7 or TLR3 signaling. To address this possibility directly, we pretreated TLR2×4×9-KO BMMs with bafilomycinA1, an inhibitor of the vacuolar ATPase (V-ATPase) that prevents activation of endosomal TLRs. BafilomycinA1 treatment inhibited TNF production in TLR2×4-KO and TLR2×4×9-KO BMMs to almost background levels, suggesting that TLR7 and/or TLR3 are responsible for the remaining TNF production in response to", "(", "D). Collectively, these data indicate that TLR2, TLR4, TLR9, and TLR7 (and/or TLR3) each contribute to the recognition of", "in infected BMMs." ], "italic": [ "S. typhimurium", "S. typhimurium", "S. typhimurium", "S. typhimurium" ], "cross-ref": [ { "@refid": "fig1", "#text": "Figure 1" }, { "@refid": "fig1", "#text": "Figure 1" }, { "@refid": "fig1", "#text": "Figure 1" }, { "@refid": "figs1", "#text": "Figure S1" }, { "@refid": "fig1", "#text": "Figures 1" }, { "@refid": "fig1", "#text": "Figure 1" } ], "float-anchor": { "@refid": "figs1" } } ] }, { "@id": "sec2.2", "section-title": { "#text": [ "TLR Signaling Is Required for", "Virulence" ], "italic": "S. typhimurium" }, "para": [ { "@id": "p0050", "#text": [ "Having established which TLRs respond to ligands derived from", "in BMMs, we sought to test the effect of TLR deficiency on bacterial virulence in vivo. We crossed a functional allele of the", "gene onto the C57BL/6 background and generated TLR-deficient or TLR-adaptor-deficient mice with functional Nramp1 (see", "). We expected that reduced TLR function would lead to greater susceptibility to infection. Indeed, all TLR2×4-KO mice died within 16 days when challenged orally with", ", whereas 75% of the wild-type mice survived for the duration of the experiment (", "A", "). By contrast, TLR2×4×9-KO mice were", "susceptible to infection than TLR2×4-KO mice, despite a greater impairment in TLR function (", "A). This increased survival was not a consequence of reduced immunopathology due to reduced TLR function. In fact, TLR2×4×9-KO mice had lower numbers of bacteria 4 days post-infection in spleens, livers, ceca, and mesenteric lymph nodes (MLNs) relative to TLR2×4-KO mice (", "B). Thus, despite less robust innate immune function,", "was less virulent in TLR2×4×9-KO mice." ], "italic": [ "S. typhiumurium", "nramp1", "S. typhimurium", "less", "S. typhimurium" ], "cross-ref": [ { "@refid": "sec4.9", "#text": "Extended Experimental Procedures" }, { "@refid": "fig2", "#text": "Figure 2" }, { "@refid": "fig2", "#text": "Figure 2" }, { "@refid": "fig2", "#text": "Figure 2" } ], "float-anchor": { "@refid": "fig2" } }, { "@id": "p0055", "#text": [ "The difference in susceptibility between TLR2×4-KO and TLR2×4×9-KO mice could indicate that TLR9 plays a negative role in immunity to", ". To test this possibility, we challenged mice lacking TLR4 and TLR9 (TLR4×9-KO). We reasoned that if TLR9 were playing a negative role in immunity, then any genotype lacking TLR9 would be resistant to infection. Instead, TLR4×9-KO mice were as susceptible to infection as TLR2×4-KO mice, indicating that lack of TLR9 by itself does not confer increased resistance to infection (", "A). Thus, the data presented suggest that overall TLR signaling is in some way required for", "virulence. Despite this apparent requirement, MyD88-KO and MyD88×TRIF-KO mice (with wild-type Nramp1) were highly susceptible to", "infection (", "). As discussed earlier, the extreme sensitivity of these mice relative to TLR2×4×9-KO mice is likely due to the role of MyD88 downstream of the IL-1, IL-18, and IL-33 receptors (", "). Thus, to examine the role for TLR signaling in", "virulence, we must use TLR-deficient mice, not mice lacking common signaling adaptors." ], "italic": [ "S. typhimurium", "S. typhimurium", "S. typhimurium", "S. typhimurium" ], "cross-ref": [ { "@refid": "fig2", "#text": "Figure 2" }, { "@refid": "figs2", "#text": "Figure S2" } ], "float-anchor": { "@refid": "figs2" }, "cross-refs": { "@refid": "bib38 bib46", "#text": "Mayer-Barber et al., 2010; Raupach et al., 2006" } }, { "@id": "p0060", "#text": [ "One potential caveat of these in vivo studies is that the commensal flora may be different between TLR2×4-KO and TLR2×4×9-KO mice. Recent studies have reported alterations in commensal communities in mice lacking certain TLRs or TLR-signaling adaptors (", "). To address this possibility, we challenged mice with a different gram-negative enteric pathogen,", "(", "), which shares a similar route of intestinal colonization but remains extracellular after crossing the intestinal epithelia. In contrast to our experiments with", ", TLR2×4×9-KO mice were equally, if not more, susceptible relative to TLR2×4-KO mice (", "C). The differential sensitivity of TLR2×4×9-KO mice to these two enteric bacteria argues that alterations in commensal flora are not contributing to the phenotypes of TLR2×4-KO and TLR2×4×9-KO mice. Instead, the reduction in TLR signaling in TLR2×4×9-KO mice appears to specifically impact the virulence of", "." ], "cross-refs": { "@refid": "bib62 bib66", "#text": "Vijay-Kumar et al., 2008; Wen et al., 2008" }, "italic": [ "Yersinia enterocolitica", "Y. enterocolitica", "S. typhimurium", "S. typhimurium" ], "cross-ref": { "@refid": "fig2", "#text": "Figure 2" } } ] }, { "@id": "sec2.3", "section-title": "TLR Signaling Is Required for Intracellular Growth of Bacteria", "para": [ { "@id": "p0065", "#text": [ "Because survival within macrophages is required for systemic infection (", "), we next used a gentamicin protection assay to examine survival and replication in BMMs lacking various TLRs. Consistent with our in vivo experiments,", "was able to replicate in TLR2×4-KO but not TLR2×4×9-KO BMMs (", "A", "). When we counted bacteria in individual BMMs by immunofluorescence microscopy (IF), the number of bacteria per cell in TLR2×4-KO BMMs accumulated over time, whereas the number of bacteria per cell in TLR2×4×9-KO BMMs remained constant, indicating that bacterial replication was responsible for the differences in colony-forming units (CFU) between genotypes (", "B and 3C). We observed a similar lack of bacterial replication in MyD88×TRIF-KO BMMs (", "A). Unlike our in vivo experiments, the phenotype of MyD88×TRIF-KO BMMs is most likely due to a deficiency in TLR signaling, as the IL-1 receptor family is not involved in the initial recognition of", "within BMMs in vitro. Furthermore, TLR4×9-KO BMMs supported bacterial replication similarly to TLR2×4-KO BMMs, corroborating the conclusions from our in vivo experiments (", "B and 3C). TLR2×4×9-KO and MyD88×TRIF-KO BMMs did support replication of", "and", "(", "). In addition,", "replicated well in MyD88×TRIF-KO BMMs lacking functional Nramp1 (", "). These results indicate that phagosomes of TLR-deficient cells are formally capable of supporting bacterial growth, but the combination of functional Nramp1 and lack of TLR signaling prevents", "replication." ], "cross-refs": { "@refid": "bib19 bib34", "#text": "Fields et al., 1986; Leung and Finlay, 