Scs system links copper and redox homeostasis in bacterial pathogens.

The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu+-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.

The multifarious MerR family of transcriptional regulators.

The MerR family of transcriptional regulators includes a variety of bacterial cytoplasmic proteins that respond to a wide range of signals, including toxins, metal ions, and endogenous metabolites. Its best-characterized members share similar structural and functional features with the family founder, the mercury sensor MerR, although most of them do not respond to metal ions. The group of "canonical" MerR homologs displays common molecular mechanisms for controlling the transcriptional activation of their target genes in response to inducer signals. This includes the recognition of distinctive operator sequences located at suboptimal σ70 -dependent promoters. Interestingly, an increasing number of proteins assigned to the MerR family based on their DNA-binding domain do not match in structure, sequence, or mode of action with any of the canonical MerR-like regulators. Here, we analyzed several members of the family, including this last group. Based on a phylogenetic analysis, and similarities in structural/functional features and position of their target operators relative to the promoter elements, we propose to assign these "atypical/divergent" MerR regulators to a phylogenetically separated group. These atypical/divergent homologs represent a new class of transcriptional regulators with novel regulatory mechanisms.

Balance between bacterial extracellular matrix production and intramacrophage proliferation by a Salmonella-specific SPI-2-encoded transcription factor.

Biosynthesis and secretion of a complex extracellular matrix (EM) is a hallmark of Salmonella biofilm formation, impacting on its relationship with both the environment and the host. Cellulose is a major component of Salmonella EM. It is considered an anti-virulence factor because it interferes with Salmonella proliferation inside macrophages and virulence in mice. Its synthesis is stimulated by CsgD, the master regulator of biofilm formation in enterobacteria, which in turn is under the control of MlrA, a MerR-like transcription factor. In this work, we identified a SPI-2-encoded Salmonella-specific transcription factor homolog to MlrA, MlrB, that represses transcription of its downstream gene, orf319, and of csgD inside host cells. MlrB is induced in laboratory media mimicking intracellular conditions and inside macrophages, and it is required for intramacrophage proliferation. An increased csgD expression is observed in the absence of MlrB inside host cells. Interestingly, inactivation of the CsgD-controlled cellulose synthase-coding gene restored intramacrophage proliferation to rates comparable to wild-type bacteria in the absence of MlrB. These data indicate that MlrB represses CsgD expression inside host cells and suggest that this repression lowers the activation of the cellulose synthase. Our findings provide a novel link between biofilm formation and Salmonella virulence.

Compartment and signal-specific codependence in the transcriptional control of Salmonella periplasmic copper homeostasis.

Copper homeostasis is essential for bacterial pathogen fitness and infection, and has been the focus of a number of recent studies. In Salmonella, envelope protection against copper overload and macrophage survival depends on CueP, a major copper-binding protein in the periplasm. This protein is also required to deliver the metal ion to the Cu/Zn superoxide dismutase SodCII. The Salmonella-specific CueP-coding gene was originally identified as part of the Cue regulon under the transcriptional control of the cytoplasmic copper sensor CueR, but its expression differs from the rest of CueR-regulated genes. Here we show that cueP expression is controlled by the concerted action of CueR, which detects the presence of copper in the cytoplasm, and by CpxR/CpxA, which monitors envelope stress. Copper-activated CueR is necessary for the appropriate spatial arrangement of the -10 and -35 elements of the cueP promoter, and CpxR is essential to recruit the RNA polymerase. The integration of two ancestral sensory systems-CueR, which provides signal specificity, and CpxR/CpxA, which detects stress in the bacterial envelope-restricts the expression of this periplasmic copper resistance protein solely to cells encountering surplus copper that disturbs envelope homeostasis, emulating the role of the CusR/CusS regulatory system present in other enteric bacteria.

CpxR/CpxA Controls scsABCD Transcription To Counteract Copper and Oxidative Stress in Salmonella enterica Serovar Typhimurium.

