(C) PLOS One
This story was originally published by PLOS One and is unaltered.
 . . . . . . . . . .



Copper microenvironments in the human body define patterns of copper adaptation in pathogenic bacteria [1]

['Francesca Focarelli', 'Biosciences Institute', 'Faculty Of Medical Sciences', 'Newcastle University', 'Newcastle Upon Tyne', 'United Kingdom', 'Andrea Giachino', 'Kevin John Waldron']

Date: 2022-11 

Abstract Copper is an essential micronutrient for most organisms that is required as a cofactor for crucial copper-dependent enzymes encoded by both prokaryotes and eukaryotes. Evidence accumulated over several decades has shown that copper plays important roles in the function of the mammalian immune system. Copper accumulates at sites of infection, including the gastrointestinal and respiratory tracts and in blood and urine, and its antibacterial toxicity is directly leveraged by phagocytic cells to kill pathogens. Copper-deficient animals are more susceptible to infection, whereas those fed copper-rich diets are more resistant. As a result, copper resistance genes are important virulence factors for bacterial pathogens, enabling them to detoxify the copper insult while maintaining copper supply to their essential cuproenzymes. Here, we describe the accumulated evidence for the varied roles of copper in the mammalian response to infections, demonstrating that this metal has numerous direct and indirect effects on immune function. We further illustrate the multifaceted response of pathogenic bacteria to the elevated copper concentrations that they experience when invading the host, describing both conserved and species-specific adaptations to copper toxicity. Together, these observations demonstrate the roles of copper at the host–pathogen interface and illustrate why bacterial copper detoxification systems can be viable targets for the future development of novel antibiotic drug development programs.

Author summary Copper is required by both animals and bacteria in small quantities as a micronutrient. During infection, the mammalian immune system increases the local concentration of copper, which gives rise to copper toxicity in the pathogen. In turn, bacterial pathogens possess specialized systems to resist this copper toxicity. Copper also plays important, indirect roles in the function of the immune system. In this review, we explain the diverse roles of copper in the human body with a focus on its functions within the immune system. We also describe how bacterial pathogens respond to the copper toxicity that they experience within the host during infection, illustrating both conserved copper homeostasis and detoxification systems in bacteria and species-specific adaptations that have been shown to be important to pathogenicity. The key role of copper at the host–pathogen interface and the essential requirement for pathogenic bacteria to resist copper toxicity makes the protein components that confer resistance on pathogens potential targets for future development of novel antibiotic drugs.

Citation: Focarelli F, Giachino A, Waldron KJ (2022) Copper microenvironments in the human body define patterns of copper adaptation in pathogenic bacteria. PLoS Pathog 18(7): e1010617. https://doi.org/10.1371/journal.ppat.1010617 Editor: N.Luisa Hiller, Carnegie Mellon University, UNITED STATES Published: July 21, 2022 Copyright: © 2022 Focarelli et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: KJW was supported by the Biotechnology and Biological Sciences Research Council (BB/S006818/1). FF was supported by a John William Luccock and Ernest Jeffcock Research Studentship from Newcastle University’s Faculty of Medical Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Copper is an essential micronutrient for both prokaryotes and eukaryotes. Its redox properties are exploited in biochemical reactions by copper-dependent enzymes. Therefore, during infection, invading microorganisms must acquire copper from the host to maintain copper supply to essential cuproenzymes. On the other hand, too much copper is toxic [1]. Copper’s high affinity for protein metal-binding sites (MBS) can result in the displacement of iron from enzyme catalytic sites, particularly iron-sulfur clusters [2]. Moreover, copper inhibits peptidoglycan cross-linking by LD-transpeptidases [3] and the maturation of lipoproteins in the gram-negative bacterial cell envelope [4], and induces protein aggregation by catalyzing the cross-linking of cysteine residues and the formation of non-native disulfide bonds [5]. Finally, copper participates in Fenton-like chemistry, leading to the production of reactive oxygen species and other toxic radicals [6]. Thus, both host and microorganism possess homeostatic systems that carefully regulate cellular copper bioavailability to prevent toxicity. Together, this presents an opportunity for the immune system to utilize elevated local copper concentrations as a biocidal weapon with which to attack invading pathogens [7,8]. Research studies have long supported a role for copper in the function of the immune system. It has been known for some time that copper deficiency impairs immune function [9,10]. Mammal models fed a copper-deficient diet are more prone to infection, and copper-rich diets increase their ability to fend off invading pathogens [11–15]. Copper deficiency in humans causes a number of symptoms, including neutropenia [16]. Recent evidence has begun to explain the reason for these observations mechanistically. Copper is used as a bactericidal agent within macrophages, accumulating in phagolysosomes during infection [8,17,18], and both systemic and localized copper concentrations in mammals are increased during infection [11,12]. The general principles of copper handling in bacteria and in mammals have been recently separately reviewed [1,19]. Here, we focus on the role of copper at the host–pathogen interface, summarizing the evidence for a crucial role for copper excess in host immunity to infection and illustrating the opposing role of copper detoxification systems encoded by human pathogens. We highlight the importance of copper microenvironments at different host/pathogen interfaces and show that pathogens colonizing the same niche adopt similar copper response strategies.

