Dataset Information

Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

ABSTRACT: Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow-spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a metal-dependent DNase while pyocin SX2 kills cells through inhibition of protein synthesis. Mapping the uptake pathways of SX1 and SX2 shows these pyocins utilize a combination of the common polysaccharide antigen (CPA) and a previously uncharacterized TonB-dependent transporter (TBDT) PA0434 to traverse the outer membrane. In addition, TonB1 and FtsH are required by both pyocins to energize their transport into cells and catalyze their translocation across the inner membrane, respectively. Expression of PA0434 was found to be specifically regulated by copper availability and we have designated PA0434 as Copper Responsive Transporter A, or CrtA. To our knowledge these are the first S-type pyocins described that utilize a TBDT that is not involved in iron uptake.

SUBMITTER: Premsuriya J

PROVIDER: S-EPMC10422930 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

Premsuriya Jiraphan J Mosbahi Khedidja K Atanaskovic Iva I Kleanthous Colin C Walker Daniel D

The Biochemical journal 20230701 14

Pseudomonas aeruginosa is a common cause of serious hospital-acquired infections, the leading proven cause of mortality in people with cystic fibrosis and is associated with high levels of antimicrobial resistance. Pyocins are narrow-spectrum protein antibiotics produced by P. aeruginosa that kill strains of the same species and have the potential to be developed as therapeutics targeting multi-drug resistant isolates. We have identified two novel pyocins designated SX1 and SX2. Pyocin SX1 is a ...[more]

PMID: 37399084

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Project description:The envelope of Gram-negative bacteria is composed of two membranes separated by the periplasmic space. This organization imposes geometrical and distance constraints that are key for the mechanism of action of multicomponent systems spanning the envelope. However, consideration of all three compartments by experimental approaches is still elusive. Here, we have used the state-of-the-art molecular dynamics simulation in an Escherichia coli envelope model to obtain a dynamic view of molecular interactions between the outer membrane heme transporter HasR and the inner membrane TonB-like protein HasB. Their interaction allows the transfer of the inner membrane proton-motive force derived energy to the transporter for heme internalization. The simulations that incorporate both membranes show the key role of periplasmic domains of both proteins and their dynamics in complex formation and stability. They revealed a previously unidentified network of HasR-HasB protein-protein interactions in the periplasm. Experimental validation (mutations, in vivo phenotypic and biophysical assays) provides support for the simulation-predicted interactions. Based on structural and sequence conservation, the network of interaction revealed in this study is expected to occur in other nutrient import systems.ImportanceGram-negative bacteria import scarce nutrients such as metals and vitamins by an energized mechanism involving a multicomponent protein system that spans the cell envelope. It consists of an outer membrane TonB-dependent transporter (TBDT) and a TonB complex in the inner membrane that provides the proton motive force energy for the nutrient entry. Despite the intense research efforts focused on this system (a) from structural and fundamental microbiology perspectives and (b) for the interest in the development of new antibacterial strategies, the molecular mechanism of the system is not at all well understood. The lack of understanding comes from incomplete structural data and the experimental difficulties of studying an inherently flexible multicomponent complex that resides within the heterogeneous environment of the double membrane bacterial cell envelope. To address these challenges and obtain a comprehensive view of the molecular interactions at atomic level, here, we have used the combined power of advanced molecular simulations and complementary microbiology and biochemical experiments. Our results represent a significant step forward in understanding the structural and molecular bases of this vital mechanism.

Project description:Intracellular zinc concentration needs to be maintained within strict limits due to its toxicity at high levels, and this is achieved by a finely regulated balance between uptake and efflux. Many bacteria use the Zinc Uptake Regulator Zur to orchestrate zinc homeostasis, but little is known regarding the transport of this metal across the bacterial outer membrane. In this work we determined the C. crescentus Zur regulon by global transcriptional and in silico analyses. Among the genes directly repressed by Zur are those encoding a putative high affinity ABC uptake system (znuCBA), three TonB-dependent receptors (znuD, znuE and znuF) and one new putative transporter of a family not yet characterized (zrpW). Zur is also directly involved in the activation of a RND and a P-type ATPase efflux systems, as revealed by Î²-galactosidase and site-directed mutagenesis assays. Several genes belonging to the Fur regulon were also downregulated in the zur mutant, suggesting a putative cross-talk between Zur and Fur regulatory networks. Interestingly, a phenotypic analysis of the znuD and znuE mutants has shown that these genes are essential for growth under zinc starvation, suggesting that C. crescentus uses these TonB-dependent outer membrane transporters as key zinc scavenging systems. The DNA microarray experiments were performed as described in (da Silva Neto et al., 2013). Briefly, cDNA was generated from 12.5 Âµg of total RNA and labeled with either Cy3 or Cy5 fluorescent dyes (FairPlay III Microarray Labeling System, Stratagene). Labeled cDNA samples were hybridized to a Caulobacter DNA oligo microarray (Agilent), and the arrays were scanned with an Agilent High Resolution Microarray Scanner. RNA from three independent biological cultures was used for DNA microarray analysis. We considered as differentially expressed genes those showing a minimum of 2-fold change relative to the control, considering at least three out of the four last probes for each gene (that are downstream of the translational start site) in at least two of three biological replicates. The values for the relative expression of each gene were obtained as the average of the four last probes.

Dataset Information

Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

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Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA.

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