Signals behind Listeria monocytogenes virulence mechanisms

ABSTRACT

The tight and coordinated regulation of virulence gene expression is crucial to ensure the survival and persistence of bacterial pathogens in different contexts within their hosts. Considering this, bacteria do not express virulence factors homogenously in time and space, either due to their associated fitness cost or to their detrimental effect at specific infection stages. To efficiently infect and persist into their hosts, bacteria have thus to monitor environmental cues or chemical cell-to-cell signaling mechanisms that allow their transition from the external environment to the host, and therefore adjust gene expression levels, intrinsic biological activities, and appropriate behaviors. Listeria monocytogenes (Lm), a major Gram-positive facultative intracellular pathogen, stands out for its adaptability and capacity to thrive in a wide range of environments. Because of that, Lm presents itself as a significant concern in food safety and public health, that can lead to potentially life-threatening infections in humans. A deeper understanding of the intricate bacterial virulence mechanisms and the signals that control them provide valuable insights into the dynamic interplay between Lm and the host. Therefore, this review addresses the role of some crucial signals behind Lm pathogenic virulence mechanisms and explores how the ability to assimilate and interpret these signals is fundamental for pathogenesis, identifying potential targets for innovative antimicrobial strategies.

KEYWORDS:

• Listeria monocytogenes
• Virulence mechanisms
• Virulence signals

Introduction

Pathogen adaptability is pivotal to the infection outcome and is determined by bacterial factors allowing the establishment and progression of the infection. Importantly, the tight and coordinated regulation of these virulence genes is crucial and strategic for their expression in specific time-frames, within particular tissues, or by bacteria subsets. Moreover, the untimely expression of virulence factors represents a major fitness burden that can be detrimental to a successful survival strategy, once bacteria do not express virulence factors homogenously in time and space. Infections caused by Gram-positive bacteria are challenging and still remain a major public health. Listeria monocytogenes (Lm) is a major Gram-positive intracellular foodborne pathogen which causes a life-threatening infection, listeriosis, acquired from the ingestion of contaminated food. It is the most severe human systemic infection among zoonotic diseases under EU surveillance. It can be particularly severe in specific high-risk groups, including pregnant women, elderly and immunocompromised people. Despite the availability of antibiotics-based treatment, listeriosis remains the most frequent cause of death due to the consumption of contaminated food in Europe, with the higher proportion of hospitalized cases (~96%) and a case mortality rate of ~18.1%.^1,2 Lm is ubiquitously found in a wide range of environments, such as soil, water, animal feces and vegetation, being constantly exposed to a number of stress adaptations both throughout its saprophytic life and transiting to infect human tissues. Once inside the host, it is capable to invade and colonize the gut, transverse the intestinal barrier and disseminate, establishing a systemic infection, and replicating within target organs.^3,4 Importantly, the expression of virulence factors is directly dependent on the Lm ability to detect and process signals from the surrounding environment adjusting its physiology accordingly.

The coordinated phenotypic expression of Lm virulence factors and the underlying mechanisms of their regulation provide a competitive advantage for the bacterial community in different contexts, such as biofilm formation, host infectionor antibiotic resistance. This review focuses on the role of different signals behind Lm virulence gene expression and how they impact bacterial communication throughout host infection and therefore pathogenesis. Since the successful host colonization by pathogens is conditioned by the signals they integrate, it is crucial to understand how these signals act to further modulate bacterial colonization and therefore control disease. Importantly, most of the signals required to control virulence mechanisms are transversal to different pathogens. This review provides an overview of both the signaling molecules generated by Lm itself for its own profit and the nutritional and physical external signals that control bacterial virulence (Table 1).

Table 1. Summary of signals sensed by Lm to modulate virulence.

Category	Signal	Function/related processes	Associated genes	Refs
Lm signaling molecules	S-adenosylmethionine (SAM)	Involved in PrfA regulation via SreA and SreB riboswitches	metK, lmo2417, lm0135, sreA-B	5–11
	AI-2	Involved in S-adenosylhomocysteine (SAH) detoxification		12
	S-ribosylhomocysteine (SRH)	Involved in biofilm formation and production of AI-2		9
	Autoinducer peptide (AIP)	Triggers the induction of the Agr system	agrD	13
	Peptide-pheromone pPplA	Signals the presence within the host vacuole and facilitates vacuolar escape by promoting PrfA activation	lmo2637	14
	c-di-AMPs	Maintains intracellular GTP levels required for CodY expression		15–17
	Glutathione	Mediates PrfA activation		18–20
	Vitamin B12 /cobalamin	Impairs aspocR and rli55 transcription to control propanediol and ethanolamine metabolism	rli55, eutV, rli39, aspocR, pdu, cob, cbi	21–24
Lm signaling RNAs	prfA-5’UTR	Thermosensor controlling prfA expression		25
	Anti0677	Downregulates flagellum biosynthesis via MogR	mogR, fliNPQ	3,26
	LhrA	Regulates ChiA activity required to control iNOS ativity Modulates host immune response via INF- β induction	chiA	27 28
	LhrC1–5	Prevents host immune recognition and facilitates adaptation in heme-rich conditions Protects against cell wall-acting antibiotics	oppA, tcsA, lapB, lmo2185, lmo2186 lmo0484	29–31
	Rli60	Modulates gene expression in response to BCAAs	ilvD	32,33
	RliB	Plays a role in Lm adhesion and invasion		34
	SbrE	Involved in isoleucine biosynthesis control	ilvA	35
	Rli22	Promotes survival within the intestinal lumen	lisRK	36
	Rli27	Modulates levels of a cell wall surface proteins	lmo0514	37
	Rli31	Protects against lysozyme activity	spoVG	38,39
	Rli32	Protects against cell envelope stresses Modulates genes involved in tryptophan biosynthesis	virR, lmo1958, lm1960, hbp1-2 lmo1627–33	28,40
	Rli38	Related with organ colonization	fur, lmo0460, lmo2752	3,41
	Rli50	Promotes macrophage proliferation and intracellular survival		28,42
Lm signaling via TCSs	β-lactams, lysozyme, ethanol Antimicrobial agents	Sensed by CesK	lmo2420 lmo0441, lmo2812	43 44
	β-lactams, acidic conditions, hemoglobulin, ethanol, bile salts	Sensed by LisK	htrA, lhrC1–5	31,45–47
	Human defensins, cationic peptides	Sensed by VirS	dltP, mprF	48
	Gastric conditions	Sensed by PieS	prsA1-2	49,50
Host physical signals	Temperature	Triggers prfA and PrfA-controlled genes expression Impairs Gmar activity, allowing the expression of mogR	gmaR, mogR	25,51,52
	pH	Activates Lm stressosome and σ^β to maintain pH homeostasis Controls LLO activity	rsbR1-2-3-4, rsbL-S-T, gadD3 hly	53–58 59–62
Host nutritional signals	L-cysteine	Required for GSH biosynthesis	gshF	63,64
	Cysteine containing peptides	Organic nitrogen source used to induce the activation of PrfA	opp	65
	Bile salts	Promotes Lm adaptation to the gastrointestinal tract	bsh, brtA, mdrT, cadC	4,66–69
	BCAAs	Modulate virulence gene expression and protein synthesis in a CodY-dependent manner	codY, ilvC, purH, prfA, rli60	32,70–73
	FFAs	Impair PrfA-dependent gene activation within the gut		74,75
	L-glutamine	Induces hly, plcA, plcB and actA	glnQ, glnP	76,77
	Non-dependent PTS	Carbon sources sensed and metabolized to improve bacteria growth within the host	hpt, prfA, glpF, inlC, hly, actA	78,79
Host chemical signals	Iron	Mitigates ROS-mediated damage in phagosomes Induces InlA, InlB and LAP expression during early infection stages Required for the upregulation of FrvA	inlA-B, lap frvA	80,81 82–84 85,86
	ROS	Trigger regulatory pathways to allow survival within the host	perR, kat, fur, hemA, fri, frvA, prfA, hly	87–89

Listeria monocytogenes signaling molecules regulating virulence

Pathogenic bacteria have developed several strategies to survive and persist within host cells and tissues. In Lm, most of these strategies are controlled and/or influenced by cell-to-cell communication, a process mediated by molecules produced and secreted by bacteria themselves. These signaling molecules are part of a dialogue between bacteria, and can be either chemical and cell-population density related (e.g quorum-sensing, pheromones) or generated by bacteria at different infection stages (e.g RNAs, second messengers, and glutathione), being both essential to ensure the successful establishment of infection.⁹⁰

