Research Article

CodY-Mediated c-di-GMP-Dependent Inhibition of Mammalian Cell Invasion in Listeria monocytogenes

Ahmed M. Elbakush, Kurt W. Miller, Mark Gomelsky
George O'Toole, Editor
DOI: 10.1128/JB.00457-17

ABSTRACT

Elevated levels of the second messenger c-di-GMP suppress virulence in diverse pathogenic bacteria, yet mechanisms are poorly characterized. In the foodborne pathogen Listeria monocytogenes, high c-di-GMP levels inhibit mammalian cell invasion. Here, we show that invasion is impaired because of the decreased expression levels of internalin genes whose products are involved in invasion. We further show that at high c-di-GMP levels, the expression of the entire virulence regulon is suppressed, and so is the expression of the prfA gene encoding the master activator of the virulence regulon. Analysis of mechanisms controlling prfA expression pointed to the transcription factor CodY as a c-di-GMP-sensitive component. In high-c-di-GMP strains, codY gene expression is decreased, apparently due to the lower activity of CodY, which functions as an activator of codY transcription. We found that listerial CodY does not bind c-di-GMP in vitro and therefore investigated whether c-di-GMP levels affect two known cofactors of listerial CodY, branched-chain amino acids and GTP. Our manipulation of branched-chain amino acid levels did not perturb the c-di-GMP effect; however, our replacement of listerial CodY with the streptococcal CodY homolog, whose activity is GTP independent, abolished the c-di-GMP effect. The results of this study suggest that elevated c-di-GMP levels decrease the activity of the coordinator of metabolism and virulence, CodY, possibly via lower GTP levels, and that decreased CodY activity suppresses L. monocytogenes virulence by the decreased expression of the PrfA virulence regulon.

IMPORTANCE Listeria monocytogenes is a pathogen causing listeriosis, a disease responsible for the highest mortality rate among foodborne diseases. Understanding how the virulence of this pathogen is regulated is important for developing treatments to decrease the frequency of listerial infections in susceptible populations. In this study, we describe the mechanism through which elevated levels of the second messenger c-di-GMP inhibit listerial invasion in mammalian cells. Inhibition is caused by the decreased activity of the transcription factor CodY that coordinates metabolism and virulence.

INTRODUCTION

Listeria monocytogenes is a firmicute capable of causing listeriosis, the disease known for the highest mortality rate among foodborne pathogens. L. monocytogenes virulence components are encoded in a virulence regulon that encodes products involved in host cell invasion, intracellular replication, and bacterial spread from infected to adjacent cells. Several bacterial surface proteins contribute to the invasion of host cells, among which the internalins InlA and InlB play the major roles (1, 2). InlA binds to the E-cadherin receptor, whereas InlB binds to the hepatocyte growth factor receptor (3). Following internalization by endocytosis, bacteria escape from membrane-bound vacuoles into the host cell cytoplasm, where they replicate and subsequently invade neighboring cells via actin-based motility. In healthy individuals, infection is limited to the gastrointestinal tract. However, in people with compromised immune systems, the elderly, infants, and pregnant women, listeria can cross the gut epithelial barrier into the blood, causing systemic disease with possible complications that contribute to a case fatality rate of 20 to 25% (4). One possible avenue for decreasing mortality is to identify a means to reduce listeriosis rates in susceptible populations by decreasing the ability of listeria to infect the host.

We showed previously that L. monocytogenes strains with elevated levels of the second messenger c-di-GMP are severely impaired in human cell invasion (5), and this observation prompted us to investigate the mechanism involved. c-di-GMP, which was first described as an allosteric activator of cellulose synthase in Gluconacetobacter xylinus (6), is now known to regulate a variety of physiological processes in bacteria. Most commonly, elevated c-di-GMP levels are associated with a transition of bacteria from a single-cell, motile lifestyle to a surface-attached, biofilm lifestyle. The biofilm lifestyle is often associated with suppressed motility but increased production of exopolysaccharides, surface adhesive proteins, and/or pili (7–9). Our previous work revealed that L. monocytogenes synthesizes a unique c-di-GMP-inducible exopolysaccharide, Pss (10); however, invasion defects of the strains with elevated intracellular c-di-GMP levels (high-c-di-GMP strains) proved to be largely independent of this exopolysaccharide (5).

c-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGCs) and degraded by c-di-GMP-specific phosphodiesterases (PDEs) (7). All sequenced L. monocytogenes strains contain 3 DGCs and 3 PDEs (5). Elevated c-di-GMP levels in L. monocytogenes strains impaired in invasion were caused either by the expression of a constitutive heterologous DGC, Slr1143 (recently designated Cip1 [11]), or by the deletion of all 3 PDE genes (5). Furthermore, bioinformatics analysis of listerial genomes revealed no homologs of known c-di-GMP-binding receptors/effector proteins, beyond the activator of exopolysaccharide synthesis, PssE (5). Therefore, it remained enigmatic as to which proteins could mediate c-di-GMP-dependent invasion inhibition.

