Elucidating the relationships between antimicrobial resistance and virulence is key to understanding the evolution and population dynamics of resistant pathogens. Here, we show that the susceptibility of the gram-positive bacterium Listeria monocytogenes to the antibiotic fosfomycin is a complex trait involving interactions between resistance and virulence genes and the environment. We found that a FosX enzyme encoded in the listerial core genome confers intrinsic fosfomycin resistance to both pathogenic and non-pathogenic Listeria spp. However, in the genomic context of the pathogenic L. monocytogenes, FosX-mediated resistance is epistatically suppressed by two members of the PrfA virulence regulon, hpt and prfA, which upon activation by host signals induce increased fosfomycin influx into the bacterial cell. Consequently, in infection conditions, most L. monocytogenes isolates become susceptible to fosfomycin despite possessing a gene that confers high-level resistance to the drug. Our study establishes the molecular basis of an epistatic interaction between virulence and resistance genes controlling bacterial susceptibility to an antibiotic. The reported findings provide the rationale for the introduction of fosfomycin in the treatment of Listeria infections even though these bacteria are intrinsically resistant to the antibiotic in vitro.

Epistasis, or interactions between genes, is the phenomenon where the phenotypic effect of a locus is altered or masked by other loci in a given genomic context. Working with Listeria bacteria, we show that the effect of an intrinsic resistance determinant that protects these organisms against fosfomycin, a natural, microbial-derived antibiotic, is epistatically cancelled by virulence determinants present in the pathogenic species, L. monocytogenes. Since these virulence determinants are specifically activated within the host, the epistatic effect only manifests during infection, not when the bacteria are living saprophytically. Our study dissects the underlying mechanism, substantiating at the molecular level that virulence and resistance can be closely intertwined via gene-gene epistatic phenomena, with strong effects on the antimicrobial susceptibility phenotype. The findings are significant because any functional interaction between resistance and virulence may inextricably link the evolution of these two key pathogen traits. Understanding in detail these interactions is essential for predicting the evolutionary dynamics of resistance among pathogenic microbes or the impact of antimicrobial policies on drug-resistant virulent strains. In addition to their fundamental interest, our study provides the science-based evidence needed for the use of fosfomycin to treat listeriosis, a severe foodborne infectious disease, despite the causative bacteria showing strong resistance to the antibiotic.

Funding: This study was supported by the Wellcome Trust (Programme Grant WT074020MA to JVB) and in part by core BBSRC funding from the Roslin Institute Strategic Programme (BB/J004227/1). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2018 Scortti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we show that fosX is a core trait of the Listeria genus that confers high levels of resistance to fosfomycin unless epistatically controlled, in the pathogenic species L. monocytogenes, by members of the in vivo-activated PrfA virulence regulon. Our work demonstrates that epistatic interactions between virulence and resistance genes can have dramatic effects on the antimicrobial susceptibility phenotype of bacterial pathogens.

Despite the above evidence, a potential obstacle for the clinical use of fosfomycin in the treatment of listeriosis is the reported presence in L. monocytogenes of a fosfomycin hydrolyzing enzyme, FosX [ 22 ]. The fosX gene was originally discovered in the soil bacteria Mesorhizobium loti and Desulfitobacterium hafniense and in the L. monocytogenes reference genome strain EGDe (lmo1702) by in silico mining for homologs of the fosfomycin resistance proteins FosA and FosB [ 23 ]. However, the actual distribution of fosX, and whether this gene actually confers fosfomycin resistance in L. monocytogenes had not been established.