Quorum sensing

Quorum sensing (QS) is a microbial cell-to-cell communication process that relies on the production, secretion, detection, and group-wide accumulation of extracellular signaling molecules named autoinducers. Throughout this process, bacteria share information concerning the population density to accordingly regulate the expression of genes involved in virulence, competition, stress response, and resistance.^91,92

Pathogenic Gram-positive bacteria typically produce autoinducing peptides (AIPs) as autoinducers, being initially synthesized as precursor peptides (pro-AIPs), modified, and then exported through a specialized protein transport system.^93,94 AIPs are generally sensed and recognized by a two-component signal transduction system. High cell density communities accumulate higher concentrations of extracellular AIP, which binds to a membrane-bound histidine kinase receptor triggering its own phosphorylation and transferring the phosphate group to a cognate cytoplasmic response-regulator, that ultimately regulate the expression of QS related-genes.^93,95,96 Moreover, AIPs can be transported back to the bacterial cytoplasm where they are capable to interact with cytoplasmic transcriptional factors, thus modulating their activity and gene expression pattern.^97,98 The Agr system and its orthologs are the most well-studied circuits of bacterial QS, mainly in Staphylococcus aureus, and play a fundamental role in the spatiotemporal expression of virulence mechanisms of Gram-positive human pathogens, including Enterecoccus faecalis, Clostridium perfringens and Lm.^5,99-102

Lm QS systems have been associated with bacterial communication and survival providing competitive advantages in adhesion to surfaces, biofilm formation, invasion of mammalian cells, mouse infection, and changes in gene expression (Figure 1).^5,99,107,108 Two QS systems, Lux, and Agr, widespread among different bacterial species, were already established for Lm. The Lux system (Figure 1C) involves the conversion of S-adenosylhomocysteine (SAH) into S-ribosylhomocysteine (SRH) that is further processed to give rise to both homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD). This latter reaction is achieved by two Lm enzymes, Pfs (S-adenosylhomocysteine nucleosidase) and LuxS (S-ribosylhomocysteinase). Thereon, DPD rearranges into a furanone derivative called autoinducer AI-2, which is then released to the extracellular media.^6,12 AI-2 was found to be important only for the detoxification of SAH, while its precursor SRH is important for biofilm formation.¹² Importantly, both SAH and SRH are by-products generated in the activated methyl cycle (AMC) from S-adenosylmethionine (SAM) processing. SAM is an intermediate metabolite generated from methionine in the MetK synthase cascade pathway, and is recognized as a sentinel metabolite capable of bind to 5’ UTRs riboswitches controlling the transcription of their downstream genes, mainly involved in the biosynthesis, transport, and utilization of amino acids, oligopeptides and SAM itself: between them, the adenosylmethionine synthetase (metK) and the methionine (lmo2417) and cysteine (lm0135) transporters.^6-9

Figure 1. Signalling of Lm virulence gene expression during the infection of the gastrointestinal (GI) tract. (a) The temperature within the mammalian host is key for Lm to modulate the expression of virulence genes. This thermal cue is sensed both by the Lm pleiotropic regulator prfA, whose ribosome binding region becomes accessible enabling the translation of downstream mRNA,²⁵ and by the anti-repressor GmaR that suffers a conformational shift, allowing the expression of MogR and consequently the repression of genes involved in flagellum biosynthesis. Moreover, the Anti0677 is capable of inducing the transcription of mogR by generating a longer anti0677 transcript, positioned on the opposite strand of the flagellum operon coding sequence (fliN, fliP and fliQ).⁴¹ within the gastrointestinal tract, Lm encounters bile salts. It uses Bsh, which is crucial for Lm resistance to bile and virulence, to catalyze the hydrolysis of GDCA- and TDCA-conjugated bile salts into free cholic acids, that are exported by the MdrT efflux pump of the bacterium.⁶⁶ Consequently, in the presence of cholic acids, the bile sensor BrtA, which controls mdrT, is no longer able to repress Lm MdrT.⁶⁷ (b) the ability of Lm to internalize intestinal epithelial cells relies, in part, on its capacity to form biofilms at the surface of the intestinal villi, which is dependent on MouR that positively regulates the agr operon by binding to its promoter region.¹⁰³ AgrD is processed by AgrB into a mature AIP, that is subsequently secreted to the extracellular medium. The secreted AIP is then recognized by the sensor kinase AgrC present within the neighboring bacteria, triggering a phosphorylation cascade that culminates in the activation of the response regulator AgrA. In turn, AgrA induces the auto-activation circuit. Therefore, the Agr system induces chitinase activity by the down-regulation of the ncRNA lhrA, which blocks translation by binding itself to chitinase mRNA (chiA). In turn, chitinase represses the inducible nitric oxide synthase (iNOS), known to be part of the immune response.^27,104 (c) the activated methyl cycle (AMC) also plays a role in biofilm formation by the generation of the S-ribosylhomocysteine (SHR) by-product, through the processing of S-adenosylmethionine (SAM). Concomitantly, SAM is used to bind and activate SreA riboswitch, that in turn increases the expression of agrD.^5,8,10 Simultaneously, the activation of Lm SreA riboswitch by SAM inhibits the prfA transcription in the intestine.^3,10 (d) Additionally, within the GI Lm comes across ethanolamine, a metabolite derived from the phospholipids of eukaryotic cell membranes, using it as carbon source to support bacterium growth.¹⁰⁵ in presence of ethanolamine, the sensor kinase EutW induces the phosphorylation and activation of the anti-terminator EutV, that together with Rli55 repression, upregulate the expression of the ethanolamine operon eut. The repression of Rli55 is dependent on a B₁₂-riboswitch, located upstream of the eut operon, which in the presence of vitamin B₁₂ blocks the transcription of Rli55 allowing the expression of eut genes. Vitamin B₁₂ is also used as cofactor in propanediol metabolism: in the presence of propanediol, B₁₂ riboswitch ends prematurely the transcription of AsPocR, allowing the transcription of pocR, which is important to activate the pdu and cob operons, respectively involved in propanediol utilization and B₁₂ biosynthesis respectively.¹⁰⁶

The Lm Agr QS is encoded by the agr locus agrBDCA (Figure 1B). agrD encodes a ribosomal peptide precursor that is proteolytic processed into a mature AIP by the integral membrane-bound peptidase AgrB.¹³ At this point, in Staphylococcus aureus, it is described the formation of an intermediate enzyme-bound thiolactone, which is then transported across the membrane.¹⁰⁹ However, in Lm there is no evidence about the formation of such intermediate. On the other hand, a recent study presented strong evidences that the native Lm AIP might be an homodetic pentamer peptide arising from thiolactone.¹¹⁰ Once the processed AIP is out of the cell, it binds the sensor membrane-bound histidine kinase AgrC, triggering the phosphorylation of the response regulator AgrA, together acting as a two-component system for downstream gene regulation.¹³ The Lm Agr QS system was found to be positively regulated by MouR, a transcriptional regulator belonging to the VanR class of the GntR superfamily of regulatory proteins.¹⁰³ The GntR family consists of helix-turn-helix transcriptional regulators that control several metabolic and biological processes in bacteria. These proteins share a conserved DNA-binding domain at the N-terminal, allowing them to modulate gene expression in response to signals, and an effector binding/oligomerization domain at the C-terminal The diversity of the C-terminal domain is used to divide GntR proteins in different sub-classes, where the proteins composed by six α-helices in the C-terminal are allocated to the VanR class.^111–113 MouR binds directly to the agr promoter region, modulating both chitinase activity and biofilm formation, thus favoring Lm virulence.¹⁰³

Previous findings have demonstrated a cooperation between the Agr QS and the SAM signal in the regulation of biofilm formation. The generation of SAM induces the expression of the agr locus, with agrD appearing as the most expressed gene. In contrast, the absence of the Agr QS system significantly impairs SAM-enhanced biofilm formation, but does not affect the expression of the genes involved in SAM biosynthesis.⁸

