The Global Reciprocal Reprogramming between Mycobacteriophage SWU1 and Mycobacterium Reveals the Molecular Strategy of Subversion and Promotion of Phage Infection

Abstract

Bacteriophages are the viruses of bacteria, which have contributed extensively to our understanding of life and modern biology. The phage-mediated bacterial growth inhibition represents immense untapped source for novel antimicrobials. Insights into the interaction between mycobacteriophage and Mycobacterium host will inform better utilizing of mycobacteriophage. In this study, RNA sequencing technology (RNA-seq) was used to explore the global response of Mycobacterium smegmatis mc(2)155 at an early phase of infection with mycobacteriophage SWU1, key host metabolic processes of M. smegmatis mc(2)155 shut off by SWU1, and the responsible phage proteins. The results of RNA-seq were confirmed by Real-time PCR and functional assay. 1174 genes of M. smegmatis mc(2)155 (16.9% of the entire encoding capacity) were differentially regulated by phage infection. These genes belong to six functional categories: (i) signal transduction, (ii) cell energetics, (iii) cell wall biosynthesis, (iv) DNA, RNA, and protein biosynthesis, (v) iron uptake, (vi) central metabolism. The transcription patterns of phage SWU1 were also characterized. This study provided the first global glimpse of the reciprocal reprogramming between the mycobacteriophage and Mycobacterium host.

Keywords: interaction; ion channel; mycobacteriophage; mycobacterium; transcriptome.

Figure 1

Transcription of the SWU1 genome determined by RNA-seq. (A) Transcription patterns in the SWU1 genome determined by RNAseq. RNA was isolated during early SWU1 infection (green), middle SWU1 infection (blue), and late SWU1 infection (red). At all time points, one sample was taken for sequencing (Illumina HiSeqTM2000). The Y-axis is the number of RNAseq reads. The X-axis is the map of the SWU1 genome. (B) Cluster analysis of differentially expressed genes of SWU1. Based on log₁₀ (RPKM+1), we cluster the differentially expressed genes in different phase of SWU1 infection. Red stands for high expressed genes; blue stands for low expressed genes. In early phase of SWU1 infection, a lesser number of genes were transcribed. Most of genes transcribed were located on the right arm of SWU1 genome. In middle and late phase of SWU1 infection, most of genes transcribed were located on the left arm of SWU1 genome.

Figure 2

The scatter plot of KEGG pathways enriched for differential expression genes. Panel (A) shows up-regulated pathways. Panel (B) shows down-regulated pathways. The Y-axis is different KEGG pathway. The X-axis is the number of rich factor. The color of circles stands for the q-value of pathway. The range of q value is from 0 to 1. The size of circles stands for gene number. Some up-regulated genes associated to ribosome, protein export, bacterial secretion system, glycerophospholipid metabolism, and RNA degradation were enriched. Some down-regulated genes associated to siderophore non-ribosomal peptides and nitrotoluene degradation were enriched.

Figure 3

Effect of the SWU1 infection on sodium and ferrum fluxes. (A) Positive value stands for the concentration of ions increased after SWU1 infected 30 min. (B) Negative value stands for the concentration of ions decreased after SWU1 infected 30 min. The capacity of sodium efflux was suppressed after SWU1 infected. Iron uptake system was disrupted by SWU1. The data were averaged from two independent experiments ± s.d. Significant differences (^*p < 0.05).

Figure 4

Verification of RNA-seq results by real-time PCR. Numbers means the numbering of gene in M. smegmatis mc²155. The data were averaged from three independent experiments ± s.d. Significant differences (^***p < 0.001).

Figure 5

Putative working model of M. smegmatis mc²155 response to phage SWU1 infection. Numbers stand for host response to SWU1 infection, which is described in summary. Hollow-out wide arrows stand for those regulated genes. Dotted lines stand for the process of regulatory network. Forks represent that the process is inhibited.