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Title: 27. PhAMP-induced modulation of biofilm related mutants in Pseudomonas aeruginosa
Authors: Asma Ramzan, Uzma Qaisar
Journal: Pure and Applied Biology (PAB)
Publisher: Bolan Society for Pure and Applied Biology
Country: Pakistan
Year: 2023
Volume: 12
Issue: 1
Language: English
Pseudomonas aeruginosa is an opportunist bacterium which grows at the infection site of immunocompromised patients and secretes virulence factors. It produces antibiotic resistant biofilms which are very hard to eradicate and a major problem for clinicians. Biofilm formation involves initial attachment to solid surface which require combined action of flagella and pili. These attached bacteria form sessile colonies by cell-cell attachment using fimbriae and by secreting exopolysaccharides and other proteins conferring resistance. Peganum harmala antimicrobial peptides (PhAMP) are known to inhibit the biofilm formation. In order to find out which component of biofilm formation is affected by PhAMP, transposon mutants of flagella, pili, fimbriae and exopolysaccharide production were used. We grew MPAO1 parent and its mutant strains (ΔflgK, ΔPilA, ΔcupA1 and ΔpslC) in the presence and absence of PhAMP. It was found that in the absence of PhAMP, the growth of planktonic cells was significantly affected in flagella and pili mutants and was not affected in fimbriae and exopolysaccharide mutants in comparison with MPAO1 strain while biofilm development was reduced in all mutants except ΔpslC. In the presence of PhAMP, the growth of both planktonic and biofilm cells was significantly reduced in all parent and mutant strains indicating that PhAMP effects bacterial growth even in the absence of a functional component. Gene expression analysis indicated that the transcriptional inhibition was observed in flgK, pilA, cupA1 and pslC genes by PhAMP. This study imparts important information about the molecular mechanism of action of PhAMP in bacterial biofilm production and bacterial growth.
Keywords: Antimicrobial peptides; Biofilm; Peganum harmala; Pseudomonas aeruginosa;
Transcription
http://dx.doi.org/10.19045/bspab.2023.120032
To investigate the effect of Peganum harmala antimicrobial peptides (PhAMP) on biofilm formation in Pseudomonas aeruginosa and to determine which components of biofilm formation are affected by PhAMP using transposon mutants.
The study used MPAO1 parent and mutant strains (flgK, PilA, cupA1, and pslC) of Pseudomonas aeruginosa. The strains were grown in the presence and absence of PhAMP. Planktonic cell growth and biofilm formation were quantified. Gene expression analysis was performed using real-time RT-PCR to determine the transcriptional inhibition of flgK, pilA, cupA1, and pslC genes by PhAMP.
graph TD
A[Obtain P. aeruginosa strains MPAO1, flgK, PilA, cupA1, pslC] --> B[Grow strains with and without PhAMP];
B --> C[Measure planktonic cell growth and biofilm formation];
C --> D[Perform RNA extraction and cDNA synthesis];
D --> E[Real-time RT-PCR for gene expression analysis];
E --> F[Analyze gene expression dataflgK, pilA, cupA1, pslC];
F --> G[Draw conclusions about PhAMP's effect on biofilm formation];
The study suggests that PhAMP affects bacterial growth even in the absence of a functional component. The downregulation of genes involved in the formation of extracellular appendages in the presence of PhAMP indicates a mechanism by which PhAMP inhibits biofilm formation.
In the absence of PhAMP, flagella and pili mutants showed significantly affected planktonic cell growth, while biofilm development was reduced in all mutants except pslC. In the presence of PhAMP, the growth of both planktonic and biofilm cells was significantly reduced in all parent and mutant strains. Gene expression analysis indicated transcriptional inhibition in flgK, pilA, cupA1, and pslC genes by PhAMP.
PhAMP is a promising antibiotic agent that can inhibit planktonic cell growth and biofilm formation in Pseudomonas aeruginosa by hampering the gene expression of biofilm-related genes.
1. The study used 18.5µg/ml of PhAMPs: Confirmed by the text in the "Effect of PhAMP on the growth of planktonic and biofilm cells of mutants of P. aeruginosa" section.
2. flgK and pili mutant strain PilA showed significant decrease (7.3% and 14% respectively) in the growth of planktonic cells: Confirmed by the text in the "Growth comparison of parent and mutant strains of P. aeruginosa" section.
3. Expression of cupA gene was 200 fold reduced in presence of PhAMPs: Confirmed by the text in the "PhAMP-based expression modulation of biofilm related genes in the MPAO1 strain of P. aeruginosa" section.
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