You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Introduction {#s1}
============

*Salmonella enterica* serovar Typhimurium (*S*. Typhimurium) is a Gram-negative enterobacterium capable of colonizing and causing infections in a wide variety of organisms ranging from Man and mice to nematodes, amoebae and plants [@pone.0045417-Schikora1]--[@pone.0045417-TezcanMerdol1]. It thereby follows that salmonellae must be equipped with virulence strategies, which allow infection of such a broad evolutionary range of hosts [@pone.0045417-Kingsley1]. Still, most of the details that govern *Salmonella* infections have been obtained from murine infection models.

Upon oral infection of mice, *S*. Typhimurium invades the small intestine [@pone.0045417-Hohmann1], [@pone.0045417-Jones1], followed by dissemination to the mesenteric lymph nodes, and eventually to the liver and spleen [@pone.0045417-VazquezTorres1]. Subsequently, the bacterium acts as a facultative intracellular pathogen and replicates in macrophages [@pone.0045417-Fields1]--[@pone.0045417-RichterDahlfors1]. This complex infection cycle relies on several sets of virulence genes, many of which are contained on horizontally acquired genetic inserts termed *Salmonella* pathogenicity islands, or SPIs [@pone.0045417-HansenWester1]. SPI1 and SPI2 code for two separate type III protein secretion systems (T3SS). With the aid of the T3SS, *S*. Typhimurium injects effector proteins into host cells; SPI1 effector proteins orchestrate actin reorganization in epithelial cells, leading to internalization and translocation of *S*. Typhimurium at the intestinal lining [@pone.0045417-Clark1], while SPI2 effector proteins manipulate vesicular trafficking in infected cells, allowing the bacteria to replicate intracellularly [@pone.0045417-Ibarra1].

SPI1 and SPI2 do not act independently from the rest of the chromosome. The highly regulated expression of the SPIs, for example, is intimately linked to many evolutionarily conserved gene regulatory systems, such as the sensor regulator systems EnvZ-OmpR and PhoP-PhoQ [@pone.0045417-Aguirre1], [@pone.0045417-Bijlsma1]. In addition, the intracellular induction of SPI2 strongly relies on the highly conserved bacterial cytoplasmic reductase thioredoxin 1 (TrxA, *trxA*) [@pone.0045417-Negrea1]. Thus mutations in selected "house-keeping" genes, such as *trxA*, result in attenuations that equal SPI2 mutants in the murine infection model [@pone.0045417-Negrea1], [@pone.0045417-Bjur1].

The soil nematode *Caenorhabditis elegans* has been used as a biological model to study several aspects of vertebrate biology, including signal transduction, neuronal development, behavioral responses, senescence and innate immunity [@pone.0045417-Brenner1]--[@pone.0045417-Kenyon1]. The high genetic tractability and large number of available tools have made *C. elegans* an emerging model to dissect the molecular basis of many mammalian diseases [@pone.0045417-Xu1]. Furthermore, a large number of mammalian bacterial species [@pone.0045417-Alegado1], including *S*. Typhimurium , is pathogenic to the nematode. Interestingly, the EnvZ-OmpR and PhoP-PhoQ regulatory systems, as well as the SPI1 T3SS, contribute to the virulence of *S*. Typhimurium in *C. elegans* [@pone.0045417-Aballay1], [@pone.0045417-Labrousse1], [@pone.0045417-Tenor1]. The SPI1 T3SS appears to generate pro-apoptotic signals in germline cells outside the intestine [@pone.0045417-Tenor1]. However, the mechanisms by which *S*. Typhimurium elicits death in *C. elegans* have not been fully clarified.

Production of reactive oxygen species (ROS) represents one of the most primordial innate defense mechanisms against many invading microbes [@pone.0045417-Torres1]--[@pone.0045417-Apel1]. Furthermore, lack of an efficient *phox*-mediated oxidative burst response sensitizes both Man and mice to infections with *Salmonella* spp. [@pone.0045417-DeGroote1]--[@pone.0045417-Shiloh1]. On the other hand, overwhelming ROS production has been implicated as a crucial pathological effector during septic shock in Man and other animals [@pone.0045417-Andrades1]--[@pone.0045417-Cadenas1]. *C*. *elegans* also has the ability to mount a protective oxidative response upon infection with pathogens such as *Enterococcus faecalis* [@pone.0045417-Chvez1], [@pone.0045417-Chvez2]. In this study, we sought to elucidate the pathogenesis of *S*. Typhimurium infection in *C. elegans*. We showed that *S*. Typhimurium kills the nematode by inducing a disseminated and overwhelming host oxidative response that depends on active bacterial protein synthesis and the bacterial TrxA. Importantly, our data thereby also suggest a novel and unique aspect of TrxA\'s role in *S*. Typhimurium.

