A systemic network for Chlamydia pneumoniae entry into human cells

Abstract

Bacterial entry is a multistep process triggering a complex network, yet the molecular complexity of this network remains largely unsolved. By employing a systems biology approach, we reveal a systemic bacterial-entry network initiated by Chlamydia pneumoniae, a widespread opportunistic pathogen. The network consists of nine functional modules (i.e., groups of proteins) associated with various cellular functions, including receptor systems, cell adhesion, transcription, and endocytosis. The peak levels of gene expression for these modules change rapidly during C. pneumoniae entry, with cell adhesion occurring at 5 min postinfection, receptor and actin activity at 25 min, and endocytosis at 2 h. A total of six membrane proteins (chemokine C-X-C motif receptor 7 [CXCR7], integrin beta 2 [ITGB2], platelet-derived growth factor beta polypeptide [PDGFB], vascular endothelial growth factor [VEGF], vascular cell adhesion molecule 1 [VCAM1], and GTP binding protein overexpressed in skeletal muscle [GEM]) play a key role during C. pneumoniae entry, but none alone is essential to prevent entry. The combination knockdown of three genes (coding for CXCR7, ITGB2, and PDGFB) significantly inhibits C. pneumoniae entry, but the entire network is resistant to the six-gene depletion, indicating a resilient network. Our results reveal a complex network for C. pneumoniae entry involving at least six key proteins.

FIG. 1.

Systemic network and modules involved in C. pneumoniae attachment/entry. Enhanced genes that were overlapped from C. pneumoniae attachment to entry (5 min, 25 min, and 2 h postinfection) were clustered into functional groups and combined into a network. This composition was treated as a C. pneumoniae entry network. Only the primary functions for each gene are indicated and colored in the index. Cellular components are shown on the left. (A) C. pneumoniae entry network, including the functional groups (highlighted). (B) Unique network activated at 2 h, in which genes are not overlapped with those upregulated at other time points.

FIG. 2.

Functional modules dynamically activated during C. pneumoniae entry. Average gene expression for each functional module altered by time (D_expression/D_t) was plotted against time, and results were clustered into three groups (A to C) on the basis of their patterns. (A) Attachment; (B) activation; (C) endocytosis.

FIG. 3.

Key proteins identified for C. pneumoniae entry. Key proteins in the network were predicted in silico, and then RNAi was used for knockdown of the predicted proteins. (A) Key proteins (network hubs) were predicted by using the percentage of alterations in the diameter, and the connectivity of the network individual hub was knocked out. (B) A total of six predicted hubs were successfully knocked down by RNAi, which significantly reduced C. pneumoniae infectivity. Data are averages of at least replicates of each cell line, as for Fig. 4.

FIG. 4.

Robustness of the C. pneumoniae entry network. (A) Gene combination knockout. The network diameter was dramatically changed after a 6-gene knockdown. (B) A combination of genes significantly reduced C. pneumoniae (CPN) infectivity. Knockdown of six genes almost entirely ablated C. pneumoniae infectivity, as measured by C. pneumoniae 16S rRNA expression using real-time qRT-PCR as described in Materials and Methods.