Recent efforts have been done to modulate QS and virulence outcome with the goal of designing novel antimicrobial therapeutics. Chemical antagonists able to diminish Lm Agr activity and thus leading to the reduction of biofilm formation by over 90% were recently discovered.¹¹⁴ Moreover, in silico analysis revealed different proteins secreted by lactic acid bacteria working as a promising anti-QS strategy, by inhibiting the translocation of cyclic AIP via AgrB and its recognition by the AgrC active site.¹¹⁵

Of note, some Lm strains like the well-studied laboratory reference strain 10403S harbor mutation in agr genes, being still virulent in mouse infection models.¹¹⁶

Peptide-pheromone pPplA

In addition to the Agr system discussed above, an additional peptide-signaling system has been associated with Lm virulence. Lm displays a small peptide pheromone, pPplA, that is used to signal its presence within the host cell vacuole, facilitating its vacuolar escape possibly by promoting PrfA activation. pPplA is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide, been then secreted through the general secretory pathway. When confined into the vacuole, Lm is able to sense and import previously produced and accumulated pPlpA, thus initiating a signaling cascade that favors vacuolar disruption and bacterial escape to the host cytosol. This pheromone signaling appears to be linked to PrfA activation.¹⁴

RNA-mediated regulation

RNAs have emerged as prime regulators of adaptive response and pathogenesis. This type of regulation is quite present in pathogenic bacteria and provides new information not only about how bacteria can regulate their virulence gene expression but also about their survival associated-phenotypes.^117-119 RNA molecules can regulate stress response, metabolism and virulence post-transcriptionally, both through translation and stability of mRNAs encoding genes or interacting with specific proteins.^118,120 Importantly, the RNA-mediated regulation presents an advantage for the bacteria throughout infection, since it provides quick responses with minimal consumption of energy.¹²¹ Non-coding RNAs (ncRNAs) are functional RNA molecules not translated into a protein. They are crucial gene regulatory elements for Lm, both within or outside the host, and were found to play essential roles in many biological processes including signaling, RNA processing, gene regulation and protein synthesis, which condition virulence and host-immune response.³ Transcriptomics studies of Lm have already identified 46 cis-acting riboswitches, 104 antisense RNAs and 154 putative small trans-acting RNAs.¹²² In this section, we will focus on the most relevant categories of Lm ncRNAs governing virulence.

The first category of Lm ncRNAs comprises the regulatory elements, usually located in the 5’-untranslated regions (UTRs), and capable to control the expression of virulence genes at the post-transcriptional level.¹²³ Their secondary structure generally suffers a conformational change in response to cellular and/or environmental signals including temperature (thermosensors) or ligand binding (riboswitches).¹²⁴ The thermosensors regulation depends on temperature to generate an alternative conformation in which the ribosome-binding site is hidden by the thermosensing stretch of the RNA element, thus modulating protein translation.^125,126 The most prominent example of this type of regulation in Lm, that signals its presence in mammalian hosts allowing the activation of most virulence mechanisms, is the 5’-UTR of prfA, the major transcriptional activator of Lm adhesins, internalins, phagosome-escaping factors and immune modulating factors.^127–130 At low temperatures (<30°C), the prfA-UTR forms a secondary structure that masks the ribosomal region of prfA, avoiding the ribosome binding and consequently inhibiting the translation of PrfA and the expression of PrfA-regulated virulence genes. Temperature rise (>37°C) induces a structural change in the prfA 5’-UTR, allowing ribosome access, the translation of prfA and the expression of PrfA-dependent genes.¹³⁰ Nonetheless, this thermocontrol appears to act in parallel with other PrfA-activating mechanisms since the temperature increase to 37°C per se is not sufficient to induce PrfA-dependent gene expression.¹³¹ In that regard, the regulation of PrfA is also dependent on two SAM riboswitches, SreA and SreB. Interestingly, these riboswitches, due to their high complementary with prfA-5’UTR, may function as trans-ncRNAs, inhibiting the transcription of prfA mRNA.¹⁰ Likewise, the absence of SreA decreases the amount of AgrD,^8,10 which is crucial for invasion of intestinal Caco2-epithelial cells and biofilm formation.⁵ SAM riboswitches being usually activated by the binding of their effector molecule S-adenosylmethionine, allow the formation of a termination structure that ends up gene transcription and inhibits the synthesis of downstream mRNA.^9,11 However, the SreA-prfA-5’UTR interaction is possible only at temperatures permissive for infection (i.e., 37°C), and does not necessarily require the presence of SAM.¹⁰ Altogether, these findings pointed for a role of SAM riboswitches in Lm virulence, downregulating prfA expression in the intestine, where it seems to be less critical than in the blood, keeping active other regulatory pathways such as the Agr system.^3,10 Recently, a chimeric antisense oligonucleotide (ASO) was designed as a novel antibacterial agent targeting SAM riboswitch.¹³²

Another regulation dependent on a riboswitch (Figure 1D) concerns the ncRNA Rli55, nearby the eut operon involved in the consumption of ethanolamine, an abundant molecule in the human intestine that contributes to Lm mouse infection. Vitamin B₁₂, also named cobalamin, binds to the B₁₂ riboswitch that controls Rli55 expression by ending prematurely its transcription.²¹ Rli55 functions by binding and sequestering the two-component response regulator EutV. Defects in ethanolamine utilization, or in its regulation by Rli55, significantly attenuate Lm virulence in mice.^3,21 The availability of vitamin B₁₂ also regulates the Rli39 riboswitch to control the transcription of AspocR, an antisense RNA. Truncated transcripts of aspocR arise whenever vitamin B₁₂ binds to the riboswitch; contrarily, a full-length transcript is generated in the absence of vitamin B₁₂, inhibiting pocR mRNA translation, through an antisense mechanism. In turn, PocR up-regulate the expression of the pdu genes, which are essential for the propanediol metabolism involved in the pathogenesis of many intestinal pathogens.²² PocR was also proposed to control the expression of Lm cob and cbi genes, that are involved in vitamin B₁₂ de novo biosynthesis, in an attempt to maintain the production of vitamin B₁₂ derived cofactors.^23,24

The second category of Lm ncRNAs comprises the antisense RNAs (asRNAs), including long antisense RNAs (lasRNAs), that act as major negative regulators of gene expression and are encoded on the opposite strand in relation with the genes they regulate.¹²³ These type of ncRNAs have full complementary sequence with the mRNA, providing not only strong and specific bindings, but also more efficient action, thus rapidly conditioning mRNA stability and translational activity.^120,133 Anti0677 is a lasRNA, named excludon, crucial for Lm infection, that negatively regulates the flagellum biosynthesis, in particular through the control of the MogR transcriptional repressor of flagellum genes. mogR transcription occurs from two alternative promoters: the P1 promoter is located upstream the mogR start codon and produces a shorter and constitutive transcript, whereas the P2 promoter is σ^B-dependent and generates a longer anti0677 transcript that contains mogR and originates on the opposite strand to the coding sequence of the fliNPQ flagellum operon.³ The expression of anti0677 leads to higher transcription of mogR and decreased levels of fliNPQ transcripts, in turn inducing a strong repression of flagellum genes and reduced Lm motility.²⁶

The third category of ncRNAs involved in Lm virulence embraces the trans regulatory RNAs (trans-ncRNAs), which are encoded in trans in relation to the genes they regulate, and are capable both to regulate multiple mRNAs playing with their translation and stability, and to interact with proteins.¹²³ Trans-ncRNAs can act either as antisense or sense RNAs, and this regulation may happen by two different ways: either the trans-ncRNA and its target mRNA are encoded on the same DNA locus, but transcribed in opposite directions leading to the formation of extended base-pairings or, the trans-ncRNA is encoded in a different location of the target mRNA, generating a weaker, short and imperfect complementarity with the mRNA, but allowing to control more than one target mRNA.^133-136 Lm displays several trans-ncRNA involved in its pathogenicity, namely LhrA, LhrC1–5, Rli33–1 (LhrC6), Rli22 (LhrC7), RliB, Rli47 (SbrE), Rli27, Rli31, Rli32, Rli38 and Rli50.³

