Jianjun Dai

Effects of ibeA Deletion on Virulence and Biofilm Formation of Avian Pathogenic Escherichia coli

ABSTRACT The ibeA gene is located on a genomic island, GimA, which is involved in the pathogenesis of neonatal meningitis Escherichia coli (NMEC) and avian pathogenic E. coli (APEC). The prevalence of ibeA in the APEC collection in China was investigated, and 20 of 467 strains (4.3%) were positive. In addition, analysis of the association of the E. coli reference (ECOR) groups with positive strains revealed that ibeA was linked to group B2. The ibeA gene in DE205B was analyzed and compared to those of APEC and NMEC, which indicated that the specificity of ibeA was not consistent along pathotypes. The invasion of chicken embryo fibroblast DF-1 cells by APEC DE205B and RS218 was observed, which suggested that DF-1 cells could be a model to study the mechanism of APEC invasion. The inactivation of ibeA in APEC DE205B led to the reduced capacity to invade DF-1 cells, defective virulence in vivo, and decreased biofilm formation compared to the wild-type strain. In addition, strain AAEC189 expressing ibeA exhibited enhanced invasion capacity and biofilm formation. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the loss of ibeA decreased the colonization and proliferation capacities of APEC in the brain during system infection.

Suppression subtractive hybridization identifies an autotransporter adhesin gene of E. coli IMT5155 specifically associated with avian pathogenic Escherichia coli (APEC)

BackgroundExtraintestinal pathogenic E. coli (ExPEC) represent a phylogenetically diverse group of bacteria which are implicated in a large range of infections in humans and animals. Although subgroups of different ExPEC pathotypes, including uropathogenic, newborn meningitis causing, and avian pathogenic E. coli (APEC) share a number of virulence features, there still might be factors specifically contributing to the pathogenesis of a certain subset of strains or a distinct pathotype. Thus, we made use of suppression subtractive hybridization and compared APEC strain IMT5155 (O2:K1:H5; sequence type complex 95) with human uropathogenic E. coli strain CFT073 (O6:K2:H5; sequence type complex 73) to identify factors which may complete the currently existing model of APEC pathogenicity and further elucidate the position of this avian pathoype within the whole ExPEC group.ResultsTwenty-eight different genomic loci were identified, which are present in IMT5155 but not in CFT073. One of these loci contained a gene encoding a putative autotransporter adhesin. The open reading frame of the gene spans a 3,498 bp region leading to a putative 124-kDa adhesive protein. A specific antibody was raised against this protein and expression of the adhesin was shown under laboratory conditions. Adherence and adherence inhibition assays demonstrated a role for the corresponding protein in adhesion to DF-1 chicken fibroblasts. Sequence analyses revealed that the flanking regions of the chromosomally located gene contained sequences of mobile genetic elements, indicating a probable spread among different strains by horizontal gene transfer. In accordance with this hypothesis, the adhesin was found to be present not only in different phylogenetic groups of extraintestinal pathogenic but also of commensal E. coli strains, yielding a significant association with strains of avian origin.ConclusionsWe identified a chromosomally located autotransporter gene in a highly virulent APEC strain which confers increased adherence of a non-fimbriated E. coli K-12 strain to a chicken fibroblast cell line. Even though flanked by mobile genetic elements and three different genetic regions upstream of the gene, most probably indicating horizontal gene transfer events, the adhesin gene was significantly linked with strains of avian origin. Due to the nucleotide sequence similarity of 98% to a recently published adhesin-related gene, located on plasmid pAPEC-O1-ColBM, the name aatA (APEC autotransporter adhesin A) was adopted from that study.Our data substantiate that AatA might not only be of relevance in APEC pathogenicity but also in facilitating their reservoir life style in the chicken intestine, which might pave the way for future intestinal preventive strategies.

Comparative Genomic Analysis Shows That Avian Pathogenic Escherichia coli Isolate IMT5155 (O2:K1:H5; ST Complex 95, ST140) Shares Close Relationship with ST95 APEC O1:K1 and Human ExPEC O18:K1 Strains