Copper in systemic infection Copper resistance contributes to tissue colonization and inflammation for many pathogens, including uropathogenic Escherichia coli [31], Staphylococcus aureus [13], Streptococcus pneumoniae [32], Mycobacterium tuberculosis [12,33], Listeria monocytogenes [34], Neisseria gonorrhoeae [35], Klebsiella pneumoniae [36], and Pseudomonas aeruginosa [37]. Therefore, all these pathogens are thought to encounter significant concentrations of toxic copper at least in some phases of their pathogenesis. Copper toxicity at the host–pathogen interface can occur in 2 ways: either the pathogen is exposed to copper in the extracellular space, such as on the surface of an epithelium, or intracellularly, after being engulfed by phagocytes. An increase in circulating copper during infection, and other pathological conditions, has been recognized for more than 60 years [23,38], such that copper accumulates at high levels in the blood and peripheral tissues during systemic infection [38]. This increase is driven by the acute phase overexpression and secretion of ceruloplasmin (Cp) from the liver, which is induced by pro-inflammatory agents such as interferon gamma (IFNγ), interleukin (IL) 1-β, and bacterial lipopolysaccharide (LPS) [39–41]. Epithelial cells also overexpress ceruloplasmin during infection, driving its accumulation in airways [40] and the urinary tract [11]. Acute phase ceruloplasmin overexpression upon inflammation is thought to mediate increased copper availability to other cytotypes to support the cell-mediated response to invading organisms [11,13]. In addition, increased extracellular copper also contributes to pathogen clearance through phagocyte-independent mechanisms, whose precise molecular mechanisms are currently under investigation [17,42]. Another copper-binding protein that is expressed during inflammation is the pro-inflammatory mediator calgranulin C (S100A12, also known as EN-RAGE) [43], which is produced by granulocytes and mediates the activation of endothelium, mononuclear phagocytes, and lymphocytes [43]. It has been suggested that the copper-S100A12 complex might sequester copper as part of the nutritional immunity response, similarly to how calprotectin (S100A8/S100A9, also known as the cystic fibrosis antigen, calgranulin A/B, and MRP-8/9) sequesters metals [44]. However, these interesting possibilities currently await experimental confirmation. Importantly, the need for copper tolerance varies across different infection sites. S. pneumoniae encounters copper toxicity in the nasopharynx and lungs, but not in blood [32], and M. tuberculosis strains impaired in copper tolerance are less virulent in the lung, but not the spleen [12]. Copper tolerance plays a role in Salmonella Typhimurium colonization of liver and spleen, but not of lymph nodes [42]. Moreover, copper abundance and the presence of other bactericidal agents also varies across infection sites. Consequently, pathogens colonizing different environments have evolved distinct mechanisms to deal with copper toxicity.

