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Open Access

Peer-reviewed

Research Article

Listeria monocytogenes requires phosphotransferase systems to facilitate intracellular growth and virulence

  • Matthew J. Freeman,

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

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  • Noah J. Eral,

    Roles Conceptualization, Investigation, Methodology, Writing – review & editing

    Affiliation Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

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  • John-Demian Sauer

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

    sauer3@wisc.edu

    Affiliation Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America

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This is an uncorrected proof.

Abstract

The metabolism of bacterial pathogens is exquisitely evolved to support virulence in the nutrient-limiting host. Many bacterial pathogens utilize bipartite metabolism to support intracellular growth by splitting carbon utilization between two carbon sources and dividing flux to distinct metabolic needs. For example, previous studies suggest that the professional cytosolic pathogen Listeria monocytogenes (L. monocytogenes) utilizes glycerol and hexose phosphates (e.g., Glucose-6-Phosphate) as catabolic and anabolic carbon sources in the host cytosol, respectively. However, the role of this putative bipartite metabolism in L. monocytogenes virulence has not been fully assessed. Here, we demonstrate that when L. monocytogenes is unable to consume either glycerol (ΔglpD/ΔgolD), hexose phosphates (ΔuhpT), or both (ΔglpD/ΔgolD/ΔuhpT), it is still able to grow in the host cytosol and is 10- to 100-fold attenuated in vivo suggesting that L. monocytogenes consumes alternative carbon source(s) in the host. An in vitro metabolic screen using BioLog’s phenotypic microarrays unexpectedly demonstrated that WT and PrfA* (G145S) L. monocytogenes, a strain with constitutive virulence gene expression, use phosphotransferase system (PTS) mediated carbon sources. These findings contrast with the existing metabolic model that cytosolic L. monocytogenes expressing PrfA does not use PTS mediated carbon sources. We next demonstrate that two independent and universal phosphocarrier proteins (PtsI [EI] and PtsH [HPr]), essential for the function of all PTS, are critical for intracellular growth and virulence in vivo. Constitutive virulence gene expression using a PrfA* (G145S) allele in ΔglpD/ΔgolD/ΔuhpT and ΔptsI failed to rescue in vivo virulence defects suggesting phenotypes are due to metabolic disruption and not virulence gene regulation. Finally, in vivo attenuation of ΔptsI and ΔptsH was additive to ΔglpD/ΔgolD/ΔuhpT, suggesting that hexose phosphates and glycerol and PTS mediated carbon source are relevant metabolites. Taken together, these studies indicate that PTS are critical virulence factors for the cytosolic growth and virulence of L. monocytogenes.

Author summary

Listeria monocytogenes is an important bacterial pathogen and the causative agent of listeriosis, a foodborne infection associated with significant morbidity and mortality. L. monocytogenes lives in a diverse set of environments including as a saprophyte in the soil and in the cytosol of host cells during infection. Understanding the metabolic crosstalk between host cells and their bacterial invaders can illuminate mechanisms of bacterial pathogenesis and the host response and could lead to the development of novel treatments in a world of ever-increasing antibiotic resistance. Here we use bacterial genetics combined with metabolic screens to identify how L. monocytogenes acquires nutrients from the host during infection. We find that L. monocytogenes uses a combination of host cell derived hexose phosphates, glycerol, and free sugars to support its metabolic needs during infection. Specifically, we find L. monocytogenes requires its arsenal of phosphotransferase systems (PTS), a set of free sugar importers, to survive and replicate during infection. Together our results broaden our understanding of how cytosolic pathogens acquire nutrients to support their metabolic needs during infection and highlight novel targets for future therapeutic intervention.

Citation: Freeman MJ, Eral NJ, Sauer J-D (2025) Listeria monocytogenes requires phosphotransferase systems to facilitate intracellular growth and virulence. PLoS Pathog 21(4): e1012492. https://doi.org/10.1371/journal.ppat.1012492

Editor: Mary X. O'Riordan, University of Michigan Medical School, UNITED STATES OF AMERICA

Received: August 9, 2024; Accepted: March 27, 2025; Published: April 15, 2025

Copyright: © 2025 Freeman 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.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: MJF was supported by T32 Funding from the University of Wisconsin Madison Medical Scientist Training Program from the National Institute of General Medical Sciences (https://www.nigms.nih.gov/) T32GM140935 and T32GM008692 and from the University of Wisconsin Microbes in Health and Disease from the National Institute of Allergy and Infectious Diseases (https://www.niaid.nih.gov/)T32AI055397. Additional funding supporting this work was obtained from National Institute of Allergy and Infectious Diseases (https://www.niaid.nih.gov/) awarded to JDS as R01AI184369, R21AI173738 and R01AI137070. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The mammalian cytosol is a stringent and hostile environment that restricts the growth of bacteria not specifically adapted to that niche [1–5]. One mediator of bacterial growth restriction in the host cytosol is nutrient availability whereby cells actively or passively limit access to vital nutrient resources preventing bacterial growth. For example, intracellular pathogens are metabolically restricted through limited access to metal ions, vitamin cofactors, and amino acid pools [6–9]. More specifically, macrophages contribute to this metabolic foray by shifting metabolism between M1 or M2 states, altering concentrations of metals, and rewiring glycolysis and the tricarboxylic acid cycle to control bacterial pathogens [10–12]. Despite this well-orchestrated defense, canonical cytosolic pathogens such as Listeria monocytogenes (L. monocytogenes) can replicate in this environment at a rate equivalent to that in rich media [13]. This rapid growth allows L. monocytogenes to overtake host defenses and disseminate to distant sites of infection from the intestine (spleen, liver, & meninges), resulting in a mortality rate approaching 30% [14–16]. Defining bacterial metabolism can reveal novel targets for antibiotics and a better understanding of host-pathogen interactions. Despite significant progress, there are significant unknowns about what metabolites pathogens such as L. monocytogenes are using in their respective host environments, how these nutrients are acquired, and what impact this has on the host response to infection [17–20]. Key challenges to progress in understanding host-pathogen metabolisms include an incomplete understanding of the metabolic profile of host cells during infection and redundant metabolic pathways in both the host and the pathogen.

In addition to L. monocytogenes’ role as an important pathogen, it serves as a powerful model organism to answer questions about host-pathogen interactions [21]. L. monocytogenes’ virulence genes and intracellular lifecycle have been well studied including in macrophages which serve as a primary replicative niche and host dissemination vehicle [13,22]. L. monocytogenes encodes the master transcriptional virulence regulator, PrfA, that controls many key virulence factors to mediate the L. monocytogenes cytosolic lifecycle including Listeriolysin O (LLO) that facilitates cytosolic access, the phospholipase Cs (PLCs) that contribute to secondary vacuole escape, ActA which mediates intracellular motility and cell-to-cell spread, and the hexose phosphate transporter UhpT which contributes to cytosolic metabolism [13,22]. PrfA is activated upon entry into the cytosol via allosteric regulation by glutathione, however PrfA* mutations such as the G145S mutation result in constitutive virulence gene expression and upregulation of uhpT, the L. monocytogenes hexose phosphate transporter [23–25]. Finally, well-defined ex vivo and in vivo models of L. monocytogenes infection and its genetic tractability allow for a thorough examination of how metabolic perturbation might impact its virulence and host response to that metabolic perturbation [26].