1991" }, "italic": [ "S. typhimurium", "S. typhimurium", "Listeria monocytogenes", "Legionella pneumophila", "S. typhimurium", "S. typhimurium" ], "cross-ref": [ { "@refid": "fig3", "#text": "Figure 3" }, { "@refid": "fig3", "#text": "Figures 3" }, { "@refid": "fig3", "#text": "Figure 3" }, { "@refid": "fig3", "#text": "Figures 3" }, { "@refid": "figs3", "#text": "Figure S3" }, { "@refid": "figs3", "#text": "Figure S3" } ], "float-anchor": [ { "@refid": "fig3" }, { "@refid": "figs3" } ] }, { "@id": "p0070", "#text": [ "Collectively, these data suggest that", "requires TLR signaling for replication in macrophages. However, the lack of replication in wild-type BMMs would appear to contradict this conclusion, as TLR function is normal in these cells. When we counted the number of bacteria per cell by IF, though, we observed a similar increase in bacteria per cell over time as in TLR2×4-KO BMMs (", "C). This contradiction was resolved when we measured cell death of BMMs after infection. Wild-type BMMs exhibited greater cell death relative to each of the other genotypes (", "D and 3E). Because only wild-type BMMs express functional TLR4, the increased death of these cells seems likely to be due to a previously described TLR4-dependent cell death that occurs in", "infected cells (", "). Thus, the apparent lack of replication as measured by CFU in wild-type BMMs is the result of macrophage death followed by gentamicin-mediated killing of the bacteria. In contrast, the inability of", "to replicate in TLR2×4×9-KO or MyD88×TRIF-KO BMMs is due to a different mechanism." ], "italic": [ "S. typhimurium", "S. typhimurium", "S. typhimurium" ], "cross-ref": [ { "@refid": "fig3", "#text": "Figure 3" }, { "@refid": "fig3", "#text": "Figures 3" } ], "cross-refs": { "@refid": "bib13 bib29 bib42", "#text": "Cook et al., 2007; Hsu et al., 2004; Park et al., 2002" } } ] }, { "@id": "sec2.4", "section-title": "TLR Signaling Is Required for Establishment of the SCV", "para": { "@id": "p0075", "#text": [ "To better understand why", "is unable to replicate in TLR2×4×9-KO and MyD88×TRIF-KO BMMs, we used transmission electron microscopy (EM) to investigate the fate of bacteria in infected BMMs. At 2 hr post-infection, bacteria were clearly visible in well-defined vacuoles in BMMs of all three genotypes (", "A", ", black triangles). By 8 hr and 22 hr post-infection, bacteria in TLR2×4-KO BMMs remained largely unchanged, although evidence of replication was evident, especially at 22 hr (", "B and 4C). In contrast, phagosomes containing bacteria in TLR2×4×9-KO and MyD88×TRIF-KO BMMs were quite distinct. The bacteria often appeared mottled or irregular in shape, and in many cases bacteria were surrounded by electron-dense staining material consistent with lysosomal fusion (", ", open triangles). In some instances, bacteria were no longer surrounded by membrane, suggesting that they entered the cytosol (", ", white triangles). Cytosolic bacteria have been described when bacteria fail to secrete certain SPI-2 effectors (", "). In total, the images clearly demonstrate a defect in the ability of", "to establish a replicative compartment in TLR2×4×9-KO and MyD88×TRIF-KO BMMs." ], "italic": [ "S. typhimurium", "S. typhimurium" ], "cross-ref": [ { "@refid": "fig4", "#text": "Figure 4" }, { "@refid": "fig4", "#text": "Figures 4" }, { "@refid": "fig4", "#text": "Figure 4" }, { "@refid": "fig4", "#text": "Figure 4" }, { "@refid": "bib6", "#text": "Beuzon et al., 2000" } ], "float-anchor": { "@refid": "fig4" } } }, { "@id": "sec2.5", "section-title": "Induction of SPI-2 Genes by TLR Signaling", "para": [ { "@id": "p0080", "#text": [ "Our studies thus far indicate that intracellular growth of", "is impaired in TLR2×4×9-KO and MyD88×TRIF-KO BMMs and suggest that this defect may be related to inefficient SCV formation. We next sought to define the underlying basis for impaired growth in BMMs lacking TLR function by profiling gene expression of bacteria isolated from BMMs of each genotype. To overcome the lack of sensitivity of microarray-based approaches, we performed quantitative RT-PCR to measure expression of all genes in the", "genome (", "A", ")." ], "italic": [ "S. typhimurium", "S. typhimurium" ], "cross-ref": { "@refid": "fig5", "#text": "Figure 5" }, "float-anchor": { "@refid": "fig5" } }, { "@id": "p0085", "#text": [ "Using K-means clustering analysis, we identified subsets of genes with differential expression profiles between the BMM genotypes. Genes within the SPI-2 locus were upregulated in bacteria in wild-type and TLR2×4-KO BMMs but not in bacteria in TLR2×4×9-KO or MyD88×TRIF-KO BMMs. For validation, we reanalyzed expression of each gene within the SPI-2 locus and adjacent to the locus (as controls), using independent RNA samples from infected BMMs of all genotypes. As shown in", "B, 13 genes within the SPI-2 locus were upregulated in wild-type and TLR2×4-KO BMMs but not in TLR2×4×9-KO and MyD88×TRIF-KO BMMs. These 13 genes most likely underestimate the extent to which the entire SPI-2 locus is differentially expressed between BMM genotypes, as many genes were statistically excluded due to extremely low levels of message in TLR2×4×9-KO or MyD88×TRIF-KO samples. For most SPI-2 genes, induction was higher in wild-type BMMs relative to TLR2×4-KO BMMs (", "C), suggesting that induction correlates with the strength of TLR signaling. Thus, the lack of intracellular replication in TLR-deficient cells may be due to a failure to upregulate SPI-2 genes." ], "cross-ref": [ { "@refid": "fig5", "#text": "Figure 5" }, { "@refid": "fig5", "#text": "Figure 5" } ] }, { "@id": "p0090", "#text": [ "These expression-profiling studies indicated that transcription of SPI-2 genes within BMMs depends on signals downstream of TLR activation. To view SPI-2 induction at the protein level, we utilized a strain of", "(12023) with an HA-tagged allele of", ", a SPI-2 effector. 12023 displays the same dependence on TLR signaling for intracellular growth as SL1344 (", "D). PipB2 was strongly induced and secreted in infected TLR2×4-KO BMMs (", "D). In contrast, the levels of PipB2 were significantly reduced in TLR2×4×9-KO BMMs and barely detectable in MyD88×TRIF-KO BMMs, despite equivalent numbers of bacteria in all samples (indicated by DnaK levels). These data are consistent with our transcriptional analyses and indicate that TLR signaling is required for the induction of SPI-2 genes." ], "italic": [ "S. typhimurium", "pipB2" ], "cross-ref": [ { "@refid": "figs3", "#text": "Figure S3" }, { "@refid": "fig5", "#text": "Figure 5" } ] } ] }, { "@id": "sec2.6", "section-title": "SPI-2 Genes Are Required for Intracellular Growth", "para": [ { "@id": "p0095", "#text": [ "We hypothesized that the impaired induction of SPI-2 genes in bacteria isolated from TLR2×4×9-KO and MyD88×TRIF-KO BMMs was responsible for the defect in SCV formation and intracellular replication in these cells. To test this hypothesis, we compared the fates of bacteria