Periplasmic thiol/disulfide oxidoreductases participate in the formation and isomerization of disulfide bonds and contribute to the virulence of pathogenic microorganisms. Among the systems encoded in the Salmonella genome, the system encoded by the scsABCD locus was shown to be required to cope with Cu and H2O2 stress. Here we report that this locus forms an operon whose transcription is driven by a promoter upstream of scsA and depends on CpxR/CpxA and on Cu. Furthermore, genes homologous to scsB, scsC, and scsD are always detected immediately downstream of scsA and in the same genetic arrangement in all scsA-harboring enterobacterial species. Also, a CpxR-binding site is detected upstream of scsA in most of those species, providing evidence of evolutionarily conserved function and regulation. Each individual scs gene shows a different role in copper and/or H2O2 resistance, indicating hierarchical contributions of these factors in the defense against these intoxicants. A protective effect of Cu preincubation against H2O2 toxicity and the increased Cu-mediated activation of cpxP in the ΔscsABCD mutant suggest that the CpxR/CpxA-controlled transcription of the ScsABCD system contributes to prevent Cu toxicity and to restore the redox balance at the Salmonella envelope.IMPORTANCE Copper intoxication triggers both specific and nonspecific responses in Salmonella The scs locus, which codes for periplasmic thiol/disulfide-oxidoreductase/isomerase-like proteins, has been the focus of attention because it is necessary for copper resistance, oxidative stress responses, and virulence and because it is not present in nonpathogenic Escherichia coli Still, the conditions under which the scs locus is expressed and the roles of its individual components remain unknown. In this report, we examine the contribution of each Scs factor to survival under H2O2 and copper stress. We establish that the scs genes form a copper-activated operon controlled by the CpxR/CpxA signal transduction system, and we provide evidence of its conserved gene arrangement and regulation in other bacterial pathogens.

A novel peptidoglycan D,L-endopeptidase induced by Salmonella inside eukaryotic cells contributes to virulence.

Bacteria remodel peptidoglycan structure in response to environmental changes. Many enzymes are involved in peptidoglycan metabolism; however, little is known about their responsiveness in a defined environment or the modes they assist bacteria to adapt to new niches. Here, we focused in peptidoglycan enzymes that intracellular bacterial pathogens use inside eukaryotic cells. We identified a peptidoglycan enzyme induced by Salmonella enterica serovar Typhimurium in fibroblasts and epithelial cells. This enzyme, which shows γ-D-glutamyl-meso-diaminopimelic acid D,L-endopeptidase activity, is also produced by the pathogen in media with limited nutrients and in resting conditions. The enzyme, termed EcgA for endopeptidase responding to cessation of growth', is encoded in a S. Typhimurium genomic island absent in Escherichia coli. EcgA production is strictly dependent on the virulence regulator PhoP in extra- and intracellular environments. Consistent to this regulation, a mutant lacking EcgA is attenuated in the mouse typhoid model. These findings suggest that specialised peptidoglycan enzymes, such as EcgA, might facilitate Salmonella adaptation to the intracellular lifestyle. Moreover, they indicate that readjustment of peptidoglycan metabolism inside the eukaryotic cell is essential for host colonisation.

The CpxR/CpxA system contributes to Salmonella gold-resistance by controlling the GolS-dependent gesABC transcription.

Several regulatory systems contribute to bacterial resistance to heavy metals controlling the expression of factors required to eliminate the intoxicant and/or to repair the damage caused by it. In Salmonella, the response to Au ions is mediated by the specific metalloregulator GolS that, among other genes, controls the expression of the RND-efflux pump GesABC. In this work, we demonstrate that CpxR/CpxA, a main cell-envelope stress-responding system, promotes gesABC transcription in the presence of Au ions at neutral pH. Deletion of either cpxA or cpxR, or mutation of the CpxR-binding site identified upstream of the GolS-operator in the gesABC promoter region reduces but does not abrogate the GolS- and Au-dependent activation of gesABC. Au also triggers the activation of the CpxR/CpxA system and deletion of the cpxRA operon severely reduces survival in the presence of the toxic metal. Our results indicate that the coordinated action of GolS and CpxR/CpxA contribute to protecting the cell from severe Au damage.

A single serine residue determines selectivity to monovalent metal ions in metalloregulators of the MerR family.

