Zinc homeostasis in Pseudomonas.

In the genus Pseudomonas, zinc homeostasis is mediated by a complete set of import and export systems, whose expression is precisely controlled by three transcriptional regulators: Zur, CzcR and CadR. In this review, we describe in detail our current knowledge of these systems, their regulation, and the biological significance of zinc homeostasis, taking Pseudomonas aeruginosa as our paradigm. Moreover, significant parts of this overview are dedicated to highlight interactions and cross-regulations between zinc and copper import/export systems, and to shed light, through a review of the literature and comparative genomics, on differences in gene complement and function across the whole Pseudomonas genus. The impact and importance of zinc homeostasis in Pseudomonas and beyond will be discussed throughout this review.

Bile Salts Regulate Zinc Uptake and Capsule Synthesis in a Mastitis-Associated Extraintestinal Pathogenic Escherichia coli Strain.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are major causes of urinary and bloodstream infections. ExPEC reservoirs are not completely understood. Some mastitis-associated E. coli (MAEC) strains carry genes associated with ExPEC virulence, including metal scavenging, immune avoidance, and host attachment functions. In this study, we investigated the role of the high-affinity zinc uptake (znuABC) system in the MAEC strain M12. Elimination of znuABC moderately decreased fitness during mouse mammary gland infections. The ΔznuABC mutant strain exhibited an unexpected growth delay in the presence of bile salts, which was alleviated by the addition of excess zinc. We isolated suppressor mutants with improved growth in bile salts, several of which no longer produced the K96 capsule made by strain M12. The addition of bile salts also reduced capsule production by strain M12 and ExPEC strain CP9, suggesting that capsule synthesis may be detrimental when bile salts are present. To better understand the role of the capsule, we compared the virulence of mastitis strain M12 with that of its unencapsulated ΔkpsCS mutant in two models of ExPEC disease. The wild-type strain successfully colonized mouse bladders and kidneys and was highly virulent in intraperitoneal infections. Conversely, the ΔkpsCS mutant was unable to colonize kidneys and was unable to cause sepsis. These results demonstrate that some MAEC strains may be capable of causing human ExPEC illness. The virulence of strain M12 in these infections is dependent on its capsule. However, capsule may interfere with zinc homeostasis in the presence of bile salts while in the digestive tract.

The Zinc Transporter ZnuABC Is Critical for the Virulence of Chromobacterium violaceum and Contributes to Diverse Zinc-Dependent Physiological Processes.

Chromobacterium violaceum is a ubiquitous environmental bacterium that causes sporadic life-threatening infections in humans. How C. violaceum acquires zinc to colonize environmental and host niches is unknown. In this work, we demonstrated that C. violaceum employs the zinc uptake system ZnuABC to overcome zinc limitation in the host, ensuring the zinc supply for several physiological demands. Our data indicated that the C. violaceum ZnuABC transporter is encoded in a zur-CV_RS15045-CV_RS15040-znuCBA operon. This operon was repressed by the zinc uptake regulator Zur and derepressed in the presence of the host protein calprotectin (CP) and the synthetic metal chelator EDTA. A ΔznuCBA mutant strain showed impaired growth under these zinc-chelated conditions. Moreover, the deletion of znuCBA provoked reductions in violacein production, swimming motility, biofilm formation, and bacterial competition. Remarkably, the ΔznuCBA mutant strain was highly attenuated for virulence in an in vivo mouse infection model and showed low capacities to colonize the liver, grow in the presence of CP, and resist neutrophil killing. Overall, our findings demonstrate that ZnuABC is essential for C. violaceum virulence, contributing to subversion of zinc-based host nutritional immunity.

Zinc Acquisition Mechanisms Differ between Environmental and Virulent Francisella Species.

