Impact of phage predation on bacterial transcriptome under simulated human airway conditions

Bacteriophages have been proven to be efficient in the combat of bacterial multidrug-resistant infections, including those caused by Pseudomonas aeruginosa. Nevertheless, the interactions of phages with bacteria in the human body remains unexplained and its disclosure could lead to advance research and development in phage-based therapies. In this work, RNA-sequencing of phage-infected P. aeruginosa PAO1 adhered to a human epithelial cell monolayer (Nuli-1 ATCC® CRL-4011) was performed to assess bacterial transcriptional processes occurring in phagebacteriahuman cells, i.e., mimicking phage predation under more realistic settings. To achieve that, adhered bacteria were infected with phage LUZ19, and total RNA was extracted from the complex cell mixture. Thereafter, bacterial rRNA/human RNA was depleted and cDNA libraries were prepared to sequence. The differentially expressed genes (DEGs) were quantified using uninfected bacteria as control. In human airway-simulated conditions, there were 21, 39, and 129 bacterial DEGs after 5, 10, and 15 min-post infection, respectively. From DEGs, some genes were identified as part of LUZ19 typical induced responses (prophage, glycerol metabolism, and spermidine synthesis genes). However, unique responses were also captured including upregulation of pyochelin syntheses, LPS modification, sulfate starvation, exopolysaccharide-related genes, and downregulation of bacterial global regulators. These changes are associated with starvation-like conditions (iron and sulfate) and bacteria adaptation to the host, but its role in phage infection progression is still unknown. The study of its impact on bacterial virulence or phage efficient infectivity under human physiology is of most importance. This comprehensive study allows the comparison of bacterial and phage transcripts in the presence of host cells, contributing to a better understanding of phage-bacteria-host interactions, which are relevant in a phage therapy context.info:eu-repo/semantics/publishedVersio

The phage-encoded protein PIT2 impacts Pseudomonas aeruginosa quorum sensing by direct interaction with LasR

Summary: In recent decades, there has been a notable increase in antibiotic-resistant Pseudomonas aeruginosa isolates, necessitating the development of innovative treatments to combat this pathogen. This manuscript explores the potential of different phage proteins to attenuate virulence factors of P. aeruginosa, particularly the type II secretion system (T2SS). PIT2, a protein derived from the lytic Pseudomonas phage LMA2 inhibits the T2SS effectors PrpL and LasA and attenuates the bacterial virulence toward HeLa cells and Galleria mellonella. Using RNAseq-based differential gene expression analysis, PIT2’s impact on the LasR regulatory network is revealed, which plays a key role in bacterial quorum sensing. This discovery expands our knowledge on phage-encoded modulators of the bacterial metabolism and offers a promising anti-virulence target in P. aeruginosa. As such, it lays the foundation for a new phage-inspired anti-virulence strategy to combat multidrug resistant pathogens and opens the door for SynBio applications

Impact of phage predation on P. aeruginosa adhered to human airway epithelium: major transcriptomic changes in metabolism and virulence-associated genes

Phage therapy is a promising adjunct therapeutic approach against bacterial multidrug-resistant infections, including Pseudomonas aeruginosa-derived infections. Nevertheless, the current knowledge about the phage-bacteria interaction within a human environment is limited. In this work, we performed a transcriptome analysis of phage-infected P. aeruginosa adhered to a human epithelium (Nuli-1 ATCC® CRL-4011). To this end, we performed RNA-sequencing from a complex mixture comprising phagebacteriahuman cells at early, middle, and late infection and compared it to uninfected adhered bacteria. Overall, we demonstrated that phage genome transcription is unaltered by bacterial growth and phage employs a core strategy of predation through upregulation of prophage-associated genes, a shutdown of bacterial surface receptors, and motility inhibition. In addition, specific responses were captured under lung-simulating conditions, with the expression of genes related to spermidine syntheses, sulphate acquisition, biofilm formation (both alginate and polysaccharide syntheses), lipopolysaccharide (LPS) modification, pyochelin expression, and downregulation of virulence regulators. These responses should be carefully studied in detail to better discern phage-induced changes from bacterial responses against phage. Our results establish the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, being obvious the phage versatility on bacterial cell invasion.The work was supported by the Fundação para a Ciência e a Tecnologia [SFRH/BD/133193/2017]; Fundação para a Ciência e a Tecnologia [UIDB/04469/2020]; Fundação para a Ciência e a Tecnologia [LA/P/ 0029/2020]; H2020 European Research Council [819800]; H2020 European Research Council [[819800]]; Portuguese Foundation for Science and Technology (FCT) [EXPL/EMD-EMD/1142/2021]; “la Caixa” Foundation [LCF/PR/HP21/52310017].info:eu-repo/semantics/publishedVersio