UNLABELLED: MerR metalloregulators alleviate toxicity caused by an excess of metal ions, such as copper, zinc, mercury, lead, cadmium, silver, or gold, by triggering the expression of specific efflux or detoxification systems upon metal detection. The sensor protein binds the inducer metal ion by using two conserved cysteine residues at the C-terminal metal-binding loop (MBL). Divalent metal ion sensors, such as MerR and ZntR, require a third cysteine residue, located at the beginning of the dimerization (α5) helix, for metal coordination, while monovalent metal ion sensors, such as CueR and GolS, have a serine residue at this position. This serine residue was proposed to provide hydrophobic and steric restrictions to privilege the binding of monovalent metal ions. Here we show that the presence of alanine at this position does not modify the activation pattern of monovalent metal sensors. In contrast, GolS or CueR mutant sensors with a substitution of cysteine for the serine residue respond to monovalent metal ions or Hg(II) with high sensitivities. Furthermore, in a mutant deleted of the Zn(II) exporter ZntA, they also trigger the expression of their target genes in response to either Zn(II), Cd(II), Pb(II), or Co(II). IMPORTANCE: Specificity in a stressor's recognition is essential for mounting an appropriate response. MerR metalloregulators trigger the expression of specific resistance systems upon detection of heavy metal ions. Two groups of these metalloregulators can be distinguished, recognizing either +1 or +2 metal ions, depending on the presence of a conserved serine in the former or a cysteine in the latter. Here we demonstrate that the serine residue in monovalent metal ion sensors excludes divalent metal ion detection, as its replacement by cysteine renders a pan-metal ion sensor. Our results indicate that the spectrum of signals detected by these sensors is determined not only by the metal-binding ligand availability but also by the metal-binding cavity flexibility.

Survival in amoeba--a major selection pressure on the presence of bacterial copper and zinc resistance determinants? Identification of a "copper pathogenicity island".

The presence of metal resistance determinants in bacteria usually is attributed to geological or anthropogenic metal contamination in different environments or associated with the use of antimicrobial metals in human healthcare or in agriculture. While this is certainly true, we hypothesize that protozoan predation and macrophage killing are also responsible for selection of copper/zinc resistance genes in bacteria. In this review, we outline evidence supporting this hypothesis, as well as highlight the correlation between metal resistance and pathogenicity in bacteria. In addition, we introduce and characterize the "copper pathogenicity island" identified in Escherichia coli and Salmonella strains isolated from copper- and zinc-fed Danish pigs.

Protein signatures that promote operator selectivity among paralog MerR monovalent metal ion regulators.

Two paralog transcriptional regulators of the MerR family, CueR and GolS, are responsible for monovalent metal ion sensing and resistance in Salmonella enterica. Although similar in sequence and also in their target binding sites, these proteins differ in signal detection and in the set of target genes they control. Recently, we demonstrated that selective promoter recognition depends on the presence of specific bases located at positions 3' and 3 within the operators they interact with. Here, we identify the amino acid residues within the N-terminal DNA-binding domain of these sensor proteins that are directly involved in operator discrimination. We demonstrate that a methionine residue at position 16 of GolS, absolutely conserved among GolS-like proteins but absent in all CueR-like xenologs, is the key to selectively recognize operators that harbor the distinctive GolS-operator signature, whereas the residue at position 19 finely tunes the regulator/operator interaction. Furthermore, swapping these residues switches the set of genes recognized by these transcription factors. These results indicate that co-evolution of a regulator and its cognate operators within the bacterial cell provides the conditions to avoid cross-recognition and guarantees the proper response to metal injury.

Identification of a Salmonella ancillary copper detoxification mechanism by a comparative analysis of the genome-wide transcriptional response to copper and zinc excess.

Copper and zinc are essential metal ions, but toxic in excess. Bacteria have evolved different strategies to control their intracellular concentrations, ensuring proper supply while avoiding toxicity, including the induction of metal-specific as well as non-specific mechanisms. We compared the transcriptional profiles of Salmonella Typhimurium after exposure to either copper or zinc ions in both rich and minimal media. Besides metal-specific regulatory networks many global stress-response pathways react to an excess of either of these metal ions. Copper excess affects both zinc and iron homeostasis by inducing transcription of these metal-specific regulons. In addition to the control of zinc-specific regulons, zinc excess affects the Cpx regulon and the σ(E) envelope-stress responses. Finally, novel metal-specific upregulated genes were detected including a new copper-detoxification pathway that involves the siderophore enterobactin and the outer-membrane protein TolC. This work sheds light onto the transcriptional landscape of Salmonella after copper or zinc overload, and discloses a new mechanism of copper detoxification.

Dissecting the metal selectivity of MerR monovalent metal ion sensors in Salmonella.