Results {#s2}
=======

*S*. Typhimurium perturbs intestinal morphology but does not disseminate beyond the intestinal border {#s2a}
-----------------------------------------------------------------------------------------------------

When we compared the lifespans of wild-type N2 *C. elegans* propagated either on *Escherichia coli* strain OP50 or on the virulent *S*. Typhimurium strain 14028, we noted a pathogenicity of *S*. Typhimurium as evidenced by a significantly shortened lifespan (*p*\<0.0001, [Figure 1A](#pone-0045417-g001){ref-type="fig"}), consistent with previous reports.

![*S*. Typhimurium strain 14028 is pathogenic to *C. elegans*.\
(A) Survival of wild-type N2 nematodes was compared when fed *E. coli* OP50 or *S*. Typhimurium 14028 (*p*\<0.0001). (B) Survival of nematodes was compared when infected with non-fluorescent *S*. Typhimurium 14028, 14028::GFP (*p* = 0.2251) or 14028::RFP (*p* = 0.9384; all *p* values as compared to the non-fluorescent strain). Each survival curve is representative of 3 independent assays, each with 3 plates per strain and 20 nematodes per plate.](pone.0045417.g001){#pone-0045417-g001}

In murine models for typhoid fever, virulence of *S.* Typhimurium depends on its ability to invade the intestinal epithelium and subsequent dissemination to the liver and spleen. Expectedly, non-invasive mutants of *S*. Typhimurium are avirulent in mice challenged orally [@pone.0045417-Lostroh1]. To test if *S*. Typhimurium pathogenicity in *C. elegans* also depends on intestinal invasion, we infected wild-type *C. elegans* with a GFP-expressing strain of *S*. Typhimurium 14028, shown to have comparable virulence as the non-fluorescent strain in mice [@pone.0045417-Hautefort1] as well as in the nematode (*p* = 0.2251, [Figure 1B](#pone-0045417-g001){ref-type="fig"}).

When observed using fluorescence microscopy 24 h post infection, wild-type nematodes exposed to either GFP-expressing *S*. Typhimurium 14028 or *E. coli* OP50 showed a diffuse fluorescent pattern within the intestinal lumen ([Figures 2A and 2B](#pone-0045417-g002){ref-type="fig"}). At later time points though, the fluorescent luminal content expanded substantially in *S*. Typhimurium-infected nematodes and consisted of intact bacterial cells ([Figures 2C and 2D](#pone-0045417-g002){ref-type="fig"}). In contrast, nematodes fed on *E. coli* retained a diffuse string-like staining pattern, without significant luminal expansion ([Figures 2E and 2F](#pone-0045417-g002){ref-type="fig"}). At no time point of the infection could we detect any significant amount of *S*. Typhimurium bacteria in the surrounding tissues. Here we also provide evidence for the first time, that SU159 nematodes, expressing the GFP-tagged apical epithelial marker AJM-1, displayed a continuous and unbreached staining of the intestinal epithelium upon infection with *S*. Typhimurium ([Figures 2G and 2H](#pone-0045417-g002){ref-type="fig"}).

![*S*. Typhimurium colonizes the nematode gut and does not disseminate across the intestinal epithelium.\
(A--F) Wild-type N2 nematodes were fed *S*. Typhimurium (*S*. Tm) 14028::GFP or *E. coli* OP50::GFP. At each time point, live nematodes were harvested and mounted for microscopy in PBS with NaN~3~. In these merged images, *S*. Typhimurium 14028 or *E. coli* OP50 is shown in green and intestinal autofluorescence in blue. (G--H) SU159 nematodes expressing AJM-1::GFP were infected with *S*. Typhimurium 14028::RFP. At each time point, live nematodes were mounted for microscopy. In these merged images, *S*. Typhimurium 14028 is shown in red and both the apical epithelial marker AJM-1 and intestinal autofluorescence in green. The intestinal border is marked by white arrowheads. Images are shown at 100× magnification and the pharynx (p) is indicated. Scale bar represents 0.01 mm. Images are representative of at least 20 nematodes from 3 independent assays.](pone.0045417.g002){#pone-0045417-g002}

These observations corroborate and extend the observations from previous studies that reported lack of invasiveness of *S*. Typhimurium in *C. elegans* and that in autophagy-competent nematodes, any invading *S*. Typhimurium was observed to be effectively targeted to lysosomes for degradation and thus not significantly detected nor able to cause disruption to the intestinal epithelial cells [@pone.0045417-Jia1].

Infection with *S. enterica* evokes a pathogen-specific dissemination of oxidative stress {#s2b}
-----------------------------------------------------------------------------------------

As ROS plays a protective role during infection of *C. elegans* by *E. faecalis* [@pone.0045417-Chvez1], [@pone.0045417-Chvez2], we hypothesized that the differential virulence of *S*. Typhimurium and *E. coli* in the nematode could relate to their abilities to trigger an oxidative host defense. To test this possibility, we stained wild-type nematodes fed on either non-fluorescent *S*. Typhimurium or *E. coli* with 2′,7′-dichlorodihydrofluorescein diacetate (H~2~DCFDA), a dye that becomes cleaved intracellularly and fluoresces green upon exposure to various intracellular ROS [@pone.0045417-Oyama1], [@pone.0045417-LeBel1].