Avian pathogenic E. coli and human extraintestinal pathogenic E. coli serotypes O1, O2 and O18 strains isolated from different hosts are generally located in phylogroup B2 and ST complex 95, and they share similar genetic characteristics and pathogenicity, with no or minimal host specificity. They are popular objects for the study of ExPEC genetic characteristics and pathogenesis in recent years. Here, we investigated the evolution and genetic blueprint of APEC pathotype by performing phylogenetic and comparative genome analysis of avian pathogenic E. coli strain IMT5155 (O2:K1:H5; ST complex 95, ST140) with other E. coli pathotypes. Phylogeny analyses indicated that IMT5155 has closest evolutionary relationship with APEC O1, IHE3034, and UTI89. Comparative genomic analysis showed that IMT5155 and APEC O1 shared significant genetic overlap/similarities with human ExPEC dominant O18:K1 strains (IHE3034 and UTI89). Furthermore, the unique PAI I5155 (GI-12) was identified and found to be conserved in APEC O2 serotype isolates. GI-7 and GI-16 encoding two typical T6SSs in IMT5155 might be useful markers for the identification of ExPEC dominant serotypes (O1, O2, and O18) strains. IMT5155 contained a ColV plasmid p1ColV5155, which defined the APEC pathotype. The distribution analysis of 10 sequenced ExPEC pan-genome virulence factors among 47 sequenced E. coli strains provided meaningful information for B2 APEC/ExPEC-specific virulence factors, including several adhesins, invasins, toxins, iron acquisition systems, and so on. The pathogenicity tests of IMT5155 and other APEC O1:K1 and O2:K1 serotypes strains (isolated in China) through four animal models showed that they were highly virulent for avian colisepticemia and able to cause septicemia and meningitis in neonatal rats, suggesting zoonotic potential of these APEC O1:K1 and O2:K1 isolates.

Novel roles for autotransporter adhesin AatA of avian pathogenic Escherichia coli: colonization during infection and cell aggregation.

Systemic infections in avian species caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. To unravel factors possibly involved in APEC pathogenicity, suppression subtractive hybridization was applied, leading to the identification of a putative APEC autotransporter adhesin gene aatA in our previous study. In this study, pathogenic mechanism of AatA was further determined. A deletion mutant of aatA was constructed in the APEC DE205B, which results in the reduced capacity to adhere to DF-1 cells, defective virulence in vivo, and decreased colonization capacity in lung during the systemic infection compared with the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatA makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, aggregation assays for strain AAEC189 expressing aatA indicated that AatA mediates cell aggregation and settling of cells. However, this cell aggregation is blocked by Type I fimbriae. This study illustrates the first examination of the role of AatA in aggregation and systemic infection.

DotU expression is highly induced during in vivo infection and responsible for virulence and Hcp1 secretion in avian pathogenic Escherichia coli

Type VI secretion systems (T6SSs) contribute to pathogenicity in many pathogenic bacteria. Three distinguishable T6SS loci have been discovered in avian pathogenic Escherichia coli (APEC). The sequence of APEC T6SS2 locus is highly similar to the sequence of the newborn meningitis Escherichia coli (NMEC) RS218 T6SS locus, which might contribute to meningitis pathogenesis. However, little is known about the function of APEC T6SS2. We showed that the APEC T6SS2 component organelle trafficking protein (DotU) could elicit antibodies in infected ducks, suggesting that DotU might be involved in APEC pathogenicity. To investigate DotU in APEC pathogenesis, mutant and complemented strains were constructed and characterized. Inactivation of the APEC dotU gene attenuated virulence in ducks, diminished resistance to normal duck serum, and reduced survival in macrophage cells and ducks. Furthermore, deletion of the dotU gene abolished hemolysin-coregulated protein (Hcp) 1 secretion, leading to decreased interleukin (IL)-6 and IL-8 gene expression in HD-11 chicken macrophages. These functions were restored for the complementation strain. Our results demonstrated that DotU plays key roles in the APEC pathogenesis, Hcp1 secretion, and intracellular host response modulation.

Characterization and Functional Analysis of AatB, a Novel Autotransporter Adhesin and Virulence Factor of Avian Pathogenic Escherichia coli

ABSTRACT Autotransporter (AT) proteins constitute a large family of extracellular proteins that contribute to bacterial virulence. A novel AT adhesin gene, aatB, was identified in avian pathogenic Escherichia coli (APEC) DE205B via genomic analyses. The open reading frame of aatB was 1,017 bp, encoding a putative 36.3-kDa protein which contained structural motifs characteristic for AT proteins: a signal peptide, a passenger domain, and a translocator domain. The predicted three-dimensional structure of AatB consisted of two distinct domains, the C-terminal β-barrel translocator domain and an N-terminal passenger domain. The prevalence analyses of aatB in APEC indicated that aatB was detected in 26.4% (72/273) of APEC strains and was strongly associated with phylogenetic groups D and B2. Quantitative real-time reverse transcription-PCR analyses revealed that AatB expression was increased during infection in vitro and in vivo. Moreover, AatB could elicit antibodies in infected ducks, suggesting that AatB is involved in APEC pathogenicity. Thus, APEC DE205B strains with a mutated aatB gene and mutated strains complemented with the aatB gene were constructed. Inactivation of aatB resulted in a reduced capacity to adhere to DF-1 cells, defective virulence capacity in vivo, and decreased colonization capacity in lung during systemic infection compared with the capacities of the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatB makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, biofilm formation assays with strain AAEC189 expressing AatB indicated that AatB mediates biofilm formation.