Previously, L. monocytogenes has been employed as a tool to understand bacterial cytosolic metabolism through isotopologue analysis. Specifically, this work examined the metabolism of L. monocytogenes during the infection of macrophages, a necessary step in disseminated infection in vivo [27,28]. The Eisenreich and Goebel groups previously identified glycerol and hexose phosphates (e.g., Glucose-6-Phosphate) as the primary carbon metabolites used by L. monocytogenes during cytosolic replication [29–32]. These findings were supported by the fact that Listeria innocua, a non-pathogenic Listeria species, lacks the transporter necessary for use of hexose phosphates (UhpT) and that glycerol is a common metabolite used by a variety of intracellular pathogens [33,34]. Further, these analyses suggested that glycerol and hexose phosphates are primarily funneled into lower glycolytic catabolism and pentose phosphate pathway anabolism, respectively. Despite this, L. monocytogenes mutants lacking the ability to use glycerol (ΔglpD) or hexose phosphates (ΔuhpT) individually maintain intracellular growth and significant virulence [29,32]. The lack of robust virulence phenotypes could be due to incomplete perturbation of specific carbon source use, a lack of physiologic relevancy of the ex vivo models, or the ability of L. monocytogenes to utilize alternative, yet to be defined carbon sources [35–39].

In addition to being able to replicate in the host cytosol following infection, L. monocytogenes also lives as a saprophyte in the environment and in food production facilities where its metabolic potential has also been studied intensely [40–42]. Like many other bacteria, L. monocytogenes can utilize phosphotransferase systems (PTS) in these environments to acquire free sugar [43]. The L. monocytogenes 10403s strain used in this study encodes 29 complete PTS, encoded by a collection of 86 genes [39,44,45]. Interestingly, other L. monocytogenes strains as well as different Listeria species such as L. innocua and L. welshimeri vary in the specific PTS they encode suggesting flexibility in the use of PTS [46–49]. Early work has shown these differences may be important for virulence, but a global understanding of their importance remains unknown [46,49,50]. Mechanisms of PTS function are well-defined and reviewed elsewhere [51]; yet, there are many open questions about the relationship between PTS components’ structure, sugar specificity, regulatory inputs/outputs, and ultimately their importance during infection [51]. PTS mediate carbon source import and phosphorylation, with secondary functions on transcriptional regulation having been reported [52]. PTS import free sugars following binding of a sugar to pre-phosphorylated import permeases. This pre-phosphorylation is tied to the lower glycolytic conversion of phosphoenolpyruvate (PEP) to pyruvate through two phosphocarrier proteins (PtsI [EI] & PtsH [HPr-His]). The result of this phospho-cycling is that free sugars are phosphorylated during import and readied for direct funneling to glycolysis. It has previously been reported that PrfA-dependent virulence gene expression is repressed when L. monocytogenes is utilizing primarily PTS-dependent carbon sources [42,53,54]. Conversely, when PTS function is blocked via deletion of the HPr phosphocarrier protein (ΔptsH), PrfA-dependent virulence gene expression is significantly increased in vitro [20,32,54,55]. Because of these observations, PTS have been widely believed to be inactive during L. monocytogenes intracellular growth and virulence [39,44,45,56,57].

In this work, we assessed the contribution of glycerol and hexose phosphate metabolism to L. monocytogenes virulence in vivo finding that glycerol and hexose phosphates are neither sufficient nor essential to support intra-macrophage growth and although they do contribute to virulence of L. monocytogenes in vivo. A metabolic screen of carbon sources revealed highly similar metabolite utilization between WT and PrfA* (G145S) L. monocytogenes, including the use of PTS mediated carbon sources. Ablation of all PTS-mediated carbon acquisition via deletion of the phosphocarrier proteins EI (ΔptsI) or HPr (ΔptsH) revealed that L. monocytogenes requires PTS function to support intracellular growth and virulence. In vivo virulence defects could not be rescued by constitutive virulence gene expression with the PrfA* (G145S) allele in ΔptsI or ΔglpD/ΔgolD/ΔuhpT suggesting that the virulence defects are due to metabolic disruption and not altered virulence gene expression. Further, ΔptsI and ΔglpD/ΔgolD/ΔuhpT mutants both showed basally increased levels of hemolytic activity relative to WT L. monocytogenes suggesting increased PrfA activity. Finally, the phenotypes associated with loss of PTS function are additive with the inability to utilize glycerol and hexose phosphates (ΔglpD/ΔgolD/ΔuhpT/ΔptsI or ΔglpD/ΔgolD/ΔuhpT/ΔptsH) demonstrating that L. monocytogenes uses a highly complex and varied network of metabolites to promote rapid intracellular replication during infection.

Results

glpD/golD and uhpT genes are required for L. monocytogenes to consume glycerol and hexose phosphate, respectively

L. monocytogenes uses host-derived glycerol and hexose phosphates during cytosolic replication, as defined using isotopologue metabolomics analysis by the Goebel and Eisenreich labs [29,31,32], however whether these are the predominant carbon sources used during in vivo infection has not been fully explored. Although uhpT, glpD and golD mutants have been studied in isolation, the phenotype of combination metabolic mutants of parallel glycerol utilization pathways (ΔglpD/ΔgolD) and of glycerol with hexose phosphate pathways (ΔglpD/ΔgolD/ΔuhpT) have not been assessed (Fig 1A). We aimed to test the hypothesis that combination metabolic mutants of ΔglpD/ΔgolD, ΔuhpT, and combination ΔglpD/ΔgolD/ΔuhpT would completely ablate L. monocytogenes growth on glycerol, hexose phosphates, and both, respectively. To do this we generated these metabolic mutants and assessed growth in Listeria synthetic media (LSM) with glucose, glycerol, glucoe-6-phosphate (+glutathione), or glycerol/glucose-6-phosphate (+glutathione) as the sole carbon source (Fig 1B–E). LSM with defined sole carbon sources was inoculated with WT L. monocytogenes or the indicated mutants, and growth was assessed via OD600 absorbance every 15 minutes. Importantly, any LSM containing hexose phosphates required further supplementation with 10 mM reduced glutathione to induce prfA, and therefore uhpT, expression [23,58]. Of note, we found that a ΔglpD mutant was still able to grow in LSM supplemented with glycerol and a second glycerol utilization pathway required ablation (ΔgolD) to limit in vitro growth [38]. We found that glycerol (ΔglpD/ΔgolD), hexose phosphate (ΔuhpT), and combined (ΔglpD/ΔgolD/ΔuhpT) mutants were not defective for in vitro growth when compared to WT L. monocytogenes in LSM with glucose as the sole carbon source (Fig 1B). In contrast, when ΔglpD/ΔgolD or ΔuhpT mutants were supplied with glycerol and hexose phosphates, respectively, they were unable to grow while WT L. monocytogenes sustained growth (Fig 1E). Mutants lacking only the hexose phosphate transporter (ΔuhpT) showed sustained growth in LSM with glycerol alone, but they were only able to growth modestly in LSM with glycerol and hexose phosphates (Fig 1C and 1E). Finally, a mutant defective for glycerol and hexose phosphate (ΔglpD/ΔgolD/ΔuhpT) utilization was fully capable of growing in defined media with glucose as the carbon source but was unable to grow on glycerol, hexose phosphates, or glycerol/hexose phosphates combined when compared to WT L. monocytogenes (Fig 1B–E). Taken together, these data demonstrate that there are not additional unknown glycerol or hexose-phosphate utilization pathways and that the ΔglpD/ΔgolD/ΔuhpT mutant is incapable of growing on glycerol and hexose phosphates as primary carbon sources. Similarly, the ΔglpD/ΔgolD/ΔuhpT mutant shows no growth defects when supplied with glucose as its sole carbon source.

