Methylthioadenosine reduces host inflammatory response by suppressing Salmonella virulence

In order to deploy virulence factors at appropriate times and locations, microbes must rapidly sense and respond to various metabolite signals. Previously we showed transient elevation of the methionine-derived metabolite methylthioadenosine (MTA) in serum during systemic Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Here we explored the functional consequences of increased MTA concentrations on S. Typhimurium virulence. We found that MTA—but not other related metabolites involved in polyamine synthesis and methionine salvage—reduced motility, host cell pyroptosis, and cellular invasion. Further, we developed a genetic model of increased bacterial endogenous MTA production by knocking out the master repressor of the methionine regulon, metJ. Like MTA treated S. Typhimurium, the ΔmetJ mutant displayed reduced motility, host cell pyroptosis, and invasion. These phenotypic effects of MTA correlated with suppression of flagellar and Salmonella pathogenicity island-1 (SPI-1) networks. ΔmetJ S. Typhimurium had reduced virulence in oral infection of C57BL/6 mice. Finally, ΔmetJ bacteria induced a less severe inflammatory cytokine response in a mouse sepsis model. These data provide a possible bacterial mechanism for our previous findings that pretreating mice with MTA dampens inflammation and prolongs survival. Together, these data indicate that exposure of S. Typhimurium to MTA or disruption of the bacterial methionine metabolism pathway is sufficient to suppress SPI-1 mediated processes, motility, and in vivo virulence. Significance Salmonella enterica serovar Typhimurium (S. Typhimurium) is a leading cause of gastroenteritis and bacteremia worldwide. Widespread multi-drug resistance, inadequate diagnostics, and the absence of a vaccine for use in humans, all contribute to the global burden of morbidity and mortality associated with S. Typhimurium infection. Here we find that increasing the concentration of the methionine derived metabolite methylthioadenosine, either in S. Typhimurium or in its environment, is sufficient to suppress virulence processes. These findings could be leveraged to inform future therapeutic interventions against S. Typhimurium aimed at manipulating either host or pathogen methylthioadenosine production.

pretreating mice with MTA dampens inflammation and prolongs survival. Together, these data indicate that exposure of S. Typhimurium to MTA or disruption of the bacterial methionine metabolism pathway is sufficient to suppress SPI-1 mediated processes, motility, and in vivo virulence.

Introduction
Microbial communities within a mammalian host are bombarded by an array of intercellular, interspecies, and cross-kingdom metabolites and proteins. Cross-kingdom signaling plays important roles in Salmonella pathogenesis. For instance, in order to invade non-60 phagocytic host cells, Salmonella must deploy a secretion system encoded the Salmonella Pathogenicity Island-1 (SPI-1) (1, 2), which is regulated by many signals such as pH, bile, and short chain fatty acids (3)(4)(5)(6)(7). Together, these factors spatially limit the bacteria so that most invasion occurs in the ileum (8). Furthermore, recent work demonstrates that a host mimic of the bacterial AI-2 quorum molecule can directly impact S. Typhimurium gene expression in vitro by 65 activating the lsr operon (9). Understanding how the bacteria's environment influences Salmonella pathogenesis is important as it could help to inform future therapeutic interventions to suppress virulence.
One signal that may facilitate cross-talk between host and pathogen during infection is methylthioadenosine (MTA), a key metabolite in methionine metabolism. In addition to its role 70 in protein synthesis, methionine is used in both eukaryotic and prokaryotic systems to generate S-Adenosyl methionine (SAM), which is a critical methyl donor for a number of reactions (10,11). SAM catabolism results in a number of metabolic byproducts, including MTA and Sadenosylhomocysteine (SAH). In many eukaryotic and prokaryotic systems, MTA is recycled back into methionine, however, E. coli and S. Typhimurium cannot salvage methionine from 75 MTA (12). Instead, E. coli and Salmonella spp. regulate intracellular MTA concentrations by using an MTA/SAH nucleosidase (pfs) to cleave MTA into 5'methythioribose and excreting it (13,14). MTA regulation is considered to be critical for the bacterial cell as deletion of pfs impairs growth (15), but the effects of MTA on Salmonella virulence remain unknown.
Previously, our lab determined that MTA plays a multifaceted role in Salmonella 80 infection. We originally identified MTA as a positive regulator of host cell pyroptosis, a rapid, proinflammatory form of cell death, during Salmonella infection (16). More recently, we showed that host MTA is released into plasma during S. Typhimurium infection, and that high plasma MTA levels are associated with poor sepsis outcomes in humans (17). Paradoxically, we showed that exogenous treatment with MTA suppressed sepsis-associated cytokines and extended the 85 lifespan of mice infected with a lethal dose of S. Typhimurium (17). While consistent with previous reports that MTA acts as an anti-inflammatory molecule (18)(19)(20), this was in contrast to our findings that MTA primes cells to undergo pyroptosis. Together, these data led us to hypothesize that increased extracellular concentrations of MTA could potentially have independent effects on both the host and pathogen during infection. 90 Here we show that fluctuations in MTA levels regulate S. Typhimurium virulence in vitro and in vivo. Treatment of S. Typhimurium with exogenous MTA prior to infection or increasing endogenous bacterial production of MTA through genetic deletion of the methionine regulon suppressor, metJ, reduced the induction of pyroptosis and invasion in vitro. Furthermore, we report that both ∆ metJ mutants and MTA treated bacteria demonstrate transcriptional, 95 translational, and functional reductions in SPI-1 activity and motility. Finally, we find that ∆ metJ mutants have reduced virulence in vivo and that disrupting the methionine metabolism pathway in the bacteria can influence the inflammatory state of the host. Together, these data reveal the importance of MTA and bacterial methionine metabolism for regulating S. Typhimurium virulence and host inflammation, and provide a possible example of host-pathogen metabolite 100 cross-talk during infection.