lacking a functional SPI-2 secretion system (", ") in BMMs of each genotype. As expected, SPI-2 mutant bacteria were unable to replicate in BMMs of any genotype (", "A", "). Moreover, EM analysis of SPI-2 mutant bacteria in TLR2×4-KO BMMs revealed the same lack of SCV formation observed for wild-type bacteria in TLR2×4×9-KO and MyD88×TRIF-KO BMMs (", "B)." ], "italic": "ssaV::Kan", "cross-ref": [ { "@refid": "fig6", "#text": "Figure 6" }, { "@refid": "fig6", "#text": "Figure 6" } ], "float-anchor": { "@refid": "fig6" } }, { "@id": "p0100", "#text": [ "If the lack of intracellular growth in TLR2×4×9-KO and MyD88×TRIF-KO BMMs is due to failure to induce SPI-2 genes, then an", "strain with constitutive expression of SPI-2 genes should regain the ability to grow in these cells. To test this possibility directly, we constructed a strain lacking", "(Δ", "), a negative regulator of SPI-2 genes. Previous work has demonstrated that the Δ", "mutant strain expresses SPI-2 genes constitutively (", "). Remarkably, Δ", "mutant bacteria replicated equivalently in BMMs of all genotypes, except wild-type cells where the lack of growth is due to TLR4-dependent cell death (", "C). Although Hha probably negatively regulates additional", "virulence genes, restoration of growth in TLR-deficient BMMs is consistent with the conclusion that constitutive expression of SPI-2 genes can bypass the requirement for TLR signaling." ], "italic": [ "S. typhimurium", "hha", "hha, hha::Cm", "hha", "hha", "S. typhimurium" ], "cross-ref": [ { "@refid": "bib51", "#text": "Silphaduang et al., 2007" }, { "@refid": "fig6", "#text": "Figure 6" } ] } ] }, { "@id": "sec2.7", "section-title": "Induction of SPI-2 Genes Requires TLR-Dependent Acidification of the SCV", "para": [ { "@id": "p0105", "#text": [ "Our results thus far indicate that TLR signaling provides a cue used by", "to regulate SPI-2 expression. TLR activation induces host transcription as well as more proximal effects, such as production of ROS and RNS and phagosome maturation and acidification, although this last aspect remains controversial. Using pharmacological inhibitors to block each of these potential signals we measured the effect on PipB2 induction and secretion. Treatment of TLR2×4-KO BMMs with cyclohexamide (CHX) had no effect on PipB2 induction, indicating that host translation was not required for generation of the signal sensed by", "(", "A", ", bottom panel). Similarly, blocking ROS or RNS production did not prevent PipB2 induction. However, inhibition of the V-ATPase with bafilomycinA1 blocked", "induction of PipB2 in both TLR2×4-KO and wild-type BMMs. The block in TLR2×4-KO cells could be due to an inhibition of TLR signaling, as bafilomycinA1 almost completely inhibits the residual response to", "(", "D). In wild-type cells, though, TLR2 and TLR4 signaling is largely unaffected by bafilomycinA1, suggesting that TLR-dependent acidification of the SCV may be the signal required by", "for SPI-2 gene induction (", "A, top panel). Experiments analyzing the induction of SPI-2 genes at the transcriptional level also indicated a requirement for phagosome acidification (data not shown)." ], "italic": [ "S. typhimurium", "S. typhimurium", "S. typhimurium", "S. typhimurium", "S. typhimurium" ], "cross-ref": [ { "@refid": "fig7", "#text": "Figure 7" }, { "@refid": "fig1", "#text": "Figure 1" }, { "@refid": "fig7", "#text": "Figure 7" } ], "float-anchor": { "@refid": "fig7" } }, { "@id": "p0110", "#text": [ "Based on these data, we hypothesized that the lack of SPI-2 induction in TLR2×4×9-KO and MyD88×TRIF-KO BMMs is due to failure of SCVs to acidify. The issue of whether TLR signaling influences the kinetics of phagosomal maturation remains controversial (", "). To investigate this issue, we used ratiometric imaging to measure the pH of", "-containing phagosomes in BMMs of each genotype. Whereas the mean pH of SCVs in wild-type and TLR2×4-KO BMMs dropped below 6 within 60 min post-infection, SCVs in TLR2×4×9-KO and MyD88×TRIF-KO BMMs failed to acidify to the same extent and exhibited slower acidification kinetics (", "B). By 30 min post-infection, over 70% of SCVs in wild-type and TLR2×4-KO BMMs had reached pH 6, whereas less than 35% of SCVs in TLR2×4×9-KO and MyD88×TRIF-KO BMMs had similarly acidified (", "B). Consistent with the lower transcriptional induction of SPI-2 genes in TLR2×4-KO BMMs (relative to wild-type), the rate of acidification in TLR2×4-KO cells was slower than in wild-type cells, despite ultimately reaching pH 6 by 60 min. Collectively, these data support a model in which TLR signaling accelerates phagosomal acidification, which is used by", "as a cue for SPI-2 gene induction." ], "cross-refs": { "@refid": "bib7 bib8 bib9 bib49 bib71", "#text": "Blander and Medzhitov, 2004, 2006a, 2006b; Russell and Yates, 2007; Yates and Russell, 2005" }, "italic": [ "Salmonella", "S. typhimurium" ], "cross-ref": [ { "@refid": "fig7", "#text": "Figure 7" }, { "@refid": "fig7", "#text": "Figure 7" } ] } ] } ] }, { "@id": "sec3", "section-title": "Discussion", "para": [ { "@id": "p0115", "#text": [ "Biological interactions are strong drivers of evolution, and the dynamics of host-pathogen interactions provide some of the clearest examples of this principle. Hosts have evolved resistance mechanisms, such as TLRs, that work by reducing pathogen fitness and drive the evolution of pathogen virulence (", "). Although virulence genes provide a fitness advantage, they can be energetically costly and often serve as targets of host sensors (", "). Therefore, the ability to regulate expression of virulence genes based on changing environments is a key feature of microbial pathogenesis. In this study, we report the requirement of TLR signaling for", "to establish a successful infection and cause disease. We demonstrate that this requirement stems, at least in part, from the need for TLR-dependent phagosome acidification to induce SPI-2 genes, resulting in replication and virulence of the microbe. These data demonstrate that a pathogen can evolve to require innate immune signaling for full virulence." ], "cross-refs": [ { "@refid": "bib28 bib47 bib70", "#text": "Hedrick, 2004; Rausher, 2001; Woolhouse et al., 2002" }, { "@refid": "bib40 bib57", "#text": "Miao et al., 2010; Vance et al., 2009" } ], "italic": "S. typhimurium" }, { "@id": "p0120", "#text": [ "Previous studies have demonstrated that host genetic variation can result in prolonged survival upon infection (", "). However, these phenotypes are generally attributable to reduced inflammation and immunopathology, suggesting that the host is more tolerant to an increased pathogen burden (", "). By contrast, our work demonstrates that mice lacking sufficient TLR signaling are less susceptible to an", "infection due to reduced bacterial growth. A similar relationship has been described in", ", where mutations in the melanization arm of the innate immune response render flies less susceptible to", "with reduced