CRISPR can push Pseudomonas aeruginosa into new horizon

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CRISPR can push Pseudomonas aeruginosa into new horizon



Pseudomonas aeruginosa is a encapsulated, Gram-negative, rod shaped bacterium. P. aeruginosa is a major human pathogen and is capable of causing severe infections in vulnerable patients hospitalized with cystic fibrosis, burns, acquired immunodeficiency syndrome, or cancer. One of the most worrisome characteristics of P. aeruginosa is its low antibiotic susceptibility, which is attributable to a concerted action of multidrug efflux pumps with chromosomally encoded antibiotic resistance genes (e.g., mexAB, mexXY, etc.) and the low permeability of the bacterial cellular envelopes. So, the increasing prevalence of multidrug-resistant (MDR), extensively drug resistant (XDR), and pandrug-resistant (PDR) P. aeruginosa poses a grim challenge to antimicrobial therapy. Extensive research has been focused to dissect the molecular basis of the infection mechanisms and to develop novel therapeutic means against P. aeruginosa infections. Meanwhile, P. aeruginosa represents a model organism for biological study of biofilm formation, quorum sensing, drug target and metabolic engineering.

  Research Direction of P. aeruginosa

CRISPR/Cas 9 based genome editing will accelerate versatility of investigations on Pseudomonas aeruginosa.
· Genome, Proteome and Biology
· Metabolism and Maintenance of Extracellular matrices · Drug Resistance
· Secretory System
· Cell Polarity and Epithelial Barrier

 CRISPR-B™ for efficient gene editing in P. aeruginosa

Genome editing and manipulation often revolutionizes the understanding, exploitation, and control of microbial species. P. aeruginosa represents a model organism for biological study of biofilm formation, quorum sensing, drug target and metabolic engineering. However, traditional method for P. aeruginosa genetic manipulation required multi-steps selection process to generate mutant and leave a scar sequence in the place of deleted gene. So, the traditional genetic manipulation methods for major human pathogen P. aeruginosa, are still time consuming and laborious.

Ubigene developed CRISPR-B™ for gene manipulation of P. aeruginosa. Thereby, possible to achieve a one-step seamless genome editing in Pseudomonas species with the utilization of the CRISPR/Cas9 system. The CRISPR/Cas9 and the λ-Red recombination systems used for rapid, precise, and seamless genetic manipulation in P. aeruginosa. Ubigene can customize the gene-editing in P. aeruginosa as well as can generate various genes modification in microbes.

 CRISPR/Cas9 mediated microbes gene editing facilitates antimicrobial drug development

RAW264.7 is the most commonly used in vitro model for the selection of active compounds with anti-inflammatory and studying inflammation. RAW264.7 cells can mimic inflammatory reaction and release or upregulate a variety of inflammatory mediators, such as nitric oxide (NO), cyclooxygenase-2 (COX-2), tumor necrosis factor - α (TNF - α), interleukin-6 (IL-6) and so on.

 CRISPR/cas9 mediated miRNA-155 knockout model helps to explore the inhibition of proinflammatory cytokines production in rheumatoid arthritis

Gallium nitrate (Ganite) is a potential drug for the treatment of Pseudomonas aeruginosa infection. CRISPR/Cas9-based gene mutagenesis studies reveal that siderophore pyochelin-facilitated uptake and an ABC transporter are two major Ga3+ internalization pathways in Pseudomonas aeruginosa (P. aeruginosa). The researcher revealed that Ga3+ can inhibitor of P. aeruginosa growth and biofilm formation in vitro.


Gallium nitrate could improve lung function in patients with multidrug resistant CF clinical isolates and chronic P. aeruginosa lung infection without signs of any serious adverse effects, indicative of a potential clinical use of gallium nitrate for treatment of P. aeruginosa infection.


To investigate the molecular mechanism of gallium uptake by P. aeruginosa, the researcher utilized CRISPR/Cas9-based genome editing tools to mutate essential genes in P. aeruginosa model strain PAO1 to block each iron acquisition pathway individually. In total, 5 different PAO1 mutants were constructed. The PAO1ΔhitA mutant was constructed to block the classical high affinity iron acquisition system ABC transporter. Two mutants PAO1ΔpvdA and PAO1ΔpchF abolished the biosynthesis pathways of two siderophores PVD and PCH, respectively, while PAO1ΔfeoB mutant lost the high affinity inner membrane ferrous ion transporter and PAO1Δtesf mutant abrogated the Tesf-mediated iron acquisition pathway.


Fig 1: The sequencing results of P. aeruginosa mutants.

The researcher analyses the effects of Ga(III) on the growth of wild-type (WT) and different PAO1 mutants. The minimal inhibitory concentrations (MICs) of Ga(NO3)3 against different strains in iron-deficient medium. They also analyze the effect of different iron acquisition pathways on gallium uptake of PAO1 and PAO1Δtesf mutants. A substantial decline of the intracellular iron level was also observed in this double mutant strain (Fig 3B) indicate that HitABC transporter is the major contributor for iron uptake in P. aeruginosa.



Fig 2: MIC values of Ga(NO3)3 against wild-type (WT) P. aeruginosa and different mutants.

HitA is a soluble ferric iron-binding protein located in the bacterial periplasm and binds a single Fe3+ with high affinity. HitA could exert a similar role in the Ga3+ uptake process. To uncover the phenomena, they apply cellular thermal shift assay (CETSA) observed that Ga3+ can binding of HitA in vivo. Afterward, they performed metal competition assay and observed that Fe3+ could readily replace Ga3+ from Ga3+-HitA protein. The researcher generates high resolution crystal structures of apo-HitA and Fe3+-bound and Ga3+-bound HitA proteins. The structure of Ga3+-HitA resembles that of Fe3+-HitA with backbone root-mean-square deviation (RMSD) of 0.268 Å. The coordination sphere of Fe3+ is almost the same as that identified in the Ga3+ site, consisting of the four residues (His39, Glu87, Tyr223, and Tyr224), one water molecule, and a phosphate ion as the monodentate ligand. The structural data confirmed that Ga3+ could mimic Fe3+ to occupy the metal-binding site of HitA.



Fig 4: Superimposition of two chains of Fe3+ -HitA and Ga3+ -HitA from one asymmetric unit. The metal coordination spheres are identical in two chains.

Ubigene developed CRISPR-B™ which optimizes the microbial gene-editing vectors and process. The efficiency and accuracy are 20x higher than traditional methods. CRISPR-B™ can be used in gene editing of bacteria and fungi. Contact us immediately to know about your research related services!


References

Identification and characterization of a metalloprotein involved in gallium internalization in Pseudomonas aeruginosa. ACS Infect Dis 2019, 5(10):1693-1697.

Research Topic on Pseudomonas aeruginosa, Biology, Genetics, and Host-Pathogen Interactions. Front Microbiol 2012, 3:20