Previous work from our laboratory identified fosfomycin (disodium salt for parenteral use) [ 11 – 13 ] as one such potentially useful anti-listerial drugs. Fosfomycin [(1R,2S)-epoxypropylphosphonic acid] is a low-molecular-weight bactericidal molecule discovered in 1969 in Streptomyces fradiae [ 14 ] that inhibits peptidoglycan biosynthesis through covalent inactivation of UDP-N-acetylglucosamine-3-enolpyruvyl transferase (MurA) [ 11 ]. Although known to be resistant to fosfomycin by standard in vitro testing [ 9 , 15 , 16 ], we found that L. monocytogenes was actually susceptible to this antibiotic in infected cells and in vivo in mice [ 17 ]. The efficacy of fosfomycin against intracellular L. monocytogenes was independently confirmed by others [ 18 ]. The basis of this in vitro-in vivo paradox is the PrfA-regulated expression of the listerial sugar phosphate permease Hpt, a homolog of the enterobacterial hexose phosphate transporter UhpT that also transports fosfomycin [ 17 ]. Hpt is a virulence factor that promotes rapid replication in the cytosol by allowing bacterial utilization of host-cell hexose phosphates as a carbon source [ 19 ]. However, Hpt remains unexpressed outside the host due to PrfA On-Off switching [ 17 , 20 ], preventing Hpt-mediated fosfomycin import into the listerial cell [ 17 ]. Importantly, we also showed that L. monocytogenes spontaneous fosfomycin resistance was mostly due to mutations in the prfA (56%) or hpt (41%) genes [ 17 ]. Since prfA is essential for pathogenesis [ 6 , 21 ] and hpt is required for full in vivo virulence [ 19 ], L. monocytogenes fosfomycin resistant mutants were consequently found to be counterselected in infected macrophages [ 17 ].

The facultative intracellular pathogen Listeria monocytogenes is the causative agent of listeriosis, a foodborne infection characterized by severe clinical manifestations including meningoencephalitis, bacteremia, miscarriage and neonatal sepsis or meningitis [ 1 – 3 ]. The pathogenesis of listeriosis relies on a group of virulence genes that are co-ordinately regulated by the PrfA transcriptional activator [ 4 ]. PrfA-regulated genes are selectively induced within host cells through a mechanism involving cofactor-mediated allosteric switching of PrfA between weakly active (“Off”) and strongly active (“On”) states [ 5 , 6 ]. PrfA regulation is both essential for the activation of the listerial virulence program within the host and for preventing the costly production of unneeded virulence factors when L. monocytogenes is living as an environmental saprotroph [ 7 , 8 ]. Listeriosis is the foodborne infection with the highest mortality in the Western hemisphere despite hospital-based therapy (20–50%) [ 2 ]. This is partly attributable to the intracellular lifestyle of L. monocytogenes and the location of lesions, e.g. the brain, which render these bacteria relatively inaccessible to drugs thereby limiting the therapeutic choices [ 9 , 10 ]. Cell-permeant antimicrobials able to penetrate the blood-brain barrier (BBB) and other listerial infection sites at bactericidal concentrations may therefore significantly aid in the treatment of listeriosis.