levels of bacteria, but the mechanism behind this observation remains unclear (", "). Our work clearly shows that", "fails to induce virulence genes when deprived of innate immune signals." ], "cross-ref": [ { "@refid": "bib44", "#text": "Raberg et al., 2007" }, { "@refid": "bib2", "#text": "Ayres and Schneider, 2008" } ], "cross-refs": { "@refid": "bib24 bib63", "#text": "Gowen et al., 2006; Wang et al., 2004" }, "italic": [ "S. typhimurium", "Drosophila", "Streptococcus pneumoniae", "S. typhimurium" ] } ], "section": [ { "@id": "sec3.1", "section-title": "Crosstalk between Nramp-1 and TLR Signaling", "para": [ { "@id": "p0125", "#text": [ "Two aspects of our approach were crucial for our ability to observe the requirement for TLR-dependent signals in", "virulence. First, by using mice that are deficient in multiple TLRs, as opposed to mice lacking MyD88 and TRIF, we were able to circumvent the susceptibility associated with lack of IL-1R family function. Indeed, the difference that we observe in susceptibility between MyD88×TRIF-KO and TLR2×4×9-KO mice underscores the importance of the IL-1R family in defense against infection. We were somewhat surprised by the role for nucleic acid-sensing TLRs in innate recognition of", ", although TLR9 and TLR7 have been implicated in recognition of bacterial nucleic acid (", "). Although the simplest explanation for this observation is that some bacteria are degraded, it is also possible that a nucleic acid ligand is secreted by", "or present on the bacterial surface (", ")." ], "italic": [ "Salmonella", "Salmonella", "Salmonella" ], "cross-refs": [ { "@refid": "bib4 bib36", "#text": "Bafica et al., 2005; Mancuso et al., 2009" }, { "@refid": "bib67 bib69", "#text": "Whitchurch et al., 2002; Woodward et al., 2010" } ] }, { "@id": "p0130", "#text": [ "A second critical aspect of our study is that we used mice with a functional allele of", ". Why the lack of this protein renders mice so susceptible to intracellular pathogens remains unclear, but this heightened sensitivity may simply obviate any requirement for TLR-dependent SPI-2 induction. The presence of functional Nramp1 has been shown to enhance SPI-2 expression as well as TLR-dependent responses (", "). Regardless of the precise mechanism responsible for the strong TLR dependence when Nramp1 is functional, it is important to recognize that infection of cells with functional Nramp1 represents the “wild-type” scenario. Indeed, mutations in the human Nramp1 gene are associated with increased susceptibility to several intracellular pathogens (", "). Therefore, examining virulence in the presence of functional Nramp1 most accurately reflects the host-pathogen interactions between", "and the mammalian immune system." ], "italic": [ "nramp1", "S. typhimurium" ], "cross-refs": [ { "@refid": "bib21 bib55 bib72", "#text": "Fritsche et al., 2003; Valdez et al., 2008; Zaharik et al., 2002" }, { "@refid": "bib5 bib35", "#text": "Bellamy et al., 1998; Malik et al., 2005" } ] } ] }, { "@id": "sec3.2", "section-title": { "#text": [ "TLR Signaling Alters the pH of the", "-Containing Vacuole" ], "italic": "Salmonella" }, "para": [ { "@id": "p0135", "#text": [ "Our studies indicate that the difference in susceptibility of TLR-deficient mice is due to lack (or substantial delay) of SPI-2 induction. We show that SPI-2 induction requires phagosome acidification, and our measurements of phagosomal pH indicate that acidification is impaired and/or delayed in TLR-deficient cells. The extent to which TLR signaling influences phagosomal maturation (including increasing phagolysosomal fusion, acidification, and proteolytic activity) has remained a contentious issue (", "). Although our studies were not designed to address this controversy, we clearly show that TLR signaling is required for rapid acidification of the SCV and has profound implications for the fate of intracellular bacteria and disease outcome. The mechanism is likely similar to the acidification of lysosomes during DC maturation, when TLR signaling leads to recruitment of the V1 subunit of the vacuolar ATPase to the lysosomal membrane (", "). The precise signaling pathways that lead to assembly of this machinery are unknown. Moreover, whether bacteria sense pH directly or utilize other phagosomal features that require acidic pH remains unclear." ], "cross-refs": { "@refid": "bib7 bib8 bib9 bib49 bib71", "#text": "Blander and Medzhitov, 2004, 2006a, 2006b; Russell and Yates, 2007; Yates and Russell, 2005" }, "cross-ref": { "@refid": "bib53", "#text": "Trombetta et al., 2003" } }, { "@id": "p0140", "#text": [ "Importantly, we are not suggesting that phagosome maturation cannot occur without TLR signaling. Indeed, our EM images of infected TLR2×4×9-KO and MyD88×TRIF-KO BMMs at late time points show bacteria within electron-dense compartments, suggestive of phagolysosomal fusion. Due to technical limitations, we have not extended our pH measurements beyond 60 min post-infection, but our images suggest that phagosomes in TLR-deficient cells eventually mature. In fact, we do observe a small percentage of SCV in TLR2×4×9-KO and MyD88×TRIF-KO cells with significant reductions in pH within 1 hr (", "). The lack of bacterial replication in TLR-deficient cells suggests that the eventual maturation of phagosomes is not sufficient to induce SPI-2 genes or that the induction occurs too late to prevent bacterial killing by lysosomal contents. It is also possible that the phagosome breaks down in the absence of SPI-2 function, and bacteria enter the cytosol where they are unable to replicate (", "). Our EM analyses suggest that both of these possibilities may contribute to the lack of bacterial replication." ], "cross-ref": [ { "@refid": "figs4", "#text": "Figure S4" }, { "@refid": "bib6", "#text": "Beuzon et al., 2000" } ], "float-anchor": { "@refid": "figs4" } } ] }, { "@id": "sec3.3", "section-title": "Innate Immune Signaling as an Environmental Cue for Virulence Gene Regulation", "para": [ { "@id": "p0145", "#text": [ "These findings have important implications for our understanding of the evolution of host-pathogen interactions and virulence mechanisms. Many pathogens trigger these mechanisms by utilizing signals downstream of innate receptors, most likely as a reliable mechanism to properly induce genes necessary for survival in the presence of antimicrobial mechanisms. For example, the PhoP/PhoQ two-component system, when activated by AMPs, induces expression of genes that modify lipidA and render the bacterial membrane more resistant to AMPs (", "). By a potentially similar mechanism, prior activation of cells with TLR ligands can increase replication of", "(", "). Our finding that", "has evolved to require host-resistance signals for proper expression of virulence genes is conceptually distinct from these previously described antagonistic strategies. Notably,", "is unable to replicate in TLR-deficient cells, despite the absence of the antimicrobial