Analysis of the PA3 Gene Dynamic Expression after Phage Infection.
According to the one-step growth curve of PaP3 and the dynamic infection cycle between phage and host, we examined the transcriptional changes at five time points after phage infection (5, 10, 20, 30, and 80 min) by using the RNA-seq analysis, and phage-uninfected host cells (0 min) were used as controls. These time points contain the entire cycle of phage infection in the host and thus fully represent host gene expression. The six sequenced reads covered between 70% and 100% of genes, with an average of 4,298 genes, which accounted for 80% of all reference genes.
Relative to the uninfected host, a total of 2,962 DEGs were obtained according to the gene expression analysis; among these DEGs 2,891 and 71 were PA3 and PaP3 genes, respectively. The 2,891 DEGs (fold change ≥2, P < 0.05), included 1,550 up-regulated and 1 368 down-regulated DEGs (Fig. 1). Moreover, most upregulated and downregulated genes are detected at 80 mins, when most cells are lysed and phages are released. Thus, the results from 80 mins might not be accurate.
Host phage regulation mainly occurred in the early logarithmic phase (10-20 min in this experiment). In the DEG analysis, 1,550 genes were upregulated, while 1,368 genes were downregulated. Analysis of the functions of these suppressed PA3 genes indicate that except for PA3 gene itself and a large proportion of undefined gene, the rest of the genes are mainly distributed in the transcriptional regulators (339/3863, 8.7%), amino acid synthesis and metabolism (185/3863, 4.7%), post-translational modification and degradation (166/3863, 4.3%), and energy metabolism (150/3863 3.8%) (Fig. 2). These results indicate that PaP3 exerts a general inhibitory effect on host genome transcription.
A total of 71 phage genes were clustered by hierarchical cluster analysis based on the result of RNA-Seq analysis. It gathered into 3 clusters of genes, including 12 early genes (cluster1, at 5-10 min), 21 late genes (cluster 2, at 10-20 min) and 38 middle genes (cluster3, at 30-80 min) (Fig. 3). The results are consistent with microarray analysis results (Zhao et al., 2016b).
RT-qPCR Validation of Selected DEGs.
To validate DGEs identified by RNA-seq, we selected nine DEGs for RT-qPCR (Table 1), including hfq, katA, dadA, flgK, groEL, flgL, lecA, rpoS, and rpoZ. Two Up-regulated genes involved stress reaction and Quorum sensing (like oxidation-reduction process(oxidation-reduction process)through the entire infection process. While seven down-regulated included host biosynthetic process, Virulence factors, transcription and translation regulators, and metabolism-related genes,occurs mainly in the middle and late stages of phage infection(30min, 80min). All RT-qPCR results show consistent directional changes compared with the RNA-seq results.
Table 1. RT-qPCR validation of the selected DEGs.
|
Expression multiple
|
Gene name
|
RNA-seq
|
RT-qPCR
|
5min
|
10min
|
20min
|
30min
|
80min
|
5min
|
10min
|
20min
|
30min
|
80min
|
hfq
|
-
|
0.322
|
0.204
|
0.332
|
0.105
|
-
|
0.448
|
0.222
|
0.018
|
0.020
|
katA
|
-
|
15.709
|
10.997
|
-
|
-
|
-
|
21.547
|
16.749
|
-
|
-
|
dadA
|
0.203
|
0.141
|
0.108
|
0.159
|
0.148
|
0.368
|
0.251
|
0.039
|
0.039
|
0.041
|
flgK
|
-
|
0.348
|
0.191
|
0.334
|
0.350
|
-
|
0.484
|
0.181
|
0.031
|
0.040
|
groEL
|
-
|
0.400
|
0.414
|
0.493
|
0.295
|
-
|
0.442
|
0.304
|
0.062
|
0.034
|
flgL
|
-
|
0.371
|
0.354
|
0.346
|
0.396
|
-
|
0.460
|
0.421
|
0.087
|
0.119
|
lecA
|
-
|
-
|
-
|
9.781
|
5.708
|
-
|
-
|
-
|
2.231
|
2.102
|
ropS
|
-
|
-
|
0.292
|
0.371
|
0.230
|
-
|
-
|
0.093
|
0.032
|
0.073
|
ropz
|
-
|
-
|
0.369
|
0.038
|
0.089
|
-
|
-
|
0.294
|
0.021
|
0.022
|
* 7 downregulated genes and 2 upregulated genes of P. aeruginosa were selected from the RNA-seq data set (P‹0.05). The genes expression of DEGs are indicated as Log2(fold change) values.