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Fig 1. Mutants defective for consumption of glycerol (ΔglpD/ΔgolD) and/or hexose phosphates (ΔuhpT) require alternative carbon sources to support growth in a defined medium.

(A) Simplified model of glycerol being imported and funneled into two parallel glycerol utilization pathways (glpD and golD) for entry into central catabolic glycolysis. Hexose phosphate imported and funneled into the anabolic pentose phosphate pathway. Indicated strains were grown in LSM at 37°C, shaking at 250 r.p.m. with the addition of 55mM glucose (B) or carbon equivalent amounts of glycerol (C), hexose phosphates (+10mM glutathione) (D), and glycerol and hexose phosphates (+10mM glutathione) (E). OD600 was monitored every 15 minutes for 24 hours. Data represents average of three technical replicates from one representative of three biological replicates.

https://doi.org/10.1371/journal.ppat.1012492.g001

ΔglpD/ΔgolD/ΔuhpT L. monocytogenes mutants unable to use hexose phosphates and glycerol replicate in macrophages and are attenuated in vivo

Glycerol and hexose phosphates have been demonstrated via isotopologue metabolomic flux analysis to be utilized by L. monocytogenes during intra-macrophage replication [30,31]. We hypothesized that mutants deficient in both pathways would be attenuated for virulence. To determine if glycerol, hexose phosphates, or both were necessary for cytosolic replication of L. monocytogenes we performed intra-macrophage growth curves in murine bone marrow-derived macrophages (BMDMs). Intracellular growth curves in BMDM demonstrated that individual (ΔglpD/ΔgolD and ΔuhpT) and combined (ΔglpD/ΔgolD/ΔuhpT) metabolic mutants unable to use glycerol and/or hexose phosphates were still able to grow in the host cytosol with kinetics like WT L. monocytogenes (Fig 2A). This data suggests that in a single cycle infection in primary BMDMs, L. monocytogenes does not require glycerol or hexose phosphate to support cytosolic replication. Further, this data suggests that L. monocytogenes must be able to use alternate undefined carbon source(s) to support cytosolic growth.

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Fig 2. Mutants defective for metabolism of glycerol (ΔglpD/ΔgolD) and/or hexose phosphates (ΔuhpT) are readily able to grow in the host cytosol and maintain virulence.

(A) Intracellular growth of WT, ΔglpD/ΔgolD, ΔuhpT, ΔglpD/ΔgolD/ΔuhpT was determined in BMDMs following infection at an MOI of 0.2. Growth curves are representative of at least three independent experiments. Error bars represent the standard deviation of the means of technical triplicates within the representative experiment. (B) L2 fibroblasts were infected with indicated L. monocytogenes strains at an MOI of 0.5 and were examined for plaque formation 4 days post infection. Assays were performed in biological triplicate and data displayed is the median and SEM of a strain’s plaque size relative to WT in one of three representative biological replicates. (C) Bacterial burdens from the spleen and liver were enumerated at 48 hours post-intravenous infection with 1x105 bacteria. Data are representative of results from two separate experiments. Horizontal dashed lines represent the limits of detection, and the bars associated with the individual strains represents the mean and SEM of the group.

https://doi.org/10.1371/journal.ppat.1012492.g002

In vivo, L. monocytogenes not only replicates in the primary infected cell but must spread to neighboring cells using ActA mediated actin-based motility [22]. This intracellular replication and spread is modeled ex vivo using a plaquing assay [59,60]. Mutants with replication defects, impaired cytosolic survival, or defects in actin-based motility and secondary vacuole escape all produce small plaques as this assay measures both replication and cell-to-cell spread over multiple infectious cycles and days. Thus, plaque assays can further elucidate minor defects due to their more stringent conditions. To further test the hypothesis that glycerol and hexose phosphates contribute to cytosolic replication and cell-to-cell spread, we measured the plaque sizes of the metabolic mutants relative to WT L. monocytogenes. Hexose phosphate acquisition deficient L. monocytogenes (ΔuhpT) formed statistically significantly smaller plaques (Fig 2B). In contrast, mutants defective for glycerol utilization (ΔglpD/ΔgolD) generated plaques indistinguishable from WT. L. monocytogenes. Finally, a mutant defective for both glycerol and hexose phosphate utilization (ΔglpD/ΔgolD/ΔuhpT) phenocopied a hexose phosphate mutant (ΔuhpT) alone suggesting no additive role for glycerol in the context of multicycle infection and cell to cell spread in fibroblasts (Fig 2B). This data indicates that while glycerol utilization is dispensable in single cycle infections in BMDMs and plaque multi-cycle plaque assays, hexose phosphates contribute to L. monocytogenes fitness in the context of multi-cycle infections and/or cell-to-cell spread, but not single cycle BMDM growth curves.

Given the apparent role for hexose phosphates in multicycle infections in the plaquing assay, we wanted to test the hypothesis that glycerol and hexose phosphates would be required for virulence in vivo. Mutants with replication and survival defects will sometimes show more robust virulence defects in murine models compared to ex vivo assays given the more restrictive physiology and multicycle infectious nature [61]. To test this hypothesis, we utilized a murine disseminated listeriosis model and assessed virulence via bacterial burdens in the spleen and liver 48 hours after intravenous infection (Fig 2C). Consistent with results from the intracellular growth curves and the plaquing assay, glycerol mutants (ΔglpD/ΔgolD) were not statistically significantly attenuated, although there was a trend towards lower bacterial burdens 48 hours post infection. Additionally, hexose phosphate mutants (ΔuhpT) showed statistically significant attenuation in both organs with approximately 1.5-2 logs of virulence reduction. Finally, failure to use glycerol in addition to hexose phosphates (ΔglpD/ΔgolD/ΔuhpT) resulted in more significant attenuation than hexose phosphate mutants alone (ΔuhpT) in the spleen, while a similar trend was observed in the liver (Fig 2C). Taken together this data suggests that hexose phosphates, and not glycerol, are essential for multi-cycle replication ex vivo and full virulence during in vivo infection. Importantly, even though a mutant defective for utilization of both glycerol and hexose phosphates is attenuated in vivo relative to wild-type L. monocytogenes, it is unexpected that it can grow the host cytosol, productively invade neighboring cells, and maintain significant bacterial burdens in a murine model. These observations suggest that additional, yet to be defined, carbon source(s) are major contributors to replication and virulence of L. monocytogenes in vivo.