Copper-mediated pathogen killing in the phagolysosome The mature phagolysosome is a copper-rich environment, accumulating copper in the range of tens to hundreds of micromolar [18]. Copper accumulation is mediated by the overexpression of ATP7A and its vesicular delivery from the trans-Golgi to the surface of maturing phagolysosomes [7,8]. Copper enters engulfed bacteria [53] and contributes to phagocytic killing through a variety of mechanisms, which have been extensively reviewed elsewhere [1]. In response to phagosomal copper overload, engulfed bacteria upregulate copper-homeostasis genes, such as that encoding the copper-efflux pump CopA [54]. Bacterial resistance to copper toxicity is a key virulence determinant, and it contributes to bacterial survival within macrophages. Bacterial strains lacking copper-tolerance genes are more sensitive to phagosomal killing, as demonstrated in E. coli [8], S. enterica serovar Typhimurium [7,53], S. pneumoniae [55], and S. aureus [56,57]. Conversely, in pathogenic strains that possess mobile genetic elements (MGE) encoding for copper hypertolerance, these MGE further enhance intraphagosomal survival [56,57]. Other intracellular pathogens also require copper homeostasis to survive within host cells, as is the case for N. gonorrhoeae within cervical epithelial cells [35]. The mechanisms of copper-mediated killing within phagocytes are multifaceted. Following engulfment, the phagosome matures into a bactericidal environment by the regulated fusion of vesicles, particularly lysosomes and proteases, progressive pH acidification (to final pH 4.5) and nutrient deprivation [58]. Eventually, the mature phagolysosome contains high concentrations of copper and zinc [18,59,60], low concentrations of iron [61], radical-inducing species such as reactive oxygen, nitrogen, and chlorine species [58,62,63], and other bactericidal proteins such as lysozyme, defensins, phospholipases, and proteases [58]. Copper ions participate in Fenton-like chemistry with ROS and RNS, amplifying the generation of reactive radicals [6]. Moreover, both copper and zinc can mismetalate ferroenzymes [2,64]; as a consequence, copper and zinc toxicity further enhance the effects of iron removal from the phagolysosome [61] and inflamed tissues [65] as part of nutritional immunity [59]. Interestingly, several pathogens display elegant mechanisms to escape the macrophage intracellular killing. For example, pathogenic S. Typhimurium inhibits the delivery of NADPH oxidase and inducible nitric oxide synthase (iNOS) to the maturing phagosome, preventing both the early oxidative burst [66] and long-term RNS toxicity [67]. In addition, this bacterium counteracts high copper concentrations in the phagolysosome by successfully preventing copper entry into the bacterial cell [59]. Similarly, M. tuberculosis also inhibits iNOS recruitment to the phagolysosome [68] and also interferes with vesicle fusion with the phagosomal compartment, ultimately resulting in the creation of a vacuole that is permissive for bacterial growth [69]. In other words, pathogens can avoid phagosomal toxicity in two different ways: either by preventing the maturation of the bactericidal compartment or by coping with bactericidal factors after phagolysosomal maturation.