It is important to note that a negative correlation between elevated c-di-GMP levels and acute virulence is common among bacterial pathogens (7, 12). For example, higher concentrations of c-di-GMP reduce the expression levels of key virulence factors, tcdA and tcdB, in Clostridium difficile (13); diminish virulence gene expression in Borrelia burgdorferi (14); inhibit invasion by Salmonella enterica serovar Typhimurium in epithelial cells (15); decrease the expression of the cholera toxin ctxAB operon in Vibrio cholerae (16); downregulate the production of the type III secretion system and its effectors in Pseudomonas aeruginosa (17); decrease virulence gene expression in Burkholderia cenocepacia (18); and suppress intramacrophage replication and virulence of Francisella novicida in mice (19). While the phenomenon of c-di-GMP-dependent virulence suppression is common, mechanisms appear to be organism specific, and their understanding remains incomplete for most pathogens.

In our search for the cause of host cell invasion inhibition by high c-di-GMP levels in L. monocytogenes, we focused on changes in surface proteins. Once the first clues emerged, we methodically worked up the signal transduction cascades. In the process, we discovered that the entire virulence regulon in L. monocytogenes is downregulated by high c-di-GMP levels and that the virulence master regulator PrfA (1, 20) and the coordinator of metabolism and virulence, CodY (21, 22), are involved.

RESULTS

Investigation of potential causes of c-di-GMP-dependent impairment of L. monocytogenes invasion of mammalian cells.We repeated invasion assays using HT-29 human colon adenocarcinoma cells and L. monocytogenes strains with low (WT [wild type]::yhjH) and high (WT::slr and ΔpdeB ΔpdeC ΔpdeD [ΔpdeBCD] ΔpssC) c-di-GMP levels (Table 1), where the WT::yhjH strain expresses a potent Escherichia coli PDE, the WT::slr strain expresses a potent Synechocystis sp. DGC, and the ΔpdeBCD ΔpssC strain lacks all c-di-GMP-specific PDEs and, in addition, is impaired in exopolysaccharide biosynthesis (5). This assay confirmed that invasion by high c-di-GMP strains is strongly suppressed (Fig. 1A). Note that our use of two high-c-di-GMP strains diminishes the possibility that strain-specific, as opposed to c-di-GMP-specific, factors are responsible for the observed phenomenon.

View this table:
TABLE 1

Strains and plasmids used in this studya

FIG 1
FIG 1

Effects of c-di-GMP levels and codY mutation on listerial invasion of mammalian cells. (A) Impaired invasion by high-c-di-GMP strains of L. monocytogenes in HT-29 human colon carcinoma cells. Shown are values for invasion relative to the value for the WT. (B) Restoration of the prfA and inlA mRNA levels in the high-c-di-GMP strains to the levels of the wild type negates the inhibitory effect of elevated c-di-GMP levels on listerial invasion. L. monocytogenes was grown in the presence of 60 mM IPTG, where indicated. (C) The ΔcodY deletion abolishes the inhibitory effect of elevated c-di-GMP levels on listerial invasion. Shown are values for invasion relative to the value for the ΔcodY mutant. Data are from 3 biological replicates, each done in 3 technical replicates. Error bars indicate standard deviations. The average invasion efficiency for the WT strain was 0.085 ± 0.009, and the average invasion efficiency for the ΔcodY mutant was 0.010 ± 0.002. Data were analyzed by using Student's t test, with P values of <0.01 (**) and <0.05 (*) indicating statistical significance.

To gain insight into the cause of suppressed invasion, we tested the effect of c-di-GMP on the cell surface properties of listeria. First, we measured potential surface hydrophobicity changes using N-hexadecane extraction of hydrophobic proteins but found no significant difference among strains with various c-di-GMP levels (see Fig. S1 in the supplemental material). We then extracted listerial surface proteins using a number of detergents and chaotropic reagents {Tris, KSCN, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), maltoside, and SDS} in anticipation of finding readily visible differences. However, visual inspection of protein profiles on SDS-PAGE gels revealed no drastic changes in surface protein abundances (not shown), suggesting that high c-di-GMP levels do not grossly modify listerial cell surface proteins.