LhrA is a trans-ncRNA with a role in the transition from exponential to stationary phase of Lm growth, whose stability is dependent on the presence of the Hfq chaperone¹³⁷ Transcriptomic studies clearly showed that lhrA inactivation alters the expression levels of more than 300 Lm genes, including the chiA gene that encodes Lm chitinase A important for Lm pathogenesis in mice, possibly by targeting host proteins involved in immune signaling.^27,138,139 Lm LhrA may modulate host immune response throughout infection by inducting interferon-beta (IFN-β) production.²⁸ In addition, the QS Agr system promotes chitinase activity through negative regulation of this short ncRNA lhrA, which itself binds to chitinase mRNA (chiA) blocking ribosome access and translation.¹⁰⁴ In turn, ChiA secretion by Lm downregulates the inducible nitric oxide synthase (iNOS) activity in infected mice, enhancing bacterial survival.²⁷ MouR, a transcriptional activator of the Lm Agr system, was also shown to repress the expression of lhrA.¹⁰³

trans-ncRNAs belonging to the LhrC family, including LhrC1–5, Rli33–1 and Rli22, were shown to be induced when Lm reaches the human bloodstream.³ Such is the case of LhrC1–5, which is upregulated when bacteria are exposed to the heme group of the human hemoglobulin and positively activated by the response regulator LisR, part of the two-component system LisRK.^29,30 Moreover, LhrCs were shown to repress, at the post-transcriptional level, different Lm virulence-associated genes (oppA, tcsA and lapB), acting by base pairing to the Shine-Dalgarno (SD) region, thus decreasing mRNA levels or inhibiting translation. This regulation by LhrC1–5 is a competitive advantage throughout human blood infection under heme-rich conditions. The downregulation of these surface exposed proteins prevents immune recognition and enhances bacterial survival within the host.^29–31 Additionally, in the presence of the β-lactam antibiotic cefuroxime, LhrC1–5 downregulates the expression of a heme oxygenase-like protein Lmo0484, by base paring to the ribosomal binding site and consequently inhibiting its translation. This repression was found to protect Lm against this cell wall-acting antibiotic.^29,30 Other examples of ncRNAs belonging to the LhrC family and playing a role in Lm virulence are Rli33–1 and Rli22. The lack of rli33–1 has been found to significantly impair Lm survival within macrophages and virulence both in murine and insect models of infection, suggesting a critical role for this ncRNA to favor Lm persistence within host cells.⁴² The expression of rli22 was found to be upregulated in bacteria inside the intestinal lumen of mice or upon bacteria exposure to cell envelope stress in a LisRK two-component system manner.^3,36

Furthermore, there is a significant correlation between the ncRNA RliB and Lm virulence, as demonstrated by the upregulation of this ncRNA in key infection sites such as the mouse intestinal lumen and the blood.³ RliB plays a role in the adhesion and invasion ability of Lm but mechanisms underlying its upregulation and functional role during infection require further research.³⁴

Rli47, also named SbrE, is a σ-dependent ncRNA highly induced in the intestinal lumen and in macrophages.^3,42 In silico and in vivo analysis of SbrE structure reveal its role in binding the SD region of ilvA mRNA, encoding a threonine deaminase involved in the biosynthesis of branched-chain amino acids.³⁵ This interaction occludes the ilvA ribosome binding site, thus repressing its translation. Consequently, the threonine deaminase enzymatic activity is repressed and the initial step of the L-isoleucine biosynthetic pathway blocked. These observations highlight the role of SbrE in the isoleucine biosynthesis repression under harsh conditions, inhibiting Lm growth and impacting virulence.³⁵

Rli27, which is upregulated in the mouse intestinal lumen and in blood,³ was implicated in the post-transcriptional regulation of lmo0514 during the infection process.³⁷ Lmo0514 is an internalin-like protein covalently linked to the bacteria cell wall by a LPXTG motif. It is upregulated during Lm colonization of eukaryotic cells in a PrfA and σ^B-independent manner,¹⁴⁰ and is required for bacterial survival in plasma, having a role in virulence at early infection stages.¹⁴¹ Rli27 interacts with lmo0514 long 5’-UTR, inducing its translation and modulating the cell wall surface protein levels during intracellular infection. Within the eukaryotic niche, this regulation depends on the activity of two promoters of the target gene, generating a short and a long 5′-UTR transcripts, whose relative abundance varies from extra- to intra-cellular bacteria. Importantly, the sRNA binding site is only present in intracellular bacteria, once they are enriched with the long 5’-UTR version.³⁷

The transcription of both rli31 and rli32 ncRNAs increases significantly during Lm infection of macrophages.⁴² These ncRNAs were also found to play a role in Lm virulence.^28,38,39 The absence of rli31 renders Lm more susceptible to the action of lysozyme, leading to a significant attenuation of Lm virulence upon intravenous mice infection. These findings highlight the critical role of Rli31 in protecting Lm from the enzymatic activity of lysozyme present in the bloodstream.³⁸ In addition, in vitro experiments confirmed that Rli31 is capable to bind to the 5’-UTR of spoVG mRNA, a regulator implied in lysozyme resistance, motility and virulence. However, the absence of rli31 does not impact spoVG mRNA or protein levels. Curiously, the absence of both Rli31 and SpoVG induces a contrary effect on the regulation of lysozyme resistance and virulence, probably implying an antagonistic regulatory relationship between Rli31 and SpoVG, that allows bacteria to fine-tune their growth in different environments.³⁹ Rli32, which also binds to SpoVG protein, was found to be secreted from bacteria to the host cytosol upon infection, triggering the expression of β-interferon.^28,39 Additionally, the transcription of rli32 was found to be dependent on VirR, a transcriptional regulator of Lm virulence, suggesting that Rli32 plays a role in virulence and/or resistance to cell envelope stress underscoring its importance in bacterial adaptation and survival strategies.⁴⁰ The absence of rli32 leads to a decrease in Lm ability to survive within macrophages and promotes the downregulation of the ncRNA Rli60 and genes responsible for the tryptophan biosynthesis (lmo1627–33). Conversely, overexpression of rli32 favors Lm intracellular growth and renders the bacteria more susceptible to the action of cefuroxime. The Rli32 overexpression induces Lm resistance to H₂O₂-induced stress and also increases the expression of the ncRNAs LhrC1–4, genes encoding the ferrichrome ABC transport (lmo1958 and lmo1960), and the heme-binding proteins Hbp1 and Hbp2.²⁸ These intricate findings highpoint the multifaceted roles of Rli32 in Lm, impacting various cellular processes, including host interactions, stress responses, and antibiotic susceptibility.

Rli38 was shown to be associated with Lm colonization of mouse organs.^3,41 This trans-ncRNA is capable to target mRNAs of three different genes, including the global iron uptake regulator Fur, related with bacterial adaptation in blood, the membrane associated lipoprotein Lmo0460 that belongs to the internalin family, and the ABC transporter ATP-binding protein Lmo2752. During an oral infection of mice, the absence of Rli38 attenuates Lm virulence.³

Another example of a trans-ncRNAs impacting Lm virulence is Rli50 which exhibits higher transcription levels intracellularly. The absence of rli50 not only impairs the bacterium capacity to proliferate within macrophages but also results in virulence attenuation in both insects and mice.^28,42 These findings highlight the role of Rli50 to promote Lm intracellular survival, pathogenicity and an adequate host immune response.

The last category of ncRNAs is composed by the cis-regulatory RNAs (cis-ncRNAs). Unlike trans-ncRNAs which can act on genes located at different genomic locus, cis-ncRNAs mostly exert their regulatory effects on the same genomic region where they are transcribed from. These ncRNAs can adopt two alternative RNA structures, leading either to transcription termination or transcription antitermination of downstream genes.^118,120 Such is the case of rli60, a cis-ncRNA located upstream of an operon responsible for the biosynthesis of branched-chain amino acids (ilvD), that is transcribed whenever levels of these amino acids are limited.³² Rli60 was implicated in Lm virulence in a mouse model, especially due to the capability of Lm to invade and proliferate within the liver, spleen and macrophages.³³ Interestingly, the absence of rli60 was correlated with increased adhesiveness to macrophages and decreased ability to form biofilms.^33,142 It was also suggested that Lm Rli60 is able to sense the available branched-chain amino acids, that are essential for its optimal growth, downregulating downstream genes accordingly. This regulation favors bacterial pathogenicity by ensuring the proper levels of internal branched-chain amino acids, which are per se a signal for the CodY regulator to bind prfA, thus allowing the expression of a number of major Lm virulence factors.³²

Second messengers

Interpreting extracellular information also requires signal transduction across the bacterial cell membrane. Therefore, intracellular small molecules such cyclic dinucleotides were found to participate in gene transduction through the transmission of signals/stimulus to effector molecules.^143,144 Importantly, second messenger systems can assimilate several sensory inputs modulating the microorganism response output.¹⁴³ Lm uses two major types of intracellular second messengers to modulate its virulence: the cyclic di-adenosine monophosphate (c-di-AMP) and the cyclic di-guanosine monophosphate (c-di-GMP) (Figure 2).