Bacterial adaptations to host copper flux In the past decade, the role of metal ions at the host-microbe interface has attracted increasing attention. Copper toxicity, in particular, is an important emerging field due to copper’s powerful antimicrobial properties, especially in the age of rapidly spreading antibiotic resistance. Yet, despite recent advances, the role of copper at the host–pathogen interface is far from fully understood. One challenge in investigating copper homeostasis stems from the key differences in host microenvironments that we discussed earlier in this work: copper distribution in the human body is not uniform, and pathogens can be exposed to significant fluctuations in copper concentration both in space (different bodily niches) and time (e.g., as a consequence of immune system activation). Furthermore, copper tolerance in bacteria is not limited to the physical handling of copper, but also includes mechanisms to adapt microbial metabolism to repair, or bypass, the toxic effects of copper [1]. Yet, our increasing understanding of the bacterial copper response has highlighted some important similarities across human pathogens, as well as some key differences (Fig 4). In the following sections, we compare the copper response of six important human pathogens (S. aureus, S. pneumoniae, M. tuberculosis, uropathogenic E. coli, S. enterica serovar Typhimurium, and P. aeruginosa) to highlight its key role in the host–pathogen interaction. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Copper resistance in human pathogens. Despite individual variations, most human pathogens share a common pattern of copper resistance proteins, involving four key functions: copper sensing (green), export (blue), chelation (ochre), and oxidation (magenta). The first three functions (sensing, export, and chelation) are common to the majority of pathogens, and many also include the fourth function (oxidation). In some cases, the copper-homeostasis suite can also vary across different strains or serotypes (most notably in S. aureus and S. enterica). Copper oxidation only occurs outside of the cytosol, and it may contribute to slowing down copper entry into the cytosol. All P-type ATPases belong to a single family (CopA) which is conserved across the tree of life (including eukaryotes). Different subfamilies of the ATPase may feature different terminal metal-binding domains, and these are homologous to that organism’s cytosolic chaperone. (A) Streptococcus pneumoniae contains a minimal set of copper-homeostasis proteins, including the CopY sensor, the CopA exporter, and the CupA chaperone. (B) The common USA300 strain of Staphylococcus aureus contains two copper-efflux pumps (CopA and CopB/X) as well as a cell-surface associated chelator (CopL) and multicopper oxidase (Mco). (C) Mycobacterium tuberculosis has a minimum copper-homeostasis system with the CtpV efflux pump, the MymT metallothionein, and the MmcO oxidase; however, this system is controlled by two separate (but homologous) regulators: CsoR for CtpV, and RicR for MymT and MmcO. (D) As a gram-negative organism, Escherichia coli features a rich suite of periplasmic copper-homeostasis proteins, including the CusSR two-component sensory system, the CusCBA efflux pump, the CusF chaperone, and the CueO oxidase. Copper homeostasis in the E. coli cytoplasm is minimal, with only the CueR regulator, the CopA pump, and the CopZ chaperone dealing with copper homeostasis in this compartment. (E) Although related to E. coli, Salmonella enterica sv. Typhimurium lacks systems for periplasmic copper sensing or efflux; conversely, it carries a duplication of the cytoplasmic copper-homeostasis system (the Gol proteins) and an additional copper-storage protein (Csp3). (F) Pseudomonas aeruginosa features copper sensing and efflux both in the cytosol and the periplasm, but no periplasmic copper chelator or oxidase have been identified to date. https://doi.org/10.1371/journal.ppat.1010617.g004 Importantly, this group of pathogens includes diverse organisms from the gram-positive, gram-negative, and mycobacterial groups (Fig 4). Some of these organisms are closely related (E. coli and S. enterica) while others are not. Finally, these pathogens colonize different niches in the body, yet some of them can occupy the same or similar niches: for example, both S. Typhimurium and M. tuberculosis are intracellular pathogens, while S. pneumoniae and M. tuberculosis both occupy the human respiratory tract. By linking these pathogens’ copper response with their distinctive lifestyle, we show how different bacterial adaptations can be understood in terms of their respective adaptation to different host niches. Crucially, the suite of copper resistance proteins in human pathogens is remarkably similar, despite individual variations (Fig 4). Three core functions of the copper response are universally conserved across pathogens: copper sensing, copper export, and copper chelation. A fourth mechanism, copper oxidation, is also present in many but not all organisms.