Results

fosX is part of the Listeria core genome Analysis of a collection of genomic sequences from 1,696 L. monocytogenes isolates from 13 countries, representing the four lineages of the species, 164 sublineages and 1,013 core-genome MLST subtypes [24], showed that the fosX gene is universally conserved in L. monocytogenes (Fig 1A). L. monocytogenes strains encoded a 133-residue FosX protein between 92 and 100% identical to the product of the 402-bp fosX gene of strain EGDe [23] (S1 Table). No other putative fosfomycin resistance enzyme genes were identified in L. monocytogenes. FosX orthologs were also encoded in Listeria innocua, Listeria welshimeri (89% identity), Listeria marthii (91% identity) and Listeria ivanovii (73% identity) (S1 Table). These species belong together with L. monocytogenes to one of the main phylogenetic subdivisions of the genus, clade (i) or Listeria “sensu stricto” [25]. With the exception of Listeria seeligeri, in which the gene appears to have been lost, fosX was present at the same chromosomal location in all members of the Listeria sensu stricto clade (Fig 1C). This, and the fact that a phylogenetic tree based on the FosX protein sequence closely mirrored the genus’ phylogenomic structure (Fig 1B), indicated that fosX is an ancient Listeria trait that evolved with the core genome of these bacteria. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. fosX evolved with the Listeria core genome. (A) Single linkage clustering of 1,696 L. monocytogenes isolates based on core-genome MLST profiles [24] showing the conservation of foxX in the species. Main L. monocytogenes lineages I to IV [24] are indicated in different colors. The presence of complete fosX coding regions is marked in gray in the outer ring (where gaps indicate strains with frameshift mutations leading to a truncated FosX protein; see text). (B) Neighbor-joining tree of FosX enzymes from Listeria spp. rooted with Brucella melitensis FosX (NCBI RefSeq WP_004687281.1). The topology of the tree mirrors the Listeria genus whole-genome phylogeny (which currently includes a number of Listeria-like groups) [25] and L. monocytogenes diversification into lineages (color coded as in panel A). The Listeria sensu stricto clade is indicated with thick lines. More distant fosX homologs (possibly paralogs) are present in the Listeria sensu lato “Paenilisteria” clade (represented by L. cornellensis, L. rocourtiae, L. weihenstephanensis and L. riparia in the tree; 63–65% amino acid sequence identity to L. monocytogenes FosX vs 73–91% for Listeria sensu stricto orthologs) in a different chromosomal location. See S1 Table for details. (C) Genomic organization around the fosX gene (lmo1702) in the Listeria sensu stricto clade [25]. Orthologs are in the same color, non-core genes are in gray. fosX is present in all members of the clade except L. seeligeri. L.m., L. monocytogenes. Coding sequence numbers according to L. monocytogenes EGDe nomenclature. https://doi.org/10.1371/journal.pgen.1007525.g001

Evidence for OPA permease-independent fosfomycin import The fosfomycin susceptibility of P14ΔfosX in BHI raises the question of how fosfomycin might enter the listerial cell at inhibitory concentrations in conditions where PrfA is “Off” and Hpt is completely downregulated [17]. A double ΔfosXΔhpt mutant had the same MIC as the fosX mutant (P = 0.399) (Fig 2, left panel), excluding leaky expression of the Hpt transporter as the cause. Although a very small molecule (138 Da), fosfomycin is hydrophilic and unlikely to permeate into the bacterial cell unless through facilitated diffusion via a carrier protein(s). The only known bacterial fosfomycin transporters are two types of organophosphate:inorganic phosphate antiporters (OPA) [35], exemplified by the hexose phosphate transporter UhpT (and listerial homologue Hpt) and the GlpT glycerol-3-phosphate permease [11, 36]. Genome searches confirmed that Hpt is the only OPA permease in Listeria spp. This indicates that the susceptibility of the L. monocytogenes ΔfosXΔhpt mutant (and the L. innocua fosX mutant) must depend on another, as yet unknown fosfomycin uptake pathway (Fig 5). We suggest that the selective pressure imposed by this uncharacterized transport mechanism is a major driver underlying fosX acquisition and maintenance in Listeria. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Model of virulence-resistance gene epistatic interaction determining L. monocytogenes fosfomycin phenotype. In saprophytic (in vitro) conditions (left section of figure), the FosX enzyme inactivates the (relatively low) concentrations of fosfomycin that enter the bacterial cell via an uncharacterized transport mechanism (indicated with an inward arrow; see text for details). This fosfomycin uptake mechanism operates in both obligate saprotrophic and pathogenic Listeria spp. However, in L. monocytogenes, two of its virulence determinants, prfA encoding the central virulence regulator PrfA [4], and the PrfA-regulated hpt gene encoding the sugar phosphate permease Hpt [19] (which also transports fosfomycin) [17], suppress the effect of FosX. This occurs during infection, where PrfA is activated upon sensing of host signals [5, 6, 41] and PrfA-promoted expression of Hpt leads to increased fosfomycin influx. In these conditions, FosX is overwhelmed and a fraction of incoming fosfomycin reaches its MurA target at inhibitory concentrations (right section of figure). Fosfomycin is shown in molecular structure representation. MurA catalyzes the first committed step in peptidoglycan biosynthesis (ligation of phosphoenolpyruvate to the 3’-hydroxy group of UDP-N-acetylglucosamine) [11, 36]. https://doi.org/10.1371/journal.pgen.1007525.g005

