INTRODUCTION
Many pathogens utilize type III secretion systems (T3SSs) to deliver effectors into eukaryotic cells, which facilitates infections [Reference Galan1, Reference Raymond2]. Most bacteria contain only one T3SS, but some pathogens harbour multiple T3SSs, which function independently in different aspects of pathogenesis. Salmonella utilize the Salmonella pathogenicity island 1 (SPI-1) T3SS to invade host cells, while the SPI-2 T3SS is required for intracellular survival [Reference Moest and Meresse3]. Intestinal pathogenic Escherichia coli, such as enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC), use the locus of enterocyte effacement (LEE)-encoded T3SS to cause attaching/effacing lesions and diarrhoeal disease [Reference Franzin and Sircili4]. A second T3SS, designated the E. coli type III secretion system 2 (ETT2), was identified by sequencing the genome of an EHEC O157 strain [Reference Hayashi5]; however, the role of ETT2 in E. coli infections is less clear.
ETT2 is unable to encode a functional secretion system in most E. coli because it has undergone widespread mutational attrition, but it plays a role in regulating bacterial virulence [Reference Ren6, Reference Zhou7]. ETT2 can affect the expression of virulence genes outside the ETT2 cluster, and it is indirectly involved in the virulence of intestinal pathogenic E. coli [EHEC and enteroaggregative E. coli (EAEC)] [Reference Zhang8, Reference Sheikh9]. However, ETT2 is crucial for the virulence of extraintestinal pathogenic E. coli (ExPEC) strains. The ETT2 locus in avian pathogenic E. coli (APEC), although degenerate, contributes to virulence and reduced serum survival [Reference Ideses10]. Moreover, some evidence suggests that intact ETT2 might deliver effector proteins into human brain microvascular endothelial cells (HBMECs) to enhance bacterial invasion, intracellular survival and virulence of newborn meningitis E. coli (NMEC) strains [Reference Yao11].
ETT2 is known to be present, in whole or in part, in the majority of E. coli strains; it is most frequent in intestinal pathogenic E. coli strains [Reference Cheng12–Reference Osawa14], but of low prevalence in uropathogenic E. coli (UPEC) strains [Reference Miyazaki15]. ExPEC strains (NMEC, UPEC, APEC) share a broad range of similar virulence factors and pathogenic mechanisms and APEC is thought to be a potential reservoir for human ExPEC [Reference Tivendale16–Reference Johnson18]. Thus, this study investigated the prevalence and characteristics of ETT2 in APEC isolates to elucidate the potential zoonotic risk of APEC.
MATERIALS AND METHODS
Bacterial strains, growth conditions and DNA preparation
A strain collection containing 245 APEC isolates obtained from chickens, ducks or geese with typical colibacillosis symptoms in Eastern China was screened for the ETT2 locus (Supplementary Table S1). The ETT2-positive EHEC O157 strain ATCC43999 and the ETT2-negative strain DE719 [Reference Wang19] were used as controls in polymerase chain reaction (PCR) amplifications. All E. coli isolates were routinely grown in Luria–Bertani broth at 37 °C with aeration. Bacterial genomic DNA was prepared using the TIANamp Bacterial DNA kit (Tiangen, China) according to the manufacturer's guidelines.
Serotyping
The serotype of each APEC isolate was identified by an allele-specific PCR assay as described previously [Reference Wang20] and confirmed by agglutination with specific hyperimmune rabbit antisera to O1, O2 and O78 (Statens Serum Institut, Denmark) according to the manufacturer's guidelines.
Phylogenetic grouping
All isolates were classified into phylogenetic groups (A, B1, B2, D) according to the presence of the chuA and yjaA genes, and an anonymous DNA fragment, TSPE4.C2 by a triplex PCR as described previously [Reference Clermont, Bonacorsi and Bingen21].
Detection of ETT2
Isolates were tested for the presence of ETT2 genes by eight tiling-path PCR (TP-PCR), consisting of several interlocking, PCR-based approaches [Reference Ren6]. A series of primers was designed to amplify long fragments that spanned the entire ETT2 region in EHEC O157:H7 (GenBank accession no. NC002695·1) (Table 1). The structure of ETT2 cluster of EHEC O157:H7, EAEC 042 and NMEC CE10 are displayed in Figure 1a . If the long-fragment PCR was negative, a relevant short overlap PCR with the same primers and/or a deletion-scanning, long-fragment PCR employing primers flanking the lost segments was performed (Fig. 1b ). The distribution of the eip locus, which encodes homologues of SPI-1 translocators and additional T3SS-related proteins, was determined by PCR with the respective primers (Table 1).