Nematodes fed on either *S.* Typhimurium or *E. coli* revealed green ROS fluorescence clearly contained within the intestinal lumen at 24 h post infection ([Figures 3A and 3B](#pone-0045417-g003){ref-type="fig"}). At later time points, *S.* Typhimurium-infected nematodes first exhibited strong green ROS signals contained within foci along the extra-intestinal tissues of the nematode, mainly the hypodermis ([Figures 3C and 3D](#pone-0045417-g003){ref-type="fig"}). Subsequently, the signals culminated together in the whole of these tissues ([Figures 3E and 3F](#pone-0045417-g003){ref-type="fig"}). In contrast, no escalation or spread of the ROS signal was observed in *E. coli*-fed nematodes at later time points ([Figures 3G and 3H](#pone-0045417-g003){ref-type="fig"}).

![*S*. Typhimurium induces an emergence of ROS from the nematode intestine to the surrounding tissues.\
(A--H) Wild-type N2 nematodes were fed non-fluorescent *S*. Typhimurium 14028 or *E. coli* OP50. At each time point, nematodes were harvested, stained with H~2~DCFDA to detect intracellular ROS and mounted for microscopy in PBS with NaN~3~. In these merged images, ROS is shown in green and intestinal autofluorescence in blue. The pharynx (p) and ROS foci (white arrowheads) are indicated. Images are shown at 40× magnification and scale bar represents 0.01 mm. Images are representative of at least 20 nematodes from 3 independent assays.](pone.0045417.g003){#pone-0045417-g003}

To assess whether the dissemination of ROS staining in *S.* Typhimurium-infected nematodes was a generic response to bacterial pathogens or unique to *S.* Typhimurium, we repeated the experiments with two other pathogens, *Burkholderia thailandensis* strain 700388 and *S. enterica* serovar Dublin (*S.* Dublin) strain STM75. In these experiments, the ROS staining pattern appeared rather similar between nematodes fed *B. thailandensis* or *E. coli* ([Figures 4A and 4B](#pone-0045417-g004){ref-type="fig"}), despite the fact that *B. thailandensis* infection substantially decreased the lifespan of *C. elegans* (*p*\<0.0001, [Figure 4E](#pone-0045417-g004){ref-type="fig"}). This was in contrast to infection with *S*. Dublin, which was also pathogenic (*p*\<0.0001, [Figure 4E](#pone-0045417-g004){ref-type="fig"}), and generated a ROS staining pattern comparable to that caused by *S*. Typhimurium ([Figures 4C and 4D](#pone-0045417-g004){ref-type="fig"}). Thus, the emergence of ROS in the extra-intestinal tissues of the nematode appears to be a consequence specific to *S. enterica*.

![Induction of ROS response is specific to *S. enterica*.\
(A--D) Wild-type nematodes were infected with non-fluorescent *B. thailandensis* 700388 or *S*. Dublin STM75. At each time point, infected nematodes were harvested, stained with H~2~DCFDA and mounted for microscopy. In these merged images, ROS is shown in green and intestinal autofluorescence in blue. Images are shown at 40× magnification. (E) Survival of wild-type nematodes was compared when fed *E. coli* OP50, *B. thailandensis* 700388 (*p*\<0.0001) or *S*. Dublin STM75 (*p*\<0.0001; all *p* values as compared to *E. coli*).](pone.0045417.g004){#pone-0045417-g004}

Ablation of ROS increases host survival upon infection with *S*. Typhimurium {#s2c}
----------------------------------------------------------------------------

The above observations indicate that either *C. elegans* relies on a ROS response to defend against selected pathogens such as *S. enterica*, or that the emergence of ROS constitutes a facet of *S. enterica* pathogenicity in the nematode. To distinguish between these possibilities we repeated the infection experiments in the presence of the reductant ascorbic acid [@pone.0045417-King1] or catalase, an enzyme that converts hydrogen peroxide (H~2~O~2~) to water and molecular oxygen [@pone.0045417-Chelikani1].

At a 50 mM concentration of ascorbic acid, nematodes infected with *S.* Typhimurium showed a drastic reduction in the pathogen-induced ROS signal ([Figures 5A to 5D](#pone-0045417-g005){ref-type="fig"}) and a significant increase in the lifespan (*p* = 0.0006, [Figure 5E](#pone-0045417-g005){ref-type="fig"}). The lifespan of infected nematodes was also enhanced when ascorbic acid was replaced by catalase (*p* = 0.03, [Figure 5F](#pone-0045417-g005){ref-type="fig"}). In comparison, neither ascorbic acid nor catalase increased the lifespan of nematodes fed on *E. coli* (*p* = 0.5406, [Figure 5E](#pone-0045417-g005){ref-type="fig"} and data not shown).

