Karrera Y. Djoko

The role of copper and zinc toxicity in innate immune defense against bacterial pathogens.

Zinc (Zn) and copper (Cu) are essential for optimal innate immune function, and nutritional deficiency in either metal leads to increased susceptibility to bacterial infection. Recently, the decreased survival of bacterial pathogens with impaired Cu and/or Zn detoxification systems in phagocytes and animal models of infection has been reported. Consequently, a model has emerged in which the host utilizes Cu and/or Zn intoxication to reduce the intracellular survival of pathogens. This review describes and assesses the potential role for Cu and Zn intoxication in innate immune function and their direct bactericidal function.

Phenotypic Characterization of a copA Mutant of Neisseria gonorrhoeae Identifies a Link between Copper and Nitrosative Stress

ABSTRACT NGO0579 is annotated copA in the Neisseria gonorrhoeae chromosome, suggesting that it encodes a cation-transporting ATPase specific for copper ions. Compared to wild-type cells, a copA mutant was more sensitive to killing by copper ions but not to other transition metals. The mutant also accumulated a greater amount of copper, consistent with the predicted role of CopA as a copper efflux pump. The copA mutant showed a reduced ability to invade and survive within human cervical epithelial cells, although its ability to form a biofilm on the surface of these cells was not significantly different from that of the wild type. In the presence of copper, the copA mutant exhibited increased sensitivity to killing by nitrite or nitric oxide. Therefore, we concluded that copper ion efflux catalyzed by CopA is linked to the nitrosative stress defense system of Neisseria gonorrhoeae. These observations suggest that copper may exert its effects as an antibacterial agent in the innate immune system via an interaction with reactive nitrogen species.

Copper Resistance in E. coli: The Multicopper Oxidase PcoA Catalyzes Oxidation of Copper(I) in CuICuII‐PcoC

es that three soluble proteins (PcoA, PcoC, PcoE in E. coli) are expressed to the periplasm, that two copper pumps (PcoB, PcoD) are present in the outer and inner membranes, and that there is a copper sensing system (PcoS, PcoR). PcoC and CopC are highly homologous b-barrels (~11 kDa) that bind both Cu and Cu at sites separated by about 30 6. These sites are tailored to bind their individual ions with high affinity (KD~10 m): CuACHTUNGTRENNUNG(His) ACHTUNGTRENNUNG(Met)2or3 (trigonal or tetrahedral), Cu ACHTUNGTRENNUNG(His)2ACHTUNGTRENNUNG(N-term) ACHTUNGTRENNUNG(OH2) (square planar). [5, 6] Each shows little affinity for the other ion. All possible types of intermolecular copper transfer reactions have been demonstrated: oxidative transfer from the Cu site to the Cu site, reductive transfer from the Cu site to the Cu site, and non-redox transfers between Cu and Cu sites. This versatile chemistry is consistent with a role for PcoC as a copper carrier (chaperone) in the oxidizing periplasm, but which functions are employed remains unknown. apo-PcoA (63.9 kDa) was over-expressed in E. coli and isolated in high purity in the presence of ethylenediaminetetraacetate (EDTA), dithiothreitol (DTT), and glycerol (Figure S1). The protein ran as a monomer on a Superdex-75 size exclusion column (Figure S1). Incubation with more than five equiv of Cuaq in the presence of glutathione and removal of unbound ions by gel filtration led to optimal development of an absorbance spectrum characteristic of a multicopper oxidase (type 1 center : 600 nm (e, 4000m 1 cm ) ; OH-bridged type 3 binuclear center : ~340 nm sh; A280/A600, 23.0; Figure S2). Both type 1 and type 2 Cu centers were also detected in the frozen EPR spectrum (type 1 center: g?=2.07, gk =2.28, Ak =8.5G 10 3 cm ; type 2 center: g?=2.07, gk=2.30, Ak =17G 10 3 cm ; Figure S3). Isolated holo-PcoA contained 4.3 (0.4s) equiv of copper and exhibited phenol oxidase activity with substrate 2,6-dimethoxyphenol (DMP) at pH 7 (Figure S4; 550 mm DMP per mm PcoA per min), a feature of most multicopper oxidases. Unexpectedly, this enzyme exhibited maximal activity in the absence of added Cuaq, whereas related multicopper oxidases require this ion in excess to induce maximal phenol oxidase activity. Reaction of holo-PcoA with air-stable CuCu-PcoC under catalytic conditions (1:50) in air-saturated MOPS buffer (pH 7) led to quantitative generation of product Cu-PcoC (represented as C Figure 1); this is consistent with oxidation of bound Cu. The catalysis was suppressed dramatically in deoxygenated buffer (Figure S5), consistent with O2 acting as the oxidant. The product protein &Cu II has little affinity for co-product Cuaq, which appeared to be ACHTUNGTRENNUNGreleased into solution. Clean regeneration of CuCu-PcoC occurred upon addition of reductant NH2OH (that is, Figure 1D converted to Figure 1A). This is a robust catalyst : there was negligible loss of activity after four cycles of oxidation and ACHTUNGTRENNUNGreduction. The product PcoC protein &Cu suppressed cuprous oxidase activity, consistent with a previous suggestion that PcoC interacts with PcoA. Inclusion of increasing concentrations of &Cu in the initial reaction mixture in Figure 1 led to proportional decreases in the rate of the catalytic reaction, whereas addition of generic proteins such as lysozyme had no effect (Figure 2). These results, when coupled with the high affinity of PcoC for Cu (KD~10 m), demonstrate that PcoA catalyzes the oxidation of Cu bound in CuCu-PcoC. They suggest that PcoA and PcoC cooperate to convert Cu into the less toxic Cu in the O2-rich periplasm. This system exhibits two new features: cuprous oxidase ACHTUNGTRENNUNGactivity with a likely biological partner and maximal phenol ACHTUNGTRENNUNGoxidase activity in the absence of excess Cuaq. In seeking a model for this behavior, it was noted that PcoA has some homology (23%) with the tolerance enzyme CueO and, impor[a] K. Y. Djoko, Dr. Z. Xiao, Prof. A. G. Wedd School of Chemistry and Bio21 Research Institute, University of Melbourne Parkville, Victoria 3010 (Australia) Fax: (+61)3-9347-5180 E-mail : z.xiao@unimelb.edu.au