BioLog phenotype microarray screening reveals WT and PrfA* (G145S) L. monocytogenes equivalently respire on PTS mediated carbon sources

The unexpected results that ΔglpD/ΔgolD/ΔuhpT L. monocytogenes can grow the host cytosol and maintain virulence in a murine model led us to hypothesize that L. monocytogenes must use additional carbon sources during infection. To identify potential metabolites used by L. monocytogenes that could support cytosolic growth, we employed BioLog’s phenotypic carbon microarrays (PM1 and PM2A) to screen for differential carbon source respiration between WT and PrfA* (G145S) L. monocytogenes [25]. PrfA* mutants contain a G145S mutation in the virulence regulator PrfA that results in constitutive virulence gene expression, including upregulation of uhpT for the use of hexose phosphates [23–25]. We hypothesized that PrfA* (G145S) L. monocytogenes may differentially use carbon sources relative to WT L. monocytogenes, similar to its differential use of hexose phosphates, which could reveal targets used to support cytosolic growth. Inoculation and setup of phenotypic microarray plates were performed as previously described, assays were performed in triplicate and plates were incubated at 37° stationary for 48 hours [62,63]. At 48 hours post-inoculation, plates were assessed for change in tetrazolium dye color, indicating bacterial respiration on the carbon source. OD490 values were normalized to glucose, a carbon source known to be used by both strains, for each respective strain to account for potential global metabolic variance between strains. 190 total carbon sources were assessed for use by L. monocytogenes relative to glucose respiration (Fig 3 and Tables 1 and 2). WT L. monocytogenes was able to use 51 carbon sources at or above the level of glucose, of which, 35 are hypothesized to be available in the host cytosol according to the human metabolome database [64]. PrfA* was able to consume all these same carbon sources and had significantly increased respiration on hexose phosphates as expected based on its known upregulation of uhpT (Fig 3 and Table 1). PrfA* also showed a statistically significant decreased respiration on a single PTS mediated carbon source, α-D-Lactose. Notably, PrfA* L. monocytogenes had overall similar use of most carbon sources, including PTS mediated carbon sources (Fig 3 and Table 1). The glycerol and hexose phosphate (ΔglpD/ΔgolD/ΔuhpT) mutant was similarly tested using PM1 and PM2A and phenocopied WT L. monocytogenes except for the loss of the ability to respire glycerol and α-Methyl-D-Glucoside (Fig 3 and S4 Tables and 2). Contrary to our hypothesis that PrfA* mutants would utilize metabolites differentially relative to WT L. monocytogenes, the only metabolites utilized more readily in PrfA* L. monocytogenes were hexose phosphates. Nevertheless, we were surprised to find that PrfA* L. monocytogenes readily utilized PTS-dependent carbon sources nearly identically to WT L. monocytogenes. This was striking given the established model in the field that PTS are not used during infection. However, PrfA* L. monocytogenes using PTS-mediated carbon sources is not mutually exclusive to the existing literature, in which, PTS transcripts are repressed during PrfA activation and use of PTS-mediated carbon sources’ reciprocally repress prfA expression [42,54].

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Fig 3. Carbon metabolite respiration of WT, ΔglpD/ΔgolD/ΔuhpT, and PrfA* L. monocytogenes is globally similar, identified using Biolog’s Phenotypic Microarrays.

Clustered heatmaps indicating level of tetrazolium dye color change as measured by OD490 at 48 hours in response to ΔglpD/ΔgolD/ΔuhpT (Top), WT (Middle), and PrfA* (Bottom) respiration of carbon metabolites (PM1 & PM2A) at 37°C stationary. Each bar indicates the average of 3 biologic replicates. Samples were normalized to readings of a α-D-glucose control (~1 on scale and labeled) and sorted based on cluster analysis. Select differentially used metabolites from Tables 1 and 2 of hexose phosphates, glycerol, and lactose are labeled above.

https://doi.org/10.1371/journal.ppat.1012492.g003

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Table 1. Statistically significant and greater than 2-fold differentially used metabolites between WT and PrfA* L. monocytogenes.

https://doi.org/10.1371/journal.ppat.1012492.t001

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Table 2. Statistically significant and greater than 2-fold differentially used metabolites between WT and ΔglpD/ΔgolD/ΔuhpT L. monocytogenes.

https://doi.org/10.1371/journal.ppat.1012492.t002

PTS are necessary for intracellular L. monocytogenes growth and virulence in vivo

Based on the similar utilization of PTS-mediated carbon sources between WT and PrfA* L. monocytogenes and the incomplete attenuation of the ΔglpD/ΔgolD/ΔuhpT mutant, we hypothesized that PTS-dependent sugars could be an alternative carbon source during infection. The L. monocytogenes strain 10403s used in this study encodes 29 complete PTS systems encoded by 86 genes making it difficult to test individual PTS for importance during infection [44]. Therefore, to test the hypothesis that PTS contribute to L. monocytogenes virulence we instead targeted the universally conserved phosphocarrier proteins essential for the function of all PTS, HPr (ptsH) and EI (ptsI) (Fig 4A). ΔptsI mutants were constructed in WT and ΔglpD/ΔgolD/ΔuhpT L. monocytogenes backgrounds to assess the relative contribution of hexose phosphates and glycerol vs PTS-dependent metabolites during cytosolic growth and virulence. To assess the role of PTS in cytosolic growth we infected primary murine BMDMs with WT, ΔglpD/ΔgolD/ΔuhpT, ΔptsI, and ΔglpD/ΔgolD/ΔuhpT/ΔptsI L. monocytogenes. While ΔglpD/ΔgolD/ΔuhpT and WT L. monocytogenes were able to replicate in the macrophage cytosol as previously demonstrated (Figs 2B and 4B), PTS deficient strains were completely unable to replicate, independent of the presence or absence of glycerol and hexose phosphate utilization pathways. Taken together these data demonstrate that PTS-dependent metabolites are both necessary and sufficient to support replication in the cytosol of BMDMs.

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Fig 4. ΔptsI mutants are impaired for intramacrophage growth and virulence, with more decreased virulence in a ΔglpD/ΔgolD/ΔuhpT background and can be trans-complemented with ptsI over expression for intracellular growth and virulence.

(A) PTS mediated free sugar import and phosphorylation by phosphocarrier protein phospho-cycling from the terminal conversion of phosphoenol-pyruvate (PEP) to pyruvate. (B) Intracellular growth of WT, ΔglpD/ΔgolD/ΔuhpT, ΔptsI, and ΔglpD/ΔgolD/ΔuhpT/ΔptsI was determined in BMDMs following infection at an MOI of 0.2. Growth curves are representative of at least three independent experiments. Error bars represent the standard deviation of the means of technical triplicates within the representative experiment. (C) L2 fibroblasts were infected with indicated L. monocytogenes strains at an MOI of 0.5 and were examined for plaque formation 4 days post infection. Assays were performed in biological triplicate and data displayed is the median and SEM of a strain’s plaque size relative to WT in one of three representative biological replicates. (D) Bacterial burdens from the spleen and liver were enumerated at 48 hours post-intravenous infection with 1x105 bacteria. Data are representative of results from two separate experiments. Horizontal dashed lines represent the limits of detection, and the bars associated with the individual strains represents the mean and SEM of the group.

https://doi.org/10.1371/journal.ppat.1012492.g004

The striking lack of cytosolic replication of PTS deficient L. monocytogenes in BMDMs, led us to ask whether PTS systems were similarly required for replication and cell-to-cell spread in more protracted multi-cycle infections by performing a plaquing assay in L2 fibroblasts. As previously demonstrated, ΔglpD/ΔgolD/ΔuhpT mutants had a statistically significant plaquing defect relative to WT L. monocytogenes; however, in contrast to the BMDM growth phenotype of PTS deficient mutants, L. monocytogenes strains lacking only ptsI demonstrated no plaquing defects (Fig 4C). Combining PTS deficiency with glycerol and hexose phosphate deficiency led to a complete loss of plaque formation (Fig 4C). Taken together these data suggest that while PTS are absolutely required for replication in the macrophage cytosol, PTS are only conditionally required in fibroblasts when glycerol and hexose phosphates are not available. These data suggest that there are differences in nutrient availability in different cell types, and further, that L. monocytogenes uses metabolic flexibility to grow in diverse cell types.