IbeB is involved in the invasion and pathogenicity of avian pathogenic Escherichia coli

The ibeB gene in neonatal meningitis Escherichia coli (NMEC) contribute to the penetration of human brain microvascular endothelial cells (HBMECs). However, whether IbeB plays a role in avian pathogenic E. coli (APEC) infection remains unclear. Thus, this study was conducted to investigate the distribution of the ibeB gene in Chinese APEC strains and examine whether IbeB is involved in APEC pathogenicity. The ibeB gene was found in all 100 detected E. coli isolates with over 97% sequence homology. These results indicated that ibeB is a conserved E. coli gene irrelevant of pathotypes. To determine the role of ibeB in APEC pathogenicity, an ibeB mutant of strain DE205B was constructed and characterized. The inactivation of ibeB resulted in reduced invasion capacity towards DF-1 cells and defective virulence in animal models as compared to the wild-type strain. Animal infection experiments revealed that loss of ibeB decreased APEC colonization and invasion capacity in brains and lungs. These virulence-related phenotypes were partially recoverable by genetic complementation. Reduced expression levels of invasion- and adhesion-associated genes in ibeB mutant could be major reasons as evidenced by reduced ibeA and ompA expression. These results indicate that IbeB is involved in APEC invasion and pathogenicity.

Development of an Allele-Specific PCR Assay for Simultaneous Sero-Typing of Avian Pathogenic Escherichia coli Predominant O1, O2, O18 and O78 Strains

Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. E. coli strains belonging to serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster controlling O antigen synthesis is usually various among different E. coli serotypes. In present study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared. Based on the serotype-specific genes in rfb gene cluster, an allele-specific polymerase chain reaction (PCR) assay was developed. This PCR assay was highly specific and reliable for sero-typing of APEC O1, O2, O18 and O78 strains. The sensitivity of the assay was determined as 10 pg DNA or 10 colony forming units (CFUs) bacteria for serotypes O2 and O18 strains, and 500 pg DNA or 1,000 CFUs bacteria for serotypes O1 and O78 strains. Using this PCR system, APEC isolates and the infected tissue samples were categorized successfully. Furthermore, it was able to differentiate the serotypes for the samples with multi-agglutination in the traditional serum agglutination assay. Therefore, the allele-specific PCR is more simple, rapid and accurate assay for APEC diagnosis, epidemiologic study and vaccine development.

Prophage lysin Ply30 protects mice from Streptococcus suis and Streptococcus equi subsp. zooepidemicus infections.

ABSTRACT Streptococcus suis and Streptococcus equi subsp. zooepidemicus are capable of infecting humans and various animals, causing significant problems for the worldwide swine industry. As antibiotic resistance has increased, lysosomal enzymes encoded by phages have shown potential for use against pathogenic bacteria. In this study, a novel bacteriophage lysin, Ply30, encoded by the S. suis prophage phi30c, was recombinantly expressed and purified. Ply30 showed high bacteriolysis activity on S. suis and S. equi subsp. zooepidemicus in vitro. The ratio of the optical density at 600 nm (OD600) with treatment versus the OD600 with no treatment for most tested S. suis and S. equi subsp. zooepidemicus strains decreased from 1 to <0.3 and <0.5, respectively, within 1 h. The results of plate viability assays showed that treated bacteria suffered a 1- to 2-log decrease in CFU within 1 h. The optimal concentration of Ply30 was 50 μg/ml, and the optimal pH was 7. Moreover, Ply30 maintained high activity over a wide pH range (pH 6 to 10). The MICs of Ply30 against Streptococcus strains ranged from 16 to 512 μg/ml. In vivo, a 2-mg dose of Ply30 protected 90% (9/10 mice) of mice from infection with S. equi subsp. zooepidemicus and 80% (8/10 mice) of mice from infection with S. suis. Seven days after lysin Ply30 treatment, bacterial loads were significantly decreased in all tested organs and blood compared with those at 1 h postinfection without Ply30 treatment. Ply30 showed in vitro and in vivo antimicrobial efficiency and protected mice against two kinds of bacterial infections, indicating that Ply30 may be an effective therapeutic against streptococci.

IbeR Facilitates Stress-Resistance, Invasion and Pathogenicity of Avian Pathogenic Escherichia coli

Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. IbeR, located on genomic island GimA, was shown to serve as an RpoS-like regulator in rpoS gene mutation neonatal meningitis E. coli (NMEC) RS218. However, the role of IbeR in pathogenicity of APEC carrying active RpoS has not yet been investigated. We showed that the APEC IbeR could elicit antibodies in infected ducks, suggesting that IbeR might be involved in APEC pathogenicity. To investigate the function of IbeR in APEC pathogenesis, mutant and complementation strains were constructed and characterized. Inactivation of ibeR led to attenuated virulence and reduced invasion capacity towards DF-1 cells, brains and cerebrospinal fluid (CSF) in vitro and in vivo. Bactericidal assays demonstrated that the mutant strain had impaired resistance to environmental stress and specific pathogen-free (SPF) chicken serum. These virulence-related phenotypes were restored by genetic complementation. Quantitative real-time reverse transcription PCR revealed that IbeR controlled expression of stress-resistance genes and virulence genes, which might led to the associated virulence phenotype.