mechanisms normally induced by TLRs. One implication of this remaining dependence is that the virulence genes induced by TLR signaling are required for purposes other than simply evading TLR-induced antimicrobial mechanisms to promote", "fitness." ], "cross-refs": { "@refid": "bib3 bib26", "#text": "Bader et al., 2005; Guo et al., 1998" }, "italic": [ "S. typhimurium", "S. typhimurium", "S. typhimurium", "S. typhimurium" ], "cross-ref": { "@refid": "bib68", "#text": "Wong et al., 2009" } }, { "@id": "p0150", "#text": [ "Why would", "use signals downstream of TLRs to broadly coordinate expression of virulence genes required for intracellular growth? These signals may be the most reliable contextual cues that", "can use to sense its presence within a macrophage phagosome. In general, a fundamental problem faced by", "is the need to interact with multiple cell types through the course of an infection. Unique sets of virulence genes are required to survive each of these stages, and", "must recognize its environment and induce the appropriate genes. For example,", "must detect when it has encountered a macrophage and induce SPI-2 genes, which are necessary for formation of the SCV and maintenance of the integrity of the phagosome. Precise regulation of such virulence genes is clearly essential for optimal growth, as mutant bacteria with constitutive expression of SPI-2 genes (e.g.,", "mutants) are attenuated in vivo (", "). Inappropriate expression of certain virulence genes could result in decreased fitness due to recognition by innate sensors or may disrupt proper regulation of other virulence genes required at specific stages of infection. Therefore,", "utilizes TLR-dependent signals within the phagosome to detect its presence within a macrophage. Linking the induction of virulence genes (including SPI-2) to phagosomal signals downstream of TLRs may be an efficient way of coordinating multiple virulence mechanisms in response to a unifying contextual cue." ], "italic": [ "Salmonella", "Salmonella", "Salmonella", "Salmonella", "Salmonella", "Δhha", "Salmonella" ], "cross-refs": { "@refid": "bib14 bib51", "#text": "Coombes et al., 2005; Silphaduang et al., 2007" } } ] } ] }, { "@id": "sec4", "@role": "materials-methods", "section-title": "Experimental Procedures", "section": [ { "@id": "sec4.1", "section-title": "Cell Culture", "para": { "@id": "p0155", "#text": [ "BMMs were differentiated from bone marrow for 5 days using macrophage colony-stimulating factor (M-CSF) as previously described (", "). See", "for details." ], "cross-ref": [ { "@refid": "bib17", "#text": "Ewald et al., 2008" }, { "@refid": "sec4.9", "#text": "Extended Experimental Procedures" } ] } }, { "@id": "sec4.2", "section-title": "Bacterial Strains and Infections", "para": { "@id": "p0160", "#text": [ "Overnight cultures of", "were opsonized for infection. BMMs were spin-infected, incubated at 37°C, then washed with PBS before the addition of 10 μg/ml gentamicin media. For intracellular CFU determination, cells were washed with PBS and lysed in 1% Triton X-100 in PBS. Lysates were plated on LB agar plates containing 200 μg/ml streptomycin (Life Technologies). See", "for strain details and descriptions of assays with", "and", "." ], "italic": [ "S. typhimurium", "L. monocytogenes", "L. pneumophila" ], "cross-ref": { "@refid": "sec4.9", "#text": "Extended Experimental Procedures" } } }, { "@id": "sec4.3", "section-title": "Measurement of Cell Death", "para": { "@id": "p0165", "#text": "Lactate dehydrogenase (LDH) release was quantified using the CytoTox 96 Nonradioactive cytotoxicity kit (Promega) according to manufacturer's instructions. Annexin V staining was performed using Annexin V-FITC (BD Pharmingen) in Annexin V staining buffer according to manufacturer's instructions." } }, { "@id": "sec4.4", "section-title": "Measurement of BMM Activation", "para": { "@id": "p0170", "#text": "NO was quantified in supernatants from BMMs treated overnight with 100 U/ml recombinant IFN-γ (R&D Systems) using the Griess assay (all reagents from Sigma Aldrich). TNF-α production was measured by intracellular cytokine staining using anti-TNF-α antibody (eBioscience) according to manufacturer's instructions (eBioscience). All steps prior to fixation were performed in the presence of 10 μg/ml gentamicin. Cells were analyzed on an FC500 flow cytometer (Beckman Coulter)." } }, { "@id": "sec4.5", "section-title": { "italic": "S. typhimurium", "#text": "Effector Secretion" }, "para": { "@id": "p0175", "#text": [ "BMMs were infected (multiplicity of infection [moi] of 25) with", "-2xHA (12032). At the indicated time points, cells were washed with PBS and lysed in 1% NP-40 in PBS with protease-inhibtor cocktail (Roche) and EDTA (Fisher), and lysates were subjected to immunoprecipitation with rat anti-HA agarose beads (Roche). Cells were pretreated with inhibitors for 1 hr before infection. See", "for a more detailed explanation of sample processing and detection of effector secretion." ], "italic": "S. typhimurium pipB2", "cross-ref": { "@refid": "sec4.9", "#text": "Extended Experimental Procedures" } } }, { "@id": "sec4.6", "section-title": "Mice and In Vivo Infections", "para": { "@id": "p0180", "#text": [ "All animal experiments were performed in accordance with University of California Animal Care and Use Committee guidelines. See", "for descriptions of strains and backcrossing analyses. For survival and CFU enumeration experiments, age-matched mice were fasted for 14 hr followed by oral gavage with 100 μl", "(SL1344) or", "(8081) in PBS (see figure legends for CFU). For CFU enumeration, organs were harvested, homogenized in PBS using a Polytron PT2100 homogenizer (Kinematica), diluted, and plated on streptomycin (for", ") or irgasan (1 μg/ml) (for", ") LB-agar plates." ], "cross-ref": { "@refid": "sec4.9", "#text": "Extended Experimental Procedures" }, "italic": [ "S. typhimurium", "Yersinia enterocolitica", "Salmonella", "Yersinia" ] } }, { "@id": "sec4.7", "section-title": "Gene Expression Analyses", "para": { "@id": "p0185", "#text": [ "RNA from infected BMMs (moi of 5) was extracted with Trizol RNA reagent, purified using PureLink Micro-to-Midi total RNA purification system, DNase-treated, reverse transcribed using random hexamers (all reagents from Life Technologies), and processed for quantitative PCR. Sample processing, primer sequences/design, and description of data analysis can be found in the", "and", "." ], "cross-ref": [ { "@refid": "sec4.9", "#text": "Extended Experimental Procedures" }, { "@refid": "mmc1", "#text": "Table S1" } ] } }, { "@id": "sec4.8", "section-title": "Microscopy", "para": [ { "@id": "p0190", "#text": [ "For IF, BMMs on coverslips were stained with FITC-conjugated mouse anti-", "antibody (clone 1E6, Santa Cruz Biotechnology) and Cy3-conjugated wheat germ agglutinin (Life Technologies) at the indicated time points. Cells were imaged on a Nikon E800 fluorescent microscope and bacteria per cell were