Given the differential requirements of PTS in macrophages versus fibroblasts ex vivo, we wanted to determine the role of PTS during in vivo infection in a murine disseminated listeriosis model. As we previously demonstrated, ΔglpD/ΔgolD/ΔuhpT mutants had 10–100-fold lower bacterial burdens relative to wild type L. monocytogenes 48 hours post infection (Fig 4D). In contrast, ΔptsI mutants were 500-fold and 5000-fold attenuated in the spleens and livers of infected mice respectively (Fig 4D). Furthermore, loss of PTS function in the absence of glycerol and hexose phosphate utilization led to an additive virulence defect whereby the ΔglpD/ΔgolD/ΔuhpT/ΔptsI mutants were more attenuated than the ΔglpD/ΔgolD/ΔuhpT mutants or the ΔptsI mutants alone (Fig 4D). Intra-macrophage growth, cell-to-cell spread, and virulence in vivo were proportionally rescued with constitutive overexpression trans-complementation of ptsI (Fig 4C and 4D). Together this data suggests that PTS mediated carbon acquisition is an important contributor to in vivo virulence. Furthermore, the additive attenuation of the ΔglpD/ΔgolD/ΔuhpT/ΔptsI mutant relative to ΔptsI mutant alone demonstrates that glycerol, hexose phosphates and PTS mediated carbon sources are all significant contributors to L. monocytogenes virulence in vivo.

Virulence defects of ΔglpD/ΔgolD/ΔuhpT and ΔptsI mutants are not due to lack of PrfA activation

Given the intertwined nature of L. monocytogenes’ metabolism and its virulence gene regulation it is possible that all or part of ΔglpD/ΔgolD/ΔuhpT and ΔptsI virulence defects could be attributed to virulence gene mis-regulation. To test the hypothesis that ΔglpD/ΔgolD/ΔuhpT and ΔptsI are attenuated due to incomplete PrfA activation, a constitutively active PrfA* (G145S) allele was cloned in situ into WT, ΔglpD/ΔgolD/ΔuhpT and ΔptsI L. monocytogenes. These strains [PrfA* (G145S), ΔglpD/ΔgolD/ΔuhpT::PrfA* (G145S) and ΔptsI::PrfA* (G145S)] were compared to each other and their isogenic counterparts (WT, ΔglpD/ΔgolD/ΔuhpT and ΔptsI) in a murine burden assay. As previously demonstrated, ΔglpD/ΔgolD/ΔuhpT mutants display a 1–2 log defect in both the spleen and liver, while ΔptsI mutants were 500-fold and 5000-fold attenuated in the spleens and livers of infected mice respectively (Fig 5A). Meanwhile, PrfA* (G145S), ΔglpD/ΔgolD/ΔuhpT::PrfA* (G145S), and ΔptsI::PrfA* (G145s) strain showed no restoration of virulence compared to WT L. monocytogenes and no statistically significant benefit compared to their isogenic counterparts (Fig 5A). Taken together this suggests that defective PrfA-regulon expression does not account for the virulence defects observed in ΔglpD/ΔgolD/ΔuhpT and ΔptsI L. monocytogenes and supports that these virulence defects are due to metabolic deficiencies.

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Fig 5. ΔglpD/ΔgolD/ΔuhpT and ΔptsI mutants are not rescued by constitutively active PrfA and show basally higher levels of virulence protein activity.

(A) Bacterial burdens from the spleen and liver were enumerated at 48 hours post-intravenous infection with 1x105 bacteria. Data are representative of results from two separate experiments. Horizontal dashed lines represent the limits of detection, and the bars associated with the individual strains represents the mean and SEM of the group. (B) Median and SEMs of percent hemolysis interpolated at OD600 of 0.025 from biological triplicate for displayed strains. (C) Representative toxin dose-response curves from a single biological replicate used for interpolation and generation of continuous percent hemolysis data as a function of OD600.

https://doi.org/10.1371/journal.ppat.1012492.g005

Overall, the lack of rescue associated with the PrfA* (G145S) allele in a ΔptsI mutant was consistent with prior literature showing that disruption of PTS should result in increased PrfA-regulon expression and terminal activity [54]. In contrast, based on the literature suggesting that PTS function is inversely related to PrfA activation, it was somewhat surprising that ΔglpD/ΔgolD/ΔuhpT L. monocytogenes, a strain dependent on PTS for virulence (Fig 4C and 4D), showed no benefit from the PrfA* (G145S) allele [23,32,54]. To assess PrfA activation in our metabolic mutants we assessed Listeriolysin-O (LLO)-dependent hemolysis of sheep red blood cells (RBCs), an established surrogate of PrfA activity [65], using supernatants from ΔglpD/ΔgolD/ΔuhpT and ΔptsI L. monocytogenes compared to WT as previously described [65]. Strains were grown overnight at 37°C shaking to overcome RNA thermosensor repression of PrfA, supernatants harvested, serially diluted and incubated with sheep RBCs, and hemoglobin release quantified via OD420 [65,66]. A standard curve of detergent driven hemolysis was used to calculate percent hemolysis across dilutions of each strain in each biological replicate. From these values, percent hemolysis was interpolated as a function of OD600 for each strain and the resultant equation was used to calculate percent hemolysis at an OD600 of 0.025. An OD600 of 0.025 was selected as it was where ~50% hemolysis occurred in our experimental strains. Hemolysis from the supernatants of ΔglpD/ΔgolD/ΔuhpT and ΔptsI L. monocytogenes showed significantly more activity than that of WT L. monocytogenes (Fig 5B). This was further visualized and verified by left shift of toxin dose-response curves from a single representative biological replicate relative to WT L. monocytogenes (Fig 5C). Taken together these data indicate that both ΔglpD/ΔgolD/ΔuhpT and ΔptsI L. monocytogenes have excess LLO activity, and therefore PrfA activity, relative to WT. Further this demonstrates why PrfA* alleles did not rescue virulence of these strains and indicates that the primary defect of these mutants is metabolic in nature.

Loss of HPr (ptsH) Phenocopies loss of EI (ptsI)

To further confirm that the loss of virulence in the ΔptsI mutant was due to loss of function of PTS and not another unknown function of EI, we created deletion mutants in the other conserved essential phosphocarrier protein HPr (ptsH). HPr is known to have a secondary role in genetic regulation via CcpA through phosphorylation by HPr kinase, therefore we hypothesized that ΔptsH should, at minimum, phenocopy ΔptsI, with the potential for further attenuation. Consistent with PTS being essential for growth in the macrophage cytosol, ΔptsH mutants were unable to replicate in BMDM alone or when combined with hexose phosphate and glycerol (ΔglpD/ΔgolD/ΔuhpT/ΔptsH) mutants (Fig 6A). Similarly, ΔptsH mutants were significantly attenuated for virulence in vivo and when combined with glycerol and hexose phosphate mutants, ΔglpD/ΔgolD/ΔuhpT/ΔptsH mutants were essentially avirulent in vivo (Fig 6B). Taken together, these data suggest that PTS are major contributors to virulence of L. monocytogenes in vivo and that a loss of PTS combined with loss of glycerol and hexose phosphate utilization leads to almost complete attenuation of L. monocytogenes in vivo.