Figure 2. The interplay between host nutritional signals and Lm signaling molecules in virulence. Within the host, Lm imports G6P via the PrfA-regulated permease Hpt, to support its intracellular growth.⁷⁹ Additionally, Lm uses host L-glutamine whose uptake is dependent on the ABC transporter GlnPQ, to further induce the expression of PrfA-controlled virulence genes. Lm is also capable to synthesize L-glutamine, even not being enough to induce virulence genes, in the presence of ammonia and glutamate by using the glutamate synthase GnlA.⁷⁷ the expression of prfA is also dependent on glutathione (GSH), which is synthesized from the host cytosol imported L-cysteine, via the glutathione synthase GshF. L-cysteine uptake is mediated by three different transport systems: the substrate-binding CtaP, the Opp transporter and the ABC transporter TcyKLMN. Once synthesized, GSH binds PrfA, inducing a conformational change that enables its binding to the the PrfA box within the promoter regions of PrfA-regulated genes, thereby activating their expression.¹⁰⁶ Importantly, prfA-transcription is enhanced in response to the low availability of branched-chain amino-acids (BCAAs) through the binding of CodY to its coding sequence.⁷³ Under high concentrations of BCAAs, CodY binds to isoleucine repressing the expression of both ilv-leu operon and purH gene, involved in BCAA and purine biosynthesis respectively, and also rli60. In contrast, isoleucine-unbound CodY can bind to DNA, allowing its own expression and inducing the expression of prfA and consequently PrfA-regulated genes. When challenged with low BCAA levels rli60 is transcribed forming a ribosome-mediated attenuator that restricts the expression of genes belonging to the ilv-leu operon, in turn increasing Lm virulence through CodY expression.³² CodY-mediated regulation is also dependent on c-di-AMP levels. c-di-AMP is synthesized from two ATP molecules via DacA and degraded by PDEs, PdeA and PgpH, giving rise to pApA. High c-di-AMP concentrations induce the up-regulation of CodY regulon, via CbpB-mediated inhibition of RelA activity.¹⁶ This inhibition ensures the maintenance of GTP levels derived from the degradation of c-di-GMP, that induce the expression of CodY.¹⁴⁵

The synthesis of c-di-AMP is required for full Lm viability both in in vitro conditions and within the host.^15,146 This process is catalyzed by a diadenylate cyclase, DacA, from two molecules of ATP and degraded by two phosphodiesterases (PDEs), PdeA and PgpH.¹⁴⁷ Mutants lacking the dacA gene have been found to exhibit reduced infection capability in a mouse model, increased susceptibility to cell wall antibiotics and inability to growth in rich media without salt supplementation.^146,148,149 Moreover, a decrease in c-di-AMPs levels upon DacA depletion or PDEs overexpression results in decreased bacterial replication into primary bone marrow-derived macrophages.¹⁴⁶ DacA has been associated with the induction of IFN-β expression during infection, triggering the innate immune response thereby leading to the activation of host cellular defense mechanisms to control infection.¹⁵⁰ The c-di-AMP binding protein CbpB acts as a c-di-AMP sensor and activates the synthesis of the stringent response regulator (p)ppGpp (guanosine penta-phosphate) by binging to the bifunctional (p)ppGpp synthetase/hydrolase RelA. High c-di-AMP concentrations preventes RelA activation by binding and sequestering CbpB. (p)ppGpp is also a second messenger that binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. CodY is a pleiotropic transcriptional regulator that represses nutrient uptake and amino acid biosynthesis genes in response to the levels of GTP and branched-chain amino acids. c-di-AMP appears essential to regulate (p)ppGpp levels because accumulated (p)ppGpp altered GTP concentrations, thereby inactivating CodY.^15,16 c-di-AMP was also shown to bind and regulate the enzyme pyruvate carboxylase allowing bacterial proliferation and subsistence within the host.¹⁷

c-di-GMP synthesis is accomplished by diguanylate cyclases (DGCs) from two GTP molecules. Beyond being degraded by PDEs, c-di-GMP is able to inhibit its own expression by binding itself to its inhibitory site in order to avoid an excessive GTP consumption.^151-153 Low c-di-GMP levels were found to be required for bacteria to induce an acute infection, while high levels of c-di-GMP are necessary for a long-term host colonization (chronic infections).¹⁵⁴ By binding to effector molecules, c-di-GMP regulates several cellular processes including virulence, QS systems, cell morphology, differentiation, cell–cell communication, exopolysaccharide matrix production, flagellum biosynthesis and pilus assembly.^155-158 Specifically, for its activity at the gene expression level, c-di-GMP interacts with RNAs such as riboswitches and DNA-binding proteins.¹⁵⁹ In Lm, it was demonstrated that high c-di-GMP levels play a negative role on virulence, by inducing the synthesis of an exopolysaccharide that was associated with motility inhibition, cell aggregation and enhancement of tolerance to disinfectants and desiccation. Moreover, high levels of c-di-GMP were found to alter the expression of internalin genes by downregulating the entire virulence regulon, including the master regulator PrfA, thus diminishing not only enterocyte invasiveness in vitro but also the bacterial load in the liver upon oral mice infection.^145,158 It was proposed that elevated c-di-GMP levels induce lower GTP levels, decreasing the activity of CodY, the coordinator of metabolism and virulence, in turn suppressing Lm virulence via decreased PrfA expression.¹⁴⁵

Additionally, the degradation of both c-di-AMP and c-di-GMP can result on the formation of two linear dinucleotides, pApA and pGpG, respectively, that are important products of the RNA metabolism.^151,160,161 Both molecules can also be hydrolyzed by the oligoribonucleotidase NrnA, generating the mononucleotides AMP and GMP, respectively. Concomitantly, the nrnA gene was also found to be up-regulated during Lm biofilms formation.¹⁶²

Two-component systems

Beyond the signaling molecules previously discussed, Lm employs other regulatory mechanisms that contribute to establish infection. Among them, two-component transduction systems (TCSs) stand as paramount players enabling bacteria adaptation to the conditions encounter within the host. These bacterial systems are typically composed by a transmembrane sensor histidine kinase and a cytoplasmic response regulator, both working together to sense and process external stimuli including nutrients, metal ions, host-derived metabolites and gut specific conditions. External cues usually trigger the activation and autophosphorylation of the histidine kinase, and the phosphoryl group is transferred to the cognate response regulator, which in turn is activated to work as a transcription factor that regulates gene expression.^163,164 So far, among the 14 TCSs of Lm, four have been related to its pathogenicity: CesRK, LisRK, ViRS, and PieRS (Figure 3).

Figure 3. Schematic representation of the two-component transduction systems governing virulence gene expression in Lm.