Antimicrobial Action of Copper Is Amplified via Inhibition of Heme Biosynthesis

Copper (Cu) is a potent antimicrobial agent. Its use as a disinfectant goes back to antiquity, but this metal ion has recently emerged to have a physiological role in the host innate immune response. Recent studies have identified iron-sulfur containing proteins as key targets for inhibition by Cu. However, the way in these effects at the molecular level translate into a global effect on cell physiology is not fully understood. Here, we provide a new insight into the way in which Cu poisons bacteria. Using a copA mutant of the obligate human pathogen Neisseria gonorrhoeae that lacks a Cu efflux pump, we showed that Cu overloading led to an increased sensitivity to hydrogen peroxide. However, instead of promoting disproportionation of H2O2 via Fenton chemistry, Cu treatment led to an increased lifetime of H2O2 in cultures as a result of a marked decrease in catalase activity. We showed that this observation correlated with a loss of intracellular heme. We further established that Cu inhibited the pathway for heme biosynthesis. We proposed that this impaired ability to produce heme during Cu stress would lead to the failure to activate hemoproteins that participate in key processes, such as the detoxification of various reactive oxygen and nitrogen species, and aerobic respiration. The impact would be a global disruption of cellular biochemistry and an amplified Cu toxicity.

Novel Bacterial MerR-Like Regulators: Their Role in the Response to Carbonyl and Nitrosative Stress

Recognition of the diversity of transcriptional regulators of the MerR family has increased considerably over the last decade and it has been established that not all MerR-like regulators are involved in metal ion recognition. A new type of MerR-like regulator was identified in Neisseria gonorrhoeae that is distinct from metal-binding MerR proteins. This novel transcription factor, the Neisseria merR-like regulator (NmlR) is related to a large and diverse group of MerR-like regulators. A common feature of the majority of the genes encoding the nmlR-related genes is that they predicted to control the expression of adhC, which encodes a glutathione-dependent alcohol dehydrogenase. The function of the NmlR regulon appears to be to defend the bacterial cell against carbonyl stress and in some cases nitrosative stress. A potential role for NmlR in bacterial pathogenesis has been identified in Neisseria gonorrhoeae and Streptococcus pneumoniae. Although it is not known how NmlR is activated it is suggested that conserved cysteine residues may be involved in thiol-based signaling.