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Fig 6. ΔptsH mutants are more attenuated for intracellular growth and virulence than in ΔptsI in all backgrounds.

(A) Intracellular growth of wild-type, ΔglpD/ΔgolD/ΔuhpT, ΔptsH, and ΔglpD/ΔgolD/ΔuhpT/ΔptsH was determined in BMDMs following infection at an MOI of 0.2. Growth curves are representative of at least three independent experiments. Error bars represent the standard deviation of the means of technical triplicates within the representative experiment. (B) Bacterial burdens from the spleen and liver were enumerated at 48 hours post-intravenous infection with 1x105 bacteria. Data are representative of results from two separate experiments. Horizontal dashed lines represent the limits of detection, and the bars associated with the individual strains represents the mean and SEM of the group.

https://doi.org/10.1371/journal.ppat.1012492.g006

Discussion

Mechanisms of carbon acquisition, catabolism, and anabolism by cytosolic pathogens remain incompletely defined, but are vitally important virulence factors in driving pathogenesis. A mechanistic understanding of bacterial metabolism during infection can help identify novel anti-microbial targets and host targeted metabolic interventions. Despite L. monocytogenes being both an important pathogen and a model organism that has been studied for decades, we still have a relatively limited understanding of its metabolism during infection. In this work, we demonstrated that although hexose phosphate and glycerol contribute to L. monocytogenes infection in vivo, they are dispensable for cytosolic replication in macrophages in contrast to previous suggestions from isotopologue metabolomics. Further, combining an unbiased screen using BioLog’s Carbon Phenotypic Microarrays (PM1 and PM2A) with genetic deletions of conserved PTS phosphocarrier proteins, we demonstrate that PTS are essential for replication in the macrophage cytosol and are critical for virulence in vivo. We then separated L. monocytogenes’ metabolism from virulence gene regulation using a PrfA* (G145S) allele in the ΔglpD/ΔgolD/ΔuhpT and ΔptsI backgrounds and found no rescue of in vivo virulence and further demonstrate that ΔglpD/ΔgolD/ΔuhpT and ΔptsI mutants have increased LLO mediated hemolytic activity relative to WT L. monocytogenes. Together our data suggest that L. monocytogenes is utilizing a previously underappreciated and more diverse metabolic strategy to replicate in the cytosolic environment and potentiate infection. These findings illuminate a generally overlooked contributor to virulence, PTS, and point to a system not previously identified as necessary for successful intracellular growth and virulence.

Despite PTS’ broad and well-defined role in carbon acquisition and previous demonstrated roles in the growth of Shigella flexneri and Streptococcus pyogenes during infection, they have largely been viewed as either minor contributors or in some cases active inhibitors of bacterial pathogenesis more broadly [40,42,44,52,54,67–69]. In L. monocytogenes, work demonstrating that PTS activity represses prfA expression lead to a widespread and often repeated assumption that PTS are not used during infection, as virulence gene repression was considered detrimental to pathogenesis [39,44,45,56]. Nevertheless, our findings demonstrate that a ΔglpD/ΔgolD/ΔuhpT mutant L. monocytogenes with presumed obligate use of PTS, can still readily cause infection [32,54]. Interestingly, this mutant even showed increased LLO-mediated hemolysis relative to WT L. monocytogenes, however the mechanism by which this is mediated remains unclear. These results indicate that more must be done for the field to understand how more specific metabolic mutants’, such as ΔuhpT or metabolite-specific PTS mutants, regulate their virulence gene expression through PrfA. Our results demonstrate that over reliance on PTS, and consequential PrfA regulon repression is not the driver of virulence defects of ΔglpD/ΔgolD/ΔuhpT L. monocytogenes observed in vivo as a PrfA* (G145S) allele does not rescue in vivo virulence. Like ΔglpD/ΔgolD/ΔuhpT, ΔptsI mutant L. monocytogenes shows elevated PrfA-regulon activity and are not rescued by PrfA* (G145S) allele. Interestingly while not statistically significant, ΔptsI mutant L. monocytogenes subjectively appear harmed by PrfA-regulon over expression. We hypothesize this may be due to the substantial metabolic demand of constitutive PrfA activity, hexose phosphate toxicity as described in other organisms, or some unknown regulatory effects of PrfA* on bacterial metabolism [70]. One remaining question is why L. monocytogenes would use this complex metabolic strategy utilizing such a wide array of carbon sources including those with potentially negative impacts on virulence gene expression? We hypothesize that L. monocytogenes may be using a balance of carbon sources to optimize virulence gene expression while maximizing carbon acquisition for metabolism and growth potential while avoiding host cell detection. One indicator in our data that this hypothesis might be correct is that ΔglpD/ΔgolD/ΔuhpT mutants show mild declines during intra-macrophage growth and virulence defects in plaque assays and murine burdens, This strain may be attenuated later during its intracellular life cycle due to starvation from depletion of host metabolites through PTS, resultant host cell death, bacterial physiologic defects, or even host cell detection and elimination. As such, we conclude the phenotypes uncovered through these studies represent the relative metabolic insufficiencies due to failure of glycerol, hexose phosphates, and PTS mediated carbon source acquisition and use in the host.

Bacterial PTS encode two phosphocarrier proteins that are necessary for the function of all PTS regardless of sugar: PtsI (EI) and PtsH (HPr) [43]. Despite these two proteins sharing universal roles in PTS carbon acquisition, they have diverse functions, and per our results, phenotypes. ΔptsI L. monocytogenes are defective for only carbon acquisition through PTS as PtsI has no described role as a transcriptional regulator. In contrast, HPr (ptsH) has been shown previously to not only impact carbon acquisition through PTS but also to play a central role in transcriptional regulation of virulence through the phosphorylation of the HPr-Ser-46 residue [54,69]. The HPr-Ser residue is phosphorylated by HPr Kinase (HprK) dependent on upper glycolytic flux and the abundance of fructose-1,6-bisphosphate and ATP. Importantly, these functions are not linked to lower glycolytic PEP to pyruvate conversion or PtsI (EI) function [68]. The exact mechanism by which HPr-Ser-P enacts this repression is unknown [42,53]. Consistent with this, ΔptsH (HPr) mutants in the WT and ΔglpD/ΔgolD/ΔuhpT L. monocytogenes backgrounds show decreased virulence relative to those of ΔptsI in an isogenic background suggesting that that the transcriptional dysregulation due to loss of ptsH is key to the additional virulence defects. Our favored hypothesis is that ΔptsH L. monocytogenes is failing to modulate expression of genes necessary for alternate carbon source acquisition and/or deal with stresses unique to the host cytosol and these functions are retained in ΔptsI. For example prior work has shown that HprK mediated phosphorylation of CcpA plays an important role in hierarchal carbon source utilization and stress responses in L. monocytogenes [71,72]. Because the exact transcriptional changes mediated by HPr-Ser-P are not well defined, it would be valuable to test how phospho-ablative and -mimetic HPr-Ser and HPr-His mutants behave differently through virulence assays and transcriptomic profiling. Overall, PtsI (EI) and PtsH (HPr) have separate but overlapping, functions that once characterized could unveil how and why bacteria connect the function of PTS to gene expression.