We therefore turned to investigating the effect of c-di-GMP on the abundances of specific invasion factors, changes in which may be undetectable by SDS-PAGE. Several listerial surface proteins contribute to host cell invasion, with the internalins InlA and InlB playing major roles (1, 2, 23). We assessed the levels of these proteins by analyzing the abundances of the corresponding inlA and inlB mRNAs via quantitative reverse transcription-mediated PCR (qRT-PCR). As shown in Fig. 2A, high-c-di-GMP strains had >4-fold-lower levels of the inlA transcript, and somewhat lower inlB transcript levels, than those of the wild-type strain. Consistent with the inhibitory role of c-di-GMP, the abundances of the inlA and inlB transcripts were increased in the low-c-di-GMP WT::yhjH strain compared to the wild type (Fig. 2A). Given that InlA is expected to be the primary invasion factor responsible for entry into HT-29 cells (3) and that the changes in invasion efficiency mirrored the changes in the inlA transcript levels, we concluded that lower InlA levels in high-c-di-GMP strains were likely responsible for their suppressed cell invasion potential.

FIG 2
FIG 2

Effect of intracellular c-di-GMP levels on virulence gene expression in L. monocytogenes. (A) mRNA abundances of the PrfA regulon genes. (B) mRNA abundance of codY. Shown are relative mRNA abundances, measured by qRT-PCR, compared to the value for the WT. (C) mRNA abundances of prfA in the ΔcodY deletion strains. prfA mRNA levels in codY mutants were 3.9-fold lower than the levels in the wild type. Data are from 3 biological replicates, each done in at least 3 technical replicates, after normalization to the values for the comparison strain. Values shown represent the averages ± standard deviations.

Elevated c-di-GMP levels result in decreased expression of the prfA virulence regulon in L. monocytogenes.inlA and inlB form an operon whose expression is regulated mainly by PrfA, the master activator of virulence (24, 25), while inlB has an internal, PrfA-independent promoter (26). This information encouraged us to look closer at PrfA as a factor potentially mediating the c-di-GMP effect. To test the possibility that the DNA-binding activity of PrfA depends on c-di-GMP, we purified PrfA as a His6 fusion using Ni-nitrilotriacetic acid (NTA) affinity chromatography and assayed PrfA binding to c-di-GMP via equilibrium dialysis (27) and binding to a fluorescently labeled c-di-GMP probe using a differential radial capillary action of ligand assay (DRaCALA) (28, 29). No c-di-GMP binding was detected in these assays (data not shown), which suggested that instead of affecting PrfA activity, c-di-GMP levels may have affected the abundance of the PrfA protein.

To explore the possibility that the abundance of PrfA was affected through changes in prfA gene expression, we measured the prfA mRNA levels in high- and low-c-di-GMP strains by qRT-PCR and found that they were decreased at high c-di-GMP concentrations (Fig. 2A). If this decrease is responsible for the downregulation of PrfA levels, we would expect mRNA levels of all members of the PrfA regulon to be downregulated, similar to the downregulation of the inlAB operon. We therefore measured the transcript levels of several virulence regulon genes, including hly, plcA (both of which are responsible for the escape of listeria from the primary vacuole), and actA (responsible for bacterial cell-to-cell spread). Transcript levels of all of these genes were decreased in high-c-di-GMP strains, compared to the wild-type and WT::yhjH strains (Fig. 2A).

To test whether decreased prfA mRNA levels were the main cause of poor invasion, we constructed a high-c-di-GMP strain in which prfA expression was inducible by isopropyl-β-d-1-thiogalactopyranoside (IPTG) (ΔpdeBCD ΔpssC::pIMK4-prfA) (Table 1). We tested several IPTG concentrations and identified the one (60 μM) that restored prfA mRNA levels in this high-c-di-GMP strain to the levels of the wild type (see Fig. S2 in the supplemental material). When the invasion efficiency of this prfA-restored strain was compared to the invasion efficiency of the wild type, they turned out to be identical (Fig. 1B). Similarly, invasion by the high-c-di-GMP strain with induced inlA expression (ΔpdeBCD ΔpssC::pIMK4-inlA) (Table 1) was restored to wild-type levels (Fig. 1B and Fig. S2). Based on these results, we conclude that high c-di-GMP levels inhibit the expression of the entire PrfA-controlled virulence regulon by lowering prfA gene expression levels and that the PrfA-mediated decrease in inlA expression levels is responsible for poor invasion by high-c-di-GMP strains.