Zinc is an essential nutrient for bacterial growth. Because host cells can restrict pathogen access to zinc as an antimicrobial defense mechanism, intracellular pathogens such as Francisella must sense their environment and acquire zinc in response. In many bacteria, the conserved transcription factor Zur is a key regulator of zinc acquisition. To identify mechanisms of zinc uptake in Francisella novicida U112, transcriptome sequencing of wild-type and putative zur mutant bacteria was performed. Only three genes were confirmed as directly regulated by Zur and zinc limitation by quantitative reverse transcription-PCR. One of these genes, FTN_0879, is predicted to encode a protein with similarity to the zupT family of zinc transporters, which are not typically regulated by Zur. While a putative znuACB operon encoding a high-affinity zinc transporter was identified in U112, expression of this operon was not controlled by Zur or zinc concentration. Disruption of zupT but not znuA in U112 impaired growth under zinc limitation, suggesting that ZupT is the primary mechanism of zinc acquisition under these conditions. In the virulent Francisella tularensis subsp. tularensis Schu S4 strain, zupT is a pseudogene, and attempts to delete znuA were unsuccessful, suggesting that it is essential in this strain. A reverse TetR repression system was used to knock down the expression of znuA in Schu S4, revealing that znuA is required for growth under zinc limitation and contributes to intracellular growth within macrophages. Overall, this work identifies genes necessary for adaptation to zinc limitation and highlights nutritional differences between environmental and virulent Francisella strains.IMPORTANCEFrancisella tularensis is a tier 1 select agent with a high potential for lethality and no approved vaccine. A better understanding of Francisella virulence factors is required for the development of therapeutics. While acquisition of zinc has been shown to be required for the virulence of numerous intracellular pathogens, zinc uptake has not been characterized in Francisella This work characterizes the Zur regulon in F. novicida and identifies two transporters that contribute to bacterial growth under zinc limitation. In addition, these data identify differences in mechanisms of zinc uptake and tolerance to zinc limitation between F. tularensis and F. novicida, highlighting the role of znuA in the growth of Schu S4 under zinc limitation.

Salmonella enterica serovar Typhimurium growth is inhibited by the concomitant binding of Zn(II) and a pyrrolyl-hydroxamate to ZnuA, the soluble component of the ZnuABC transporter.

BACKGROUND: Under conditions of Zn(II) deficiency, the most relevant high affinity Zn(II) transport system synthesized by many Gram-negative bacteria is the ZnuABC transporter. ZnuABC is absent in eukaryotes and plays an important role in bacterial virulence. Consequently, ZnuA, the periplasmic component of the transporter, appeared as a good target candidate to find new compounds able to contrast bacterial growth by interfering with Zn(II) uptake. METHODS: Antibacterial activity assays on selected compounds from and in-house library against Salmonella enterica serovar Typhimurium ATCC14028 were performed. The X-ray structure of the complex formed by SeZnuA with an active compound was solved at 2.15Å resolution. RESULTS: Two di-aryl pyrrole hydroxamic acids differing in the position of a chloride ion, RDS50 ([1-[(4-chlorophenyl)methyl]-4-phenyl-1H-pyrrol-3-hydroxamic acid]) and RDS51 (1-[(2-chlorophenyl)methyl]-4-phenyl-1H-pyrrol-3-hydroxamic acid) were able to inhibit Salmonella growth and its invasion ability of Caco-2 cells. The X-ray structure of SeZnuA containing RDS51 revealed its presence at the metal binding site concomitantly with Zn(II) which is coordinated by protein residues and the hydroxamate moiety of the compound. CONCLUSIONS: Two molecules interfering with ZnuA-mediated Zn(II) transport in Salmonella have been identified for the first time. The resolution of the SeZnuA-RDS51 X-ray structure revealed that RDS51 is tightly bound both to the protein and to Zn(II) thereby inhibiting its release. These features pave the way to the rational design of new Zn(II)-binding drugs against Salmonella. GENERAL SIGNIFICANCE: The data reported show that targeting the bacterial ZnuABC transporter can represent a good strategy to find new antibiotics against Gram-negative bacteria.

Virulence regulation of Zn2+ uptake system znuABC on mesophilic Aeromonas salmonicida SRW-OG1.

Psychrophilic Aeromonas salmonicida could not grow above 25°C and therefore thought unable to infect mammals and humans. In our previous study, a mesophilic A. salmonicida SRW-OG1 was isolated from Epinephelus coioides with furunculosis. Through the analysis of preliminary RNA-seq, it was found that the Zn2+ uptake related genes znuA, znuB and znuC might be involved in the virulence regulation of A. salmonicida SRW-OG1. Therefore, the purpose of this study was to explore the effect of znuABC silencing on the virulence regulation of A. salmonicida SRW-OG1. The results showed that the growth of the znuA-RNAi, znuB-RNAi, and znuC-RNAi strains was severely restricted under the Fe2+ starvation, but surprisingly there was no significant difference under the Zn2+ restriction. In the absence of Zn2+ and Fe2+, the expression level of znuABC was significantly increased. The motility, biofilm formation, adhesion and hemolysis of the znuA-RNAi, znuB-RNAi, and znuC-RNAi strains were significantly reduced. We also detected the expression of znuABC under different growth periods, temperatures, pH, as well as Cu2+ and Pb2+ stresses. The results showed that znuABC was significantly up-regulated in the logarithmic phase and the decline phase of A. salmonicida. Interestingly, the trend of expression levels of the znuABC at 18, 28, and 37°C was reversed to another Zn2+ uptake related gene zupT. Taken together, these indicated that the znuABC was necessary for A. salmonicida SRW-OG1 pathogenicity and environmental adaptability, and was cross regulated by iron starvation, but it was not irreplaceable for A. salmonicida SRW-OG1 Zn2+ uptake in the host.