Formaldehyde Stress Responses in Bacterial Pathogens

Formaldehyde is the simplest of all aldehydes and is highly cytotoxic. Its use and associated dangers from environmental exposure have been well documented. Detoxification systems for formaldehyde are found throughout the biological world and they are especially important in methylotrophic bacteria, which generate this compound as part of their metabolism of methanol. Formaldehyde metabolizing systems can be divided into those dependent upon pterin cofactors, sugar phosphates and those dependent upon glutathione. The more prevalent thiol-dependent formaldehyde detoxification system is found in many bacterial pathogens, almost all of which do not metabolize methane or methanol. This review describes the endogenous and exogenous sources of formaldehyde, its toxic effects and mechanisms of detoxification. The methods of formaldehyde sensing are also described with a focus on the formaldehyde responsive transcription factors HxlR, FrmR, and NmlR. Finally, the physiological relevance of detoxification systems for formaldehyde in bacterial pathogens is discussed.

Copper(II)-Bis(Thiosemicarbazonato) Complexes as Antibacterial Agents: Insights into Their Mode of Action and Potential as Therapeutics

ABSTRACT There is increasing interest in the use of lipophilic copper (Cu)-containing complexes to combat bacterial infections. In this work, we showed that Cu complexes with bis(thiosemicarbazone) ligands [Cu(btsc)] exert antibacterial activity against a range of medically significant pathogens. Previous work using Neisseria gonorrhoeae showed that Cu(btsc) complexes may act as inhibitors of respiratory dehydrogenases in the electron transport chain. We now show that these complexes are also toxic against pathogens that lack a respiratory chain. Respiration in Escherichia coli was slightly affected by Cu(btsc) complexes, but our results indicate that, in this model bacterium, the complexes act primarily as agents that deliver toxic Cu ions efficiently into the cytoplasm. Although the chemistry of Cu(btsc) complexes may dictate their mechanism of action, their efficacy depends heavily on bacterial physiology. This is linked to the ability of the target bacterium to tolerate Cu and, additionally, the susceptibility of the respiratory chain to direct inhibition by Cu(btsc) complexes. The physiology of N. gonorrhoeae, including multidrug-resistant strains, makes it highly susceptible to damage by Cu ions and Cu(btsc) complexes, highlighting the potential of Cu(btsc) complexes (and Cu-based therapeutics) as a promising treatment against this important bacterial pathogen.

Characterization of an ntrX Mutant of Neisseria gonorrhoeae Reveals a Response Regulator That Controls Expression of Respiratory Enzymes in Oxidase-Positive Proteobacteria

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.

A glutathione-dependent detoxification system is required for formaldehyde resistance and optimal survival of Neisseria meningitidis in biofilms.

AIM The glutathione-dependent AdhC-EstD formaldehyde detoxification system is found in eukaryotes and prokaryotes. It is established that it confers protection against formaldehyde that is produced from environmental sources or methanol metabolism. Thus, its presence in the human host-adapted bacterial pathogen Neisseria meningitidis is intriguing. This work defined the biological function of this system in the meningococcus using phenotypic analyses of mutants linked to biochemical and structural characterization of purified enzymes. RESULTS We demonstrated that mutants in the adhC and/or estD were sensitive to killing by formaldehyde. Inactivation of adhC and/or estD also led to a loss of viability in biofilm communities, even in the absence of exogenous formaldehyde. Detailed biochemical and structural analyses of the esterase component demonstrated that S-formylglutathione was the only biologically relevant substrate for EstD. We further showed that an absolutely conserved cysteine residue was covalently modified by S-glutathionylation. This leads to inactivation of EstD. INNOVATION The results provide several conceptual innovations. They provide a new insight into formaldehyde detoxification in bacteria that do not generate formaldehyde during the catabolism of methanol. Our results also indicate that the conserved cysteine, found in all EstD enzymes from humans to microbes, is a site of enzyme regulation, probably via S-glutathionylation. CONCLUSION The adhc-estD system protects against formaldehyde produced during endogenous metabolism.

Copper(II)-bis(thiosemicarbazonato) complexes as anti-chlamydial agents.

Lipophilic copper (Cu)-containing complexes have shown promising antibacterial activity against a range of bacterial pathogens. To examine the susceptibility of the intracellular human pathogen Chlamydia trachomatis to copper complexes containing bis(thiosemicarbazone) ligands [Cu(btsc)], we tested the in vitro effect of CuII-diacetyl- and CuII-glyoxal-bis[N(4)-methylthiosemicarbazonato] (Cu(atsm) and Cu(gtsm), respectively) on C. trachomatis. Cu(atsm) and to a greater extent, Cu(gtsm), prevented the formation of infectious chlamydial progeny. Impacts on host cell viability and respiration were also observed in addition to the Chlamydia impacts. This work suggests that copper-based complexes may represent a new lead approach for future development of new therapeutics against chlamydial infections, although host cell impacts need to be fully explored.