Identification of the c-di-GMP-dependent components involved in prfA gene regulation.To gain insight into the factor through which c-di-GMP affects prfA expression, we analyzed known prfA gene regulation components. prfA is expressed from 3 different promoters, P1 to P3 (Fig. 3). Its expression is controlled by the cis-acting riboswitch that is responsive to temperature changes (20, 25) and trans-acting riboswitches, SreA and SreB, coordinating prfA expression in response to S-adenosylmethionine availability (30). PrfA also autoactivates its own gene expression through promoter P3 (20, 25). Furthermore, the transcription factor CodY binds to the prfA coding region and functions as an activator of prfA expression (21). In addition, inside mammalian host cells, the activity of the PrfA protein is allosterically regulated by reduced glutathione (31).

FIG 3
FIG 3

Potential targets of c-di-GMP-dependent virulence gene regulation in L. monocytogenes. (Top) prfA gene regulation. (Bottom) codY gene regulation. P1 to P3, promoters; GSH, glutathione; X, hypothetical c-di-GMP-dependent transcription regulator. Dashed arrows point to potential targets of c-di-GMP action involved in prfA regulation (blue arrows), codY regulation (red arrows), and allosteric regulation of the CodY protein (green arrows).

We tested relevant mechanisms by deleting promoter P3 and the gshF gene, encoding a glutathione synthase, and by measuring sreA and sreB transcript levels. None of these components appeared to be responsible for the c-di-GMP-dependent inhibition of prfA expression (see Fig. S3 in the supplemental material). In contrast, codY gene deletions constructed in high- and low-c-di-GMP strains abolished the effect of c-di-GMP on prfA mRNA (Fig. 2C). Furthermore, the codY gene deletion lowered the invasion abilities of the high- and low-c-di-GMP strains to the same baseline level (Fig. 1C), thus verifying the physiological significance of the c-di-GMP–prfA–codY connection. Therefore, CodY appears to be responsible for mediating the c-di-GMP-dependent downregulation of prfA expression.

We hypothesized that CodY binds c-di-GMP and that its DNA binding is inhibited by c-di-GMP, which would impair its function as a prfA expression activator. This hypothesis seemed reasonable in light of the fact that listerial CodY is known to bind a structurally similar GTP molecule (32). However, in vitro binding assays using purified CodY::His6 and c-di-GMP revealed no c-di-GMP binding (data not shown), which did not support our hypothesis.

codY gene expression is regulated by c-di-GMP levels via CodY.We reasoned that if c-di-GMP does not bind CodY, it may affect the abundance of codY mRNA. Therefore, we examined the effect of c-di-GMP on codY gene expression. We found that the abundance of codY mRNA was decreased in high-c-di-GMP strains, compared to the wild-type and WT::yhjH strains (Fig. 2B), which led us to search for a c-di-GMP-sensitive factor that controls codY gene expression.

L. monocytogenes codY is expressed as part of the codV-clpQ-clpY-codY operon. This operon is autoregulated by CodY, which is the only known transcriptional regulator affecting the expression of this operon. The CodY-binding site was previously mapped to the upstream region of codV by chromatin immunoprecipitation (CHIP) assays (22) and electrophoretic mobility shift assays (EMSAs) (21). Furthermore, in a codY mutant, codY mRNA levels were shown to be lower than those of the wild type (22). While no other promoters and transcription factors have been reported to control codY gene expression in L. monocytogenes, we could not exclude their existence because codY regulation has never been examined at high c-di-GMP levels (Fig. 3).

To facilitate expression analysis, we constructed two transcriptional fusions: codV::lacZ, containing 284 bp upstream of codV, and codY::lacZ, containing 400 bp upstream of codY (Fig. 4A). These fusions were integrated, in a single copy, into the chromosomes of strains with different c-di-GMP levels. Since no measurable codY::lacZ expression was detected in any strain (Fig. 4A), we discounted the existence of a c-di-GMP-dependent promoter immediately upstream of the codY gene. On the other hand, the expression of codV::lacZ was responsive to c-di-GMP strains similarly to the responses observed for codY mRNA (Fig. 4B).

FIG 4
FIG 4

c-di-GMP-dependent regulation of codY expression. (A) Schemes of L. monocytogenes codY gene expression and construction of the transcriptional lacZ fusions codV::lacZ (promoter P1) and codY::lacZ (putative promoter P2). Depicted on the left is a petri dish showing the expression of codV::lacZ (colonies 1 and 2) and codY::lacZ (colonies 3 and 4) in wild-type L. monocytogenes cells grown at 37°C in BHI agar supplemented with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). Two representative colonies with each lacZ fusion were streaked. (B) Quantitative β-galactosidase assays with liquid-grown strains containing codV::lacZ. Strains were grown at 37°C in BHI medium. (C) Quantitative β-galactosidase assays with liquid-grown strains containing codV::lacZ. Statistical analysis was performed by using Student's t test, and values shown represent the averages of data from triplicate independent cultures ± standard deviations, with P values of <0.01 (**) and <0.05 (*) indicating statistical significance.

