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Serum resistance is a poorly understood but common trait of some systemic disease pathogenic strains of bacteria. In this study, we analysed the role Escherichia coli type three secretion system 2 (ETT2) of avian pathogenic Escherichia coli (APEC) in serum resistance by bacteria survival number in serum culture, mRNA Seq and Tandem Mass Tag™ (TMT™) detection, lipopolysaccharide (LPS) extraction, and biofilm formation detection. We found that the ETT2 gene cluster deletion strain (ΔETT2) is more resistant to the killing effect of serum than wild type APEC40. The analysis of ΔETT2 compared to APEC40 in the transcriptomics and proteomics data showed that ETT2 has a negative effect in the ATP-binding cassette (ABC) translator system and quorum sensing system and a positive effect in purine metabolism. ETT2 may affect the LPS, biofilm, flagella, and fimbriae which may affect the serum resistance. These results could lead to effective strategies for managing the infection of APEC. The mRNA Seq data of this study are available in the Sequence Read Archive of the National Center for Biotechnology Information under the BioProject PRJNA757182, and proteomic raw data have been deposited under the accession number IPX0003420000 at iProX.

Avian pathogenic Escherichia coli (APEC) is an important pathogen that causes avian colibacillosis in poultry. APEC infection can lead to pathological changes in chicken trachea. The type VI secretion system (T6SS) of APEC contribute to the pathogenicity of APEC. However, whether T6SS plays a role in infection of the trachea remains unclear. We constructed mutant strain Δhcp2a by the Red recombination method system. The role of hcp2a (the structural secretion components and secretory protein of the T6SS) in the infection of trachea was investigated. The mutation strain displayed a significant increase in biofilm formation and a decrease in resistance to chicken serum. Moreover, RNA sequencing analyses showed that infection of chicken tracheal epithelium by the mutant strain Δhcp2a induced differential expression of genes. The result also showed that 14 genes (13 genes were downregulated) were enriched in cytokine-cytokine receptor interaction signalling pathway at 12 and 24 h post infection. The mutation Δhcp2a resulted in significant decreases in the bacterial loads in trachea at 6 and 12 h post infection. Real-time PCR analyses showed that the hcp2a mutation downregulated the expression of IL8 and IL1β at mRNA level in chicken tracheal epithelium. Our results indicate that mutation of hcp2a influenced genes expression of the cytokine-cytokine receptor interaction pathway by decreasing APEC colonization in the trachea.

Avian pathogenic Escherichia coli (APEC) can cause the acute and sudden death of poultry, which leads to serious economic losses in the poultry industry. Biofilm formation contributes to the persistence of bacterial infection, drug resistance, and resistance to diverse environmental stress. Many transcription regulators in APEC play an essential role in the formation of biofilm and could provide further insights into APEC pathogenesis. YjjQ has an important role in the pathogenicity of bacteria by regulating the expression of virulence factors, such as flagellar and iron uptake. However, YjjQ regulates other virulence factors, and their role in the overall regulatory network is unclear. Here, we further evaluate the function of YjjQ on APEC biofilm formation and motility. In this study, we successfully constructed mutant (AE27∆yjjQ) and complement (AE27ΔyjjQ-comp) strains of the wild-type strain AE27. Inactivation of the yjjQ gene significantly increased biofilm-forming ability in APEC. Scanning electron microscopy showed that the biofilm formation of the AE27 was single-layered and flat, whereas that of the AE27∆yjjQ had a porous three-dimensional structure. Moreover, the deletion of the yjjQ gene inhibited the motility of APEC. RNA-sequencing was used to further investigate the regulatory mechanism of YjjQ in APEC. The results indicate that YjjQ regulates biofilm formation and flagellar genes in AE27∆yjjQ. RT-qPCR shows that YjjQ affects the transcriptional levels of genes, including flagella genes (flhD, flhC and flgE), and biofilm formation genes (pstA, uhpC, nikD, and ygcS). These results confirm that the transcription regulator YjjQ is involved in APEC biofilm formation and motility, and provide new evidence for the prevention and control of APEC.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR (HiTAIL-PCR). The reported genes including 3 flagella genes (fliS, fliD, fliR), 4 curli fimbriae genes (csgD, csgA, csgF, csgG) and 3 type 1 fimbriae genes (fimA, fimD, fimC). The novel genes including 3 coenzyme genes (gltA, bglX, mltF) and 4 putative protein genes (yheE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted on APEC strain APEC81. The result shown that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P csgD, csgA, csgF, csgG) and other functional genes (fimC, glxK, yehE, 07045, 11255) affected the colony morphology. Particularly, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P gltA, bglX, mltF, yheE, 07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit for further study mechanism of biofilm regulation in APEC.