Two homologous transcription factors, CueR and GolS, that belong to the MerR metalloregulatory family are responsible for Salmonella Cu and Au sensing and resistance, respectively. They share similarities not only in their sequences, but also in their target transcription binding sites. While CueR responds similarly to Au, Ag, or Cu to induce the expression of its target genes, GolS shows higher activation by Au than by Ag or Cu. We showed that the ability of GolS to distinguish Au from Cu resides in the metal-binding loop motif. Here, we identify the amino acids within the motif that determine in vivo metal selectivity. We show that residues at positions 113 and 118 within the metal-binding loop are the main contributors to metal selectivity. The presence of a Pro residue at position 113 favors the detection of Cu, while the presence of Pro at position 118 disfavors it. Our results highlight the molecular bases that allow these regulators to coordinate the correct metal ion directing the response to a particular metal injury.

The protein scaffold calibrates metal specificity and activation in MerR sensors.

MerR metalloregulators are the central components of many biosensor platforms designed to report metal contamination. However, most MerR proteins are non-specific. This makes it difficult to apply these biosensors in the analysis of real environmental samples. On-demand implementation of molecular engineering to modify the MerR metal preferences is innovative, although it does not always yield the expected results. As the metal binding loop region (MBL) of these sensors has been proposed to be the major modulator of their specificity, we surgically switched this region for that of well-characterized specific and non-specific homologues. We found that identical modifications in different MerR proteins result in synthetic sensors displaying particular metal-detection patterns that cannot be predicted from the nature of the assembled modules. For instance, the MBL from a native Hg(II) sensor provided non-specificity or specificity toward Hg(II) or Cd(II) depending on the MerR scaffold into which it was integrated. These and other evidences reveal that residues outside the MBL are required to modulate ion recognition and transduce the input signal to the target promoter. Revealing their identity and their interactions with other residues is a critical step toward the design of more efficient biosensor devices for environmental metal monitoring.

Evolution of Copper Homeostasis and Virulence in Salmonella.

Salmonella enterica sv. Typhimurium modulates the expression of factors essential for virulence, contributing to its survival against the surge of copper (Cu) in the Salmonella-containing vacuole. This bactericidal host innate immune component primarily targets the bacterial envelope, where most cuproproteins are localized. While in most enteric species periplasmic Cu homeostasis is maintained by the CusR/CusS-controlled CusCFBA efflux system encoded in the cus locus, we noticed that these genes were lost from the Salmonella-core genome. At the same time, Salmonella acquired cueP, coding for a periplasmic Cu chaperone. As cus, cueP was shown to be essential for bacterial survival in a copper-rich environment under anaerobiosis, suggesting that it can functionally substitute the CusCFBA system. In the present study, the whole Escherichia coli cus locus was reintroduced to the chromosome of the Salmonella wild-type or the ΔcueP strain. While the integrated cus locus did not affect Cu resistance under aerobic conditions, it increases Cu tolerance under anaerobiosis, irrespective of the presence or absence of cueP. In contrast to the Cus system, CueP expression is higher at high copper concentrations and persisted over time, suggesting separate functions. Finally, we observed that, regardless of the presence or absence of cus, a mutant deleted of cueP shows a deficiency in replication inside macrophages compared to the wild-type strain. Our results demonstrate that CueP and CusCFBA exert redundant functions for metal resistance, but not for intracellular survival, and therefore for the virulence of this pathogen.

Target transcription binding sites differentiate two groups of MerR-monovalent metal ion sensors.

The evolution of bacterial regulatory circuits often involves duplication of genes encoding transcription factors that may suffer both modifications in their detected signals, as well as, rewiring of their target operators. This, and subsequent horizontal gene transfer events contribute to generate a diverse array of regulatory pathways. In Salmonella, two homologous transcription factors CueR and GolS are responsible for Cu and Au sensing and resistance respectively. They share similarities not only in their sequence but also in their target binding sites, although they cluster separately among MerR-monovalent metal sensors. Here, we demonstrate that CueR and GolS can selectively distinguish their target binding sites by recognizing bases at positions 3' and 3 of their cognate operators. Swap of these bases results in switching regulator dependency. The differences in promoter architecture plus the environmentally controlled regulator's cytoplasmic availability warrant intra-regulon regulator-operator selectivity, and the proper response to metal injury. Furthermore, the presence of the distinctive operators' bases is widely extended among the two groups of MerR-monovalent metal sensors, providing evidence of the co-evolution of these factors and their target operators. This approach allows the prediction of regulator's dependency and the identification of transcription modules among groups of homologous transcription factors.