CesRK, which is composed by the cephalosporin sensitivity response regulator CesR and its sensor histidine kinase CesK, is implicated in sensing and responding to changes in the cell wall integrity and in the expression of cell envelope-related genes.^43,44 In the absence of cesR, Lm becomes less virulent in mice upon intragastric infection, but not after intraperitoneal inoculation, thus suggesting a prime role for CesRK TCS in Lm pathogenicity.⁴³ Furthermore, CesRK is capable to sense not only the presence of β-lactam antibiotics typically used to treat Lm infections, but also ethanol and lysozyme, further inducing the transcription of the downstream small open reading frame orf2420 (lmo2420), also important for bacterial pathogenesis.⁴³ Lm CesK perceives cell wall damages caused by antimicrobial agents, generating a faster response by the bacteria that activate a cascade of events that culminate in the upregulation of key genes involved in cell wall biosynthesis, such as lmo0441 and lmo2812.⁴⁴

LisRK was found to be implicated in the transcription regulation of the htrA gene, that encodes a HtrA-like serine protease involved in Lm osmoregulation and pathogenesis,⁴⁵ and conferring protection against some β-lactams antibiotics such ampicillin.^46,47 It was suggested that Lm LisRK is able to sense perturbations in the surrounding environment, such as the acidic conditions or the presence of cell wall-acting antibiotics.⁴⁵ LisRK also plays a role in the invasion capacity of the pathogen, mutant bacteria becoming significantly less virulence upon mice infection.^165,166 Additionally, this TCS together with the trans-ncRNAs LhrC1–5, are important for intracellular replication of Lm into macrophage-like cells.³¹

The VirRS system, which composes another TCS, acts as a positive transcriptional regulator of genes mainly involved in cell surface modifications, such as the dlt-operon and mprF, which are crucial for bacteria to fight human defensins and cationic peptides.⁴⁸ The absence of the virR gene is associated with the impairment of Lm cell invasion and infection capacity in mice. Interestingly, VirR activation was shown to be not totally dependent on its cognate kinase, since it also depends of an ABC transporter encoded by the genes lmo1746–47 shown to be required for optimal intracellular infection and virulence.^40,48 Contrarily to others TCS where both genes encoding the sensor and the response regulator are contiguous on the chromosome, virR and virS are not adjacent but separated by three genes. Likewise, VirS appears to interact with other response regulators, regulating genes that are not affected by the absence of virR, thus suggesting a non-exclusive interaction between VirS and VirR.⁴⁸

The TCS PieRS plays a role in promoting Lm translocation across the intestinal barrier by regulating the secretion of two chaperones, PrsA1 and PrsA2, both found to be associated to the membrane and secreted in the extracellular medium.⁴⁹ PrsA2 plays a significant role in hemolytic and phospholipase activity, being important for Lm cell-to-cell spread and full virulence following both oral and intravenous mice infection,^{49,50,167–170} while PrsA1 is required for Lm traffic from the intestine to the liver.⁴⁹ It was proposed that when Lm is faced with harsh conditions, such the gastric stress found in the gastrointestinal tract, PieS, the sensor kinase, triggers the activation of the response regulator PieR, leading to the activation of both prsA1 and prsA2, thus enabling bacterial survival and crossing the intestinal barrier to infect target organs.⁴⁹

Glutathione

In an attempt to mediate Lm transition to a pathogenic state, it can use the tripeptide glutathione (GSH) as a metabolic signaling molecule modulating its virulence (Figure 2). This process relies on the allosteric binding of GSH to the major Lm transcriptional regulator PrfA, inducing a conformational change on PrfA and therefore its binding to DNA. Consequently, virulence genes regulated by PrfA are expressed, promoting infection.^18-20 Within bacteria, GSH levels are very limited.⁶³ Even so, Lm is able to synthesize GSH by making use of its glutathione synthase (gshF).^18,64 GshF is a bifunctional enzyme able to synthesize a GSH intermediate, γ-glutamylcysteine, by binding the γ-carboxyl group of L-glutamate to L-cysteine, previously imported from the surrounding environment since Lm is not able to synthesized it. Subsequently, GshF mediates the fusion of this intermediate product with glycine to yield GSH (also known as L-γ-glutamyl-L-cysteinylglycine).^64,171 On the other hand, inside mammalian cells Lm encounters abundant concentrations of GSH being able to import and use it as a signaling molecule to regulate the expression of virulence genes. Curiously, the host-synthesized GSH cannot completely replace the GSH produced by the bacteria itself to activate PrfA and PrfA-dependent genes. Even though Lm strain defective for gshF do not suffer a general loss of fitness, it exhibits a decrease in the expression levels of some virulence factors, such as the actin assembly-inducing protein ActA required for Lm intracellular motility, suggesting that extracellular GSH could function as a signal for bacteria into the host intracellular niche.^18,172 Importantly, L-cysteine presents itself an amino acid rate-limiting in the synthesis of GSH, directly affecting the expression of prfA and PrfA-controlled genes, hence limiting Lm virulence.^171,173 Three different transport systems were already described as being involved in the acquisition of cysteine for GSH biosynthesis: (i) CtaP, a substrate-binding component of a transport system for L-cysteine (ii) the OppABCDF transporter, capable to import exogenous cysteine-containing peptides and (iii) the ABC transporter TcyKLMN capable to collect both cysteine and its reduced form cystine from the extracellular medium.^63,65,174 Even being all implicated in the bacteria virulence, only the TcyKLMN system appears to be linked to Lm growth within the host cytosol by supplying L-cysteine for GSH synthesis.⁶³ The TcyKLMN system is controlled by three metabolic regulators CodY, CymR and CysK. When faced with limited availability of branched-chain amino acids, Lm up-regulates the TcyKLMN system to uptake cysteine from the extracellular medium, thereby promoting GSH biosynthesis and consequently PrfA activation.⁶³ A gshF mutant was shown to have a defect in growth in vitro, in the presence of diamide and copper ions stress. Moreover, it was shown that gshF is required for efficient invasion and proliferation both in macrophages and mice organs. GshF plays a role in Lm response to oxidative stress, inhibiting the transcription of the lmo1997-lmo2004 operon, that encodes a phosphoenolpyruvate-carbohydrate phosphotransferase system, being lmo2002 the most critical gene to resist to oxidative stress.¹⁷⁵

Additionally, Lm is able to use inorganic sulfur thiosulfate and H₂S as L-cysteine sources, supporting its growth and virulence. This process occurs via the Lm two-step biosynthetic pathway of L-cysteine, that involves the conversion of L-serine into L-cysteine by the CysE/CysK enzymes.¹⁷⁶ Despite these observations, the mechanism by which Lm imports thiosulfate remains unclear.

Furthermore, the toxic by-product of cellular metabolism methylglyoxal, produced by both the host and the bacteria, was recently found to be associated with PrfA activation since it functions as a host cue for Lm to produce GSH. Lm displays a GSH-dependent methylglyoxal detoxification system composed by two enzymes, GloA (glyoxalase A) and GloB (glyoxalase B) that mediate the detoxification of methylglyoxal by conjugation with glutathione. During Lm growth within activated macrophages, it faces higher levels of methylglyoxal, which consequently triggers the activation of gshF, therefore resulting in increased levels of bacteria GSH and PrfA activation.¹⁷⁷

Host nutritional and physical signals triggering Listeria monocytogenes virulence

Environmental cues signaling the colonization of the bacteria into the host are implicated in the regulation of bacterial virulence factors. Among them, the host temperature, pH, iron availability, bile salts, nutrients and the oxidative stress play key roles on how Lm fine-tunes gene expression and protein translation.¹⁷⁸ This section will focus on the impact of these external signals in Lm pathogenesis.

Temperature

Temperature increase is sensed by bacteria when they shift from the external environment to mammalian hosts. Many bacteria are thermally regulated and thus may alter the expression of their virulence genes to promote infection.¹⁷⁹ Moreover, the temperature may not only influence the DNA topology, RNA structure and metabolism, as it may impact the activity and processing of proteins.¹⁷⁸

For Lm, the temperature increase to 37°C within the host is a signal to activate the expression of the PrfA thermosensor, triggering the expression of a number of major virulence genes (Figure 1A). At temperatures below 37°C, the mRNA 5’-UTR of prfA forms a secondary structure which masks the ribosome binding region, repressing the translation of the downstream mRNA. The temperature increase to 37°C makes impossible the formation of this inhibitory RNA structure, inducing the expression of prfA and PrfA-controlled virulence genes.^130,180 Moreover, the temperature rise also triggers a conformational shift in the anti-repressor GmaR, allowing the expression of MogR and consequently, the repression of genes involved in flagellar-based motility (Figure 1A). While flagellar motility is indispensable for bacterial survival in the environment for nutrient acquisition and surface colonization, within the host, flagella is highly immunogenic working as a signal that activates the immune system, and thus detrimental for the virulence.^51,52

pH

After entering the human body, Lm encounters resistance against colonization imposed either by the harsh conditions of the gastrointestinal tract or by the host immune system.