![Virulence of *S*. Typhimurium correlates with ROS dissemination in the infected nematode.\
(A--D) Wild-type nematodes were infected with non-fluorescent *S*. Typhimurium 14028 on NGM agar with or without 50 mM ascorbic acid. At each time point, infected nematodes were harvested, stained with H~2~DCFDA and mounted for microscopy. In these merged images, ROS is shown in green and intestinal autofluorescence in blue. Images are shown at 40× magnification. (E) Survival of wild-type nematodes was compared when fed *E. coli* OP50 (*p* = 0.5406) or *S*. Typhimurium 14028 (WT) (*p* = 0.0006; all *p* values as compared to its respective untreated control) on NGM agar with or without 50 mM ascorbic acid. (F) Survival of wild-type nematodes was compared when infected with *S*. Typhimurium 14028 (WT) in a liquid assay, with or without catalase added (*p* = 0.03). (G--H) Wild-type nematodes were infected with non-fluorescent *S*. Typhimurium 14028 on NGM agar with 10 μg/ml chloramphenicol. At each time point, infected nematodes were harvested, stained with H~2~DCFDA and mounted for microscopy. (I) Survival of wild-type nematodes was compared when infected with *S*. Typhimurium 14028 (WT) on NGM agar with or without 10 μg/ml chloramphenicol (*p*\<0.0001).](pone.0045417.g005){#pone-0045417-g005}

To investigate if active bacterial protein synthesis was required to trigger the oxidative response, we performed the infection experiments in the presence of the bacterial protein synthesis inhibitor chloramphenicol. Nematodes infected with *S.* Typhimurium in the presence of chloramphenicol also did not reveal any extra-intestinal staining with H~2~DCFDA ([Figures 5G and 5H](#pone-0045417-g005){ref-type="fig"}) and consequently, exhibited significantly increased lifespan (*p*\<0.0001, [Figure 5I](#pone-0045417-g005){ref-type="fig"}).

Combined, these data suggest that the ROS response in *S.* Typhimurium-infected nematodes is detrimental rather than protective against the pathogen.

Infection-associated ROS dissemination relies on the bacterial thioredoxin 1 {#s2d}
----------------------------------------------------------------------------

*S. enterica* notably differs from non-pathogenic *E. coli* through its possession of pathogenicity islands such as SPI1 and SPI2. Furthermore, virulence in the murine infection model strongly relies on the expression of many evolutionarily conserved "house-keeping" genes, including those coding for the lipopolysaccharide (LPS) O-antigen and thioredoxin 1 (TrxA) [@pone.0045417-Bjur1], [@pone.0045417-Nevola1]. We thus tested *S*. Typhimurium mutants unable to express SPI1 (*hilA*), SPI2 (*ssaV*), LPS O-antigen (*rfaL*) or thioredoxin 1 (*trxA*) for their ability to induce ROS dissemination in the infected nematode. While the *hilA*, *ssaV* and *rfaL* mutants induced ROS signals comparable to that of the wild-type strain ([Figures 6A to 6H](#pone-0045417-g006){ref-type="fig"}), induction of ROS was substantially abrogated in nematodes infected with the *trxA* mutant ([Figures 6I to 6J](#pone-0045417-g006){ref-type="fig"}).

![ROS induction in the infected nematode is dependent on the bacterial TrxA.\
(A--J) Wild-type nematodes were infected with non-fluorescent *S*. Typhimurium 14028 wild-type, *hilA, ssaV, rfaL* or *trxA*. At each time point, infected nematodes were harvested, stained with H~2~DCFDA and mounted for microscopy. In these merged images, ROS is shown in green and intestinal autofluorescence in blue. Images are shown at 40× magnification.](pone.0045417.g006){#pone-0045417-g006}

Oxidative stress induction and pathogenesis require the catalytic activity of TrxA {#s2e}
----------------------------------------------------------------------------------

As the *trxA* mutant did not induce the ROS response in the infected nematode, we compared the lifespans of nematodes infected with wild-type *S*. Typhimurium and the *trxA* mutant. In this experiment, the lifespan of nematodes infected with the *trxA* mutant was significantly prolonged (*p*\<0.0001, [Figure 7A](#pone-0045417-g007){ref-type="fig"}), further supporting the view that the emergence of ROS in nematodes confers sensitivity to *S*. Typhimurium.