Avian colibacillosis is a serious systemic infectious disease in poultry and caused by avian pathogenic Escherichia coli (APEC). Previous studies have shown that 2-component systems (TCSs) are involved in the pathogenicity of APEC. OmpR, a response regulator of OmpR/EnvZ TCS, plays an important role in E. coli K-12. However, whether OmpR correlates with APEC pathogenesis has not been established. In this study, we constructed an ompR gene mutant and complement strains by using the CRISPR-Cas9 system and found that the inactivation of the ompR gene attenuated bacterial motility, biofilm formation, and the production of curli. The resistance to environmental stress, serum sensitivity, adhesion, and invasion of DF-1 cells, and pathogenicity in chicks were all significantly reduced in the mutant strain AE17ΔompR. These phenotypes were restored in the complement strain AE17C-ompR. The qRT-PCR results showed that OmpR influences the expression of genes associated with the flagellum, biofilm formation, and virulence. These findings indicate that the regulator OmpR contributes to APEC pathogenicity by affecting the expression and function of virulence factors.

Avian pathogenic Escherichia coli (APEC) is a pathotype of extraintestinal pathogenic E. coli and one of the most serious infectious diseases of poultry. It not only causes great economic losses to the poultry industry, but also poses a serious threat to public health worldwide. Here, we examined the role of YqeH, a transcriptional regulator located at E. coli type III secretion system 2 (ETT2), in APEC pathogenesis. To investigate the effects of YqeH on APEC phenotype and virulence, we constructed a yqeH deletion mutant (APEC40-ΔyqeH) and a complemented strain (APEC40-CΔyqeH) of APEC40. Compared with the wild type (WT), the motility and biofilm formation of APEC40-ΔyqeH were significantly reduced. The yqeH mutant was highly attenuated in a chick infection model compared with WT, and showed severe defects in its adherence to and invasion of chicken embryo fibroblast DF-1 cells. However, the mechanisms underlying these phenomena were unclear. Therefore, we analyzed the transcriptional effects of the yqeH deletion to clarify the regulatory mechanisms of YqeH, and the role of YqeH in APEC virulence. The deletion of yqeH downregulated the transcript levels of several flagellum-, biofilm-, and virulence-related genes. Our results demonstrate that YqeH is involved in APEC pathogenesis, and the reduced virulence of APEC40-ΔyqeH may be related to its reduced motility and biofilm formation.

Serum resistance is a poorly understood but common trait of some systemic disease pathogenic strains of bacteria. In this study, we analysed the role

Mutation of the PhoP/Q two-component system decreases the expression of ybjX and pagP encoding outer membrane proteins, and mutation of ybjX or pagP attenuates avian pathogenic Escherichia coli (APEC) pathogenicity. However, whether ybjX/pagP mutation (double-deletion mutant) has a synergistic effect on pathogenicity remains unknown. Herein, electrophoresis mobility shift assay (EMSA) experiments showed that the PhoP/Q system regulated ybjX and pagP transcription indirectly. The APECΔybjX/pagP mutant strain, constructed using the Red recombination method, exhibited reduced invasion of chicken embryo fibroblast (DF-1) cells, but had no effect on virulence in a chicken model. Using RNA sequencing to identify differential mRNAs in APECΔybjXΔpagP and native strains, we revealed up-regulation of genes involved in the bacterial chemotaxis pathway. The ybjX/pagP mutant strain displayed significantly increased motility, suggesting that double deletion of ybjX and pagP enhances motility via the bacterial chemotaxis pathway.