counted in random, blinded, Z-stacked images." ], "italic": "Salmonella" }, { "@id": "p0195", "#text": [ "For pH determination and video microscopy, BMMs plated on 4-chamber slides (Nunc) were infected with FITC-labeled bacteria. Following infection, chambers were incubated at 37°C for 5 min, washed extensively with PBS, incubated with phenol-free DMEM containing 10 μg/ml gentamicin, then visualized on a Nikon TE2000 inverted fluorescent microscope with environmental control. Images were collected with excitation at both 440 nm and 490 nm and analyzed using Imaris Scientific 3D/4D image processing and analysis software (Bitplane) to track individual intracellular bacteria. Background-subtracted fluorescence intensity values were used to determine the 490/440 ratios for each bacterium at each time point. Absolute pH values were determined by generating a standard curve using buffered pH solutions (see", ")." ], "cross-ref": { "@refid": "sec4.9", "#text": "Extended Experimental Procedures" } }, { "@id": "p0200", "#text": [ "For EM studies, cells were infected (moi of 10) with wild-type or", "SL1344 (as described above). At each time point, cells were fixed, embedded, sectioned, and stained for EM (see", ")." ], "italic": "ssaV::Kan", "cross-ref": { "@refid": "sec4.9", "#text": "Extended Experimental Procedures" } } ] } ], "para": { "@view": "extended", "@id": "p0205", "display": { "textbox": { "@id": "dtbox1", "@role": "e-extra", "textbox-body": { "sections": { "section": { "@id": "sec4.9", "section-title": "Extended Experimental Procedures", "section": [ { "@id": "sec4.9.1", "section-title": "Cell Culture", "para": { "@id": "p0210", "#text": "HEK293 cells (ATCC) were cultured in DMEM supplemented with 10% (vol/vol) FCS (Hyclone), L-glutamine, penicillin-streptomycin, sodium pyruvate, and HEPES, pH 7.2. All supplements and base-media were purchased from Gibco/Life Technologies, unless otherwise noted. BMMs were differentiated in BMM media (RPMI-1640 supplemented with 10% (vol/vol) fetal calf serum, L-glutamine, penicillin-streptomycin, sodium pyruvate, HEPES, pH 7.2, and M-CSF containing supernatant from 3T3-CSF cells). Differentiated BMMs cells were harvested by scraping and plated in antibiotic free RPMI-1640 medium overnight prior to infection. In some cases, BMMs were frozen in 95% FCS, 5% DMSO and stored at -150°C for later use. For thawing BMM stocks, cells were plated in BMM media, expanded for 2 additional days, and then plated for experiments." } }, { "@id": "sec4.9.2", "section-title": "Bacterial Strains and Infections", "para": [ { "@id": "p0215", "#text": [ "LT2 and SL1344 were obtained from S. Falkow (Stanford University).", "-2xHA (12032) and wild-type 12032 were gifts from S. Meresse. Δ", "(LT2) and", "(LP02 Δ", ") were gifts from R. Vance (UC Berkeley).", "constitutively expressing SPI-2, Δ", "(SL1344), was constructed by recombination using the λ Red recombinase method (", ") and phage transduction.", "(10403S) was a gift from D. Portnoy (UC Berkeley)." ], "italic": [ "PipB2", "fljb/fliC", "L. pneumophila", "fla", "S. typhimurium", "hha", "L. monocytogenes" ], "cross-ref": { "@refid": "bib74", "#text": "Datsenko and Wanner, 2000" } }, { "@id": "p0220", "#text": [ "Strain SL1344 was used for all", "infections, unless otherwise noted.", "cultures were inoculated from single colonies and grown shaking at 250 rpm overnight in LB (Fisher Scientific) supplemented with 200 μg/ml streptomycin (in the case of SL1344, Life Technologies) at 37°C. The following morning, a 1:10 dilution of the quantified culture (in PBS, Gibco/Life Technologies) was shaken in 25% normal mouse serum (Jackson Immunoresearch) for 25 min, washed twice in PBS, and added to antibiotic free RPMI-1640 culture medium (above) for infection at the indicated mois. Cells were spin-infected for 5 min at 750 rpm, then incubated at 37°C for 25 min. Following incubation, cells were washed twice with PBS before the addition of 10 μg/ml gentamicin-containing media. For mois greater than 10, cells were first incubated in 100 μg/ml gentamicin-containing media for 1 hr, followed by a reduction to 10 μg/ml gentamicin for the remainder of the experiment. For intracellular CFU determination, cells were washed once with PBS at the indicated time post-infection and lysed in 1% Triton-X 100 in PBS for 5 minutes at 37°C. Lysates were diluted to 0.2% Triton-X 100 with antibiotic-free complete RPMI, and dilutions were plated on LB agar plates containing 200μg/ml streptomycin for colony enumeration. For", "infections, intracellular growth was monitored by gentamicin intracellular replication assay, as described (", "). For", "infections, BMMs were infected at an moi of 0.05 with a", "strain expressing the", "operon. Growth was monitored by increase in luminescence over time, as described (", ")." ], "italic": [ "S. typhimurium", "S. typhimurium", "L. monocytogenes", "L. pneumophila", "L. pneumophila", "lux" ], "cross-ref": [ { "@refid": "bib75", "#text": "Leber et al., 2008" }, { "@refid": "bib76", "#text": "Lightfield et al., 2008" } ] } ] }, { "@id": "sec4.9.3", "section-title": "Measurement of BMM Activation", "para": { "@id": "p0225", "#text": "For intracellular cytokine staining experiments, BMMs were treated with brefeldinA 30 min post-infection and harvested for staining 7.5 hr later. For bafilomycinA1 pretreatment, BMMs were pretreated with 100 μM bafilomycinA1 (EMD/Calbiochem) for 2 hr prior to infection/stimulation and harvested for staining 6 hr post-infection." } }, { "@id": "sec4.9.4", "section-title": "TLR Signaling in HEK293 Cells", "para": { "@id": "p0230", "#text": [ "All HEK293 cell lines (with the exception of TLR4/MD2/CD14) expressing TLRs were made by stably transfecting each TLR into a HEK293 cell line containing an ELAM NFκB-luciferase reporter. For the TLR4/MD2/CD14 HEK293 line, HEK293 cells stably expressing TLR4/MD2 were transiently transfected with plasmids encoding CD14 and the ELAM NFκB-luciferase reporter using Lipofectamine LTX (Life Technologies) the night before the assay. Heat-killed", "(60°C for 30 min) was used to stimulate each cell line at the indicated relative moi (assuming one doubling overnight). For measurement of TLR9 responses, HEK293 cells stably expressing a TLR9/TLR4 chimeric protein were stimulated with", "genomic DNA purified by phenol:chloroform extraction. This chimeric receptor is surface localized, bypassing the need for ligand internalization (", "). For all assays, cells were stimulated for 8 hr, lysed in passive lysis buffer (Promega), and luciferase activity was quantified using an LMaxII-384 luminometer (Molecular Devices). Data are shown as fold luminescence over unstimulated cells. The following TLR ligands were purchased from Invivogen: BLP (Pam", "SK", "), R848, Flagellin, and LPS. Phosphorothioate CpG oligonucleotides (5′-TCCATGACGTTCCTGACGTT-3′) were purchased from Integrated DNA Technologies." ], "italic": [ "S. typhimurium", "S. typhimurium" ], "cross-ref": { "@refid": "bib73", "#text": "Barton