Two scenarios may explain c-di-GMP-dependent codV::lacZ expression: c-di-GMP affects either the activity of CodY or the activity of an as-yet-unknown transcription regulator involved in codV operon regulation (factor X) (Fig. 3). First, we tested whether CodY indeed activates codV operon expression. This was found to be the case, as codV::lacZ expression was strongly downregulated in the codY mutant compared to the wild-type strain (Fig. 4C).

Role of CodY coactivators in mediating the effect of c-di-GMP.Since CodY does not bind c-di-GMP, we tested whether c-di-GMP levels affect known coactivators of listerial CodY, i.e., branched-chain amino acids (BCAA) and GTP. Of the BCAA, Ile shows the strongest effect on CodY DNA binding (33, 34). Our experiments were done using rich, brain heart infusion (BHI) medium; therefore, a deficiency in BCAA was unlikely. To verify that BCAA were not involved, we measured codV::lacZ expression levels in high- and low-c-di-GMP strains grown in BHI medium supplemented with extra Ile. As shown in Fig. 5A, the addition of Ile did not abolish the inhibitory effect of c-di-GMP. Similarly, the addition of Ile to cultures grown in minimal medium did not change c-di-GMP-dependent codV::lacZ expression (Fig. 5B).

FIG 5
FIG 5

Role of the CodY coactivators Ile and GTP in mediating the effect of c-di-GMP. (A to C) Quantitative β-galactosidase assays reflecting codV::lacZ expression in liquid-grown strains in rich (BHI) medium (A and C) and minimal medium (B), with or without extra Ile. Shown are data for strains with the wild-type genetic backgrounds (A and B) and codY replacement strains (C). (D) Relative invasion by the codY replacement strains in HT-29 cells showing that listerial but not streptococcal CodY is sensitive to elevated c-di-GMP levels. Statistical analysis was performed by using Student's t test, and values shown represent the averages of data from triplicate independent cultures ± standard deviations, with P values of <0.01 (**) and <0.05 (*) indicating statistical significance.

Next, we investigated the possibility that high levels of c-di-GMP resulted in decreased GTP concentrations. Because L. monocytogenes CodY mutants impaired in GTP binding have not been described, we took advantage of the fact that streptococcal CodY proteins do not bind GTP, in contrast to listerial CodY proteins (35). The L. monocytogenes and Streptococcus pneumoniae CodY proteins are highly similar (48% sequence identity); therefore, we expected them to be functionally interchangeable. We precisely replaced the listerial codY gene (codY-Lm) with the streptococcal codY homolog by inserting the streptococcal codY gene (codY-Sp) into the chromosome of the L. monocytogenes codY deletion mutant. As a control, in parallel, we inserted codY-Lm into the codY deletion mutant, thus restoring the wild-type genetic background. Such replacement strains were also made in the high-c-di-GMP (ΔpdeBCD ΔpssC) background. When codV::lacZ expression was tested in the constructed strains, we observed similar β-galactosidase levels in the strains with codY-Lm and codY-Sp, which indicated the functionality of the streptococcal homolog. However, the high-c-di-GMP strain expressing codY-Sp lost its responsiveness to c-di-GMP (Fig. 5C).

We further tested the effect of streptococcal CodY on invasion in the wild-type and high-c-di-GMP backgrounds. We found that the invasion efficiency of the replacement strain (i) was virtually identical to that of the strain with listerial codY and, most importantly, (ii) was not inhibited by high c-di-GMP levels (Fig. 5D). Since listerial and streptococcal CodY proteins differ in their sensitivities to GTP, we conclude that elevated c-di-GMP levels likely decrease GTP concentrations, which are sensed by listerial (but not streptococcal) CodY, whose activity decreases, thus resulting in lower codV operon expression levels via the positive-feedback loop (Fig. 3).