![Virulence in the infected nematode relies on the catalytic activity of TrxA.\
(A) Survival of wild-type nematodes was compared when fed *E. coli* OP50, *S*. Typhimurium 14028 wild-type (WT) or *trxA* (*p*\<0.0001 as compared to wild-type). (B) Survival of nematodes was compared when infected with *S*. Typhimurium 14028 wild-type, *trxA* or *trxA trans*-complemented with wild-type TrxA (pFA3), variants of TrxA with decreasing reducing potentials (pFA5-pFA8) or vector (pBAD33) (all *p* values as compared to *S*. Typhimurium 14028 wild-type). (C) Survival of nematodes was compared when infected with *S*. Typhimurium 14028 wild-type, *trxA* (*p*\<0.0001), *trxB* (*p*\<0.0001), *trxC* (*p* = 0.8743) or *dsbC* (*p* = 0.8875; all *p* values as compared to wild-type). (D) Survival of nematodes was compared when infected with *S*. Typhimurium 14028 *trxA* on NGM agar with or without 50 mM ascorbic acid (*p* = 0.8113).](pone.0045417.g007){#pone-0045417-g007}

Since the primary structure of TrxA is identical in both *E. coli* and *S.* Typhimurium, observations regarding TrxA in *E. coli* may also be applicable to TrxA in *S.* Typhimurium [@pone.0045417-Bjur1], [@pone.0045417-McClelland1], [@pone.0045417-slund1]. In *E. coli* TrxA acts as a cytoplasmic reductant and also indirectly contributes to proper cross-linking of disulphide bonds in the bacterial periplasmic space by providing electrons to disulphide isomerases [@pone.0045417-slund1]. In addition, TrxA functions as a protein chaperone independent of its redox activity [@pone.0045417-Kern1].

To test whether the induction of ROS in *S.* Typhimurium-infected nematodes relied on the redox activity of TrxA, we *trans-*complemented the *trxA* mutant with plasmids coding for wild-type *E. coli* TrxA having a reducing potential (E~o~ ^r^) of −270 mV (pFA3), or with a series of engineered *trxA* alleles coding for TrxA variants with decreasing reducing potentials (E~o~ ^r^ with smaller numericals) (pFA5-pFA7) [@pone.0045417-Bjur1], [@pone.0045417-Mssner1]. As a control we used plasmids coding for a catalytically inactive TrxA (pFA8) or the empty cloning vector (pBAD33) ([Figure 7B](#pone-0045417-g007){ref-type="fig"}).

We could fully complement virulence by expressing the wild-type TrxA in the *trxA* mutant (*p* = 0.6684). However, full complementation failed when the E~o~ ^r^ of engineered TrxA variants decreased to −195 mV (pFA5). Accordingly, we did not note any complementation with the catalytically inactive TrxA. Thus, *S*. Typhimurium requires a redox-active TrxA to elicit virulence in *C. elegans*, as reflected by the correlation between the degree of virulence complementation and E~o~ ^r^.

We also probed the potential contribution of other members of the thioredoxin system, which are homologous to that in *E. coli* [@pone.0045417-McClelland1], [@pone.0045417-slund1], by infecting nematodes with *S*. Typhimurium mutants lacking the cytoplasmic thioredoxin reductase TrxB (*trxB*), the cytoplasmic thioredoxin 2 TrxC (*trxC*) or the periplasmic oxidoreductase DsbC (*dsbC*). DsbC reduces and re-oxidizes improper disulphide bonds by receiving electrons from the cytoplasm with the aid of TrxA, TrxB and TrxC [@pone.0045417-slund1], [@pone.0045417-Hiniker1]. We observed that the *trxB* mutant exhibited attenuated virulence (*p*\<0.0001, [Figure 7C](#pone-0045417-g007){ref-type="fig"}), similar to the *trxA* mutant. When nematodes were infected with the *trxC* or *dsbC* mutant, we did not see any significant effect on virulence (*p* = 0.8743 and 0.8875 respectively, [Figure 7C](#pone-0045417-g007){ref-type="fig"}).

As the *trxA* mutant still appeared more virulent than *E. coli*, yet did not induce the ROS response in the infected host, we tested whether adding ascorbic acid had any effect on the lifespan of nematodes infected with the *trxA* mutant. This regimen, however, did not prolong the lifespan of these nematodes (*p* = 0.8113, [Figure 7D](#pone-0045417-g007){ref-type="fig"}).

Copper chloride restores ROS production and virulence upon infection with the *trxA* mutant {#s2f}
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Copper chloride (CuCl~2~) catalyzes disulphide bond formation for periplasmic *E. coli* proteins *in vivo* [@pone.0045417-Hiniker1]. To perturb periplasmic disulphide bond formation, we fed *C. elegans* with *E. coli,* wild-type *S.* Typhimurium or the *trxA* mutant that had been pre-exposed to a sublethal 2 mM concentration of CuCl~2~. Feeding *C. elegans* with *E. coli* (*p* = 0.5298) or wild-type *S*. Typhimurium (*p* = 0.7227) pre-exposed to CuCl~2~ did not affect the lifespan of the nematodes, as compared to unexposed bacteria ([Figure 8A](#pone-0045417-g008){ref-type="fig"}). Surprisingly, infection with the *trxA* mutant pre-treated with CuCl~2~ caused a significant shortening of the lifespan of the nematodes (*p*\<0.0001, [Figure 8A](#pone-0045417-g008){ref-type="fig"}), comparable to that of nematodes infected with wild-type *S.* Typhimurium. In parallel we also noted restoration of the ROS staining in *trxA*-infected nematodes upon CuCl~2~ treatment ([Figures 8B to 8E](#pone-0045417-g008){ref-type="fig"}).

