Integration of RNA-seq and RNAi reveals the contribution of znuA gene to the pathogenicity of Pseudomonas plecoglossicida and to the immune response of Epinephelus coioides.

Pseudomonas plecoglossicida is an important pathogen in aquaculture and causes serious economic losses. Our previous study indicated that znuA gene might play an important role in the pathogenicity of P. plecoglossicida. Five shRNAs were designed and synthesized to silence the znuA gene of P. plecoglossicida. Two of the five mutants of P. plecoglossicida exhibited significant reduction in the expression level of znuA mRNA with different efficiencies. The mutant with the highest silencing efficiency of 89.2% was chosen for further studies. Intrapleural injection of the znuA-RNAi strain at a dose of 105  cfu/fish did not cause the death of Epinephelus coioides, and no significant signs were observed at the spleen surface of infected E. coioides, while the counterpart E. coioides infected by the same dose of wild-type strain of P. plecoglossicida all died in 5 days post-infection (dpi). The expression of znuA gene of znuA-RNAi strain in E. coioides was always lower than that in wild-type strain of P. plecoglossicida. The pathogen load in the early stage of infection was higher than that in the later stage of infection. Although the infection of the znuA-RNAi strain of P. plecoglossicida could induce the production of antibodies in E. coioides, it failed to produce a good immune protection against the infection of wild-type strain of P. plecoglossicida. Compared with the transcriptome data of E. coioides infected by the wild-type strain of P. plecoglossicida, the transcriptome data of E. coioides infected by the znuA-RNAi strain of P. plecoglossicida have altered significantly. Among them, KEGG enrichment analysis showed that the focal adhesion pathway was significantly enriched and exhibited the largest number of 302 DEMs (differentially expressed mRNAs). These results showed that the immune response of E. coioides to P. plecoglossicida infection was significantly affected by the RNAi of znuA gene.

Metallochaperones Are Needed for Mycobacterium tuberculosis and Escherichia coli Nicotinamidase-Pyrazinamidase Activity.

Mycobacterium tuberculosis nicotinamidase-pyrazinamidase (PZAse) is a metalloenzyme that catalyzes conversion of nicotinamide-pyrazinamide to nicotinic acid-pyrazinoic acid. This study investigated whether a metallochaperone is required for optimal PZAse activity. M. tuberculosis and Escherichia coli PZAses (PZAse-MT and PZAse-EC, respectively) were inactivated by metal depletion (giving PZAse-MT-Apo and PZAse-EC-Apo). Reactivation with the E. coli metallochaperone ZnuA or Rv2059 (the M. tuberculosis analog) was measured. This was repeated following proteolytic and thermal treatment of ZnuA and Rv2059. The CDC1551 M. tuberculosis reference strain had the Rv2059 coding gene knocked out, and PZA susceptibility and the pyrazinoic acid (POA) efflux rate were measured. ZnuA (200 µM) achieved 65% PZAse-EC-Apo reactivation. Rv2059 (1 µM) and ZnuA (1 µM) achieved 69% and 34.3% PZAse-MT-Apo reactivation, respectively. Proteolytic treatment of ZnuA and Rv2059 and application of three (but not one) thermal shocks to ZnuA significantly reduced the capacity to reactivate PZAse-MT-Apo. An M. tuberculosis Rv2059 knockout strain was Wayne positive and susceptible to PZA and did not have a significantly different POA efflux rate than the reference strain, although a trend toward a lower efflux rate was observed after knockout. The metallochaperone Rv2059 restored the activity of metal-depleted PZAse in vitro Although Rv2059 is important in vitro, it seems to have a smaller effect on PZA susceptibility in vivo. It may be important to mechanisms of action and resistance to pyrazinamide in M. tuberculosis Further studies are needed for confirmation.IMPORTANCE Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide's mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient's individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat.

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.

Characterization of three predicted zinc exporters in Brucella ovis identifies ZntR-ZntA as a powerful zinc and cadmium efflux system not required for virulence and unveils pathogenic Brucellae heterogeneity in zinc homeostasis.