et al., 2006" }, "inf": [ "3", "4" ] } }, { "@id": "sec4.9.5", "section-title": "Sample Processing for Electron Microscopy", "para": { "@id": "p0235", "#text": [ "Following infection, cells plated on poly-D lysine-coated aclar slips were fixed with 2% gluteraldehyde (EMS) in 0.1M phosphate buffer, pH 7.2 for 2 hr. Slips were washed 3 times with phosphate buffer followed by incubation for 30 min in 1% OsO", "at room temperature in the dark. Following OsO", "treatment, slips were washed 3 times with water then stained overnight with 0.5% sterile-filtered aqueous uranyl acetate at 4°C in the dark. The following day, washed slips were dehydrated using a progressive lowering of temperature ethanol dehydration, with final incubation in acetone. Cells were infiltrated and embedded in a graded series of epon-araldite resin, and sectioned using a Reichert Ultracut E microtome. Stained sections were imaged using an FEI Tecnai 12 transmission electron microscope with an accelerating voltage of 120 kV. All EM was performed at the Robert D. Ogg EM Lab at UC Berkeley." ], "inf": [ "4", "4" ] } }, { "@id": "sec4.9.6", "section-title": "Mouse Strains", "para": { "@id": "p0240", "#text": [ "TLR2-, TLR4-, TLR9-, MyD88-, and TRIF-deficient mice were generated and provided by S. Akira (Osaka University). Mice were intercrossed to generate strains lacking multiple genes. All strains were backcrossed onto the C57Bl/6 background while maintaining the functional", "allele (G", ") from the 129S1 background (see below). TLR2, TLR4, TLR9, MyD88, and TRIF deficiency were verified by PCR. Presence of a functional", "allele was screened by sequencing for the point mutation that results in a glycine to aspartate coding change at position 169 within Nramp1. The degree of backcrossing was verified by SNP analysis comparing 129S1 to C57Bl/6. Briefly, genomic DNA isolated from tails of backcrossed mice was purified for Illumina-based 129S1 vs. C57Bl/6 SNP detection performed by the Harvard-Partners Center for Genetics and Genomics (HPCGG). A total of 510 SNPs across the genome showed greater than 90% C57Bl/6 character as indicated by at least one allele of the C57Bl/6 SNP at each locus tested. The remaining 129S1 SNPs were within regions adjacent to loci of targeted genes (and therefore unlikely to be lost through backcrossing without a rare crossover event)." ], "italic": [ "nramp1", "nramp1" ], "sup": "169" } }, { "@id": "sec4.9.7", "section-title": { "italic": "S. typhimurium", "#text": "Effector Secretion" }, "para": { "@id": "p0245", "#text": [ "BMMs were seeded in 6-well plates at 2 × 10", "cells per well and infected at an moi of 25 with", "-2xHA (12032). At the indicated timepoints post-infection, cells were washed twice with ice-cold PBS and lysed in 1% NP-40 in PBS in the presence of protease-inhibtor cocktail (Roche) and EDTA (Fisher) for 30 min on ice. Lysates were cleared by centrifugation for 20 min at 13,200 × g, and 15% of the lysate was used for detection of host tubulin while the remaining pellets were resuspended in 1X SDS buffer to monitor bacterial DnaK levels. The remaining supernatants were immunoprecipitated overnight with rat anti-HA agarose beads (blocked in 1% BSA PBS, Roche), washed four times the following morning with 1% NP-40 PBS, and run on a discontinuous 10% SDS PAGE gel. Gels were transferred onto PVDF membrane (Millipore), blocked in 5% milk, and probed with rat anti-HA (Roche) and goat anti-rat HRP (GE Healthcare). Whole-cell lysates and centrifugation pellets were similarly separated, transferred, and probed using mouse anti-tubulin (EMD-Calbiochem) and mouse anti-DnaK (Stressgen/Assay Designs), respectively. For experiments where inhibitors were used, cells were pretreated for 1 hr with either bafilomycinA1 (50 nM, EMD-Calbiochem), APDC (100 μM, Sigma Aldrich), apocynin (1 mM, EMD-Calbiochem), L-NIL (1 mM, Sigma Aldrich), cyclohexamide (10 μg/ml, EMD-Calbiochem), or vehicle control, then infected in the presence of inhibitor, and incubated with inhibitor and gentamicin for the remainder of the experiment." ], "sup": "6", "italic": "S. typhimurium pipB2" } }, { "@id": "sec4.9.8", "section-title": "Gene Expression Analyses", "para": [ { "@id": "p0250", "#text": [ "BMMs were seeded at 1.5 × 10", "cells per 75 cm", "flask and infected at an moi of 5 in 7 ml nonantibiotic complete RPMI (with gentamicin added after infection). At 2 hr and 4 hr post-infection, media was removed and cells were lysed in Trizol RNA reagent (Life Technologies). RNA was purified using PureLink Micro-to-Midi total RNA purification system (Life Technologies) according to manufacturer's instructions." ], "sup": [ "7", "2" ] }, { "@id": "p0255", "#text": [ "RNA samples were treated for residual DNA contamination using Ambion Turbo DNA-free DNase (Life Technologies) according to manufacturer's instructions. Purified RNA was quantified on a Nanodrop 1000 (Thermo Scientific) and visually inspected on a 1% agarose gel. RNA was reverse transcribe to cDNA for quantitative RT-PCR (qRT-PCR) experiments by adding 10 μg of total RNA in a mixture containing random hexamers (Life Technologies), 0.01M dithiothreitol, 25 mM dNTP mixture (Sigma), reaction buffer and 200 units of SuperScript III reverse transcriptase (Life Technologies) at 42°C for 2 hr. The RNA template then was hydrolyzed by adding NaOH and EDTA to a final concentration of 0.2 and 0.1M, respectively, and incubating at 70°C for 15 min. cDNA was purified with a Minelute column (Qiagen) as per manufacturer's protocol. cDNA was eluted into 100 μl of dH", "O, diluted 1:50 in dH", "O and mixed with an equal volume of Roche 2x SYBR master mix (Roche). cDNA/mastermix samples were aliquoted into Roche 384-well plates containing lyophilized primer pairs using a Biomek FX", "Laboratory Automation Workstation (Biomek). Plates were subjected to centrifugation at 1000 rpm for 1 min and stored at 4°C in the dark until ready for use." ], "inf": [ "2", "2" ], "sup": "P" }, { "@id": "p0260", "#text": [ "Primer pairs were designed to ensure no secondary structures, a length of roughly 20-nucelotides and a melting temperature of 60°C using the primer design software Primer 3. Primer sequences are listed in", ". Forward and reverse primers were diluted to a working concentration of 0.125 μM. Primer pairs were dispensed into Roche 384-well PCR plates using a Biomek FX", "Laboratory Automation Workstation (Biomek) in duplicates or quadruplicates. Primer pairs were then lyophilized in Roche 384-well PCR plates for downstream use. A total of 4800 primer pairs designed to 4825 ORFs were tested for the initial analyses. A subset of genes showing significant trends of differential expression between genotypes were validated by independent qRT-PCR reactions performed on biological replicates. For example, based on the initial whole-genome analysis, we reanalyzed expression of all genes within the SPI-2 pathogenicity island as well as genes flanking the locus. 