DISCUSSION

This study advances our understanding of the signal transduction cascade responsible for the poor invasiveness of high-c-di-GMP L. monocytogenes strains in human cells. It establishes the transcription factor CodY as the c-di-GMP-sensitive component. Decreased CodY activity results in a signal transduction cascade that leads to lower expression levels of codY and prfA and, therefore, lower expression levels of the entire PrfA virulence regulon, which includes proteins involved in invasion, particularly InlA. Our data further suggest that CodY senses changes in c-di-GMP levels indirectly, probably as a decrease in the intracellular GTP concentration. Direct testing of this scenario is the subject of ongoing studies.

These findings are unexpected in several ways. One unexpected outcome concerns the operation of the c-di-GMP signal transduction pathways in L. monocytogenes. Specifically, elevated c-di-GMP levels in this species bring about phenotypic changes (induced exopolysaccharide synthesis, decreased motility, and suppressed virulence [5]) as complex as those in bacteria that possess much more sophisticated c-di-GMP signaling pathways (7–9). However, L. monocytogenes appears to be able to accomplish these phenotypic and behavioral changes with only a single bona fide c-di-GMP receptor/effector protein, PssE, which acts as an activator of Pss exopolysaccharide synthesis (5, 10). We showed previously that motility in listeria is inhibited at high c-di-GMP levels due to secreted exopolysaccharide, with no involvement of the motility-specific c-di-GMP receptors/effector proteins (5). Here, we found that the c-di-GMP-dependent suppression of virulence does not involve specific c-di-GMP receptors either. If true, L. monocytogenes would appear to accomplish the responses traditionally associated with elevated c-di-GMP levels via a single c-di-GMP-binding protein receptor/effector and changes in intracellular GTP concentrations. It will be interesting to decipher whether the minimalistic version of c-di-GMP signaling in listeria represents an evolutionarily ancient signaling arrangement or is a result of reductive evolution.

How could a change in the c-di-GMP level affect the GTP concentration? Diversion of GTP, which is a substrate for c-di-GMP synthesis, toward c-di-GMP seems to be the simplest explanation for the c-di-GMP effect. However, it is unclear if the decrease in the GTP concentration can be drastic enough because neither growth rates nor other visible phenotypes in high-c-di-GMP strains were affected. At this point, we cannot exclude another possibility, i.e., that c-di-GMP inhibits a specific step(s) in GTP synthesis or accelerates GTP degradation by interacting with one of the components of these pathways. For example, a guanine-based alarmone, (p)ppGpp, which is associated with amino acid or lipid starvation, acts as an inhibitor of IMP dehydrogenase, the first enzyme in the GTP biosynthetic pathway in Bacillus subtilis (36). In the case of (p)ppGpp, however, the ultimate result is a drastic reduction in GTP levels and a slowdown in protein synthesis and growth (36). It is intriguing to envision c-di-GMP as a “mild version” of (p)ppGpp that affects only virulence but not growth.

It was also unexpected, at least at first glance, that potentially modestly lower intracellular GTP concentrations would result in the drastic changes in virulence mediated by CodY. However, if we consider that the affinity of CodY for GTP is in the millimolar range (37), it follows that CodY is meant to sense moderate deviations in the levels of GTP. It is worth mentioning that the link between GTP and virulence is not unique to L. monocytogenes but is also observed in other pathogenic firmicutes. In Bacillus anthracis, for example, a decreased GTP concentration leads to lower DNA binding of GTP-responsive CodY and increased expression of a gene whose product degrades the virulence-associated toxin AtxA (38). In Bacillus cereus, CodY activates the expression of nonhemolytic enterotoxin, phospholipases, and immune inhibitor metalloprotease 1 (39). A recent study found that a nutrient-regulated c-di-GMP phosphodiesterase in Clostridium difficile controls biofilm formation and toxin production, an observation that resembles our findings for L. monocytogenes (40).

Another unexpected aspect of this work is the realization that listerial DGCs are sufficiently active to modulate virulence even in liquid-grown cultures. Bacteria grown on surfaces tend to form biofilms, where intracellular c-di-GMP levels are high compared to the levels in liquid-grown planktonic cells (7). The nonnegligible activity of planktonic listerial DGCs is consistent with the fact that the expression of a c-di-GMP-specific E. coli PDE in the wild-type background (WT::yhjH) led to increased invasiveness (Fig. 1A). It may therefore be possible to manipulate the ability of listeria to invade human cells by affecting intracellular c-di-GMP and/or GTP levels. It is intriguing to contemplate a possibility that food additives can be found that specifically increase c-di-GMP and/or decrease GTP levels in listeria and “lock” L. monocytogenes in a less invasive state, which would be advantageous to populations particularly susceptible to listeriosis. Whether or not this possibility is feasible remains unclear; however, it is encouraging that the search for small molecules that affect nucleotide-based signaling in bacteria has begun (41).