Exogenous MTA reduces the ability for S. Typhimurium to induce pyroptosis and invade host cells in vitro. 105
Previously, we demonstrated elevated concentrations of MTA in plasma during systemic infection of mice with S. Typhimurium (17). While effects of MTA on the host inflammatory response have been documented (16-20), we asked whether elevated extracellular MTA could directly impact bacterial virulence. We examined the ability for S. Typhimurium pretreated with MTA (300 µM) to induce pyroptosis and invade human cells. As our original studies that 110 identified MTA as a modulator of pyroptosis were performed in lymphoblastoid cells (LCLs), and because B cells are a natural target of S. Typhimurium invasion in vivo (21-23), we first looked for an effect of exogenous MTA in LCLs. We assessed both pyroptosis and invasion by pairing a modified gentamicin protection assay with flow cytometry, as previously described (24) (Figure 1A). Briefly, pyroptosis was measured by quantifying the number of cells that 115 stained positive for 7AAD three hours post infection with S. Typhimurium. Independently, cellular invasion was quantified by using bacteria with a GFP plasmid, inducing GFP production after gentamicin treatment, and quantifying the number of GFP-positive host cells three hours post infection. MTA pretreatment had no effect on bacteria growth from the initial overnight culture dilution through late-log phase to induce SPI-1 gene expression ( Figure 1B). MTA 120 pretreated bacteria displayed a 30% decrease (p=0.0001) in their ability to induce pyroptosis ( Figure 1C). Furthermore we observed a 35% decrease (p=0.007) in their ability to invade LCLs ( Figure 1C). We observed similar effects of MTA on pyroptosis and invasion in THP-1 monocytes ( Figure 1D). This was in contrast to our previous finding that MTA treatment of host cells primes them to undergo higher levels of pyroptosis upon S. Typhimurium infection (16), 125 and suggests that the molecule has separate effects on both the host and pathogen.
This reduction in pyroptosis could not be reproduced by treating the bacteria with other metabolites related to the mammalian methionine salvage pathway, including methionine, SAM (also known as AdoMet), α -Keto-γ-(methylthio)butyric acid (MTOB), and phenylalanine ( Figure   1E, F). Similarly, adenosine (which is only lacking the methylthio group of MTA) was not 130 sufficient to suppress pyroptosis induction. In fact, adenosine increased pyroptosis in both LCLs and THP-1s. Of note, the bacterial cell is reportedly impervious to SAM (25, 26), so we cannot rule out that high intracellular concentrations of the molecule could suppress pyroptosis, however, our results rule out the molecule as an external signal regulating pyroptosis. Thus, these data demonstrate that MTA exposure uniquely suppresses the ability of S. Typhimurium to 135 induce pyroptosis and invade host cells.