The transcriptional regulator Zur regulates the expression of ZnuABC and T6SS4 in response to stresses in Yersinia pseudotuberculosis.

Zinc homeostasis is crucial for the development and stress resistance of bacteria in the environment. Serial zinc sensing transcriptional regulators, zinc transporters and zinc binding proteins were found to maintain the zinc homeostasis in bacteria. Zur is a zinc uptake regulator that is widely distributed in species, and ZnuABC, as well as the Type VI Secretion System (T6SS4) function in zinc acquisition. Here, we report that the regulator Zur inhibits the expression of the ZnuABC which inhibition could be eliminated at low zinc level, and upregulates the T6SS4 operon in Yersinia pseudotuberculosis to facilitate Zn2+ uptake and oxidative stress resistance. Zur regulates the expression of ZnuABC and T6SS4 by directly binding to their promoter regions. Zur senses the Zn2+ concentration and represses ZnuABC in a Zn2+-containing environment. Zur works as an auxiliary regular activator of T6SS4, facilitating oxidative stress resistance. This study revealed the dual function of regulator Zur on ZnuABC and T6SS4, and enriched the knowledge of Zn2+ homeostasis maintenance in Y. pseudotuberculosis.

Cyclic-di-GMP Regulates Autoaggregation Through the Putative Peptidoglycan Hydrolase, EagA, and Regulates Transcription of the znuABC Zinc Uptake Gene Cluster in Erwinia amylovora.

Erwinia amylovora is the causal agent of fire blight, an economically impactful disease that affects apple and pear production worldwide. E. amylovora pathogenesis is comprised of distinct type III secretion-dependent and biofilm-dependent stages. Alterations in the intracellular levels of cyclic-di-GMP (c-di-GMP) regulate the transition between the different stages of infection in E. amylovora. We previously reported that hyper-elevation of c-di-GMP levels in E. amylovora Ea1189, resulting from the deletion of all three c-di-GMP specific phosphodiesterase genes (Ea1189ΔpdeABC), resulted in an autoaggregation phenotype. The two major exopolysaccharides, amylovoran and cellulose, were also shown to partially contribute to autoaggregation. In this study, we aimed to identify the c-di-GMP dependent factor(s) that contributes to autoaggregation. We conducted a transposon mutant screen in Ea1189ΔpdeABC and selected for loss of autoaggregation. Our search identified a peptidoglycan hydrolase, specifically, a D, D-endopeptidase of the metallopeptidase class, EagA (Erwinia aggregation factor A), that was found to physiologically contribute to autoaggregation in a c-di-GMP dependent manner. The production of amylovoran was also positively affected by EagA levels. An eagA deletion mutant (Ea1189ΔeagA) was significantly reduced in virulence compared to the wild type E. amylovora Ea1189. eagA is part of the znuABC zinc uptake gene cluster and is located within an operon downstream of znuA. The znuAeagA/znuCB gene cluster was transcriptionally regulated by elevated levels of c-di-GMP as well as by the zinc-dependent transcriptional repressor Zur. We also observed that with an influx of Zn2+ in the environment, the transcription of the znuAeagA/znuBC gene cluster is regulated by both Zur and a yet to be characterized c-di-GMP dependent pathway.

Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis.

Zinc homeostasis is crucial for bacterial cells, since imbalances affect viability. However, in mycobacteria, knowledge of zinc metabolism is incomplete. Mycobacterium smegmatis (MSMEG) is an environmental, nonpathogenic Mycobacterium that is widely used as a model organism to study mycobacterial metabolism and pathogenicity. How MSMEG maintains zinc homeostasis is largely unknown. SmtB and Zur are important regulators of bacterial zinc metabolism. In mycobacteria, these regulators are encoded by an operon, whereas in other bacterial species, SmtB and Zur are encoded on separate loci. Here, we show that the smtB-zur operon is consistently present within the genus Mycobacterium but otherwise found only in Nocardia, Saccharothrix, and Corynebacterium diphtheriae By RNA deep sequencing, we determined the Zur and SmtB regulons of MSMEG and compared them with transcriptional responses after zinc starvation or excess. We found an exceptional genomic clustering of genes whose expression was strongly induced by zur deletion and zinc starvation. These genes encoded zinc importers such as ZnuABC and three additional putative zinc transporters, including the porin MspD, as well as alternative ribosomal proteins. In contrast, only a few genes were affected by deletion of smtB and zinc excess. The zinc exporter ZitA was most prominently regulated by SmtB. Moreover, transcriptional analyses in combination with promoter and chromatin immunoprecipitation assays revealed a special regulation of the smtB-zur operon itself: an apparently zinc-independent, constitutive expression of smtB-zur resulted from sensitive coregulation by both SmtB and Zur. Overall, our data revealed yet unknown peculiarities of mycobacterial zinc homeostasis.IMPORTANCE Zinc is crucial for many biological processes, as it is an essential cofactor of enzymes and a structural component of regulatory and DNA binding proteins. Hence, all living cells require zinc to maintain constant intracellular levels. However, in excess, zinc is toxic. Therefore, cellular zinc homeostasis needs to be tightly controlled. In bacteria, this is achieved by transcriptional regulators whose activity is mediated via zinc-dependent conformational changes promoting or preventing their binding to DNA. SmtB and Zur are important antagonistically acting bacterial regulators in mycobacteria. They sense changes in zinc concentrations in the femtomolar range and regulate transcription of genes for zinc acquisition, storage, and export. Here, we analyzed the role of SmtB and Zur in zinc homeostasis in Mycobacterium smegmatis Our results revealed novel insights into the transcriptional processes of zinc homeostasis in mycobacteria and their regulation.