DEG KEGG Pathway Analysis in P. aeruginosa PA3 after Phage Infection.
KEGG pathway significant enrichment analysis can identify the most important biochemical metabolic and signal transduction pathways (Table 2). The above DEGs that may be involved in the metabolic pathway were analyzed by combining the KEGG pathway database. As compared to the control group, a total of 2,962 DEGs were observed after phage PaP3 infected the host, and a total of 25 metabolic pathways were identified. The genes involved in differential expression are mainly involved in metabolic pathways, biosynthesis of secondary metabolites, and two-component systems. Pathways with enriched differential genes were mainly observed in the 30- and 80-min samples, further indicating that the regulation of phage PaP3 to the host mainly occurred in the late stage. Interestingly,Analysis of pathways enriched with differential gene expression in the late stage of phage infection show that the Cationic antimicrobial peptide (CAMP) resistance was significantly inhibited. Also, the suppressed the metabolism paths of essential amino acids (such as valine, leucine, isoleucine, tryptophan, lysine, histidine, arginine, and beta alanine), which were critical to bacterial life. In the aliphatic acid metabolic pathways, most of the relevant DEGs showed suppressed expression by inhibiting the gene expression of intermediates catalytic enzymes in aliphatic acid metabolism. This pathway involved two suppressing genes, namely, synthesizing aliphatic acid coenzyme FadD1 and FadD2. Mutations of these two enzymes not only lead to synthesis reduction of lipase, protease, rhamnolipid, and phospholipase in P. aeruginosa but also reduce the use of carbon sources in the environment [19]. The mentioned carbon sources include aliphatic acids and choline phospholipids (lung surface-active substance). Furthermore, bacterial movement and group movement are inhibited. FadD1 and FadD2 are also related to the virulence of P. aeruginosa, and the expression of these two enzymes are detected in cystic fibrosis cases [20, 21].
Table 2. The KEGG pathway in P. aeruginosa PA3 after phage infection (percentage)
KEGG pathway
|
5min
|
10min
|
20min
|
30min
|
80min
|
ABC transporters
|
|
|
14.06%
|
19.79%
|
63.02%
|
Alanine, aspartate and glutamate metabolism
|
|
|
|
32.43%
|
75.68%
|
Biosynthesis of antibiotics
|
|
|
|
19.33%
|
58.40%
|
Biosynthesis of secondary metabolites
|
|
5.64%
|
|
18.40%
|
|
Biosynthesis of unsaturated fatty acids
|
|
|
|
46.15%
|
|
Carbon metabolism
|
10.16%
|
7.81%
|
17.19%
|
25.00%
|
|
Cationic antimicrobial peptide (CAMP) resistance
|
|
|
32.14%
|
32.14%
|
75.00%
|
Degradation of aromatic compounds
|
|
|
|
|
74.07%
|
Fatty acid biosynthesis
|
|
|
|
42.31%
|
76.92%
|
Fatty acid degradation
|
15.63%
|
|
|
|
|
Fatty acid metabolism
|
14.58%
|
|
|
31.25%
|
|
Folate biosynthesis
|
|
|
|
|
77.27%
|
Glycolysis / Gluconeogenesis
|
|
|
|
32.43%
|
70.27%
|
Glyoxylate and dicarboxylate metabolism
|
|
10.53%
|
|
|
|
Microbial metabolism in diverse environments
|
8.37%
|
6.08%
|
|
20.15%
|
60.46%
|
Monobactam biosynthesis
|
|
|
|
60.00%
|
|
Nitrogen metabolism
|
18.92%
|
18.92%
|
27.03%
|
29.73%
|
|
Oxidative phosphorylation
|
|
|
|
|
69.81%
|
Propanoate metabolism
|
|
12.82%
|
|
|
|
Pyruvate metabolism
|
|
|
|
26.67%
|
|
|
|
|
|
|
|
Ribosome
|
|
|
|
|
67.65%
|
Selenocompound metabolism
|
|
|
|
50.00%
|
|
Synthesis and degradation of ketone bodies
|
|
30.00%
|
|
|
|
Two-component system
|
|
|
21.31%
|
20.22%
|
|
Valine, leucine and isoleucine degradation
|
14.58%
|
|
|
|
|
Influence of Phage Infection on Host Virulence.
