Shining a light on bacterial environmental cue integration and its relation to metabolism

SUMMARY

The ability of a bacterium to successfully colonize its host is dependent on proper adaptation to its local environment. Environmental cues are diverse in nature, ranging from ions to bacteria-produced signals, and to host immune responses that can also be exploited by the bacteria as cues. Simultaneously, bacterial metabolism must be matched to the carbon and nitrogen sources available at a given time and location. While initial characterization of a bacterium’s response to a given environmental cue or its ability to utilize a particular carbon/nitrogen source requires study of the signal in question in isolation, actual infection poses a situation where multiple signals are present concurrently. This perspective focuses on the untapped potential in uncovering and understanding how bacteria integrate their response to multiple concurrent environmental cues, and in elucidating the possible intrinsic coordination of bacterial environmental response with its metabolism.

Graphical Abstract

Bacterial host colonization is dependent on a bacterium’s ability to sense and respond to diverse environmental cues for adaptation, and to match its metabolism appropriately. This perspective focuses on the untapped potential in uncovering and understanding how bacteria integrate their response to multiple concurrent environmental cues for a coordinated adaptive output, and in elucidating the possible intrinsic coordination of bacterial environmental response with its metabolism.

INTRODUCTION

In the context of infection, all bacteria must be able to navigate changes in their local environment from the point of infection through to establishment of a replicative niche, and throughout the remainder of host colonization. Environmental signals that can be exploited by the bacteria as cues are diverse in nature, ranging from abundant (e.g. protons (H⁺, pH), chloride (Cl⁻), potassium (K⁺)) to scarce (e.g. iron, manganese, nickel) ions (Murdoch and Skaar, 2022, Tan, 2021), to bacterial-produced small molecules and outputs arising from host immune responses (e.g. nitric oxide) (Yoon and Waters, 2021, Stern and Zhu, 2014). Sensing of these cues and their transduction into adaptive responses is mediated via various regulatory systems (Matilla et al., 2022, Galperin, 2018), with two-component systems (TCSs) ubiquitous and among the best studied classes (Krell et al., 2010). Crucially, disruption of TCSs often strikingly attenuate a bacterium’s ability to colonize its host (see for example (García Véscovi et al., 1996, Richardson et al., 2006, Pérez et al., 2001)), underlining the importance of environmental sensing and response for the pathogen. However, while TCSs have been a topic of intense study, and several environmental signals such as pH, iron, and hypoxia are well-appreciated, the question of how the simultaneous presence of disparate environmental cues impact on a bacterium’s response to any one signal has been much less studied. This is an important aspect, given the complex environment a bacterium experiences throughout infection. At the same time, successful host colonization by the bacterium further requires that adaptation encompass appropriate changes to metabolism, to best utilize available carbon and nitrogen sources. Might response to environmental signals, such as different ions, be additionally intrinsically linked to changes to a bacterium’s metabolic program, as a direct conduit to ensure proper adaptation? Here, we highlight recent data that begins to explore these intriguing questions of the complexity of bacterial integration of response to different environmental cues, and its possible direct coordination to bacterial metabolism.

LINKING OF BACTERIAL RESPONSE TO DIFFERENT ENVIRONMENTAL CUES

A bacterium infecting a host is faced with a highly complex environment that further changes both temporally and spatially. For example, in the case of Mycobacterium tuberculosis (Mtb), the ion concentrations (e.g. H⁺, Cl⁻. K⁺) it is exposed to changes as the macrophage phagosome in which it often resides matures (Tan et al., 2013, MacGilvary et al., 2019, Rohde et al., 2007), even while Mtb actively prevents complete maturation of the phagosome (Sturgill-Koszycki et al., 1994). The local environment also differs spatially within the lung granulomas that are hallmark lesions of Mtb infection, with the bacteria present intracellularly within immune cells in the lesion cuff exposed to different stressors than those residing extracellularly within the necrotic lesion center (Lavin and Tan, 2022). While bacterial responses to concurrent signals can result in separate responses that each contribute independently to host adaptation, the ability of a bacterium to integrate its response to different cues provides an additional layer of coordination, potentially expanding the dynamic range of the response and opening new possibilities for precise regulatory control and adaptation. Of note, there are multiple examples of TCSs that are responsive to more than one signal. To name just a few, this includes PhoPQ (acidic pH and Mg²⁺) in Salmonella (Prost et al., 2007, García Véscovi et al., 1996), PhoPR (acidic pH and high [Cl⁻]) and DosRS(T) (hypoxia and nitric oxide) in Mtb (Tan et al., 2013, Kumar et al., 2007), and SrrAB (hypoxia and nitric oxide) in Staphylococcus aureus (Kinkel et al., 2013). While studies focusing on a single discrete signal are vital for establishing a base understanding of a bacterium’s ability to sense and respond to the signal in question, comprehension of the in vivo biology that occurs necessitates an expansion to studies that interrogate the bacterial response in the context of the multiple signals experienced concurrently.

Excitingly, the few studies that have examined this question have begun to demonstrate the synergy and biological importance of signal integration. In particular, genes upregulated by both acidic pH and high [Cl⁻] in Mtb showed a synergistic increase when both environmental cues are simultaneously present (Tan et al., 2013). With Clostridioides difficile, calcium (in the presence of taurocholate) decreased the concentration of multiple amino acids required for germination (Kochan et al., 2018). This calcium-amino acid synergy could further overcome the inhibitory effects of germination by the secondary bile salt chenodeoxycholate (Kochan et al., 2018). Most recently, it was intriguingly found that the simultaneous presence of the quorum sensing molecule autoinducer-2 (AI-2) and the polyamine norspermidine (which promotes cyclic di-GMP production) resulted in a synergistic increase in Vibrio cholerae biofilm formation greater than that observed in the presence of norspermidine alone, even though AI-2 when present alone acts to repress biofilm formation (Prentice et al., 2022). In all cases, integration of signals and synergy in transcriptional/functional outcome likely provide a mechanism for increased precision in a bacterium’s “knowledge” of its location within a host and the relative favorability for growth in that time and location, enabling adaptive responses to occur and successful host colonization. Further studies striving to uncover how bacterial responses to one signal change in the simultaneous presence of other signals will thus be vital for our complete understanding of bacterial adaptation to its local environment during infection.