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.The bacterial strains and plasmids used in these experiments are shown in Table 1. The primers used in this study are listed in Table S1 in the supplemental material. L. monocytogenes strains were grown in BHI medium (Difco) and Hsiang-Ning Tsai medium (HTM) (minimal medium; 3% glucose) (42) supplemented with appropriate antibiotics at 30°C, 37°C, and 41°C with shaking at 250 rpm. E. coli strains were grown in LB medium supplemented with appropriate antibiotics at 37°C.

Recombinant DNA techniques.L. monocytogenes genes were amplified by PCR using Phusion DNA polymerase (New England BioLabs [NEB]). Restriction enzymes were also obtained from NEB. PCR fragments were purified and concentrated by using designated kits (Zymo) and cloned into the vector pET28a (Invitrogen) in strain DH5α or in strain BL21(DE3) containing pLysS (Invitrogen). In-frame deletions in L. monocytogenes genes were generated by splicing by overlap extension (SOE) PCR. The deletion constructs were cloned into the temperature-sensitive shuttle vector pKSV7, followed by transformation into appropriate L. monocytogenes strains (42, 43).

L. monocytogenes mutant construction.L. monocytogenes genes were deleted in the wild-type EGD-e, ΔpdeBCD ΔpssC, WT::slr, and WT::yhjH backgrounds by using the SOE PCR method (43, 44). Briefly, the PCR products containing 400 bp upstream and downstream of the gene of interest were amplified and combined by PCR using EGD-e genomic DNA, Fusion DNA polymerase, and primers listed in Table S1 in the supplemental material. The 800-bp DNA in-frame deletions were cloned into suicide shuttle vector pKSV7 by using a Gibson assembly kit (NEB). Deletion constructs were transformed into and maintained in DH5α cells and then transferred to L. monocytogenes strains via electroporation (45). Two to four consecutive passages were carried out at 41°C in the presence of 10 μg chloramphenicol ml−1 to insert the constructs into the genome via homologous recombination. Five to eight passages were then performed at 30°C with no antibiotics to allow vector excision from the chromosome. Mutant strains were screened for on BHI agar plates supplemented with 10 μg chloramphenicol ml−1. In-frame deletion mutants were identified by colony PCR analysis of chloramphenicol-sensitive clones.

Invasion assays.A gentamicin-based assay was performed to test L. monocytogenes invasion of HT-29 human colon adenocarcinoma cell monolayers in 12-well plates, as described previously (46, 47). Briefly, L. monocytogenes strains were grown in BHI medium overnight (13 to 15 h) at 37°C. After centrifugation, cells were washed and resuspended in Dulbecco's modified Eagle's medium (DMEM). HT-29 cells were inoculated with L. monocytogenes suspensions (∼5 × 108 CFU ml−1) at a multiplicity of infection of 100 and kept in an incubator for 1.5 h at 37°C. HT-29 cells were then washed with phosphate-buffered saline (PBS), and fresh medium containing 100 μg gentamicin ml−1 (final concentration) was added. Cells were incubated for 1.5 h, washed with 0.1% Triton X-100, and lysed. Serial dilutions were plated onto BHI plates to count intracellular bacteria. The invasion efficiency was calculated as the ratio of the number of intracellular bacteria to the total number of bacteria used for invasion. Relative invasion was calculated by dividing the invasion efficiency of a mutant strain by that of the wild-type strain. Statistical analysis was performed with Student's t test using the PROC MIXED procedure in SAS (SAS Institute, Cary, NC). The values shown represent the averages of data from experiments ± standard deviations, with P values of <0.01 and <0.05 indicating statistical significance.

RNA purification and qRT-PCR.Total RNA extractions were performed by using methods adapted from a combined protocol (48, 49). High- and low-c-di-GMP listerial strains were grown in BHI medium overnight (13 to 15 h) at 37°C. An equal volume of a cold ethanol-acetone mixture was then added to the cultures. After centrifugation, cell pellets were resuspended in a Tris-HCl (pH 8.0)–EDTA solution, TRIzol reagent (Ambion RNA Life Technologies) was added to the suspensions, and the suspensions were vortexed. Cells were disrupted mechanically with a FastPrep cell disrupter (6.5 m s−1/45 s, three times). Total RNA was extracted and purified by using a Direct-zol RNA miniprep procedure. A second DNA digestion was further done by using the Turbo DNA-free kit according to the manufacturer's instructions. From these RNA preparations, 1 μg of purified RNA was converted to cDNA by using the iScript cDNA synthesis kit (Bio-Rad). qRT-PCRs were performed by using Bio-Rad IQ SYBR green Supermix. The transcription levels of the prfA gene and prfA-dependent genes were normalized to the gyrA reference transcript level. The primers used in these experiments are listed in Table S2 in the supplemental material.

