Pseudomonas aeruginosa is a versatile Gram-negative pathogen with intricate intracellular regulatory networks that enable it to adapt to and flourish in a variety of biotic and abiotic habitats. However, the mechanism permitting the persistent survival of P. aeruginosa within host tissues and causing chronic symptoms still remains largely elusive. By using in situ RNA sequencing, here we show that P. aeruginosa adopts different metabolic pathways and virulence repertoires to dominate the progression of acute and chronic lung infections. Notably, a virulence factor named TesG, which is controlled by the vital quorum-sensing system and secreted by the downstream type I secretion system, can suppress the host inflammatory response and facilitate the development of chronic lung infection. Mechanically, TesG can enter the intracellular compartment of macrophages through clathrin-mediated endocytosis, competitively inhibit the activity of eukaryotic small GTPase and thus suppress subsequent neutrophil influx, cell cytoskeletal rearrangement of macrophages and the secretion of cytokines and chemokines. Therefore, the identification of TesG in this study reveals a type I secretion apparatus of P. aeruginosa that functions during the host–pathogen interaction, and may open an avenue for the further mechanistic study of chronic respiratory diseases and the development of antibacterial therapy.
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Large screening datasets are provided in the Supplementary Information. The RNA-Seq data for P. aeruginosa PAO1 are deposited in the NCBI database under accession numbers SRX2662725, SRX2662726, SRX2662727 and SRX2662728. Additional data that support the findings of this study are available from the corresponding author X. Zhou upon request.
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Stover, C. K. et al. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964 (2000).
Ventre, I. et al. Multiple sensors control reciprocal expression of Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc. Natl Acad. Sci. USA 103, 171–176 (2006).
Mathee, K. et al. Dynamics of Pseudomonas aeruginosa genome evolution. Proc. Natl Acad. Sci. USA 105, 3100–3105 (2008).
Lyczak, J. B., Cannon, C. L. & Pier, G. B. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect. 2, 1051–1060 (2000).
Yonker, L. M., Cigana, C., Hurley, B. P. & Bragonzi, A. Host–pathogen interplay in the respiratory environment of cystic fibrosis. J. Cyst. Fibros. 14, 431–439 (2015).
Balasubramanian, D., Schneper, L., Kumari, H. & Mathee, K. A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence. Nucleic Acids Res. 41, 1–20 (2013).
Hauser, A. R. The type III secretion system of Pseudomonas aeruginosa: infection by injection. Nat. Rev. Microbiol. 7, 654–665 (2009).
Dietsche, T. et al. Structural and functional characterization of the bacterial type III secretion export apparatus. PLoS Pathog. 12, e1006071 (2016).
Mougous, J. D. et al. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312, 1526–1530 (2006).
Hood, R. D. et al. A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7, 25–37 (2010).
Jiang, F., Waterfield, N. R., Yang, J., Yang, G. & Jin, Q. A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells. Cell Host Microbe 15, 600–610 (2014).
Goodman, A. L. et al. A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa. Dev. Cell 7, 745–754 (2004).
Allsoppc, L. P. et al. RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 114, 7707–7712 (2017).
Pessi, G. et al. The global posttranscriptional regulator RsmA modulates production of virulence determinants and N-acylhomoserine lactones in Pseudomonas aeruginosa. J. Bacteriol. 183, 6676–6683 (2001).
Schuster, M., Lostroh, C. P., Ogi, T. & Greenberg, E. P. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J. Bacteriol. 185, 2066–2079 (2003).
Lesic, B., Starkey, M., He, J., Hazan, R. & Rahme, L. G. Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis. Microbiology 155, 2845–2855 (2009).
Rieber, N., Hector, A., Carevic, M. & Hartl, D. Current concepts of immune dysregulation in cystic fibrosis. Int. J. Biochem. Cell. Biol. 52, 108–112 (2014).
Facchini, M., De Fino, I., Riva, C. & Bragonzi, A. Long term chronic Pseudomonas aeruginosa airway infection in mice. J. Vis. Exp. 85, e51019 (2014).
Avican, K. et al. Reprogramming of Yersinia from virulent to persistent mode revealed by complex in vivo RNA-seq analysis. PLoS Pathog. 11, e1004600 (2015).
Son, M. S., Matthews, W. J. Jr, Kang, Y., Nguyen, D. T. & Hoang, T. T. In vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the lungs of cystic fibrosis patients. Infect. Immun. 75, 5313–5324 (2007).
Goo, E., An, J. H., Kang, Y. & Hwang, I. Control of bacterial metabolism by quorum sensing. Trends Microbiol. 23, 567–576 (2015).
Duong, F., Lazdunski, A., Cami, B. & Murgier, M. Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways. Gene 121, 47–54 (1992).
Duong, F. et al. The AprX protein of Pseudomonas aeruginosa: a new substrate for the Apr type I secretion system. Gene 262, 147–153 (2001).
Filloux, A. Protein secretion systems in Pseudomonas aeruginosa: an essay on diversity, evolution, and function. Front. Microbiol. 2, 155 (2011).
Gilson, L., Mahanty, H. K. & Kolter, R. Four plasmid genes are required for colicin V synthesis, export, and immunity. J. Bacteriol. 169, 2466–2470 (1987).
Gilson, L., Mahanty, H. K. & Kolter, R. Genetic analysis of an MDR-like export system: the secretion of colicin V. EMBO J. 9, 3875–3884 (1990).
Guzzo, J., Pages, J. M., Duong, F., Lazdunski, A. & Murgier, M. Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism. J. Bacteriol. 173, 5290–5297 (1991).
Schuster, M. & Greenberg, E. P. Early activation of quorum sensing in Pseudomonas aeruginosa reveals the architecture of a complex regulon. BMC Genomics 8, 287 (2007).