288 primers pairs designed to 288 ORFs (42 of which belong to SPI-2) were arrayed in quadruplicate wells for each sample." ], "cross-ref": { "@refid": "mmc1", "#text": "Table S1" }, "sup": "P" }, { "@id": "p0265", "#text": "Plates were assayed in LightCycler 480 Real-Time PCR System using the 384-well format. Reaction conditions were as follow: 1 cycle at 95°C for 5 min, 55 cycles of 95°C for 10 s, 60°C for 15 s and 72°C for 10 s. Finally, analysis was followed by a PCR melt curve analysis." }, { "@id": "p0270", "#text": [ "For data normalization, quadruplicate repeat Ct values for each sample were averaged and normalized to Ct values of control genes that were present in all samples (see", "). For each genotype, the 2 hr sample served as a baseline, allowing for normalization of the 4 hr Ct values for each corresponding genotype. The final values were multiplied by a factor of -1 such that higher expression correlated with a positive value. Sample expression data were analyzed on MeV software (TIGR). K-means clustering was used on the initial whole-genome screen to discover trends that were later validated by subset analysis." ], "cross-ref": { "@refid": "mmc1", "#text": "Table S1" } } ] }, { "@id": "sec4.9.9", "section-title": "Immunofluorescence and Fluorescent Video Microscopy", "para": [ { "@id": "p0275", "#text": [ "Coverslips were coated with poly-d-lysine (Sigma), washed with water and allowed to dry for 1 hr. BMMs were plated onto coated slips and allowed to settle overnight in non-antibiotic media and infected the following day (as above) with", "(SL1344) at an moi of 5, with addition of gentamicin. At the indicated timepoints post-infection, coverslips were washed with PBS, fixed with 4% PFA in PBS and permeabilized with 0.5% Trition-X 100 in PBS. Fixed coverslips were then washed with 0.1% Triton-X 100 in PBS, and blocked in IF blocking solution (5% goat serum, 2% BSA, 0.1% sodium azide and 0.1% Triton-X 100 in PBS). Slides were stained in IF blocking solution with FITC-conjugated mouse anti-", "antibody (clone 1E6, Santa Cruz Biotechnology) and Cy3-conjugated wheat germ agglutinin (Life Technologies). Stained cells were imaged on a Nikon E800 fluorescent microscope. For bacteria per cell enumeration, bacteria were counted in random Z-stacked images at the indicated timepoints." ], "italic": [ "S. typhimurium", "Salmonella" ] }, { "@id": "p0280", "#text": [ "For pH determination and video microscopy, BMMs were plate on poly-d-lysine coated Lab-Tek II #1.5 coverglass 4-chamber slides (Nunc) overnight followed by infection with fluorescently-labeled", "(SL1344). Bacteria were labeled by incubation with 1.5 mg/ml FITC (Sigma Aldrich) in 100 mM NaHCO", ", pH 8, rotating for 20 min in the dark at room temperature. Bacteria were then washed twice with 100 mM NaHCO", ", and added at the appropriate concentration to phenol-free DMEM (supplemented as described above but with GlutaMAX instead of L-Glutamine) for spin-infection. Following infection, chambers were incubated at 37°C for an additional 5 min, washed extensively with PBS then incubated with phenol-free DMEM containing 10 μg/ml gentamicin for the remainder of the experiment. Chambers were kept on ice prior to mounting on a Nikon TE2000 inverted fluorescent microscope with environmental control kept at 37°C and 5% CO", "." ], "italic": "Salmonella", "inf": [ "3", "3", "2" ] }, { "@id": "p0285", "#text": "Three fields for each BMM genotype containing greater than 20 intracellular bacteria were imaged for 70 min. Images were acquired at 2 min intervals with excitation at both 440 nm and 490 nm. Images were processed and individual intracellular bacteria were tracked over time using Imaris Scientific 3D/4D image processing and analysis software (Bitplane) with background subtraction. Fluorescence intensity values following excitation at 440 nm and 490nm were used to determine the 490/440 ratios for each bacterium at each timepoint." }, { "@id": "p0290", "#text": [ "To determine absolute pH values, standard curves were generated for each field and for each genotype assayed during the experiment. Briefly, pH-buffered solutions containing 145 mM KCl, 10 mM Glucose, 1 mM MgCl", ", 10 μM nigericin (added fresh), and 20 mM of either sodium acetate (pH 4.0–5.0), MES (pH 5.5–6.5), or HEPES (pH 7.0–7.4) were added to each well and images were taken of each field, as above. To account for field-specific background a standard curve was generated for each field and genotype, and the data were fit to a fourth-order polynomial equation (Microsoft Excel). Using these standard curves, ratios determined during the experiment (normalized to a ratio corresponding to pH 7 for the first timepoint) were plotted against the polynomial function to determine absolute pH using MATLAB software (MathWorks). To generate the curves shown in", "B, we used a bootstrap computation that repeatedly and randomly samples a large data set and calculates the average ratio at each time point and measured deviations for each of these values (MATLAB, MathWorks)." ], "inf": "2", "cross-ref": { "@refid": "fig7", "#text": "Figure 7" } } ] } ] } } } } } } } ] }, "acknowledgment": { "section-title": "Acknowledgments", "para": { "@id": "p0295", "#text": [ "We thank T. Machen, P. Herzmark, J. Ross, and E. Peled for assistance with ratiometric imaging; members of the Barton lab, J. Ayres, and R. Vance for critical reading of this manuscript; C. Rae and N. Meyer-Morse for assistance with", "; M. Fontana and K. Monroe for assistance with", "; R. Zalpuri and K. McDonald for assistance with EM; and H. Nolla for assistance with flow cytometry. This work was supported in part by grants from the NIH (P01-AI063302 to D.M.M. and G.M.B.; Y1-AI-8401 to S.N.P.), the Burroughs Wellcome Fund (D.M.M.), and the University of California Cancer Research Coordinating Committee (G.M.B.) and an NIH NRSA Trainee appointment on grant T32-GM007232 (N.A.)." ], "italic": [ "L. monocytogenes", "L. pneumophila" ] } }, "appendices": { "section": [ { "@view": "compact-standard", "@id": "app1", "section-title": "Supplemental Information", "para": { "@id": "p0300", "#text": [ "Supplemental Information includes Extended Experimental Procedures, four figures, and one table and can be found with this article online at", "." ], "inter-ref": { "@href": "doi:10.1016/j.cell.2011.01.031", "#text": "doi:10.1016/j.cell.2011.01.031" } } }, { "@view": "extended", "@id": "app2", "section-title": "Supplemental Information", "para": { "@id": "p0305", "display": [ { "e-component": { "@id": "mmc1", "label": "Table S1. Primer Sequences and Locus Designations for SPI-2 and Control Genes, Related to Figure 5", "link": { "@locator": "mmc1" } } }, { "e-component": { "@role": "article-plus", "@id": "mmc3", "label": "Document S1. Article Plus Supplemental Information", "link": { "@locator": "mmc3" } } } ] } } ] } } }

elastic / elasticsearch-js

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