![Copper chloride rescues attenuated virulence and lack of ROS induction in *trxA*-infected nematodes.\
(A) Survival of wild-type nematodes was compared when fed *E. coli* OP50 (*p* = 0.5298), *S*. Typhimurium 14028 wild-type (WT) (*p* = 0.7227) or *trxA* (*p*\<0.0001; all *p* values as compared to its respective untreated control), preincubated with or without 2 mM CuCl~2~. (B--E) Wild-type nematodes were infected with non-fluorescent *S*. Typhimurium 14028 *trxA*, preincubated with or without 2 mM CuCl~2~. At each time point, infected nematodes were harvested, stained with H~2~DCFDA and mounted for microscopy. In these merged images, ROS is shown in green and intestinal autofluorescence in blue. Images are shown at 40× magnification.](pone.0045417.g008){#pone-0045417-g008}

Discussion {#s3}
==========

In this study, we described the emergence of ROS as a key event in the infection pathogenesis of *S. enterica* in the soil nematode *C. elegans*. As evidenced by staining infected nematodes with the oxidative stress indicator H~2~DCFDA, we observed that ROS were initially confined to the intestinal lumen and subsequently appeared in the surrounding tissues. Still, *S.* Typhimurium remained in the intestinal lumen of the nematode throughout the infection. This phenomenon was not a general response to bacteria, as it did not occur in nematodes fed on *E. coli* or the nematocidal *B. thailandensis*.

Treatment of *S*. Typhimurium-infected nematodes with the bacterial protein synthesis inhibitor chloramphenicol not only prolonged their lifespan, but also resulted in ablation of the extra-intestinal ROS signals. This suggests that replication of *S.* Typhimurium, or a bacterial factor being actively induced during infection, triggered the ROS response. Production of ROS is considered a key facet of host antimicrobial responses [@pone.0045417-McClelland1]. However, while the use of the reductant ascorbic acid or H~2~O~2~-degrading catalase in our subsequent experiments counteracted the emergence of ROS, these regimens extended rather than shortened the lifespan of infected nematodes. This strongly implies that the ROS response did not act to protect *C. elegans* from *S. enterica* infection, unlike that observed with *E. faecalis*-infected nematodes [@pone.0045417-Mastroeni1], [@pone.0045417-Shiloh1]. Rather, the *S. enterica-*induced ROS mediated the infection pathogenesis. This is reminiscent of endotoxin-induced septic shock in rodents, where ROS have been suggested to be the mediators of the symptoms, and where the severity of disease can be eliminated by systemic application of ascorbic acid [@pone.0045417-Cadenas1], [@pone.0045417-Wilson1]. Combined, these observations implicate excessive ROS production as an effector of severe infection pathogenesis across a broad evolutionary spectrum of hosts.

When *C. elegans* were infected with *E. faecalis*, ROS were generated to protect the host and the addition of a NADPH oxidase inhibitor dampened this response [@pone.0045417-Chvez2], suggesting that members of the NOX/DUOX protein family may contribute to the phenomenon [@pone.0045417-Quinn1]. Subsequent studies revealed that the protective ROS induced during an *E. faecalis* infection was mediated by the dual oxidase Ce-DUOX/BLI-3 [@pone.0045417-Chvez1]. When we investigated the role of Ce-DUOX/BLI-3 in *S.* Typhimurium infection by reducing *bli-3* expression with RNA interference, we did not observe any significant contribution by the dual oxidase towards the induced ROS response (data not shown). Apart from the contrasting roles ROS play during the two infections, there is also the possibility that there are other NOX/DUOX protein members or even other non-related protein families responsible for generating H2DCFDA-detectable ROS in the nematode during a *S. enterica* infection.

In fact, H~2~DCFDA lacks substrate specificity and fluoresces in the presence of a wide variety of ROS including H~2~O~2~, superoxide anions, nitric oxide, peroxyl and hydroxyl radicals [@pone.0045417-Halliwell1]. H~2~DCFDA also has a spontaneous tendency to photo-oxidize if left for long periods of incubation [@pone.0045417-Halliwell1], although in our series of experiments this problem was clearly circumvented with *E. coli* and *B. thailandensis*-infected nematodes as controls. Still, that catalase prolonged nematode lifespan upon *S*. Typhimurium infection strongly implicated H~2~O~2~ as one of the mediators of *S*. Typhimurium pathogenesis in *C. elegans*. After all, H~2~O~2~ stands as a cellular endpoint metabolite derived from several reactive intermediates; free iron in the living system unavoidably also participates in redox cycling to generate ROS [@pone.0045417-LeBel1], [@pone.0045417-Halliwell1]. Thus free radical reactions resulting in significantly detectable green signals in our experiments are likely more complex than understood. Hence, the *S. enterica*-induced infection response in *C. elegans* encompasses, without doubt, the emergence of ROS which may consist of H~2~O~2~ *per se* or of reactive intermediates which have broken down into H~2~O~2~. Probes specialized for detecting individual species of ROS, such as the Griess reagent for nitrite compounds or dihydroethidum derivatives for superoxide anions, could be included in further studies to better delineate this *S. enterica*-induced phenomenon.