The VFBD database revealed 251 virulence-related genes annotated in the PAO1 strain genome and 115 genes differentially expressed after the bacteriophage PaP3 infected host PA3. Among these genes, 52 (45.2%) were downregulated, including most flagella genes involved in adhesion (adherence), type IV pili biosynthesis and twitching motility-related genes and Hcp secretion island-1 encoded type VI secretion system (H-T6SS)[22]. Further analysis showed that the transcription factors controlling these virulence genes also presented low expression levels, suggesting that the phage PaP3 can globally manipulate the expression level of the host strain PA3 transcriptome through transcription factors with broad regulation. The upregulated genes in virulence-related genes included those involved in Phenazines biosynthesis with antimicrobial activity and alginate biosynthesis with antiphagocytosis, as well as genes involved in rhamnolipid and pyoverdine biosynthesis. The upregulated virulence-related genes also include the biotypes of P. aeruginosa type Ⅲ secretion system from biosurfactants. These results suggest that the upregulated virulence genes may be involved in PA3 phage PaP3 immune resistance. In conclusion, phage infection may alter the virulence and resistance of hosts.
Comparison of the Host DEGs from RNA-seq and Microarray Platforms.
Globally, the numbers of DEGs in both microarray and RNA-seq analyses showed a considerable change in host transcription induced by phage. There were significant differences in the number of DEGs between microarray and RNA-seq. In RNA-seq there were 2,891 DEGs in the host (included 1,550 up-regulated genes and 1,368 down-regulated genes);in the microarray analysis, there were 3,037 DEGs in the host (included 109 up-regulated genes and 2,928 down-regulated genes), a total of 1,186 DEGs were overlapped. The results from the two methods showed certain heterogeneity. However, the enrichment time of DEGs was consistent, which was the middle and late stage of infection. To further estimate the correlation of these genes called by both methods, we compared the estimated log2-fold changes of the consensus genes. The resulting correlations were mapped as scatter plots, and absolute levels of correlated gene expression were estimated in terms of the correlation coefficient (r). The results showed Moderate positive correlation at each infection time point (Fig. 4). This comparison suggested that the absolute level of gene expression determined by both methods were correlated.
Interaction of PA3 and Phage PaP3 at the Proteome Level.
A total of 6 2D-GE maps were created using the samples from uninfected PaP3 cultures (0 min) and from infected PaP3 cultures at the time points of 5, 10, 20, 30, and 80 min. These maps were individually matched with a reference gel map of uninfected PA3 cultures (Fig. 5A). Degradation of the three host protein spots was identified during phage infection; these three host proteins include FlgL (PA1087), FlgK (PA1086), and GroEL (PA4385). FlgL and FlgK are both flagellar hook-associated proteins that rapidly disappear from the 2D-GE map upon infection by PaP3. This result confirms the significant inhibition of mechanisms related to motility and attachment according to the RNA-seq data. GroEL is a heat-shock protein belonging to the HSP60 family, which can only be detected on the uninfected map as FlgL and FlgK[23]. Meanwhile, two PaP3 proteins, spot 1 (ORF06) and 2 (ORF16), were differentially expressed on infection maps. A highly abundant ORF06 (putative scaffold protein) appeared after 5 min of infection and continued to accumulate until 80 min of phage infection. ORF16 is a hypothetical protein that was detected only on the infection map, which disappeared from the other maps, at 5 min. Furthermore, high sequence identity (E = 3e−109, identity = 62%) was observed between ORF16 and the phage particle protein of Pseudomonas phage TL by NCBI BlastP analysis. Both ORF06 and ORF16 were classified as phage structure proteins. Thus, the structural proteins of phage PaP3 were detected in the early phase of infection. The expression level of these proteins was listed (Fig. 5B and 5C). The trend is overall fit between RNA-seq and the proteomic content, except phage protein Orf16, which disappeared in the gel but was upregulated in the RNA-seq data after 10 min.