Conserved mechanisms of copper sensing Firstly, all bacteria that are exposed to copper stress need a way to sense it. Therefore, copper-sensing systems are nearly universally present across pathogens. These systems belong to ancient protein families, and similar gene homologues are common to multiple taxa [70]. For example, gram-negative bacteria (particularly proteobacteria) often sense cytosolic copper through a MerR-like sensor, whose prototypical member is the CueR sensor from E. coli [71]. A CueR homologue is also present in P. aeruginosa and S. Typhimurium, and the latter carries two homologues of this protein with distinct but related functions in copper tolerance [72]. By contrast, monodermic bacteria (and mycobacteria) usually sense cytosolic copper via 1 of two transcriptional repressors: the four-helix-bundle CsoR family or the helix-turn-helix CopY family [70]. In the case of M. tuberculosis, the gene for the CsoR-like regulator is duplicated, and a second homologue (RicR) is responsible for fine-tuning the copper response [33]. Therefore, even though all bacteria require a way to sense and respond to cytosolic copper overload, the precise mechanisms by which this is accomplished may differ. Another important difference in copper sensing occurs in didermic (gram-negative) bacteria. Because gram-negative cells include a periplasmic space, they also need to maintain copper homeostasis in this compartment. Consistently, many copper-homeostasis proteins can be found in this compartment. However, only some gram-negatives also encode a sensor for periplasmic copper: the two-component CopSR system (also known as CusSR in E. coli) [70]. Importantly, S. Typhimurium lacks a CopSR homologue but encodes a complex suite of periplasmic copper response proteins [53], suggesting that this bacterium has evolved to mount a cell-wide copper response instead of differentiating between different compartments. Crucially, extracytosolic copper homeostasis is not restricted to gram-negative bacteria. Both S. aureus USA300 and M. tuberculosis encode one or more surface-associated copper-homeostasis proteins, which contribute to detoxifying copper before it enters the cell [56,73]. It is significant that the CopSR system is also found in some gram-positive organisms, including Corynebacterium glutamicum [74]. This further supports the model of the cell envelope as a key player in copper homeostasis, even in the absence of a periplasmic space. Overall, the patterns of copper response regulation identify three different classes of pathogens: (a) pathogens that only encode one copper sensor, including S. pneumoniae and S. aureus; (b) pathogens that differentiate the copper response based on subcellular compartment, such as E. coli and P. aeruginosa; and (c) pathogens that fine-tune the copper response depending on the presence of additional toxicants and/or via multiple cytosolic copper sensors, such as M. tuberculosis and S. Typhimurium.

Mucosa-associated pathogens experience a stable copper environment Importantly, these patterns of copper regulation strongly correlate with the pathogen’s lifestyle, likely reflecting the unique distribution of copper in those compartments. S. pneumoniae and S. aureus are mucosal colonizers, which thrive in association with epithelia and frequently form epithelium-associated biofilms. In this scenario, the copper concentration encountered by these pathogens in the primary environment is relatively constant in time and space. Therefore, a single copper sensor is sufficient to detect (and respond to) copper abundance in the pathogen’s environment. Evidence of a constant copper pressure on mucosal epithelium-colonizing pathogens is also provided by the expansion of the copper suite in pathogenic S. aureus, which (as a clade) includes different strains adapted to varying copper levels in the host. In the case of S. aureus, only the “minimal” suite of one sensor (CsoR), one efflux pump (CopA), and one copper chaperone (CopZ), is shared by the entire clade. However, methicillin-resistant USA300 isolates carry additional loci for copper tolerance on MGEs, the same that encode antibiotic resistance in these strains [56,57,75]. These supplementary loci usually carry genes for the surface-exposed chelator CopL [56,73], and a second P-type ATPase (denoted CopX or CopB, depending on the isolate). In some cases (but not always), these are also accompanied by the Mco multicopper oxidase. Multiple lines of evidence suggest that the additional copper-homeostasis genes in USA300 are important for virulence. All three additional genes mediate copper hypertolerance [56,57,73], and this hypertolerance can be transferred horizontally to copper-sensitive strains [57]. They are also upregulated upon phagocytosis and contribute to the enhanced survival of USA300 within macrophages [56,57]. Finally, the acquisition of copB/X occurred independently in different strains, suggesting that these strains experience a common evolutionary pressure to select for additional copper-homeostasis mechanisms [75]. Importantly, the acquisition of additional copper response proteins was not accompanied by the development of a separate copper sensor, since the additional genes are all CsoR-regulated [57]. This is consistent with the fact that USA300 colonize the same body niches as the methicillin-sensitive parent; in other words, the additional genes carried by USA300 enhance the ability of S. aureus to colonize its native niche, instead of mediating a different lifestyle. Because copper inhibits the formation of S. aureus biofilms [76], it is likely that increased copper tolerance in USA300 is important for its increased ability to colonize the host.