metJ deletion in S. Typhimurium elevates levels of MTA
In order to provide independent evidence for MTA-mediated regulation of Salmonella virulence, we genetically disrupted the S. Typhimurium methionine metabolism pathway. The 140 protein MetJ is the master repressor of the methionine regulon and transcriptionally blocks multiple enzymatic steps that enable the generation of methionine and SAM ( Figure 2A) (10).
We hypothesized that deletion of metJ would relieve this transcriptional suppression and result in elevated intracellular MTA levels. Therefore, we generated a ∆ metJ mutant and performed mass spectrometry to examine how metabolites in the methionine metabolism pathway were impacted 145 by the mutation. In line with previous reports, we observed an increase in methionine levels in the ∆ metJ mutant (27) ( Figure 2B). MTA, SAM, and phenylalanine were also increased ( Figure   2C, D, E). Expressing metJ from a plasmid could reverse these increases. Consistent with MTA being an inhibitor of polyamine synthesis, the polyamine spermine was decreased in the ∆ metJ mutant, while no change was observed in another polyamine, spermidine ( Figure 2F, G). 150 Disrupting the methionine metabolism pathway by deleting metJ did not affect bacterial growth ( Figure 2H).

Elevated endogenous MTA suppresses pyroptosis and invasion in vitro
After demonstrating that metJ deletion leads to the accumulation of MTA in the bacterial 155 cell, we examined whether critical Salmonella virulence processes are suppressed in the ∆ metJ mutant. Similar to S. Typhimurium treated with MTA, ∆ metJ had a reduced ability to induce pyroptosis in LCLs and THP-1 monocytes ( Figure 3A, B). Importantly, MTA was the only measured elevated metabolite in the ∆ metJ mutant that inhibited levels of pyroptosis when added exogenously to wild-type bacteria (see Figure 1D). This reduction in pyroptosis was rescued by 160 expressing metJ from a plasmid. scanning of the host cell surface to optimize invasion (28,29). The SPI-1 secretion system not only enables the transport of effector proteins into the host cell that enable invasion (1, 2), but also acts as a trigger for pyroptosis in human cells (30)(31)(32)(33)(34). Therefore, our observation that both 170 pyroptosis and invasion are suppressed in MTA-treated and ∆ metJ S. Typhimurium led us to hypothesize that motility and/or SPI-1 are suppressed in response to increased concentrations of MTA.

Typhimurium motility
In order to determine whether motility was impaired in the ∆ metJ mutant, we performed a standard bacterial soft agar motility assay (35) and found that ∆ metJ was only able to traverse two thirds the distance of the wild-type bacteria over six hours ( Figure 4A). This motility defect was restored by expression of metJ from a plasmid. In order to confirm that increased MTA 180 levels were sufficient to drive this phenotype, we also examined S. Typhimurium motility on soft agar containing 300µM MTA. Like  Typhimurium (33). Further, while not statistically significant when taking into account multipletest correction, we saw comparable changes trending towards significance in the regulatory factor rtsA, the chaperone protein sicP, and the needle complex component prgH. ( Figure 5A). This suggests that there is at least modest suppression of SPI-1 regulated genes by MTA on the transcriptional level under standard culture conditions. Second, reduced expression of SipA, a 205 SPI-1 secreted effector, was detected in ∆ metJ cell lysates by western blot staining ( Figure 5B).
Third, this reduction in SipA was greater in ∆ metJ cell free supernatants than cell lysates, suggesting that both expression of SipA as well as its secretion by the T3SS apparatus are suppressed in the mutant ( Figure 5B). This is consistent with the suppression of invF and sipB observed by qPCR. These reductions in SipA were also detected in MTA treated S. 210 Typhimurium ( Figure 5C). Together, these data demonstrate suppression of the SPI-1 network by MTA.

Induction of inflammation and host cell invasion are critical processes that enable 215
Salmonellae to colonize and disseminate from the mouse gut (2, 8,36,[44][45][46]. Our findings that both the induction of pyroptosis and host cell invasion are attenuated in the ∆ metJ mutant led us to hypothesize that this mutant also has impaired virulence in vivo. To test this, we orally infected C57BL/6J mice with wild-type and ∆ metJ S. Typhimurium and measured the bacteria's ability to infect and disseminate to the spleen. We found that ∆ metJ had a 30-fold reduction in 220 fitness compared to wild-type S. Typhimurium at five days post infection ( Figure 6). This supports the hypothesis that metJ is important for S. Typhimurium to establish an infection in the mammalian gut and disseminate to the spleen.