Clearly, while important, the ROS response did not stand as the sole mechanism mediating pathogenesis in *S. enterica-*infected nematodes. This notion emerges from our observations that *i*) a *trxA* mutant was still more virulent than non-pathogenic *E. coli*; and *ii*) a LPS O-antigen deficient *rfaL* mutant, also attenuated in virulence in the nematode (data not shown), still evoked a ROS response similar to wild-type *S.* Typhimurium. Likewise, mutational depletion of key *Salmonella* virulence factors, such as the SPI1 and SPI2 T3SS, did not affect the ROS induction. In contrast, loss of the bacterial virulence-associated "house-keeping" gene *trxA* resulted in an ablation of the ROS response, and substantially restored nematode lifespan.

TrxA is a small evolutionarily conserved reductase. It possesses a high capability of reducing cytoplasmic proteins, and via its association with periplasmic disulphide isomerases, allows for the repair of periplasmic proteins with aberrantly oxidized sulphydryl groups [@pone.0045417-Oyama1]. Interestingly, TrxA in itself also has the potential to behave as an immune signaling molecule when released from the bacterial cytoplasm [@pone.0045417-Bertini1], and hence has the possibility to directly mediate the dissemination of ROS. However, since the primary structure of TrxA is identical in both *S.* Typhimurium and *E. coli* [@pone.0045417-McClelland1], [@pone.0045417-slund1], possession of TrxA alone clearly is not sufficient to confer *E. coli* the ROS-inducing ability when infecting nematodes. In accordance, *S.* Typhimurium TrxA is co-induced with SPI2 and needed for the proper activity of SPI2 during infection of murine models [@pone.0045417-Negrea1]. However, the fact that a SPI2 mutant still evoked a ROS response in *C. elegans* suggests that the virulence input by TrxA in the nematode infection model relies uniquely on some other *Salmonella*-specific factors other than SPI2.

Two lines of data implicate redox catalysis as an important characteristic of TrxA in mediating pathogenesis in *C. elegans*. First, the *trxA* mutant was fully *trans*-complemented with wild-type TrxA and genetically engineered TrxA variants, however only with those having an E~o~ ^r^ greater than −195 mV. Second, we could substitute TrxA simply by pre-exposing the *trxA* mutant to sublethal concentrations of CuCl~2~. As CuCl~2~ is known to catalyze disulphide bond formation for periplasmic proteins in *E. coli* [@pone.0045417-Hiniker1], our observations would connect the ROS-associated pathogenesis to periplasmic disulphide bond formation. Still, we could not attenuate virulence or ROS formation by mutating TrxC or DsbC. This suggests that attenuation of the *trxA* mutant in *C. elegans* does not relate to a general defect in periplasmic disulphide bond formation.

The Gram-positive bacterium *Streptococcus pneumoniae*, a natural human pathogen, produces an exceptionally high concentration of H~2~O~2~ *in vitro* [@pone.0045417-Ginsburg1]. Furthermore, this ability has been associated with its virulence in *C. elegans* [@pone.0045417-Bolm1], [@pone.0045417-Jansen1]. Still, we failed to demonstrate significant H~2~O~2~ production by wild-type *S*. Typhimurium or the *trxA* mutant *in vitro.* In addition, we also did not observe any hypodermal ROS staining in nematodes infected with the virulent *Str. pneumoniae* serotype I strain BHN32 (data not shown), providing evidence that H~2~O~2~ produced by the pathogen is unlikely involved in this *S. enterica-*induced ROS response. This idea was also further supported by the initial appearance of ROS signals as independent foci in the hypodermis, despite the lack of any detectable *S. enterica* at these sites. Our data thus favor the hypothesis in which *C. elegans* mounts an overwhelming pathogen-specific ROS response in the hypodermis upon infection with *S. enterica,* reminiscent to infection-associated multi-organ failure or septic shock caused by innate immune responses in higher vertebrates [@pone.0045417-Andrades1]--[@pone.0045417-Cadenas1].

Materials and Methods {#s4}
=====================

Nematode and Bacteria Strains {#s4a}
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Nematode and bacteria strains used in this study are listed in [Table S1](#pone.0045417.s001){ref-type="supplementary-material"}. Nematode strains were cultured and maintained at 20°C on modified nematode growth media (NGM, 0.35% peptone) agar and fed with *E. coli* strain OP50, as described [@pone.0045417-Brenner1]. Bacteria strains were grown in Luria-Bertani (LB) broth at 37°C. When necessary, LB broth is supplemented with ampicillin 100 μg/ml; chloramphenicol 10 μg/ml; kanamycin 50 μg/ml and/or L-arabinose 5% (w/v) (Sigma-Aldrich, St. Louis, MO).