Zhou, X. et al. MicroRNA-302b augments host defense to bacteria by regulating inflammatory responses via feedback to TLR/IRAK4 circuits. Nat. Commun. 5, 3619 (2014).
Balamayooran, G., Batra, S., Fessler, M. B., Happel, K. I. & Jeyaseelan, S. Mechanisms of neutrophil accumulation in the lungs against bacteria. Am. J. Respir. Cell Mol. Biol. 43, 5–16 (2010).
Weiss, G. & Schaible, U. E. Macrophage defense mechanisms against intracellular bacteria. Immunol. Rev. 264, 182–203 (2015).
Souza, S. T. et al. Macrophage adhesion on fibronectin evokes an increase in the elastic property of the cell membrane and cytoskeleton: an atomic force microscopy study. Eur. Biophys. J. 43, 573–579 (2014).
Li, X. et al. Lyn delivers bacteria to lysosomes for eradication through TLR2-initiated autophagy related phagocytosis. PLoS Pathog. 12, e1005363 (2016).
May, R. C. & Machesky, L. M. Phagocytosis and the actin cytoskeleton. J. Cell Sci. 114, 1061–1077 (2001).
Cromm, P. M. et al. Direct modulation of small GTPase activity and function. Angew. Chem. Int. Ed. Engl. 54, 13516–13537 (2015).
Aktories, K. Bacterial protein toxins that modify host regulatory GTPases. Nat. Rev. Microbiol. 9, 487–498 (2011).
Kristelly, R., Gao, G. & Tesmer, J. J. Structural determinants of RhoA binding and nucleotide exchange in leukemia-associated Rho guanine-nucleotide exchange factor. J. Biol. Chem. 279, 47352–47362 (2004).
Chen, Z. et al. Activated RhoA binds to the pleckstrin homology (PH) domain of PDZ-RhoGEF, a potential site for autoregulation. J. Biol. Chem. 285, 21070–21081 (2010).
Matsumura, F. & Hartshorne, D. J. Myosin phosphatase target subunit: many roles in cell function. Biochem. Biophys. Res. Commun. 369, 149–156 (2008).
Liu, J. & Lin, A. Role of JNK activation in apoptosis: a double-edged sword. Cell Res. 15, 36–42 (2005).
Marinissen, M. J. et al. The small GTP-binding protein RhoA regulates c-jun by a ROCK-JNK signaling axis. Mol. Cell 14, 29–41 (2004).
Yang, L. et al. Evolutionary dynamics of bacteria in a human host environment. Proc. Natl Acad. Sci. USA 108, 7481–7486 (2011).
Damkiaer, S., Yang, L., Molin, S. & Jelsbak, L. Evolutionary remodeling of global regulatory networks during long-term bacterial adaptation to human hosts. Proc. Natl Acad. Sci. USA 110, 7766–7771 (2013).
Jiricny, N. et al. Loss of social behaviours in populations of Pseudomonas aeruginosa infecting lungs of patients with cystic fibrosis. PLoS ONE 9, e83124 (2014).
Latifi, A., Foglino, M., Tanaka, K., Williams, P. & Lazdunski, A. A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol. Microbiol. 21, 1137–1146 (1996).
Wu, H. et al. Pseudomonas aeruginosa mutations in lasI and rhlI quorum sensing systems result in milder chronic lung infection. Microbiology 147, 1105–1113 (2001).
Schoehn, G. et al. Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas. EMBO J. 22, 4957–4967 (2003).
Ballister, E. R., Lai, A. H., Zuckermann, R. N., Cheng, Y. & Mougous, J. D. In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc. Natl Acad. Sci. USA 105, 3733–3738 (2008).
Hachani, A. et al. Type VI secretion system in Pseudomonas aeruginosa: secretion and multimerization of VgrG proteins. J. Biol. Chem. 286, 12317–12327 (2011).
Lemichez, E. & Aktories, K. Hijacking of Rho GTPases during bacterial infection. Exp. Cell Res. 319, 2329–2336 (2013).
Klesney-Tait, J. et al. Transepithelial migration of neutrophils into the lung requires TREM-1. J. Clin. Invest. 123, 138–149 (2013).
Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).
Windgassen, M., Urban, A. & Jaeger, K. E. Rapid gene inactivation in Pseudomonas aeruginosa. FEMS Microbiol. Lett. 193, 201–205 (2000).
Zhou, X. et al. Transient receptor potential channel 1 deficiency impairs host defense and proinflammatory responses to bacterial infection by regulating protein kinase Calpha signaling. Mol. Cell. Biol. 35, 2729–2739 (2015).
Huang, D. W., Sherman, B. T. & Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009).
This work is supported by the National Natural Science Foundation of China (no. 31700111 to K.Z., no. 81302371 and no. 81672675 to Jing Li, no. 81202324 to X. Zhou and no. 31570534 to X. Zhang), the Excellent Young Scientist Foundation of Sichuan University (no. 2017SCU04A16 to X. Zhou), the Innovative Spark Foundation of Sichuan University (no. 2018SCUH0032 to X. Zhou) and the National Major Scientific and Technological Special Project for ‘Significant New Drugs Development’ (no. 2018ZX09201018-013 to X. Zhou).
Supplementary Figures 1–14, Supplementary Table 1, Supplementary Table 5, Supplementary References, uncropped blots.
Significantly upregulated genes of PAO1 during acute lung infection.
Significantly upregulated genes of PAO1 during chronic lung infection.
Significance ranking of upregulated operons of PAO1 during lung infections.
Mass spectrometry analysis of proteins included exclusively in TesG group.
Small GTPase superfamily proteins included exclusively in TesG group.
Strains, plasmids and primers used in this study.
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Nature Microbiology (2019)