The Escherichia coli type III secretion system 2 (ETT2) is found in most pathogenic E. coli strains. Although many ETT2 gene clusters carry multiple genetic mutations or deletions, ETT2 is known to be involved in bacterial virulence. To date, no studies have been conducted on the role of ETT2 in the virulence of avian pathogenic Escherichia coli (APEC), which harbours ETT2. Thus, we deleted the ETT2 of APEC strain and evaluated the phenotypes and pathogenicities of the mutant. The results showed that deletion of ETT2 had no effect on APEC growth, but significantly promoted biofilm formation. In addition, as compared to the wild-type （WT） strain, the ETT2 deletion significantly promoted adherence to and invasion of DF-1 chicken fibroblasts and facilitated survival in the sera of specific-pathogen-free chickens. Analysis of the role of ETT2 in animal infection models demonstrated that the distribution of viable bacteria in the blood and organs of chicks infected with the ΔETT2 was significantly higher than those infected with WT. The results of RNA sequencing indicated that multiple genes involved in biofilm formation, lipopolysaccharide components, fimbrial genes and virulence effector proteins are regulated by ETT2. Collectively, these results implicated ETT2 is involved in the biofilm formation and pathogenicity of APEC.

The Escherichia coli type III secretion system 2 (ETT2) is present in most E. coli strains, carries a 29.9-kb ETT2 pathogenicity island (PAI) and is involved in the virulence of avian pathogenic Escherichia coli (APEC). A chaperone protein is essential for the bacterial secretion system, but the function of the ETT2 chaperone protein has not been determined. This study showed that ygeG had sequence homology with the identified bacterial chaperone protein and it possessed tetratri-copeptide repeats (TPR) containing protein. To investigate the role of ygeG in the ETT2 of APEC, ygeG mutant and complemented strains were constructed and characterized. Inactivation of ygeG had no effect on APEC growth, but significantly promoted biofilm formation, and the adherence to and invasion of DF-1 cells, especially the survival abilities in specific-pathogen-free (SPF) chicken sera serum. Analysis of the role of ygeG in chicken infection models revealed that the deletion of ygeG increased bacterial virulence. RNA Sequencing (RNA-Seq) analyses comparing the APEC wild type and the ygeG mutant indicated that multiple genes encoding biofilm formation, outer membrane proteins, fimbrial genes and virulence effector protein genes were regulated by ygeG. These results revealed the role of ygeG as a chaperone protein that affected the virulence and pathogenicity of APEC.

Antimicrobial resistance associated with colistin has emerged as a significant concern worldwide, threatening the use of one of the most important antimicrobials for treating human disease. This study aimed to investigate the prevalence of colistin-resistant avian-pathogenic Escherichia coli (APEC) and shed light on the possibility of transmission of mcr-1 (mobilized colistin resistance)-positive APEC. A total of 72 APEC isolates from Anhui Province in China were collected between March 2017 and December 2018 and screened for the mcr-1 gene. Antimicrobial susceptibility testing was performed using the broth dilution method. Pulsed-field gel electrophoresis, Southern blot analysis, and conjugation assay were performed to determine the location and conjugative ability of the mcr-1 gene. Whole-genome sequencing and analysis were performed using Illumina MiSeq and Nanopore MinION platforms. Three APEC isolates (AH25, AH62, and AH65) were found to be positive for the mcr-1 gene and showed multidrug resistance. The mcr-1 genes were located on IncI2 plasmids, and conjugation assays revealed that these plasmids were transferrable. Notably, strains AH62 and AH65, both belonging to ST1788, were collected from different places but carried the same drug resistance genes and shared highly similar plasmids. This study highlights the potential for a possible epidemic of mcr-1-positive APEC and the urgent need for continuous active monitoring. IMPORTANCE In this study, three plasmids carrying mcr-1 were isolated and characterized from APEC isolates from Anhui Province in China. The mcr-1 genes were located on IncI2 plasmids, and these plasmids were transferrable. These three IncI2 plasmids had high homology with the plasmids harbored by pathogenic bacteria isolated from other species. This finding showed that IncI2 plasmids poses a risk for the exchange of genetic material between different niches. Although colistin has been banned for use in food-producing animals in China, the coexistence of the broad-spectrum β-lactamase and mcr-1 genes on a plasmid can also lead to the stable existence of mcr-1 genes. The findings illustrated the need to improve the monitoring of drug resistance in poultry systems so as to curb the transmission or persistence of multidrug-resistant bacteria.