COORDINATION OF BACTERIAL ENVIRONMENTAL RESPONSE WITH ITS METABOLISM

Bacterial responses to the environmental signals “seen” during infection are ultimately directed towards adaptation for continued survival and growth in the host. This also requires concomitant adaptation of the bacterium’s metabolism for utilization of essential elements, including available carbon and nitrogen sources. Changes in bacterial metabolism due to specific nutrient availability is a direct relationship that is well-documented (Faucher et al., 2011, Pandey and Sassetti, 2008). Beyond this straightforward relationship however, a burgeoning number of studies are also now demonstrating that the presence/utilization of specific metabolites can affect a bacterium’s expression of “virulence factors”. From the bacterial perspective, “virulence factors” fundamentally act to aid successful colonization of the host by (or further transmission of) the bacterium. For example, pyruvate increases expression of the pore-forming leucocidins in S. aureus (Harper et al., 2018), while succinate induces expression of Salmonella pathogenicity island 2 genes (Rosenberg et al., 2021). Expression of invasion-related genes in Salmonella are also affected by short-chain fatty acids – high levels of acetate, found in the distal ileum, increased expression of invasion-related genes, while high levels of butyrate and propionate, present in the colon, decreased expression (Lawhon et al., 2002). In Borrelia burgdorferi, the expression of RpoS and virulence-associated lipoproteins that it regulates were upregulated in the presence of several short-chain fatty acids (acetate, propionate, or butyrate) (Lin et al., 2018).

Nitrogen source-related metabolites can similarly affect bacterial virulence factor gene expression. In particular, L-glutamine has been shown to induce expression of several Listeria monocytogenes virulence factors, including the listeriolysin O toxin and ActA, required for host actin recruitment and cell-to-cell spread (Haber et al., 2017). This induction functioned as an on/off switch, and was separate from nitrogen availability per se, as other nitrogen sources such as ammonia did not induce expression of these virulence factors (Haber et al., 2017). In group A Streptococcus, asparagine alters expression of ~17% of bacterial genes, including upregulation of sil (streptococcal invasion locus), while the absence of asparagine drives expression of the streptolysin O and S toxins (Baruch et al., 2014). These examples emphasize how the presence of specific nutrient sources can direct bacterial virulence gene expression.

Even more intriguingly, recent data has further begun to raise the concept of intrinsic links between a bacterium’s response to environmental cues with its metabolism. Specifically, an increase in the level of induction of genes in the Cl⁻ regulon of Mtb was observed in the simultaneous presence of cholesterol and high Cl⁻ levels (Lavin et al., 2021). Cholesterol is a vital nutrient for Mtb in vivo (Pandey and Sassetti, 2008), and given the synergistic response of Mtb to acidic pH and Cl⁻ (Tan et al., 2013), we anticipate that similar cross-regulation of the Mtb acidic pH regulon by the presence of cholesterol is also likely to occur, and studies pursuing this new facet of Mtb biology are in progress. Of note, it has also been reported that the growth of Mtb in acidic pH is improved in the presence of cholesterol or even-chain fatty acids (Gouzy et al., 2021, Baker et al., 2014), and iron limitation results in upregulated expression of Mtb cholesterol utilization genes (Kurthkoti et al., 2017, Theriault et al., 2022). Together, these findings indicate the different relationships that can exist between bacterial metabolism and environmental cue response, and future studies probing this complex aspect will undoubtedly reveal new relationships critical for bacterial adaptation to its host.

CONCLUDING REMARKS

The questions that remain to be answered in building and expanding our understanding of the concepts of bacterial integration of disparate environmental signals, and the intrinsic links between bacterial metabolism and environmental cue response, are numerous and varied. What are the mechanisms that underlie the integration of different environmental cues into a single, adaptive, response by a bacterium? Besides the ability of a single TCS to respond to multiple cues, how may cross-regulation and cross-talk between TCSs aid this integration (Laub and Goulian, 2007, Mike et al., 2014)? While the focus of this perspective has been on TCSs, it is important to note that other transcriptional regulatory systems, such as sigma factors, are also well-positioned to aid in bacterial signal integration. For example, σ^S (RpoS) has been extensively studied as a master regulator of the general stress response, particularly in enteric bacteria such as Escherichia coli and Salmonella (Battesti et al., 2011, Hengge-Aronis, 2002). How about the role of post-translational modifications mediated by other regulatory systems such as serine/threonine protein kinases (Giacalone et al., 2022, Dworkin, 2015)? Do these regulatory systems have differing importance depending on spatial location of the bacterium within a host tissue? What is the physical mechanism by which each environmental signal is sensed (an outstanding question even for well-studied TCSs such as PhoPR in Mtb)? What are the regulatory networks that underlie bacterial coordination of environmental response to metabolism? Are there (and how common) are regulators that act only in the context of the presence of multiple signals? What are the implications of the above on the heterogeneity across multiple aspects of infection observed in vivo that has become increasingly appreciated? We propose that elucidating the answers to these currently understudied questions will open research into an area of host-pathogen interactions that has untapped potential for revealing novel nodes critical to both fundamental understanding of bacterial biology, and that can be targeted for therapeutic purposes.