Disruption of methionine metabolism in S. Typhimurium reduces inflammatory cytokine 225
production We previously reported that treatment of mice with MTA before infecting with a lethal dose of S. Typhimurium resulted in reduced production of sepsis-related cytokines (IL-6 and TNFα) and modestly prolonged survival (17). We hypothesized that the previously observed effects on inflammation may be due to MTA's impact on the microbe. This hypothesis is based 230 on our findings that metJ knockout suppresses murine-detected PAMPs, specifically flagellin (47, 48) and the SPI-1 T3SS (34,49,50).
To test whether MTA reduces host inflammation by suppressing S. Typhimurium virulence, we injected mice with a lethal dose (1x10 6 CFUs) of either wild-type or However, in both these cases, the metabolic changes responsible for these phenotypes are unknown. We hypothesize that our discovery of the role of metJ in regulating intracellular MTA concentrations could help explain these findings. If exogenous MTA can drive these phenotypes, similar to what we report here in S. Typhimurium, it would suggest that modulation of MTA 270 concentrations represents a mechanism by which virulence can be manipulated across multiple bacterial species.
One mechanism by which MTA may influence the S. Typhimurium flagellar regulon and SPI-1 encoded genes is by altering methylation. In prokaryotic and eukaryotic systems, SAM provides the methyl for a variety of DNA, RNA, and protein methylation reactions (55-60). In 275 eukaryotic systems, modulation of methionine metabolism resulting in changes to the cellular MTA and SAM pools can have important consequences on protein, DNA, and RNA methylation (61)(62)(63)(64). Therefore, we hypothesize that increased MTA leads to altered methylation of critical SPI-1 and flagellar regulators, resulting in their suppression. This could be part of a metabolic stress response, as both SPI-1 secretion and swimming motility are energetically expensive 280 processes that the bacteria may downregulate in response to methionine metabolism dysregulation (65). Understanding how MTA is sensed, what process it regulates directly, and how this process influences virulence could help understand this novel example of host-pathogen communication and further inform future therapeutics targeting this process.
Since exogenous and endogenous MTA affects Salmonella virulence, both bacterial and 285 host methionine metabolism present therapeutic targets. Previous studies tested MTA nucleosidase inhibitors against bacterial pathogens based on the assumption that disrupting MTA nucleosidase would lead to an MTA accumulation, resulting in an arrest of cellular growth and reduced bacterial viability (66)(67)(68). However, MTA nucleoside inhibitors showed, at most, modest bacteriostatic potential in these studies. In contrast, studies examining the effects of these 290 compounds on quorum sensing also showed no changes in bacterial growth, but did identify suppression of AI-2 synthesis (69,70). This is in line with our observation that Salmonella growth is not impaired by increased concentrations of MTA in the ∆ metJ mutant, but that there are functional consequences on virulence. However, no study has examined the potential of these compounds to directly impact virulence, independent of growth. Our data suggest that these 295 compounds likely have antibacterial properties, because their disruption of methionine metabolism in vivo impairs virulence. Furthermore, other groups have developed S-methyl-5'-thioadenosine phosphorylase (MTAP) inhibitors, which block mammalian MTA catabolism (71)(72)(73), increasing MTA concentrations in tissues, plasma, and urine in murine models (74). Based on these results and our demonstration that high extracellular MTA suppresses virulence, we 300 hypothesize that MTAP inhibitors could be a host-directed therapy during Salmonella infection. Therefore, the findings described here suggest that MTA nucleosidase inhibitors and MTAP inhibitors could be harnessed to combat bacterial infections and improve clinical outcomes.

Mammalian cells and bacterial strains
HapMap LCLs were purchased from the Coriell Institute. LCLs and THP-1 monocytes were cultured at 37°C in 5% CO 2 in RPMI 1650 media (Invitrogen) supplemented with 10% FBS, 2 μ M glutamine, 100 U/mL penicillin-G, and 100 mg/mL streptomycin. HeLa cells were grown in DMEM media supplemented with 10% FBS, 1mM glutamine, 100 U/mL penicillin-G, 310 and 100mg/mL streptomycin. Cells used for Salmonella gentamicin protection assays were grown in antibiotic free media one hour prior to infection.
All Salmonella strains are derived from the S. Typhimurium NCTC 12023 (ATCC 14028) strain and are listed in Table 1. All knockout strains were generated by lambda red recombination (75). Recombination events were verified by PCR, and the pCP20 plasmid was 315 used to remove the antibiotic resistant cassette after recombination (76). Strains were cultured overnight in LB broth (Miller) overnight, subcultured 1:33, and grown for two hours and forty minutes shaking at 37°C before all experiments unless otherwise noted. Strains containing the temperature sensitive plasmids pKD46 or pCP20 were cultured at 30°C and removed at 42°C.