Brucella ovis causes non-zoonotic ovine brucellosis of worldwide distribution and is responsible for important economic losses mainly derived from male genital lesions and reproductive fails. Studies about the virulence mechanisms of this rough species (lacking lipopolysaccharide O-chains) are underrepresented when compared to the main zoonotic Brucella species that are smooth (with O-chains). Zinc intoxication constitutes a defense mechanism of the host against bacterial pathogens, which have developed efflux systems to counterbalance toxicity. In this study, we have characterized three potential B. ovis zinc exporters, including the ZntA ortholog previously studied in B. abortus. Despite an in-frame deletion removing 100 amino acids from B. ovis ZntA, the protein retained strong zinc efflux properties. Only indirect evidence suggested a higher exporter activity for B. abortus ZntA, which, together with differences in ZntR-mediated regulation of zntA expression between B. ovis and B. abortus, could contribute to explaining why the ΔzntR mutant of B. abortus is attenuated while that of B. ovis is virulent. Additionally, B. ovis ZntA was revealed as a powerful cadmium exporter contributing to cobalt, copper, and nickel detoxification, properties not previously described for the B. abortus ortholog. Deletion mutants for BOV_0501 and BOV_A1100, also identified as potential zinc exporters and pseudogenes in B. abortus, behaved as the B. ovis parental strain in all tests performed. However, their overexpression in the ΔzntA mutant allowed the detection of discrete zinc and cobalt efflux activity for BOV_0501 and BOV_A1100, respectively. Nevertheless, considering their low expression levels and the stronger activity of ZntA as a zinc and cobalt exporter, the biological role of BOV_0501 and BOV_A1100 is questionable. Results presented in this study evidence heterogeneity among pathogenic Brucellae regarding zinc export and, considering the virulence of B. ovis ΔzntA, suggest that host-mediated zinc intoxication is not a relevant mechanism to control B. ovis infection.

Structural and biochemical characterization of Acinetobacter baumannii ZnuA.

Acinetobacter baumannii is a Gram-negative nosocomial pathogen associated with significant disease. Crucial to the survival and pathogenesis of A. baumannii is the ability to acquire essential micronutrients such as Zn(II). Recruitment of Zn(II) by A. baumannii is mediated, at least in part, by the periplasmic solute-binding protein ZnuA and the ATP-binding cassette transporter ZnuBC. Here, we combined genomic, biochemical, and structural approaches to characterize A. baumannii AB5075_UW ZnuA. Bioinformatic analyses using a diverse collection of A. baumannii genomes determined that ZnuA is highly conserved, with the binding site comprised by three strictly conserved histidine residues. The structure of metal-free ZnuA was determined at 2.1 Å resolution, with molecular dynamics analyses revealing loop α2ß2, which harbors the putative Zn(II)-coordinating residue His41, to be highly mobile in the metal-free state. The contribution of the putative binding site histidine residues to Zn(II) interaction was further probed by mutagenesis. Analysis of ZnuA mutant variants was performed by quantitative metal binding assays, differential scanning fluorimetry, and affinity measurements, which showed that all three histidine residues contributed to Zn(II)-recruitment, albeit to different extents. Collectively, these analyses provide insight into the mechanism of Zn(II)-binding by A. baumannii ZnuA and expand our understanding of the functional diversity of Zn(II)-recruiting proteins.

Conformational flexibility in the zinc solute-binding protein ZnuA.

Zinc is an essential metal for all kingdoms of life, making its transport across the cell membrane a critical function. In bacteria, high-affinity zinc import is accomplished by ATP-binding cassette (ABC) transporters, which rely on extracellular solute-binding proteins (SBPs) of cluster A-I to acquire the metal and deliver it to the membrane permease. These systems are important for survival and virulence, making them attractive targets for the development of novel antibiotics. Citrobacter koseri is an emerging pathogen with extensive antibiotic resistance. High-affinity zinc binding to the C. koseri cluster A-I SBP ZnuA has been characterized and the structure of the zinc-bound (holo) form has been determined by X-ray crystallography. Remarkably, despite 95% sequence identity to the ZnuA homologue from Salmonella enterica, C. koseri ZnuA exhibits a different zinc-coordination environment and a closed rather than an open conformation. Comparison with structures of another close ZnuA homologue from Escherichia coli suggests a surprisingly flexible conformational landscape that may be important for efficient zinc binding and/or delivery to the membrane permease.

A novel antimicrobial approach based on the inhibition of zinc uptake in Salmonella enterica.