Metal ion homeostasis in Listeria monocytogenes and importance in host-pathogen interactions.

Listeria monocytogenes is responsible for one of the most life-threatening food-borne infections and the leading cause of food-poisoning associated deaths in the UK. Infection may be of the unborn/newly born infant where disease may manifest as listeric abortion, stillbirth or late-onset neonatal listeriosis, while in adults, infection usually affects the central nervous system causing meningitis. Crucial to the survival of L. monocytogenes, both inside and outside the host, is its ability to acquire metals which act as cofactors for a broad range of its cellular proteins. However, L. monocytogenes must also protect itself against the innate toxicity of metals. The importance of metals in host-pathogen interactions is illustrated by the restriction of metals (including zinc and iron) in vertebrates in response to infection and the use of high levels of metals (copper and zinc) as part of the antimicrobial defences within host phagocytes. As such, L. monocytogenes is equipped with various mechanisms to tightly control its cellular metal pools and avoid metal poisoning. These include multiple DNA-binding metal-responsive transcription factors, metal-acquisition, metal-detoxification and metal-storage systems, some of which represent key L. monocytogenes virulence determinants. This review discusses current knowledge of the role of metals in L. monocytogenes infections, with a focus on the mechanisms that contribute to zinc and copper homeostasis in this organism. The requirement to precisely control cellular metal levels may impose a vulnerability to L. monocytogenes which can be exploited in antimicrobials and therapeutics.

Attenuated Salmonella enterica serovar Typhimurium lacking the ZnuABC transporter: an efficacious orally-administered mucosal vaccine against salmonellosis in pigs.

We have recently demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium unable to synthesize the zinc transporter ZnuABC (S. Typhimurium ΔznuABC), is able to protect mice against systemic and enteric salmonellosis and is safe in pigs. Here, we have tested the protective effects of S. Typhimurium ΔznuABC in pigs. Resistance to challenge with the fully virulent strain S. Typhimurium ATCC 14028 was assessed in animals vaccinated with S. Typhimurium ΔznuABC (two dosages tested), in controls vaccinated with a formalin-inactivated virulent strain and in unvaccinated controls. Clinical signs of salmonellosis, faecal shedding and bacterial colonization of organs were used to assess vaccine-induced protection. After the challenge, pigs vaccinated with the attenuated S. Typhimurium ΔznuABC strain did not display clinical signs of salmonellosis (fever or diarrhoea). The vaccine also reduced intestinal tract colonization and faecal shedding of the fully virulent Salmonella strain, as compared to control groups. S. Typhimurium ΔznuABC represents a promising candidate vaccine against salmonellosis in pigs.

Competition for zinc binding in the host-pathogen interaction.

Due to its favorable chemical properties, zinc is used as a structural or catalytic cofactor in a very large number of proteins. Despite the apparent abundance of this metal in all cell types, the intracellular pool of loosely bound zinc ions available for biological exchanges is in the picomolar range and nearly all zinc is tightly bound to proteins. In addition, to limit bacterial growth, some zinc-sequestering proteins are produced by eukaryotic hosts in response to infections. Therefore, to grow and multiply in the infected host, bacterial pathogens must produce high affinity zinc importers, such as the ZnuABC transporter which is present in most Gram-negative bacteria. Studies carried in different bacterial species have established that disruption of ZnuABC is usually associated with a remarkable loss of pathogenicity. The critical involvement of zinc in a plethora of metabolic and virulence pathways and the presence of very low number of zinc importers in most bacterial species mark zinc homeostasis as a very promising target for the development of novel antimicrobial strategies.