Epistasis of the virulence genes prfA and hpt over fosX We tested the effect of fosX when Hpt-mediated fosfomycin transport is active using two “infection-mimicking” in vitro conditions: (i) supplementation of BHI medium with an adsorbent (activated charcoal or Amberlite XAD-4), which causes the partial activation of the PrfA regulation system by an as yet not fully understood mechanism [37]; and (ii) use of a constitutively activated prfA* allele, where a single amino acid substitution (e.g. PrfA*G145S) locks PrfA in “On” state, causing constitutive activation of the PrfA-regulated virulence genes [38]. A strong reduction in the fosfomycin MIC is typically observed with each of these two PrfA-activating strategies [17] (from ≥1024 to 27.3±5.3 and 12±0 μg/ml, respectively; P < 0.0001) (Fig 2, middle and right panels). In these conditions, the fosX mutation lowered further the fosfomycin MIC to virtually complete susceptibility (2.2±0.4 and 1.5±0.4 μg/ml, respectively) (P = 0.01). Complementation of the ΔfosX mutant restored the MIC to parental levels (Fig 2, middle and right panels). As expected [17], deletion of hpt or its transcriptional activator gene prfA rendered L. monocytogenes resistant to fosfomycin (MIC >1,024 μg/ml) (Fig 2, middle and right panels) but had no effect on bacteria with a wild-type prfA allele in BHI (where PrfA is “Off” and hpt is not expressed) (Fig 2, left panel). Ablation of Hpt function in the FosX−background under conditions of PrfA activation raised the MIC, from 2.2±0.4 to 36.8±7.0 μg/ml (charcoal-supplemented BHI [BHI-Ads]) or 1.5±0.4 to 35.2±6.4 μg/ml (prfA* allele) (P <0.001). These higher MIC values were similar to those for ΔfosX (or the double mutant ΔfosXΔhpt) in BHI (45.3±2.7 μg ml/ml and 41.6±3.9, respectively) (Fig 2). Overall, the above findings are consistent with a scenario where FosX: (i) can successfully inactivate the amounts of fosfomycin that enter the bacterial cell via the uncharacterized “constitutive” transport system, conferring complete resistance in in vitro (saprophytic) conditions; but (ii) is unable to process an increased influx of fosfomycin molecules via the Hpt transporter in PrfA-activating (infection) conditions (Fig 5). In other words, our data show that the intrinsic resistance conferred by the fosX gene is masked, or epistatically supressed, by the joint effect of the virulence genes prfA and hpt on fosfomycin transport.