Copper homeostasis in the gram-negative periplasm reflects copper levels in different biological niches By contrast, gram-negative bacteria such as E. coli and P. aeruginosa possess an additional subcellular compartment, the periplasm, which features different copper chemistry compared to the cytoplasm. Both E. coli and P. aeruginosa possess a 2-component sensory system for detecting periplasmic Cu(I) (CusSR and CopSR, respectively) in addition to the cytosolic sensor (CueR) [77,78]. However, the functions of these systems in the context of cellular metal homeostasis are radically different and likely reflect the different niches colonized by these bacteria. In E. coli, the CusSR system induces copper efflux from the periplasm when Cu(I) availability reaches micromolar levels [79] and is thought to constitute an adaptation to increased Cu(I) availability during anaerobic conditions, or to sudden increases in extracellular copper [79]. Importantly, mammalian gut-associated E. coli experiences drastic fluctuations in oxygen availability, changing between meals [80], along the intestinal tract [81] and from the tissue interface to the lumen [82]. Importantly, oxygen levels also influence copper availability by modulating the redox equilibrium between Cu(I) and Cu(II): anaerobic conditions increase Cu(I) availability [83], resulting in in increased copper influx and accumulation in E. coli [79]. As a consequence, the E. coli copper response is strongly induced during the transition from aerobic to anaerobic growth [84]. Taken together, the available evidence suggests that periplasmic copper homeostasis in E. coli evolved specifically to deal with copper fluctuations in the primary environment, as a consequence of variable oxygen levels. By contrast, periplasmic copper homeostasis in P. aeruginosa is believed to be more tightly controlled [85]. The P. aeruginosa periplasmic two-component system CopSR reacts to subfemtomolar periplasmic Cu(I) and is thought to maintain the P. aeruginosa periplasm devoid of free copper [78]. At the same time, P. aeruginosa is one of the few bacteria for which a periplasmic copper-import route has been identified [86] and has been shown to scavenge copper from plasma during infection [87]. Indeed, copper tolerance (and likely acquisition) plays an important role in the establishment of preinfection communities of P. aeruginosa [88] and strains defective in copper homeostasis show decreased pathogenicity [37]. One interesting model that has been recently proposed is that P. aeruginosa is subjected to copper excess and starvation outside of the human body, and that adaptation to copper-related evolutionary pressure increases this bacterium’s ability to adapt to copper fluctuations within the human host [88]. Another similarity between E. coli and P. aeruginosa is the secretion of copper-binding molecules that sequester copper outside of the cell. In P. aeruginosa, the secreted siderophores pyoverdine and pyochelin chelate copper outside the cell and increase cellular tolerance to toxic copper [89]. Similarly, many uropathogenic E. coli strains encode the siderophore yersiniabactin as part of the Yersinia high-pathogenicity island, whose expression is strongly induced during urinary tract infection [90]. Similarly to pyoverdine and pyochelin, yersiniabactin sequesters copper in the extracellular milieu, protecting uropathogenic E. coli from copper toxicity during infection [31]. At the same time, yersiniabactin also protects E. coli from the early respiratory burst in phagocytes [91] and is involved in copper acquisition during copper-limiting conditions [92], supporting the notion that successful pathogens such as P. aeruginosa and UPEC need both copper sequestration and acquisition to deal with varying copper levels within the host. Crucially, another gram-negative bacterium that is evolutionary close to E. coli, S. enterica, does not possess a periplasmic copper sensor. Instead, periplasmic copper homeostasis in S. enterica is under the control of the cytosolic copper sensor, CueR [53]. This suggests that S. enterica has a lower requirement for carefully managed periplasmic copper, but instead requires increased protection of the cytosol, as evidenced by the presence of two cytosolic efflux pumps in S. enterica sv Typhimurium [53]. This is likely linked to the different lifestyles of S. enterica, a facultative intracellular pathogen, as opposed to E. coli and P. aeruginosa, whose intracellular phase plays a less-relevant role in pathogenesis [93].