Protein overexpression and purification.For the purification of L. monocytogenes proteins, pET28a::codY and pET28a::prfA expression strains were used. PrfA and CodY were expressed in E. coli strain BL21(DE3). IPTG (final concentration, 0.2 mM) was added at an optical density at 600 nm (OD600) of 0.6. After 4 h of induction at 37°C, the cells were collected by centrifugation at 4°C. Buffer (pH 7.4) containing 300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, and protease inhibitors (P8849 protease inhibitor cocktail; Sigma-Aldrich) was used to resuspend cell pellets. The cell suspensions were lysed by using a French press minicell (Spectronic Instruments, NJ) and then sonicated by using a Sonifier 250 instrument (Branson Ultrasonics, CT). The crude cell extracts were centrifuged at 15,000 × g for 10 min. Soluble protein fractions were collected, mixed with preequilibrated Cobalt charger resin (Qiagen) for 3 h at 4°C, and then placed into a column and extensively washed with resuspension buffer containing 20 mM imidazole. The proteins were subsequently eluted by using 200 mM imidazole. Protein purity was assessed by SDS-PAGE, and the protein concentration was determined by using a bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology).

c-di-GMP binding assays in vitro.Equilibrium dialysis experiments were performed as described previously (27). Briefly, purified PrfA::His6 or CodY::His6 was loaded into one of the two chambers of a Dispo-Biodialyzer cassette (10-kDa cutoff; The Nest Group, MA) filled with dialysis buffer. c-di-GMP (concentrations from 10 to 50 μM) was injected into the opposite cell of the cassette. The cassettes were maintained for 25 h at room temperature under agitation, after which samples from each chamber were withdrawn, boiled, filtered through a 0.22-μm microfilter, and analyzed by high-performance liquid chromatography (HPLC).

DRaCALA assays were preformed as described previously (27, 29). Briefly, purified proteins were mixed with fluorescently labeled c-di-GMP (2′-fluoaminohexylcarbamoyl–c-di-GMP [2′-fluo-AHC–c-di-GMP]; Biolog) and incubated, and a fraction of these mixtures was spotted onto a nitrocellulose membrane. The membranes were scanned for fluorescence by using a GE Typhoon FLA 9500 scanner.

β-Galactosidase activity measurements.We designed a transcriptional reporter construct to assess the effect of c-di-GMP on the expression of cod operon genes (Fig. 4). We fused lacZ to the promoter region of the cod operon and cloned the construct into the pIMK-2 vector. The reporter construct was introduced into high- and low-c-di-GMP listerial strains via electroporation as described previously (45). Primers used in the constructions are listed in Table S2 in the supplemental material. β-Galactosidase activity was assayed by using o-nitrophenyl-β-d-galactopyranoside (ONPG) as the substrate and is reported as micromoles of o-nitrophenol per minute per milligram of cellular protein (50). The values shown represent the averages of data from triplicate independent cultures ± standard deviations. Statistical analysis was performed by using Student's t test, as indicated above.

ACKNOWLEDGMENTS

We are grateful to Xin (Cindy) Fang for PrfA::His6 purification, testing of PrfA–c-di-GMP binding, and help with HPLC and to Volkan Köseoğlu for help with invasion assays. We appreciate help from Carol Wilusz (Colorado State University) in providing access to the Typhoon scanner.

This work was supported in part by National Institute of Food and Agriculture award WYO-583-17. A.M.E. was supported by graduate assistantships from the Government of Libya and the Department of Molecular Biology of the University of Wyoming.

FOOTNOTES

    • Received 27 July 2017.
    • Accepted 28 November 2017.
    • Accepted manuscript posted online 11 December 2017.
  • Address correspondence to Mark Gomelsky, gomelsky{at}uwyo.edu.

  • Citation Elbakush AM, Miller KW, Gomelsky M. 2018. CodY-mediated c-di-GMP-dependent inhibition of mammalian cell invasion in Listeria monocytogenes. J Bacteriol 200:e00457-17. https://doi.org/10.1128/JB.00457-17.

  • Supplemental material for this article may be found at https://doi.org/10.1128/JB.00457-17.

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KEYWORDS

CodY
GTP
Listeria monocytogenes
PrfA
c-di-GMP
cell invasion
metabolism
transcriptional regulation
virulence regulation

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