Metabolomics
Bacteria were grown overnight as described above and subcultured 1:33 in 10mL LB and grown for 2 hours and 40 minutes. After thorough washing in PBS, samples were flash frozen, 340 thawed, and 0.5 mL PBS was added directly onto the pellets. Samples were then transferred to 2 mL CK01 bacterial lysis tubes (Bertin). These were then taken through 3 cycles of 20 second bursts at 7,500 RPM with 30 second pauses in between bursts using a Bertin Precellys (protocol as recommended by Bertin). Samples were spun at 5,000 for 5 minutes and a Bradford assay was performed on each lysate to gather protein concentration values. 100 µL from each homogenate 345 was pipetted directly into a 2 mL 96-well plate (Nunco). Bacterial RNA isolation and qPCR Bacteria were grown as described above and RNA was isolated from 5x10 8 Table 2.

Analysis of Bacterial Protein Expression
Bacteria were grown as described above. For analysis of cell lysates, bacterial cultures 385 were centrifuged at 10,000xg for 5 minutes. Supernatant was discarded and pellets were lysed in 2x laemmli buffer (Bio-Rad) with 5% 2-Mercaptoethanol. Samples were boiled for 10 minutes and analyzed on Mini-PROTEAN TGX Stain-Free gels (Bio-Rad). Bands were stained with a rabbit anti-SipA antibody overnight at 4 O C. Antibody were then detected by staining with the LI-CORE IRDye 800CW donkey anti-rabbit antibody. SipA was quantified using a LI-CORE 390 Odyssey Fc, paired with Image Studio software. Bands were quantified to total protein using the TGX stain free system. Total protein was quantified with using Fiji (77).
For secreted protein analysis, cultures were centrifuged at 10,000xg for 5 minutes, and supernatants were passed through a .2μm syringe filter. At this point, 6μL of 100ng/μL BSA was added to 600μL of supernatant as a loading control. Chilled 100% trichloroacetic acid was added 395 to a final concentration of 10% and incubated on ice for 10 minutes. 600μL of chilled 10% trichloroacetic acid was added, and the solution incubated on ice for another 20 minutes before being centrifuged at 20,000xg for 30 minutes. Pellets were washed twice with acetone and resuspended in 2x laemmli buffer (Bio-Rad) with 5% 2-Mercaptoethanol before boiling for 10 minutes. Proteins were then analyzed as described above. 400

Motility Assay
Motility assay was performed as previously described (35). Briefly, strains were cultured overnight in LB broth (Miller), subcultured 1:33, and grown for two hours and forty minutes shaking at 37°C. 2μL of the subcultured solution was plated in the center of a 0.3% agar LB 405 plate supplemented with 50μg/mL ampicillin. Metabolites or DMSO were added to the solution prior to the agar solidifying in order to allow exposure of the bacteria to the metabolite for the entirety of the assay. Plates were incubated at 37°C for 6 hours before the halo diameter was      Tables   B  a  c  t  e  r  i  a  l  S  t  r  a  i  n  s  S  t  r  a  i  n  G  e  n  o  t  y  p  e  P  l  a  s  m  i  d  R  e  s  i  s  t  a  n  c  e   D  C  K  5  4  3  N  C  T  C  1  2  0  2  3  (  A  T  C  T e t  T  C  T  T  C  T  G  C  G  C  T  T  T  C  T  C  T  G   r  t  s  A   F  w  d  A  C  C  C  G  T  G  G  T  G  A  G  C  T  T  G  A  T  G  A  G  T  (  7  8  )  R  e  v  C  C  T  G  T  C  C  A  G  G  T  G  G  G  G  A  G  C  A  T   s  i  c  P   F  w  d  A  G  A  T  G  A  T  A  T  C  T  G  G  T  T  A  T  T  G  A  A  C  G  G  T  A  T  G  (  7  8  ) T  C  A  A  A  G  G  T  T  T  T  (  7  9  )  R  e  v  C  T  T  T  C  A  C  C  G  T  T  T  T  C  C  C  G  T  T  A   f  l  h  D   F  w  d  T  G  T  T  C  C  G  C  C  T  C  G  G  T  A  T  C  A  A  C  (  8  0  ) T  G  C  T  T  T  G  C  T  A  C  A  T  A  T  G  A  A  T  A  T  C  C  T  C  C  T  T  A  G  U  p  s  t  r  e  a  m  C  A  T  C  T  G  C  G  A  C  C  G  C  T  A  A  C  T  T  D  o  w  n  s  t  r  e  a  m  T  T  T  A  T  C  C  A  C  C  G  A  G  G  G  T  T  A  T  T  C  G  p  K  D  4  6