Previous isotopologue analysis by the Eisenreich and Goebel groups have shown that L. monocytogenes uses hexose phosphates and glycerol to support its cytosolic growth [29–33,73]. These carbon substrates are logical carbon sources for an intracellular pathogen such as L. monocytogenes because not only does L. monocytogenes have dedicated transporters for these carbon sources but, equally importantly, they are available in the host cytosol [29,32]. Nearly all sugar that is brought into eukaryotic cells is phosphorylated to prevent backward diffusion to the extracellular space. Once a sugar is phosphorylated it has two primary fates: 1. Funneling into glycolysis. 2. De-phosphorylation for use as a moiety/metabolite component in more complex forms, such as glycogen in the liver [74–76]. Nevertheless, for PTS to be essential for L. monocytogenes growth in the cytosol, free sugars (monomers or polymers) must be present in abundance to support bacterial growth. Consistent with existing literature that PTS cannot facilitate transport of phosphorylated sugars, the ΔglpD/ΔgolD/ΔuhpT mutant relying on PTS was unable to grow hexose phosphates in vitro but retained ability to grow in the host cytosol. Therefore, L. monocytogenes must have access to unphosphorylated sugar in the cytosol. Whether these unphosphorylated, free sugars are liberated by the host or bacteria during the course of L. monocytogenes infection remains an unknown and critical component in understanding the use of PTS. One hypothesis is that L. monocytogenes is using a yet to be defined phosphatase to dephosphorylate and liberate free sugars. These putative phosphatases could represent high value targets for the development of antivirulence based antimicrobials [77]. Alternatively, host cells could attempt to dephosphorylate sugars to allow sugar diffusion in an act of nutritional immunity, a process that L. monocytogenes might have evolved to take advantage of. Ultimately, understanding how L. monocytogenes accesses free sugars in the cytosol of an infected cell could inform whether other cytosolic pathogens could have access to and use similar carbon sources during infection.

Answering what specific PTS and corresponding carbon source might be used by L. monocytogenes is extremely challenging given the intertwined nature of the L. monocytogenes-macrophage metabolism and the redundancy of PTS systems in the L. monocytogenes genome [78]. Importantly, while our work shows that the carbon acquisition function of PTS is an important contributor to L. monocytogenes cytosolic growth, we do not know specifically what PTS mediated sugars might be consumed. Because of the diverse arsenal of PTS encoded by L. monocytogenes, it is possible that there are multiple redundant carbon sources consumed by L. monocytogenes in the cytosol [44]. Additionally, our observation that L. monocytogenes PTS mutants have unique host cell and organ specific phenotypes suggests the presence of divergent metabolites within host cells that L. monocytogenes might be consuming. For example, ΔptsI mutants show very different phenotypes in BMDMs where no growth occurs compared to the L2 fibroblast plaquing assay where ΔptsI mutants are indistinguishable from wild type L. monocytogenes. Similarly, there are subjectively different bacterial burdens in spleens versus liver for ΔptsI during in vivo murine infection. Comparison of these growth conditions through advancing techniques in metabolomics may unveil the metabolic underpinning of these phenotypes. Identifying what specific carbon sources L. monocytogenes acquires from the host via PTS may unveil unique metabolic strategies to avoid host cell detection and support cytosolic bacterial physiology.

It is challenging to develop a complete picture of cytosolic pathogen metabolism given the ill-defined composition of the host cytosol, challenging technical methods, inherent metabolic redundancy, and diverse environments encountered in the host in different tissues. Our work demonstrates for the first time that PTS are essential for L. monocytogenes cytosolic growth and critical for virulence in contrast with the current dogma in the field [39,45,56]. These highly conserved and redundant systems are understudied, and therefore their role in carbon acquisition by bacterial pathogens within the host cell is not well understood. Further work is needed to elucidate if PTS are critical for the cytosolic growth of other pathogens. Additionally, more work is needed to identify the preferred sugars for cytosolic pathogens and how these sugars are liberated. Finally, how differential carbon source availability across cell types, organ systems and even host species impacts bacterial pathogenesis remains to be elucidated.

Methods

Ethics statement

All animal-based experiments were performed using the protocol (M005916-R01-A01) approved by the Animal Use and Care Committee of the University of Wisconsin—Madison and consistent with the standards of the National Institutes of Health.

Bacterial strains and culture

All Listeria monocytogenes strains used for experiments in this study were in a 10403s background. All L. monocytogenes strains were grown overnight in BHI and at 30°C stationary for all experiments, except as described. Escherichia coli strains were grown in Luria broth (LB) at 37°C shaking. Antibiotics used on E. coli were at a concentration of 100 µg/ml carbenicillin or 30 µg/ml kanamycin when appropriate. Antibiotics used on L. monocytogenes were at a concentration of 200 μg/mL streptomycin and/or 10 μg/mL chloramphenicol, when appropriate. Plasmids were transformed into chemically competent E. coli and further conjugated in L. monocytogenes using SM10 or S17 E.coli.

Construction of strains

pLIM (from Arne Rietsche at Case Western) suicide plasmid or pKSV7 plasmid was used for generation of in frame deletions [61,79]. The pPL2 integrative vector pIMK2 was used for constitutive expression of L. monocytogenes genes [80]. pLIM and pKSV7 knockout constructs were cloned in XL1-Blue E. coli with 100 µg/ml carbenicillin (30μg/mL Kanamycin for pIMK2) and grown for plasmid harvest using Promega MiniPrep Kit. Harvested plasmid sequences were confirmed using was performed by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation. Plasmid were then shuttled into L. monocytogenes through conjugation with SM10 (pLIM1 and pKSV7) or S17 (pIMK2) E. coli. In-frame deletions of genes in L. monocytogenes were performed by allelic exchange using suicide plasmid pLIM as previously described with p-chlorophenylalanine as a counter selectable marker [81]. De novo PrfA* (G145S) allele introduction was performed by amplifying the PrfA* allele and flanking regions and assembling into pLIM [25]. Following initial homologous recombination, enrichment of PrfA* (G145S) mutants was by performed by passaging merodiploid strains at 30°C shaking in selective Listeria Synthetic Media (LSM) with 55mM glucose-6-phosphate without glutathione (Sigma-Aldrich G7879) and plated on p-chlorophenylalanine as a counter selectable marker. Generated strains were frozen in 50:50 (glycerol:overnight culture) solution at -80°C. All mutants were confirmed via PCR, plasmid sequencing, and whole-genome sequencing using Oxford Nanopore technology from Plasmidsaurus with custom analysis and annotation.

In vitro growth assays

Bacteria were grown overnight in BHI at 30°C stationary. Overnight cultures were used to generate inoculums with ~3.7x108 CFU in PBS. 100 µLs per well of a flat bottom clear 96-well plate of Listeria synthetic media (LSM) with carbons source (supplied in 55 mM glucose carbon equivalents) was inoculated with 2 µL of inoculums. Plates were parafilmed on the edge to prevent evaporation and evaluated for OD600 in plate reader at 37°C shaking (250 r.p.m.) and reads every 15 minutes for times displayed.