In this review we discuss evidences suggesting that bacterial zinc homeostasis represents a promising target for new antimicrobial strategies. The ability of the gut pathogen Salmonella enterica sv Typhimurium to withstand the host responses aimed at controlling growth of the pathogen critically depends on the zinc importer ZnuABC. Strains lacking a functional ZnuABC or its soluble component ZnuA display a dramatic loss of pathogenicity, due to a reduced ability to express virulence factors; withstand the inflammatory response; and compete with other gut microbes. Based on this data, ZnuA was chosen as a candidate for the rational design of novel antibiotics. Through a combination of structural and functional investigations, we have provided a proof of concept of the potential of this approach.

Heterogeneous nucleation is required for crystallization of the ZnuA domain of pneumococcal AdcA.

Zn(2+) is an essential nutrient for all known forms of life. In the major human pathogen Streptococcus pneumoniae, the acquisition of Zn(2+) is facilitated by two Zn(2+)-specific solute-binding proteins: AdcA and AdcAII. To date, there has been a paucity of structural information on AdcA, which has hindered a deeper understanding of the mechanism underlying pneumococcal Zn(2+) acquisition. Native AdcA consists of two domains: an N-terminal ZnuA domain and a C-terminal ZinT domain. In this study, the ZnuA domain of AdcA was crystallized. The initial crystals of the ZnuA-domain protein were obtained using dried seaweed as a heterogeneous nucleating agent. No crystals were obtained in the absence of the heterogeneous nucleating agent. These initial crystals were subsequently used as seeds to produce diffraction-quality crystals. The crystals diffracted to 2.03 Šresolution and had the symmetry of space group P1. This study demonstrates the utility of heterogeneous nucleation. The solution of the crystal structures will lead to further understanding of Zn(2+) acquisition by S. pneumoniae.

Deletion of the znuA virulence factor attenuates Actinobacillus pleuropneumoniae and confers protection against homologous or heterologous strain challenge.

The znuA gene is known to be important for growth and survival in Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida under low Zn(2+) conditions. This gene is also present in Actinobacillus pleuropneumoniae serotype 1; therefore, the aim of this study was to investigate the existence of a similar role for the znuA gene in the growth and virulence of this organism. A precisely defined ΔznuA deletion mutant of A. pleuropneumoniae was constructed based on the sequence of the wild-type SLW01 using transconjugation and counterselection. This mutation was found to be lethal in low-Zn(2+) medium. Furthermore, the ΔznuA mutant strain exhibited attenuated virulence (≥22-fold) as well as reduced mortality and morbidity in a murine (Balb/C) model of infection. The majority of the bacteria were cleared from the lungs within 2 weeks. The ΔznuA mutant strain caused no adverse effects in pigs at doses of up to 1.0×10(9) CFU/mL. The ΔznuA mutant strain induced a significant immune response and conferred 80% and 100% protection on immunised pigs against challenge with A. pleuropneumoniae strains belonging to homologous or heterologous serovars, respectively, compared to the blank controls. The data obtained in this study indicate the potential of the mutant ΔznuA strain for development as a live vaccine capable of inducing reliable cross-serovar protection following intratracheal immunisation.

Characterization of a novel zinc transporter ZnuA acquired by Vibrio parahaemolyticus through horizontal gene transfer.

Vibrio parahaemolyticus is a clinically important foodborne pathogen that causes acute gastroenteritis worldwide. It has been shown that horizontal gene transfer (HGT) contributes significantly to virulence development of V. parahaemolyticus. In this study, we identified a novel znuA homolog (vpa1307) that belongs to a novel subfamily of ZnuA, a bacterial zinc transporter. The vpa1307 gene is located upstream of the V. parahaemolyticus pathogenicity island (Vp-PAIs) in both tdh-positive and trh-positive V. parahaemolyticus strains. Phylogenetic analysis revealed the exogenous origin of vpa1307 with 40% of V. parahaemolyticus clinical isolates possessing this gene. The expression of vpa1307 gene in V. parahaemolyticus clinical strain VP3218 is induced under zinc limitation condition. Gene deletion and complementation assays confirmed that vpa1307 contributes to the growth of VP3218 under zinc depletion condition and that conserved histidine residues of Vpa1307 contribute to its activity. Importantly, vpa1307 contributes to the cytotoxicity of VP3218 in HeLa cells and a certain degree of virulence in murine model. These results suggest that the horizontally acquired znuA subfamily gene, vpa1307, contributes to the fitness and virulence of Vibrio species.