Antimicrobial resistance associated with colistin has emerged as a significant concern worldwide, threatening the use of one of the most important antimicrobials for treating human disease. This study aimed to investigate the prevalence of colistin-resistant avian-pathogenic Escherichia coli (APEC) and shed light on the possibility of transmission of mcr-1 (mobilized colistin resistance)-positive APEC. A total of 72 APEC isolates from Anhui Province in China were collected between March 2017 and December 2018 and screened for the mcr-1 gene. Antimicrobial susceptibility testing was performed using the broth dilution method. Pulsed-field gel electrophoresis, Southern blot analysis, and conjugation assay were performed to determine the location and conjugative ability of the mcr-1 gene. Whole-genome sequencing and analysis were performed using Illumina MiSeq and Nanopore MinION platforms. Three APEC isolates (AH25, AH62, and AH65) were found to be positive for the mcr-1 gene and showed multidrug resistance. The mcr-1 genes were located on IncI2 plasmids, and conjugation assays revealed that these plasmids were transferrable. Notably, strains AH62 and AH65, both belonging to ST1788, were collected from different places but carried the same drug resistance genes and shared highly similar plasmids. This study highlights the potential for a possible epidemic of mcr-1-positive APEC and the urgent need for continuous active monitoring.IMPORTANCE In this study, three plasmids carrying mcr-1 were isolated and characterized from APEC isolates from Anhui Province in China. The mcr-1 genes were located on IncI2 plasmids, and these plasmids were transferrable. These three IncI2 plasmids had high homology with the plasmids harbored by pathogenic bacteria isolated from other species. This finding showed that IncI2 plasmids poses a risk for the exchange of genetic material between different niches. Although colistin has been banned for use in food-producing animals in China, the coexistence of the broad-spectrum β-lactamase and mcr-1 genes on a plasmid can also lead to the stable existence of mcr-1 genes. The findings illustrated the need to improve the monitoring of drug resistance in poultry systems so as to curb the transmission or persistence of multidrug-resistant bacteria.

Avian pathogenic Escherichia coli (APEC) is the leading cause of systemic infections in poultry worldwide and has a hidden threat to public health. Escherichia coli type three secretion system 2 (ETT2), similar to the Salmonella pathogenicity island SPI1, is widely distributed in APEC and associated with virulence. The function of YqeI, which is one of the hypothetical transcriptional regulators locating at the ETT2 locus of APEC, is unknown. In this study, we successfully obtained the mutant strain AE81&Delta, yqeI of the wild type strain AE81 and performed the transcriptional profiling assays. Additionally, the transcriptional sequencing results revealed that YqeI influenced localization, locomotion and biological adhesion and so on. The transmission electron microscope observation showed that the wild type strain AE81 possessed long curved flagella, whereas the mutant strain AE81&Delta, yqeI hardly had any. The strain AE81&Delta, yqeI exhibited lower motility than AE81 after culturing the dilute bacterial suspension on a semisolid medium. It was also found that the survival ability of AE81&Delta, yqeI weakened significantly when AE81&Delta, yqeI was cultured with 0%, 10%, 20%, 30%, 40% and 50% SPF serum in PBS, and AE81&Delta, yqeI had decreased adherence to DF-1 cells compared with AE81 in the bacterial adhesion assay. The bacterial colonization assay indicated that the virulence of AE81&Delta, yqeI was reduced in the heart, liver, spleen, and lung. These results confirmed that the transcription regulator YqeI is involved in APEC&rsquo, s pathogenicity, and this study provides clues for future research.

Avian pathogenic Escherichia coli (APEC), a pathotype of extraintestinal pathogenic Escherichia coli (ExPEC), can cause serious systemic infectious diseases in poultry. Escherichia coli type III secretion system 2 (ETT2) is widely distributed in E. coli strains, including ExPEC and Enterohemorrhagic Escherichia coli (EHEC). The transcriptional regulator EivF, which is located at the ETT2 cluster, affects the secretion of LEE-encoded proteins and increases bacterial adhesion to human intestinal epithelial cells in EHEC O157:H7. In a previous study, we demonstrated the transcriptional regulator can affect APEC's motility and biofilm formation. Here, we evaluated whether EivF is involved in the pathogenicity of APEC, and we found that inactivation of eivF significantly enhanced resistance to the serum, adherence to chicken embryo fibroblast (DF-1) cells, and the colonization ability of APEC in chicks. To further clarify the regulation mechanism of transcriptional regulator EivF, we performed transcriptome sequencing to analyze the differentially expressed genes and pathways, showing that EivF regulates membrane, adhesion, environmental stress, and secretion protein genes, and EivF is involved in the localization, biological adhesion, biological regulation, membrane, and toxin activity. These findings indicated that the ETT2 transcriptional regulator EivF plays a crucial role in the pathogenicity of APEC as a negative repressor.