Epistatic effect during infection We next assessed the extent to which the epistatic effect that cancels fosX-mediated resistance manifests during infection, where prfA/hpt are naturally induced [5, 6, 39–41]. To this end, the intracellular susceptibilities of wild-type L. monocytogenes P14 and isogenic ΔfosX derivative (and Δhpt mutant as a control) were compared in survival/proliferation assays in infected RAW 264.7 macrophages in the presence and absence of fosfomycin. Cell cultures were incubated with 5× the physiological concentration of D-glucose as in these conditions listerial intracellular growth is independent of Hpt-mediated hexose phosphate uptake [17]. As shown in Fig 6, both wild-type and ΔfosX L. monocytogenes were equally susceptible to fosfomycin during intracellular infection (P = 0.996). In contrast, fosfomycin had in these conditions no effect on the Δhpt mutant with disabled Hpt transport. Identical results were obtained using the human epithelial cell line HeLa (Fig 6). These data confirmed that L. monocytogenes is fully susceptible to fosfomycin in infection conditions, specifically as a consequence of the epistatic supression of fosX-mediated resistance by the PrfA-regulated (in vivo-activated) hpt gene. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. Fosfomycin susceptibility in infected cells. Intracellular survival of wild-type P14 (WT) and isogenic ΔfosX mutant demonstrating the lack of effect of FosX on L. monocytogenes susceptibility during infection. Experiments were conducted in RAW 264.7 macrophages and HeLa epithelial cells incubated with and without 180 μg/ml fosfomycin. A Δhpt mutant where PrfA-dependent fosfomycin uptake is disabled was included as a control. Mean ± SEM of at least three duplicate experiments. Relevant P values are indicated; ns, not significant (one-way ANOVA and Šídák tests of data at final time-point). https://doi.org/10.1371/journal.pgen.1007525.g006

PrfA activation does not affect fosX expression While our data are consistent with the loss of fosX-mediated intrinsic resistance under PrfA induction (infection) conditions being primarily due to increased fosfomycin influx via Hpt (Fig 5), potential effects of PrfA (or the intracellular milieu) on fosX expression could also be a contributing factor. To explore this possibility, fosX transcription was analysed by RT-QPCR in BHI and in PrfA-activating conditions in vitro (adsorbent-treated medium, prfA* allele; see above) or during intracellular infection. All three PrfA-inducing conditions caused the expected transcriptional activation of the PrfA-regulated hpt and (control) actA genes [5, 42], with no significant changes in fosX expression (P = 0.615) (Fig 7). These data excluded a potential involvement of reduced expression of fosX in the susceptibility phenotype elicited by PrfA activation. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 7. Constitutive expression of fosX. RT-QPCR transcription analysis of fosX and the PrfA-regulated hpt and (control) actA genes in L. monocytogenes P14 grown in BHI (PrfA “Off”) and under PrfA-inducing conditions (BHI-Ads, use of prfA*G145S allele in BHI, intracellular infection in RAW 264.7 macrophages). BHI-Ads is Amberlite XAD-4-supplemented BHI. Mean ± SEM of at least three independent experiments in duplicate. Relevant P values are indicated (one-way ANOVA with uncorrected Fisher’s post-hoc multiple comparison). https://doi.org/10.1371/journal.pgen.1007525.g007

