Transcriptome and proteome analysis of Pseudomonas aeruginosa during bacteriophage LUZ19 infection

Pseudomonas aeruginosa is an opportunistic pathogen, that has been a subject of interest in a number of transcriptomics and proteomics approaches, analyzing the effect of different growth conditions and stress on the organism. A yet understudied source of stress is the infection by bacterial viruses or phages. We analyzed the transcriptome and proteome of P. aeruginosa during bacteriophage infection, using DNA microarray and 2D-PAGE, respectively. Phage LUZ19 was selected for this purpose based on its high infection efficiency and constant generation time.The transcriptome analysis during infection showed that 220 genes were upregulated, whereas 131 genes were found to be downregulated. Among the strongest up- or downregulated genes were enzymes involved in the host energy metabolism, some of which belonging to the PhoPQ expression system. Moreover, a correlation was found with previous reported stress responses in P. aeruginosa. And finally, the downregulation of operons involved in LPS biosynthesis suggests a surface remodelling. After generating and validating a proteomic reference map of P. aeruginosa showing 1,200 protein spots, the host proteome was investigated during LUZ19 infection. Five bacteriophage proteins emerging at different time points could be identified, illustrating the regulation of phage expression.While no massive protein degradation or modification was observed, seven new host proteins were identified, of which 3 are involved in host stress responses, 2 correspond to ribosome-associated proteins and one has a role in energy conversion. Finally, this study revealed the modification of the α-subunit of the host RNA polymerase. Phage LUZ19 is the first phage described to use this specific strategy to inhibit host transcription.A conclusion to be made is that transcriptomic and proteomic investigations are indeed complementary and reveal different levels of regulation in an organism. Future challenges lie in the elucidation of specific influences, and in expanding this research to different phages and host organisms.status: publishe

A procedure for systematic identification of bacteriophage–host interactions of P. aeruginosa phages

AbstractImmediately after bacteriophage infection, phage early proteins establish optimal conditions for phage infection, often through a direct interaction with host-cell proteins. We implemented a yeast two-hybrid approach for Pseudomonas aeruginosa phages as a first step in the analysis of these – often uncharacterized – proteins. A 24-fold redundant prey library of P. aeruginosa PAO1 (7.32×106 independent clones), was screened against early proteins (gp1 to 9) of ϕKMV, a P. aeruginosa-infecting member of the Podoviridae; interactions were verified using an independent in vitro assay. None resembles previously known bacteriophage–host interactions, as the three identified target malate synthase G, a regulator of a secretion system and a regulator of nitrogen assimilation. Although at least two-bacteriophage infections are non-essential to ϕKMV infection, their disruption has an influence on infection efficiency. This methodology allows systematic analysis of phage proteins and is applicable as an interaction analysis tool for P. aeruginosa

A theoretical and experimental proteome map of Pseudomonas aeruginosa PAO1

A total proteome map of the Pseudomonas aeruginosa PAO1 proteome is presented, generated by a combination of two-dimensional gel electrophoresis and protein identification by mass spectrometry. In total, 1128 spotswere visualized, and 181 protein spotswere characterized, corresponding to 159 different protein entries. In particular, protein chaperones and enzymes important in energy conversion and amino acid biosynthesis were identified. Spot analysis always resulted in the identification of a single protein, suggesting sufficient spot resolution, although the same proteinmay be detected in two ormore neighboring spots, possibly indicating posttranslational modifications. Comparison to the theoretical proteome revealed an underrepresentation of membrane proteins, though the identified proteins cover all predicted subcellular localizations and all functional classes. These data provide a basis for subsequent comparative studies of the biology and metabolism of P. aeruginosa, aimed at unraveling global regulatory networks.status: publishe

Identification and comparative analysis of the structural proteomes of phiKZ and EL, two giant Pseudomonas aeruginosa bacteriophages

Giant bacteriophages phiKZ and EL of Pseudomonas aeruginosa contain 62 and 64 structural proteins, respectively, identified by ESI-MS/MS on total virion particle proteins. These identifications verify gene predictions and delineate the genomic regions dedicated to phage assembly and capsid formation (30 proteins were identified from a tailless phiKZ mutant). These data form the basis for future structural studies and provide insights into the relatedness of these large phages. The phiKZ structural proteome strongly correlates to that of Pseudomonas chlororaphis bacteriophage 201phi2-1. Phage EL is more distantly related, shown by its 26 non-conserved structural proteins and the presence of genomic inversions.status: publishe