Acidic environments trigger the activation of the Lm stressosome, a stress sensor that induces a general adaptative stress response crucial to confer acid resistance.⁵³ Lm stressosome is a dynamic signaling multi-protein complex composed by RsbR1 (lmo0889) and its paralogues RsbR2 (lmo0161), RsbR3 (lmo1642), RsbR4 (lmo1842) and RsbL (lmo0799), the scaffold protein RsbS and the serine – threonine kinase RsbT. Stressosome activation is achieved by RsbR1 and RsbS that sense and transduce the acid stress signals. This sensing is dependent on the phosphorylation of RsbR1 (at residue T209) and RsbS (at residue S56), both mediated by the RsbT kinase. Subsequently, RsbT is release from the stressosome and interacts with the RsbU phosphatase, which consequently promotes the dissociation of the anti-sigma factor RsbW from σ^β. These events culminate in the upregulation of genes involved in the general stress response and infectivity, thus favoring the host invasion.^53-56 Moreover, the activation of σ^β by the stressosome is also required for the activation of the glutamate decarboxylase enzyme gadD3, that converts L-glutamate into γ-aminobutyrate (GABA) while consuming an intracellular proton, and of the arginine (arcA) and agmatine (aguA1) deiminases, which metabolize L-arginine into citrulline and ammonia to adjust the cytoplasmic pH.^54,181 During the first process, the consumption of one proton itself is enough to increase the intracellular pH thus attenuating acidic stress.¹⁸² GadD3 together with GadD1 and GadD2 enzymes and the glutamate/GABA antiporters (GadT1 and T2) form the glutamate decarboxylase (GAD) system which is key to maintain Lm pH homeostasis in acidic environments.^57,183,184 The GAD system comprises two subsystems that differ depending on the location of the required glutamate: the first involves the uptake of extracellular glutamate by antiporters, which undergoes subsequent decarboxylation resulting in the production of GABA. Subsequently, GABA is exporter back by the same antiporter mechanism while more glutamate is being imported; for the second subsystem, Lm uses and processes intracellular glutamate to regulate its intracellular pH, in a process dependent on GadD3 and GadD1.^184,185 GAD is also involved in Lm survival under oxidative stress, namely by conferring resistance to hydrogen peroxide.⁵⁸

Macrophages are capable to recognize and phagocytose the bacteria, engulfing them into intracellular vacuoles termed phagosomes. When cells are infected by Lm, the phagosome where the bacterium is contained acidifies to a pH around 5.5 to 6. This acidification works as a switch to induce listeriolysin O (LLO) pore-forming activity per se mediating the vacuolar disruption and allowing bacteria release from the phagosome and spread.^59-62 The cytolytic activity of LLO involves the binding and subsequently oligomerization of LLO monomers within the intracellular vacuole membrane leading to the formation of pores and consequently vacuole disruption. Importantly, LLO activity is intricately linked to the pH environment and controlled by an acidic triad present in the transmembrane domain (D3) of LLO that functions as pH sensor. In stark contrast to the acidic environment encountered within the phagosome, inside the host cell cytosol Lm faces neutral pH that destabilizes the D3 domain of LLO promoting an inactivation of LLO by the formation of irreversible denatured monomers. This dual-regulation mechanism highlights the sophisticated interplay between pH dynamics and the functional modulation of LLO in the context of Lm infection.^61,186,187 Importantly, the pH-dependency also avoids uncontrolled LLO activity and consequently its insertion into the host cell membranes that would leave the bacteria constantly exposed to extracellular threats.⁶²

Iron

Iron, a vital element for the growth of almost all living cells, is involved in important cellular functions such as the transport and storage of oxygen, and plays a role on the bacteria toxicity and pathogenesis.^188-190 Although this element is abundant in the environment, the available free iron in the host is restricted. Therefore, the adaptation to shifts between high and low free iron environment is an important signal for bacteria to regulate their gene expression.¹⁸⁹ Lm needs iron for growth and survival during infection namely to establish itself in the gastrointestinal tract.¹⁹¹ To acquire iron, Lm displays different mechanisms involving the use of iron reductases and ferric citrate uptake systems. Additionally, Lm can use siderophore like-uptake systems.^192,193 Moreover, the host-derived heme found in hemoglobulin and other hemoproteins, also represents an important source for iron and can be used as metabolic cofactor for many bacteria including Lm.^82,192 Into the host cytoplasm, Lm may use iron to support its growth and proliferation and to be protected from the ROS-mediated damage. Furthermore, to mitigate ROS-mediated damage in phagosomes, Lm may be also able to sequester iron.^80,81 Some studies have reported Lm ability to use the iron available to assess its location and regulate its behavior in term of gene expression, accordingly. In the presence of higher iron concentrations found in the human gut,¹⁹⁴ the invasins InlA and InlB (both required for the invasion of non-phagocytic cells including intestinal epithelial cells)¹⁹⁵ are positively regulated, probably to enhance Lm internalization into intestinal cells.^83,84 Contrary, when bacteria are under iron starvation conditions, the expression of both LLO and ActA is increased for Lm to escape from the vacuole, reach the host cytosol, and spread to neighboring cells.^83,84,196 Additionally, when Lm encounters an heme rich environment, it induces the upregulation of FrvA, a specific Fe(II) exporter ATPase part of the PerR (a peroxide-inducible stress response regulator) and Fur (a ferric uptake regulator) regulons.⁸⁵ The absence of frvA induces a significant attenuation of virulence in mice.⁸⁶

Lm is also using heme as a host-derived signaling molecule to control the expression of the Listeria adhesion protein (LAP)⁸² a virulence factor that induces intestinal epithelial barrier dysfunction to promote Lm translocation.¹⁹⁷

Bile Salts

During the initial infection stage, Lm encounters bile salts in the gastrointestinal tract using them as signals to ascertain its location and to adapt to the surrounding environment.⁶⁷ Bile salts are synthesized in the liver, using cholesterol as precursor, being then conjugated with glycine or taurine and released into the small intestine from the gallbladder.¹⁹⁸ About 10% of the Lm genome was found to be regulated by bile. Among them, the bile sensor BrtA was found to be induced.⁶⁷ The bile salt hydrolase bsh is expressed when Lm is in the gastrointestinal tract to promote bile salts resistance catalyzing the hydrolysis of glycodeoxycholate (GDCA) and taurodeoxycholate (TDCA) conjugated bile salts into cholic acids (Figure 1A).⁶⁶ Consequently, BrtA exposure to cholic acids inhibits BrtA binding to the promoter of the multi-drug efflux pump mdrT, inducing MdrT expression and allowing the export of cholic acids.⁶⁷ Intriguingly, the overexpression of mdrT can be detrimental for Lm virulence in vivo,⁶⁸ highlighting the importance of Lm in controlling the bsh expression levels in the course of the infection. In this sense, Lm uses the transcriptional repressor CadC to repress bsh expression at late infection stages, avoiding the detrimental overexpression of mdrT during this step of the infection.^4,69 The efflux pump MdrT is also capable to transport c-di-AMP,¹⁹⁹ inducing a type I IFN-β response which enhances the susceptibility to Lm infection,^200–202 reinforcing the importance of mdrT regulation by CadC.

Peptides

Bacterial pathogenicity is significantly influenced by the availability and acquisition of nutrients from the host environment. Different Lm auxotrophic mutants for several nutrients including uracil, phenylalanine, glycine, proline or nicotinic acid were found to retain full virulence, suggesting that Lm can acquire these nutrients from the host environment. Interestingly, these strains seem to be able to utilize other nutrient sources such as peptides.²⁰³ Additionally, Lm is capable of exploiting exogeneous oligopeptides, an ubiquitous source of organic nitrogen, to sense the environment and control PrfA activity. Indeed, PrfA activation has been shown to be dependent on the presence of cysteine containing peptides (Cys/Cys-peptides) in the host environment, while being antagonized by oligopeptides lacking cysteine, such as leucine.^65,78,204 While the activation of PrfA requires occupancy of both GSH sites in the PrfA dimer, nonspecific peptide binding (such as leucine dipeptide) to one monomer is enough to change the symmetry of the two helix-turn-helix motifs, impairing DNA binding and consequently, the expression of virulence genes.⁶⁵

Branched-chain amino acids (BCAAs)