Survival Assays {#s4b}
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Bacterial strains were grown overnight in LB broth at 37°C and lawns were prepared by spreading 200 μl of overnight culture on modified NGM agar. 20 L4-staged wild-type N2 nematodes were subsequently infected as described [@pone.0045417-Powell1]. Briefly, nematodes were set down on unseeded agar before transferring to bacterial lawns to reduce, as much as possible, the transfer of *E. coli.* No visible *E. coli* growth on pathogenic lawns was observed nor was there any crowding of nematodes. Nematode survival was scored at 24°C and nematodes were considered dead upon failure to respond to gentle touch by a platinum wire. Nematodes were also transferred to fresh bacterial lawns every day. Results are representative of 3 independent assays, each with triplicates.

For experiments involving L-arabinose, ascorbic acid and chloramphenicol, modified NGM agar was impregnated with each of these compounds at the following concentration to ensure maximum exposure: L-arabinose 5% (w/v); ascorbic acid 50 mM (Sigma-Aldrich); chloramphenicol 10 μg/ml.

For experiments involving CuCl~2~, *E. coli* and S. Typhimurium strains were grown overnight in LB broth supplemented with or without 2 mM CuCl~2~ before seeding on modified NGM agar.

Immunofluorescence Assays {#s4c}
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Fluorescent *E. coli* and *S.* Typhimurium lawns were prepared by spreading 200 μl of overnight LB culture on modified NGM agar. L4-staged N2 or SU159 nematodes were added to these lawns and infected as described above. At each time point, live nematodes were harvested and mounted for microscopy in phosphate-buffered saline (PBS) with 25 mM sodium azide (NaN~3~; Sigma-Aldrich). Slides were visualized on a LEICA DMRE microscope and images were analyzed by GNU Image Manipulation Program (version 2.6.3). Images are representative of at least 20 nematodes from 3 independent assays.

Reactive Oxygen Species Detection {#s4d}
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2′,7′-dichlorodihydrofluorescein diacetate (H~2~DCFDA; Sigma-Aldrich) is used to visualize intracellular ROS in nematodes. 2 mM stock aliquots of H~2~DCFDA were prepared in dimethyl sulfoxide and stored in the dark at −80°C. L4-staged N2 nematodes were infected as described above. At each time point, infected nematodes were harvested into tubes and washed twice with M9 buffer. Nematodes were subsequently incubated with 25 μM H~2~DCFDA in 250 μl M9 buffer, in the dark for 30 min in a 20°C water bath. After staining, nematodes were washed thrice with M9 buffer and mounted for microscopy in PBS with 25 mM NaN~3~. Slides were visualized on a LEICA DMRE microscope and images were analyzed by GNU Image Manipulation Program. Images are representative of at least 20 nematodes from 3 independent assays.

Liquid Infection Assays with Catalase {#s4e}
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*E. coli* and *S.* Typhimurium strains were grown overnight in LB broth at 37°C. The overnight culture was resuspended in S Basal liquid media and 800 μl of the suspension was aliquoted into each well of a 48-well flat-bottomed plate. 190 U of catalase (Sigma-Aldrich) was added into each well and mixed thoroughly. 20 L4-staged N2 nematodes were transferred to each well, incubated at 24°C for 24 h and examined with a light microscope. Nematodes were considered dead when neither body twitching nor pharyngeal pumping could be observed. Infected nematodes were also transferred to unseeded agar to verify the phenotype. Results are representative of 3 independent assays, each with triplicates.

Statistical Analysis {#s4f}
--------------------

Survival curves were analyzed using the PRISM (version 5.0) software. Kaplan--Meier estimation of survival curves with *p* values \<0.05 were considered significantly different from the control. Student\'s *t* test was used to analyze the results from the liquid infection assays.

Supporting Information {#s5}
======================

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**List of nematode and bacteria strains used in this study.**

(PDF)

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Click here for additional data file.

We are grateful to the *Caenorhabditis* Genetics Center for providing nematode strains, Patrick Tan for providing bacteria and nematode strains, Oliver Pabst and Chee Han Lim for providing bacteria strains and plasmids, Barry Halliwell for providing chemicals, the KI Core Visualization Facility at MTC and the Birgitta Henriques-Normark Laboratory for use of equipment, and all members of the MR Laboratory for critical discussion of this work. We also thank Fredrik Åslund and Jason F. Kreisberg for critically reading and commenting on the manuscript.

[^1]: **Competing Interests:**The authors have declared that no competing interests exist.

[^2]: Conceived and designed the experiments: XS MR. Performed the experiments: XS. Analyzed the data: XS MR. Contributed reagents/materials/analysis tools: XS MR. Wrote the paper: XS MR.