Two-component systems (TCSs) are widespread regulatory systems which can help bacteria to control their cellular functions and respond to a diverse range of stimuli. The KdpD/KdpE system had been well studied for regulating potassium transport and identified as an adaptive regulator involved in the virulence of some pathogenic bacteria, but its role in avian pathogenic Escherichia coli (APEC) was still unknown. In this study, the mutant strain AE17ΔKdpDE was obtained successfully of a clinical APEC isolation AE17 using the lambda Red recombinase system and performed the transcriptional sequencing of the wild type strain AE17 and the mutant strain AE17ΔKdpDE. The transcriptional sequencing results revealed that the KdpD/KdpE two-component system mainly influenced the expression of the genes covering metabolic pathways, flagellar assembly, global transcription regulator. The expression of some flagellar-related genes detecting by quantitative real-time PCR was consistent with the results of transcriptional sequencing. Importantly, fewer flagellum of the mutant strain AE17ΔKdpDE was observed than AE17 using the transmission electron microscope and a decreased motility circle of AE17ΔKdpDE appeared in the semisolid medium. In addition, the serum bactericidal assay was carried out with the specific-pathogen-free chicken in different dilution and the survival ability in the serum of AE17ΔKdpDE was also obviously lower than that of AE17. These results suggested that in APEC, the KdpD/KdpE two-component system mainly influenced the expression of flagella-related genes, the flagellum formation, the motility and antiserum bactericidal activity.

Avian pathogenic Escherichia coli (APEC) are important pathogens that cause serious economic losses to the poultry industry worldwide. The PhoP/Q system is essential for APEC pathogenicity, and its mutation leads to a decrease in ybjX gene expression, but whether ybjX regulated by PhoP/Q plays a role in pathogenicity remains unclear. The role of ybjX in APEC pathogenicity was investigated using a ybjX mutant strain constructed from APEC strain AE17 by the Red recombination method. The mutant strain displayed significantly reduced adherence to and invasion of chicken embryo fibroblast cell line (DF-1) cells. Moreover, N-phenyl-1-naphthylamine (NPN) assays revealed an increase in outer membrane permeabilization for the ybjX mutant, suggesting ybjX affected susceptibility to avian beta-defensin through this mechanism. The ybjX mutation increased susceptibility to avian beta-defensin 9 (AvBD9), and in vivo LD50 assays revealed reduced bacterial virulence in a chicken model. Taken together, these results indicated that mutation of ybjX may attenuate APEC pathogenicity, not only by affecting bacterial adhesion and invasion, but also via reducing resistance to avian beta-defensin by increasing outer membrane permeabilization of bacteria.

Objective To express prokaryotically the non-structural protein 1 (NS1) of duck Tembusu virus (DTMUV) and prepare NS1-specific polyclonal antibodies. Methods The NS1 gene of DTMUV strain AH-F10 was amplified by PCR, followed by subcloning and expression in the prokaryotic vector pET-32a. The recombinant NS1 protein was successfully expressed in Escherichia coli BL21 (DE3), and purified with hydroxymethyl urea and renatured by gradient centrifugation. The BALB/c mice were immunized with the purified recombinant NS1 protein to prepare polyclonal antibodies against the NS1 protein. Furthermore, the titer of the polyclonal antibodies was determined by agar diffusion test (AGP), and the specificity of the polyclonal antibodies was verified by Western blotting and indirect immunofluorescence assay (IFA). Results Polyclonal antibodies against NS1 protein in serum was successfully obtained with an AGP titer of 1:8. Western blotting and IFA demonstrated that the serum with polyclonal antibodies had a high-level specificity and reactivity to the NS1 protein of DTMUV. Conclusion Polyclonal antibodies against DTMUV NS1 protein were successfully prepared and validated in this study.