Intracellular pathogens recognize copper in conjunction with other phagolysosomal antimicrobials Finally, the third class of pathogens includes the intracellular pathogens S. Typhimurium and M. tuberculosis, both of which have evolved to survive and replicate within phagocytic cells. As described earlier, the mammalian phagolysosome is a complex compartment, characterized by toxic copper levels in conjunction with a suite of other antimicrobial effectors. For intracellular pathogens, copper concentration comes as an all-or-nothing: they are either in a copper-poor environment (outside the macrophage or in a pathogen-adapted vacuole) or in a deadly, copper-rich environment (a maturing phagolysosome) [53]. Importantly, copper toxicity in the phagolysosome is accompanied by a suite of other antimicrobial factors, some of which feature potent synergistic effects with copper. It is not surprising, therefore, that intracellular pathogens would have adapted to respond to toxic copper at the same time as they are also dealing with other antimicrobial factors. Even though they are not related, both organisms evolved a duplication of the “core” copper-sensing system (CueR in S. Typhimurium, CsoR in M. tuberculosis) generating a homologous but independent sensor (GolS in S. Typhimurium and RicR in M. tuberculosis). In both organisms, the primary copper sensor responds only to copper concentration [53,94], while the additional sensor responds to copper in conjunction with other cellular stressors: the S. Typhimurium GolS responds to copper and acid stress [72], while the M. tuberculosis RicR is activated by copper, cobalt, nickel and zinc, as well as reactive oxygen and nitrogen species [94]. Copper and nitric oxide also coregulate the expression of the Rip1/PdtaSR regulon in M. tuberculosis, which mediates a coordinated response to both stressors [95], although the exact mechanisms remain to be clarified. Importantly, all of these stressors are encountered alongside copper within macrophages [58]. The duplication of the copper response system in S. Typhimurium and M. tuberculosis is likely a convergent adaptation to intraphagosomal survival. We hypothesize that by acquiring a second copy of the respective copper sensor, both organisms have evolved the ability to distinguish between copper toxicity outside and inside the phagolysosome. Importantly, this explains the “atypical” copper homeostasis in S. Typhimurium, which differs significantly from closely related organisms [53]. In fact, copper homeostasis in S. Typhimurium is more similar to M. tuberculosis than E. coli, reflecting the sharing of a similar niche with the former, but not the latter. We propose that the co-occurrence of elevated copper and other antimicrobials in the phagolysosome caused the convergent evolution of an additional copper-detoxification system in M. tuberculosis and S. Typhimurium, which specifically responds to copper and other phagolysosome-associated stress factors.

Conclusions The accumulated evidence strongly indicates diverse and important roles of copper in immune function. Some roles for copper are direct, for example, the antibacterial accumulation of copper inside the phagosome of phagocytic cells, whereas other roles are indirect, such as copper-mediated effects on endocrine signaling that modulate the immune response. In contrast to other metals, such as manganese and iron, whose abundance is restricted by the host, pathogens seem to experience elevated copper concentrations during infection. Bacterial proteins that function to detoxify copper insult are known virulence factors in several globally important human pathogens. These prokaryotic components thus have potential as targets for small-molecule inhibitors as a route to the development of novel antibiotic therapeutics. Several pathogens are observed to have undergone gene duplication events, increasing their copy number of key copper homeostasis genes. Where these duplication events were very ancient, such as in S. Typhimurium or M. tuberculosis, the duplicated genes have subsequently diverged to confer new functions such as to give fine control over the copper response to enable the bacterium to better survive inside host cells. But in the case of current epidemic lineages of S. aureus, the observed duplication was much more recent, confers hyperresistance to copper, and provides a selective benefit during interaction with immune cells. This raises the prospect that contemporary pathogen evolution is selecting for more copper-resistant phenotypes, which may have anthropogenic causes and thus be an increasing selection pressure. At the same time, metal hyperresistance has been linked to the spread of resistance to other antimicrobials, crucially antibiotics [96,97]. One way in which this could occur is the coselection of metal and antibiotic resistance genes on the same MGEs. Another mechanism is cross-resistance, in which a metal resistance determinant (e.g., a multidrug efflux pump or a particular composition of the cell envelope) is also capable of mediating antibiotic resistance, even in the absence of dedicated antibiotic resistance genes [98]. At the same time, metal resistance determinants can also increase a bacterium’s sensitivity to certain antibiotics [99]. Clearly, the potential interactions of copper and other antimicrobials are multifaceted and constitute an area of active research. If copper is to be exploited as an antimicrobial in future, monitoring of the emergence and spread of such hyperresistant phenotypes among pathogens will be essential.

[END]
---
[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010617

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/