The process of treatment of four groups of data for GO, pathway and gene network analysis.

<p>(a) The differentially expressed genes between control A/J and control B6 mice were eliminated from those between SS2-infected A/J and SS2-infected B6 mice. (b) The remain of differential genes between SS2-infected A/J and SS2-infected B6 were intersected with differentially expressed genes between SS2-infected A/J and control A/J mice. (c) The remaining set of differentially expressed genes were analyzed for inclusion in GO categories and pathways. The same process was carried out with the differentially expressed genes between SS2-infected B6 and control B6 mice.</p

Degree of key genes in gene network of SS2-infected A/J mice.

<p>Degree of key genes in gene network of SS2-infected A/J mice.</p

Confirmation of BeadChips results by qRT-PCR.

<p>Confirmation of BeadChips results by qRT-PCR.</p

Three Hcp homologs with divergent extended loop regions exhibit different functions in avian pathogenic <i>Escherichia coli</i>

Type VI secretion systems (T6SSs) contribute to the pathogenicity of avian pathogenic Escherichia coli (APEC), one of the leading causative agents of sepsis and meningitis in poultry. The Hcp protein is a core component of the T6SS tail tube and acts as an exported receptor and a chaperone of effectors. In this study, four distinct Hcp types (Ia, Ib, IIa, and IIb) were designated in Gram-negative bacteria, three of which were widely distributed in APEC. We detected divergence in transcription levels among three hcp clusters in 50% duck serum and demonstrated that hcp1 was upregulated by relieving Fur repression. Further analyses revealed that the host serum could activate the hcp2B operon by H-NS derepression to transcribe the downstream xmtU/xmtV pair for inter-bacterial antagonism. Notably, in a structural analysis based on the genetic classification, Hcp proteins exhibited significant differences in the extended loop regions, suggesting that these regions were related to their functional properties. Indeed, the variant region Vs2 (Loop L2, 3) in Hcp1 and Hcp2B was essential for the delivery of antibacterial effectors and the inhibition of macrophage phagocytosis. Further analyses using a duck model indicated that these Hcps play different roles in the pathogenic processes of APEC and immunoprotection. These results indicated that the functional differentiation of Hcp homologs was driven by differences in transcriptional regulation, extended loop regions, and effector delivery.</p

KEGG pathway analysis for significantly differentially expressed genes (A) between SS2-infected A/J and control A/J mice and (B) between SS2-infected B6 and control B6 mice.

<p><i>P</i> value<0.05 and FDR<0.05 were used as thresholds to select significant KEGG pathways. LgP is the base 10 logarithm of the <i>P</i> value.</p

LD50 of strain HA9801 on A/J mouse.

<p>LD50 of strain HA9801 on A/J mouse.</p

The excision frequency (A) and extrachromosomal copy number (B) of ICE<i>Ssu</i>HN105 in ΔOriT-attL were determined by qPCR.

All experiments were conducted independently three times. Unpaired two-tailed Student’s t-test: **** P (TIF)</p

The AbiEi antitoxin has a negative regulatory effect on SezAT transcription by directly binding to SezAT’s promoter.

(A) Sequence similarity analysis of AbiE promoter IR1 and IR2 with the SezAT promoter IR1 and IR2. (B) pTCV-lac-PAbiE was integrated to HN105 and ΔSezAT host to determine promoter activity. (C) The EMSA result shown that SezA does not bind to the AbiE promoter. The SezAT promoter served as positive control. (D) pTCV-lac-PSezAT was integrated to HN105 and ΔAbiE host to determine promoter activity. (E) EMSA result shown that AbiEi binds to the SezAT promoter. The AbiE promoter fragment served as positive control. (F) ChIP analysis was performed to detect the binding of AbiEi to SezAT promoter. The Anti-SezA antibody served as a positive control. Normal mouse serum was used as a negative control. The 16S rRNA gene PCR product was used as a negative control. (G) Transcript levels of SezA and SezT in ΔAbiEi and ΔAbiEii. All experiments were conducted independently three times. Unpaired two-tailed Student’s t-test: ns P > 0.05; *** P (TIF)</p

Epifluorescence microscopy analysis of the cryosections of the tissues obtained from the mice infected with Egfp-HA9801 or HA9801.

<p>The cryosections of liver (A-1), lung (B-1), kidney (C-1), spleen (D-1) and brain (E-1) from the Egfp-HA9801-infected mice were detected under epifluorescent microscope and the FITC images are shown in the left column. The FITC images of the cryosections of liver (A-2), lung (B-2), kidney (C-2), spleen (D-2) and brain (E-2) from the HA9801-infected mice, are showed in the right column.</p

Growth curve of Egfp-HA9801 and parent HA9801.

<p>Both Egfp-HA9801 and HA9801 were incubated in 100 ml of fresh THB at 37°C for 18 h. During the 18 h period, aliquots of 2 ml of cultures were used to monitor the bacterial concentration every hour. No significant differences were found between the two strains throughout the experiment (<i>P</i>>0.05).</p