Fosfomycin susceptibility of L. monocytogenes isolates under PrfA activation To establish whether the limited effect of fosX when the PrfA system is “On” and Hpt is expressed is a general feature of the L. monocytogenes species, the fosfomycin MIC of 142 wild-type isolates was tested in BHI and BHI-Ads. The MIC 50 and MIC 90 values shifted from ≥1,024 μg/ml in BHI to 16 and 64 μg/ml, respectively, in BHI-Ads (Fig 8A). Thus, despite fosX, in conditions of PrfA activation the fosfomycin MIC remained within the limits of susceptibility for the vast majority of the tested strains (90.33% with 64 μg/ml PK/PD breakpoint [18]; 80.64% with 32 μg/ml general fosfomycin breakpoint for gram-positive bacteria [43]). It must be noted that adsorbents only partially activate PrfA ([41] and Fig 7), and significantly lower fosfomycin MICs (median 3 μg/ml, range 2–16) are observed in L. monocytogenes prfA*G145S bacteria with the PrfA system constitutively activated to in vivo (within-host)-like levels [17, 41] (see Fig 2, right panel). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 8. Fosfomycin susceptibility of L. monocytogenes isolates. (A) MICs of a panel of 142 wild-type isolates in BHI (PrfA “Off”, Hpt–) and charcoal-supplemented BHI (BHI-Ads; PrfA “On”, Hpt+). (B) Intracellular survival of L. monocytogenes isolates in RAW 264.7 macrophages in the presence and absence of fosfomycin (180 μg/ml). Three distinct sets of bacterial isolates with different fosfomycin phenotype were included: (i) “normal” adsorbent-activable fosfomycin susceptibility (WT, n = 11, strains listed in S3 Table), (ii) constitutively susceptible fosfomycin phenotype (“low MIC(BHI) variants”, n = 9 including eight spontaneous fosX mutants, S3 Table), and (iii) isolates showing a relatively elevated MIC (96–384 μg/ml) in BHI-Ads (“high-MIC(Ads) variants”, n = 10). Data for P14 are those from Fig 6, included for reference. Mean from at least two independent experiments. Relevant P values are indicated (two-way ANOVA with Dunnett´s multitple comparison tests); ns, not significant. https://doi.org/10.1371/journal.pgen.1007525.g008 To determine if the above findings can be extrapolated to infection conditions, the intracellular susceptibility of a selection of L. monocytogenes strains with “normal” (i.e. adsorbent-activable) fosfomycin phenotype was analysed in RAW 264.7 macrophages. The tested bacteria included eight wild-type human clinical isolates plus the well-characterized strains EGDe (serovar 1/2a), 10403 (serovar 1/2a) and CLIP 80459 (serovar 4b). In addition, we also tested a representation (n = 10) of the small proportion of isolates where the fosfomycin MICs remained relatively elevated (96–384 μg/ml) despite being BHI-Ads responsive, to determine if this correlated with differences in intracellular susceptibility. Fig 8B shows that all tested strains were equally susceptible to fosfomycin in infected macrophages (P = 0.632). These data confirmed that L. monocytogenes isolates are characteristically susceptible to fosfomycin during infection, even if the MIC remains above the 64-μg/ml breakpoint as long as they exhibit the capacity to respond to PrfA-activating conditions (as tested in BHI-Ads medium).

fosX mutations in constitutively susceptible L. monocytogenes isolates A percentage of L. monocytogenes clinical isolates exhibit constitutively low fosfomycin MICs under normal in vitro testing conditions [16] (about 2 to 4.5%; data from L. monocytogenes antibiotic susceptibility surveillance at Institut Pasteur’s National and WHO Collaborating Reference Centre for Listeria and ref. [18]). We examined nine human isolates carrying a fosX gene but presenting a fosfomycin MIC ≤64 μg/ml in PrfA-non-inducing conditions (normal BHI) to determine the underlying mechanism (S3 Table). All displayed a wild-type PrfA phenotype (see Materials and Methods), excluding possible spontaneous prfA* (hpt-activating) mutations as the cause for their constitutive in vitro fosfomycin susceptibility [17]. Consistent with the key role of the FosX enzyme in the intrinsic in vitro resistance of L. monocytogenes to fosfomycin, eight of the nine strains analyzed carried fosX mutations (S3 Table). The only isolate with wild-type FosX gave a “slow-positive” sugar-phosphate acidification test (S3 Table), pointing to an increased Hpt activity as the cause. However, no differences in hpt gene expression (S3 Fig) or in the DNA sequence of the hpt region that could explain the Hpt(+) phenotype (S3 Table) were identified. Seven of the mutants had a premature stop codon at fosX triplet 128, leading to a truncated product where the lack of the six C-terminal residues most likely destabilizes FosX's catalytic site [22] (S4 Fig). The other fosX mutant carried a frameshift mutation at position 88 that introduced premature stop codons from position 89. Complementation analysis in P14ΔfosX confirmed that the fosX128stop and fosX88frameshift mutant alleles did not encode active FosX enzymes (BHI MICs: 48 and 32 μg/ml, respectively instead of ≥1,024 with wild-type fosX). As expected, similar to the P14ΔfosX (Fig 6), all the spontaneous fosX mutants showed complete susceptibility to fosfomycin in infected host cells (Fig 8B).