Intra-macrophage growth curves

Bone marrow-derived macrophages were isolated from CL57/BL6 mice and cultured as previously described in Roswell Park Memorial Institute Medium (RPMI) based media (Invitrogen: 11875093) [82]. BMDMs were plated into 60 mm dishes contain 13 degassed coverslips. BMDMs cells were infected with L. monocytogenes strains at a multiplicity of infection [MOI] of 0.2. Inoculums of L. monocytogenes were grown in 3mL of BHI at 30°C stationary until all strains had reach stationary phase. Colony forming units to OD600 ratios were determined for each strain and adjusted to ensure infection results in a comparable MOI across strains. After 30 minutes BMDM media was exchanged for media containing 50 µg/ml Gentamycin. Coverslips were harvested, cells lysed in pure water, bacteria rescued isotonically, and plated to quantify CFU at displayed time points. All strains were assayed in biological triplicate and data displayed is one representative biologic replicate.

Plaque assay

Plaque assays were conducted using a L2 fibroblast cell line grown in Dulbecco’s Minimal Essential Media (DMEM) based media (Thermo Fischer: 11965092) as previously described with minor modifications for visualization and quantification of plaques [59,60]. L2 fibroblasts were seeded at 1.2 × 106 per well of a 6-well plate, then infected at an MOI of 0.5 to obtain approximately 10–30 PFU per dish. Inoculums of L. monocytogenes were grown in 3mL of BHI at 30°C stationary until all strains had reached stationary phase. Colony forming units to OD600 ratios were determined for each strain and adjusted to ensure infection results in a comparable MOI across strains. At 4 days postinfection, cells were stained with 0.3% crystal violet for 10 min and washed twice with deionized water. Stained wells were scanned, uploaded, and areas of plaque formation were measured on ImageJ analysis software. All strains were assayed in biological triplicate and the average plaque areas of each strain (one-well per strain were normalized to wild-type plaque size within each replicate.

Murine infection and organ burdens

Infections were performed as previously described [83]. Briefly, 6–12-week-old female and male C57BL/6 mice were infected IV with 1×105 CFU logarithmically growing L. monocytogenes (optical density at 600 nm [OD600] = 0.5) in 200 µL of PBS. Colony forming units to OD600 ratios were determined for each strain and adjusted to ensure infection results in a comparable MOI across strains. 48 hours post-infection, mice were euthanized, and livers and spleens were harvested, homogenized in water with 0.1% NP-40, and plated for CFU. Two independent replicates of each experiment with 5 mice per group were performed.

Cell culture

L2 cells were all kind gifts from Daniel Portnoy (UC Berkeley) [60]. Bone marrow-derived macrophages (BMDM) were prepared from 6-to-8-week-old mice as previously described [82].

Phenotypic microarrays

Phenotype Microarrays 1 (Cat. #12111) and 2A (Cat. #12112) were obtained from BioLog (BioLog). Plates were prepared and inoculated as previously described [63,84]. OmniLog incubation was substituted with incubation at 37°C stationary. OD490 was collected for each plate at 24 and 48 hours. Data was then normalized to consumption of α-D-glucose for each strain and replicate, and averaged across triplicate. Value were clustered based on similarity using clustergrammer and plotted as a heat-map in Prism 6 [85]. Data normalized to glucose was used for statistical analysis and displayed in tables. Statistics are representative of a student’s T-test between two strains.

Hemolysis assays

Hemolysis assays were performed as described previously with minor modification to optimize assay utility and interpretation. In short, overnight cultures were grown at 37°C in a 50:50 mixture of Luria Broth (LB) and Brain Heart Infusion (BHI) to optimize growth of metabolic mutants while maintaining PrfA expression. Supernatants containing L. monocytogenes’ primary hemolytic toxin, Listeriolysin-O (LLO), were harvested by pelleting overnight cultures (15000 rcf for 1.5 minutes) and supernatants taken. These were 2-fold serially diluted with un-inoculated overnight broth 10 times to obtain robust dynamic range of sheep red blood cell (RBC) lysis. Simultaneously, a standard curve was generated by taking un-inoculated overnight broth and adding 0.1% Triton-100 and mixing in 1:1 ratio with washed 5% RBC solution and serially diluting as described above. Samples were then mixed in a 50:50 with washed 5% RBC solution and incubated for 30 minutes at 37°C stationary in round-bottom 96-well plates. After incubation, this mixture was centrifugated at 500 r.p.m. for 10 minutes to pellet unlysed RBCs. 75 µL of supernatants were transferred to a flat-bottom 96-well plate for measurement of free hemoglobin at OD420 in a plate reader. Percent hemolysis of each strains’ dilution was calculated using each biological replicates’ standard curve. Percent homolysis was then graphed as a function of OD600. Graphs for each replicate and each strain were individually interpolated using Prism 6’s (GraphPad Software) non-linear Sigmoidal, 4PL, X analysis. This provided values to fill in the sigmoidal equation and calculate percent hemolysis (‘Y’) for any given OD600 (‘X’):

The closest mid-inflection point for the majority of strains was identified (OD600 = 0.025) and input into the above equation with individual replicate values to calculate interpolated percent hemolysis for 3 replicates, averaged, and evaluated using one-way ANOVA.

Statistical analysis

Prism 6 (GraphPad Software) was used for statistical analysis of data. Means from two groups of BioLog plates were compared with unpaired two-tailed Student’s T-test. Means from more than two groups for all other assays were analyzed by one-way ANOVA test. Independently, Mann-Whitney Test was used to analyze two group comparison of non-normal data from animal experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 for all statistical tests displayed.

Supporting information

S1 Table. Bacterial strains used in this study.

Listed here are all bacterial strains used in this work. Those derived from prior works have been cited and those generated by us have been labeled under the ‘Reference’ column as ‘This Work’. Relevant strain numbers have been so that if others wish to use one of these strains in their own work it can be sourced from our strain repository.

https://doi.org/10.1371/journal.ppat.1012492.s001

(DOCX)

S2 Table. Primers used in this study.

Below are primers used to generate the complement and clean-deletion constructs in pIMK2 or pLIM and pKSV7, respectively. Restriction enzyme cut sites used for homologous overlaps for Gibson Assembly have been provided.

https://doi.org/10.1371/journal.ppat.1012492.s002

(PDF)

S3 Table. Results of BioLog PM1 and PM2A at 48 hours for WT, ΔglpD/ΔgolD/ΔuhpT, and PrfA*.

The ‘Metabolite’ column shows scientific names for all metabolites screened using BioLog Phenotypic Microarrays. Fold usage of each metabolite for each strain [WT, ΔglpD/ΔgolD/ΔuhpT, and PrfA* (G145S)] is displayed relative the α-D-Glucose usage. Finally, p-values from comparisons of average metabolite usage per strain is shown for all metabolites. Comparison data was limited to WT v.s. ΔglpD/ΔgolD/ΔuhpT and WT v.s. PrfA*.

https://doi.org/10.1371/journal.ppat.1012492.s003

(PDF)

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