Selective detection of gold using genetically engineered bacterial reporters.

Salmonella typhimurium harbours a Au-resistance system whose expression is controlled by GolS, a transcriptional regulator of the MerR family that selectively detects Au with high sensitivity. We developed both Salmonella and genetically engineered Escherichia coli strains as Au-selective whole-cell biosensors by coupling the strictly regulated GolS-dependent golB promoter to the gfp reporter gene. The bio-reporters were evaluated under different laboratory conditions and calibrated for their use as selective Au detectors. Due to the intrinsic characteristics of the regulatory protein, the transgenic E. coli sensor exhibits low background, high signal-to-noise ratio, and improved sensitivity for detection of Au ions in a wide range of concentrations (up to 470 nM) with a calculated detection limit of ∼33 nM (6 µg L(-1) or parts per billion) Au(I). The fluorescent Au-sensing bacteria exhibit also minimal interference by chemically related metals such as Cu or Ag that are commonly found in Au deposits. These highly specific and sensitive Au detectors might allow the development of rapid and robust screening tools to improve discovery and extraction procedures.

Bacterial gold sensing and resistance.

Gold ions are mobilized and disseminated through the environment and enter into the cells by non-specific intake. To avoid deleterious effect that occurs even at very low concentrations, bacteria such as Salmonella enterica and Cupriavidus metallidurans use Au-specific MerR-type transcriptional regulators to detect the presence of these toxic ions, and control the expression of specific resistance factors. In contrast to the related copper sensor CueR, the Au-selective metalloregulatory proteins are able to distinguish Au(I) from Cu(I) or Ag(I). This is achieved by finely tuning a single dithiolate metal coordination with conserved cysteine residues at the metal binding site of the proteins to lower the affinity for Cu(I) in comparison to the Cu-sensors, while maintaining or even increasing the affinity for Au(I). In Salmonella, GolS not only privileges the binding of Au(I) over Cu(I) or Ag(I), but also distinguishes its target recognition sites in its regulated promoters minimizing cross-activation of CueR-controlled operators. In this sense, the presence of a selective Au sensory devise would allow species harbouring resident Cu-homeostasis systems to eliminate the toxic ion without affecting Cu acquisition in Au rich environments.

Copper Handling in the Salmonella Cell Envelope and Its Impact on Virulence.

Copper (Cu) plays a key role at the host-pathogen interface as both an essential element and a toxic element. Intracellular strains of pathogenic Salmonella have acquired the periplasmic Cu chaperone, CueP, and the thiol oxidoreductases complex Scs, while losing the ancestral Cu-detoxification Cus system. Coregulation of these species-specific factors link Cu with redox stress and allows Salmonella to counteract Cu toxicity during infection.

Copper stress targets the rcs system to induce multiaggregative behavior in a copper-sensitive Salmonella strain.

Salmonella ΔcuiD strains form mucoid colonies on copper-containing solid media. We show here that this multiaggregative behavior is caused by the Rcs-dependent induction of colanic acid extracellular polysaccharide. Deletion of cps operon genes in a ΔcuiD strain increased the sensitivity to copper, indicating a role for colanic acid in copper resistance.

Alternative periplasmic copper-resistance mechanisms in Gram negative bacteria.

Bacteria have evolved different systems to tightly control both cytosolic and envelope copper concentration to fulfil their requirements and at the same time, avoid copper toxicity. We have previously demonstrated that, as in Escherichia coli, the Salmonella cue system protects the cytosol from copper excess. On the other hand, and even though Salmonella lacks the CusCFBA periplasmic copper efflux system, it can support higher copper concentrations than E. coli under anaerobic conditions. Here we show that the Salmonella cue regulon is also responsible for the control of copper toxicity in anaerobiosis. We establish that resistance in this condition requires a novel CueR-controlled gene named cueP. A DeltacueP mutant is highly susceptible to copper in the absence of oxygen, but shows a faint phenotype in aerobic conditions unless other copper-resistance genes are also deleted, resembling the E. coli CusCFBA behaviour. Species that contain a cueP homologue under CueR regulation have no functional CusR/CusS-dependent Cus-coding operon. Conversely, species that carry a CusR/CusS-regulated cus operon have no cueP homologues. Even more, we show that the CueR-controlled cueP expression increases copper resistance of a Deltacus E. coli. We posit that CueP can functionally replace the Cus complex for periplasmic copper resistance, in particular under anaerobic conditions.