Comparative genomic and mutational studies can be used to evaluate the role of suspected VAGs in APEC pathogenesis. Systemic E. coli from diseased birds have previously been compared to those from apparently healthy birds (often isolated from the intestinal microbiota) or to lab-attenuated strains. Genomic suppression subtractive hybridisation analysis has proven a valuable tool in allowing such comparisons to take place [139]. Suppression subtractive hybridisation has helped identify an array of potential APEC virulence genes involved in adhesion, invasion,
iron metabolism and plasmid-encoded genes in APEC strains, yet absent in non- pathogenic strains such as E. coli K-12 MG1655 [85, 134, 140-142].
Selective capture of transcribed sequences (SCOTS) allows for the identification of genes expressed by isolates taken from avian infected tissues [143]. SCOTS was used by Dozois et al. (2003) to identify essential APEC genes required during infection [144]. An advantage of SCOTS is that it does not require expansive knowledge of the genome in question.
Signature tagged transposon mutagenesis (STM) allows for large scale screening of mutant libraries to identify genes by function involved in pathogenesis [145]. Li et al. (2005) used STM to identify VAGs important in an APEC in vivo infection model which used an O2:H5 APEC (IMT5155); a series of extracellular polysaccharides and lipopolysaccharides were identified using this technique [47]. STM is perhaps one of the most powerful tools, as it allows for large-scale screening and the identification of unknown genes. Furthermore, mutants are directly linked with attenuation. A disadvantage of STM is the possibility of overlooking moderate attenuation of mutants, although this would be dependent on the sensitivity of the protocol. For example, toxins may not be required for survival in vivo but may be required for pathogenesis [146].
1.9.4 Adhesion
ExPEC are known to colonise the avian intestinal tract asymptomatically, accounting for as much as 20% of the E. coli population [24, 147]. The gastrointestinal E. coli population is a known reservoir of APEC strains [31]. APEC causes disease at various systemic sites, including the respiratory tract, liver, heart and reproductive
tract [48]. Colonisation is the first step in APEC pathogenesis and the bacteria are not thought to be particularly invasive pathogens[148].
Type 1 fimbriae have been shown to play a role in adhesion to a number of different sites including the intestinal mucus layer, enterocytes, the tracheal epithelia and lung tissue [149-151]. Type 1 fimbriae are encoded by the fim operon, a 9 gene cluster within the E. coli genome. Encoded within the fim operon is the fimH protein, which mediates mannose-sensitive binding while recombinases allow phase variation in fimbriae expression. Type 1 fimbriae have also been shown to contribute to UPEC pathogenesis [152].
E. coli are not thought to be particularly invasive [148]. Studies suggest translocation of the intestinal epithelium by APEC provides an alternative route for dissemination, but this only occurs when birds are predisposed to stress [129, 130]. A number of studies have identified ExPEC factors associated with epithelial invasion including Outer membrane protein A (OmpA), invasion barrier epithelia proteins (IbeA, B and C), fimbriae and temperature sensitive haemagglutinin (Tsh), but the exact mechanisms in many cases remain unknown [131, 149, 153-155].
Ibe proteins were first described in NMEC pathogenesis for their role in invasion of the blood-brain barrier [156, 157]. Johnson et al. (2001) estimated 33-44% of NMEC carry ibe genes [158]. Germon et al. (2005) reported 24% of APEC carry ibeA, compared to 0% of non-APEC strains tested, despite a negative correlation with O78 strains [159]. The exact role of Ibe proteins is not clear, some authors suggest ibeA encodes an extracellular protein capable of binding a 55KDa ibeA receptor (ibe10R) on bovine and human microvascular endothelial cells (BMECS/HMECS) [160]. However, Cortes et al. (2008) later proposed that ibeA may in fact encode a
cytoplasmic protein with enzymatic activity as no signal secretion sequence has been identified yet a putative flavin adenine dinucleotide binding domain has been found [154]. Cortes suggests that ibeA regulates type 1 fimbriae expression and thus indirectly contributes to the adhesion-invasive properties of ExPEC. APEC ibeA- mutants have been shown to have decreased biofilm formation potential and decreased invasiveness and virulence in both in vitro BMEC, chicken embryo DF-1 cell models and in vivo using 3-week old chickens [157, 159, 161, 162].
Autotransporter proteins are a distinct family of secreted proteins of Gram-negative bacteria. They possess an overall unifying structure composing of an N-terminal signal sequence, a C-terminal outer-membrane pore-forming translocator domain and a passenger (secreted protein) domain. The first autotransporter to be identified in APEC chi7122 was the 106KDa Tsh autotransporter protein [163]. Dozois et al. (2000) demonstrated that the Tsh protein contributed to the adherence of APEC to avian air sacs during the early stages of infection [155]. The tsh gene is carried by 46-85% of APEC and is located on a number of virulence plasmids including the pAPEC-O2-ColV plasmid [26, 164]. Ewers et al. (2007) reported a prevalence of 4- 4.5% in UPEC isolates [165].
The APEC autotransporter adhesin (aatA gene) and aatB contribute to biofilm formation and adherence to chicken embryo fibroblasts (DF-1 cells) [166, 167]. In a recent study aatB was carried by ~27% of 273 tested APEC (predominately of the B2 and D phylogroup) from China [167]. aatB was discovered following the genome sequencing of APEC DE205B, originally isolated from the brain of a Duck. aatB perhaps plays a redundant role in colonisation by some APEC, given its relatively
low prevalence and ΔaatB mutants still colonised infected birds but at lower capacity [167].
Pyelonephritis-associated pili (P pili) encoded by the pap operon may also contribute to APEC pathogenesis [29, 87, 168]. P pili are not thought to be important in the early stages of infection, but play a role during later stages leading to septicaemia and organ failure [87]. The complete pap operon is carried by the well characterised pathogenicity associated island (PAI) IAPEC-01 [169]. The PAI IAPEC-O1 pap operon, excluding papA, which showed 99% homology to porcine septicaemic E. coli, shows high sequence homology to that of UPEC CFT073 [170]. The prevalence of pap genes among APEC has been reported to be between 18.5 and 40% [171, 172]. In all, an array of adhesins has been shown to contribute to APEC pathogenicity and many are likely to still be unknown. It is possible that a combination of adhesins is required in pathogenesis and with certain adhesins playing a role at specific points during infection.