The availability of branched-chain amino acids (BCAAs), including isoleucine, leucine, and valine, constitutes another important metabolic signal sensed by Lm (Figure 2). BCAAs represent an important source of nutrients for bacteria and are crucial to remodel their virulence gene expression, protein synthesis and to control bacterial adaptation to amino acid starvation.⁷⁰ The response to BCAAs is dependent of CodY, a Lm BCAA-responsive transcriptional regulator capable to directly bind isoleucine, inducing a conformational change that promotes binding to the DNA.^71,72 This mostly happens under high concentrations of BCAAs, where CodY acts as a repressor of ilvC and purH genes, involved in BCAAs and purine biosynthesis, respectively.^32,70 Interestingly, within mammalian cells, where BCAAs are deprived (in particular isoleucine), isoleucine-unbound CodY is capable to activate prfA and therefore, PrfA-regulated virulence genes, by directly binding to the first 15 nucleotide of the prfA-coding sequence. This suggests an additional functional conformation for CodY in such conditions and thus supports the idea that the gene regulation by CodY depends on its affinity to different sites and the levels of BCAAs available.^70,73 To this end, rli60, a ncRNA encoding a ribosome-mediated transcription attenuator, cis-regulates downstream genes involved in BCAA biosynthesis. Under BCAAs limiting conditions, as occurring during infection, the ncRNA rli60 is transcribed creating a ribosome-mediator attenuator that restricts the expression of the ivl-leu operon and consequently the production of BCAA. This BCAA starvation allows Lm to control the internal levels of isoleucine thereby promoting virulence gene expression via CodY.³²

Free fatty acids (FFAs)

After ingestion, Lm needs to overcome different metabolomic landscapes throughout the mammalian gastrointestinal tract, with different metabolites produced either by the host itself or by commensal microorganisms or other microbial agents. The ability to sense and respond to these gut metabolites is therefore central for the successful colonization, since it allows bacteria to efficiently regulate gene expression accordingly. Free fatty acids (FFAs) are highly present in the gut metabolome, being important nutrient sources, not only for the resident microbiota but also for bacterial pathogens to signal gene expression by regulating DNA binding activity of virulence-related transcriptional factors.²⁰⁵ Furthermore, FFAs act as antimicrobial agents displaying a bacteriostatic or bactericidal activity mainly by targeting the cell membrane.²⁰⁶ In Lm it was found that the presence of long- or medium- FFAs impairs PrfA-dependent genes activation (Figure 2). FFAs are capable to bind PrfA, inhibiting its DNA binding activity, and preventing the activation of PrfA-regulated virulence genes.^74,75

Non-dependent phosphotransferase system host-derived sugars

The host intracellular environment displays different carbon sources that can be used by Lm. To import and use these carbon sources, Lm has evolved a non-dependent phosphotransferase system (non-dependent PTS) that allow the bacteria to make use of host phosphorylated sugars independently of the PTS system itself, thus offering a more efficient way for sugar utilization.^207,208 These carbon sources include hexose phosphates, such as glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P), and glycerol, being all signals sensed and rapidly metabolized by Lm to improve fitness and growing into the host.⁷⁹

G1P is mainly available intracellularly within the host, as a result of the primary degradation of glycogen.²⁰⁹ This sugar can be used by Lm intracellularly as a growth substrate, under the positive regulation by PrfA and therefore co-expressed with PrfA-regulated genes in response to diverse host signals.⁷⁸ Beyond G1P, Lm is also capable to proficiently import host produced G6P by using Hpt, a non-PTS permease, whose expression is tightly regulated by PrfA (Figure 2). The lack of Htp impairs Lm intracytosolic replication and proliferation within mammalian cells, and survival in mice.⁷⁹

Additionally, glycerol can be imported by the Lm uptake facilitator GlpF, sustaining its bacterial growth within the host cytosol. In the presence of glycerol, GlpF facilitates its transport across the bacterial cell membrane to be posteriorly metabolized by GlpK, a glycerol kinase that phosphorylates glycerol to produce glycerol-3-phosphate. The resulting product undergoes processing by DhaK kinase, to produce dihydroxyacetone phosphate (DHAP),²¹⁰ which is involved in the glycolysis and gluconeogenesis processes.²¹¹ Furthermore, the use of glycerol not only provides an advantageous available source of energy, but also controls Lm virulence through the upregulation of PrfA-controlled virulence factors such InlC, LLO and ActA, crucial for the bacteria to invade and spread within host cells.²¹⁰

By contrast, other carbon sources such cellobiose and glucose, typically found outside the host, are transported by phosphoenolpyruvate-dependent phosphotransferase systems and may work as signals for Lm sensing the surrounding environment, where the PrfA-dependent gene expression is no longer required.^212–214

L-glutamine

L-glutamine, an amino acid abundant in mammalian serum and cells, is used by Lm as a nitrogen source and was found to be involved in Lm virulence modulation (Figure 2).^76,77 Lm can sense the presence of extracellular L-glutamine, using the ABC transporter GlnPQ for its recognition and uptake, GlnP being a substrate binding transmembrane protein, and GLnQ an ATPase.⁷⁷ In the presence of external high concentrations of L-glutamine, GlnPQ senses L-glutamine, inducing the expression of virulence genes (including hly, plcA, plcB and actA), and promoting macrophage and mouse infection.⁷⁷ Lm has the ability to synthetize L-glutamine itself when it access to both ammonia and glutamate (via glutamate synthase, GnlA).⁷⁶ Yet, the resulting L-glutamine is not enough to induce virulence genes expression. This highlights the ability of Lm to distinguish between bacterial or host signals, thus controlling its own localization within the host.⁷⁷

Reactive oxygen species

Within the human body, bacteria must cope with reactive oxygen species (ROS), such as superoxide (O₂⁻), hydroxyl radicals (HO) and hydrogen peroxide (H₂O₂). Once exposed to ROS, Lm can activate specific regulatory pathways that contribute to its survival and virulence within the host. A regulator involved in ROS sensing and gene expression is the transcriptional factor PerR (peroxide-inducible stress response regulator). PerR represses the expression of genes involved in response to peroxide stress and metal homeostasis, including kat (hydrogen peroxide decomposition), fur (iron homeostasis regulator), hemA (heme biosynthesis), fri (iron-binding protein) and fvrA (iron efflux pump). The absence of perR is related with an increased Lm sensitivity to H₂O₂ in vitro, and decreased virulence in vivo. This strongly suggests the importance of PerR to both routine peroxide detoxification and response to ROS stress.⁸⁷

ROS generated by activated macrophages, which are fundamental to control Lm infection, seem to have also a role in the activation of Lm virulence genes prfA and hly. In activated macrophage-like cells, hly expression is enhanced. This increased hly expression can be abolished by the addition of superoxide dismutase and catalase, suggesting a role for reactive oxygen intermediates to control hly and prfA mRNAs levels.⁸⁸ Moreover, mutations leading to the constitutive activation of prfA enhance resistance to oxidative stress. Interestingly, the Lm resistance to the oxidative stress seems to be dependent on bacterial density.⁸⁹ Lm appears thus to be well equipped for both redox sensing and survival during the course of host infection.

Conclusions and Future Directions

The regulation of virulence factors in the intracellular pathogen Lm occurs in a complex and finetuned process, orchestrated by a network of regulatory systems that are able to sense and respond to various signals. These not only include signals secreted by Lm itself to promote its fitness and virulence, but also host cues that allow it to recognize the surrounding environment and modulate gene expression accordingly, to support its survival within the host and to facilitate the host colonization. The interplay between Lm signaling molecules and the host signals is also necessary to develop a proper response and to adapt to the different host environments.

Importantly, some of these signals that command virulence factors are common to several bacteria, both Gram-positive and Gram-negative. Therefore, deeply knowing and exploring these signals is certainly useful to modulate the expression of bacterial pathogen mechanisms throughout infection. Virulence genes can be essential and thus activated at specific host infection stages to promote virulence, but detrimental for infection at certain point. Furthermore, virulence factors are not expressed homogenously among the entire bacterial population, which per se also constitute a mechanism to optimize infection capacity. Controlling the signals behind these strategies can allow us to modulate pathogenesis, playing with the regulation of virulence genes in time and space, during infection.

Unravelling the bacterial and host signals controlling Lm virulence mechanisms facilitates the understanding of its pathogenesis and paves the way for the identification of new potential targets and the design next-generation therapeutic approaches to control Lm virulence.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The work was supported by the 'la Caixa' Foundation [LCF/PR/HR23-00682];  Fundação para a Ciência e a Tecnologia [2021.04806.BD].