Fig. 1. ETT2 structures and lengths of TP-PCR products. (a) Structure of the ETT2 pathogenicity island in EHEC O157:H7, EAEC 042 and NMEC CE10. Homologous genes are vertically aligned with the transcriptional regulator encoding gene shown as black and the pseudogene as a grid. (b) Bold lines indicate the size of the PCR products, and deletion-scanning PCR. (c) Length of gene deletion or insertion of ETT2 isoforms in APEC isolates.
Table 1. Primers used in the present study
Long-fragment PCRs were performed using LA Taq DNA polymerase (TaKaRa, China). For short-fragment PCRs, 1 µl template DNA was added to reaction mixtures (25 µl) containing 2·5 µl of 10× PCR buffer with MgCl2 (25 mm), 1·5 U Taq DNA polymerase (TaKaRa), 2 µl dNTPs (2·5 mm for each dNTP), and 0·5 µl (10 µ m) of each primer pair. The PCR mixtures were subjected to the following conditions in an ABI Thermal Cycler (ABI Biosystems, USA): pre-denaturation at 95 °C for 5 min, followed by 30 cycles of 95 °C for 35 s, 50 °C for 30 s and 72 °C for different times, followed by a final extension at 72 °C for 10 min. Following electrophoresis, PCR products were identified by UV illumination, sequenced, and submitted to GenBank.
Virulence genes
Isolates were screened by a previously described multiplex PCR protocol for the virulence genes fimC, papC, tsh, mat, irp2, fyuA, iroN, iucD, iss, cva/cvi, neuC, ompA, vat, ibeA and ibeB [Reference Meng22].
RESULTS
Identification and characterization of the ETT2 locus
In total, 57·6% (141/245) APEC isolates were positive for ETT2 gene sequences (Table 2). TP–PCR amplification and sequencing identified five different ETT2 isoforms among the isolates (GenBank accession nos. KU684467–KU684471). Sequence analysis indicated that the intact ETT2 gene cluster in APEC was similar to that of NMEC CE10, which was designated the type A isoform. Types B, D and E isoforms were characterized by 4·99-, 5·68- and 8·74-kb gene deletions, respectively, in the right end of the ETT2 island. Specifically, there was a 1·3-kb transposase insertion in the transcriptional regulation gene ygeH, and a 4·99-kb gene deletion in the ETT2 island of the type C isoform strains (Fig. 1c ). Of the five different ETT2 isoforms detected, type E (51·1%, 72/141) was the most widely distributed, followed by type C (14·9%, 21/141) and type B (12·8%, 18/141).
Table 2. Distribution of the ETT2 isoforms in APEC isolates
ETT2 locus in predominant APEC serotypes
As shown in Table 2, 46·9% (115/245) of the isolates were grouped as serotype O78, 22·5% (55/245) as serotype O2, and 12·7% (31/245) as serotype O1; the remaining 44 isolates (18%), were not typable with these three antisera and were classified as ‘Non-O1/O2/O78’ serotypes. The majority (64·4%, 74/115) of the O78 serotype isolates were positive for ETT2, as were 58·2% (32/55) of serotype O2, and 48·4% (15/31) of serotype O1. Correspondingly, of the 141 APEC isolates positive for ETT2, 52·5% (74) were serotype O78, 22·7% (32) serotype O2, and 10·6% (15) serotype O1. The remaining 20 (14·2%) isolates belonged to ‘Non-O1/O2/O78’ serotypes. An intact ETT2 island (type A) was found only in serotypes O1 and O2 and notably the O78 isolates always contained deleted isoforms of ETT2.
Phylogenetic groups D and B2 APEC, and ETT2 cluster
According to the Clermont E. coli phylotyping method [Reference Clermont, Bonacorsi and Bingen21], 78 (31·8%) of the APEC isolates belonged to phylogenetic group B2, while 54 (22%), 52 (21·2%) and 48 (19·6%) of the isolates fell in groups D, A and B1, respectively; 13 (5·8%) of the isolates could not be classified into a phylogenetic group (Table 2). A comparison of the distribution of the ETT2 cluster among phylogenetic groups revealed that 66·7% (32/48), 64·8% (35/54), 59·6% (31/52) and 44·9% (35/78) of the group B1, D, A and B2 isolates, respectively, and 61·5% (8/13) of the unknown group were positive for the ETT2 island. For the 17 isolates that had an intact ETT2 (type A isoform), nine fell in phylogenetic group D, five in B2, whereas types B and C isoforms were predominant in phylogenetic group A isolates, and type E in group B1.
Type III secretion-associated locus (eip locus), virulence genes and ETT2 locus
According to previous studies, the eip locus within the EAEC 042 genome is associated with the sipABCD homologue cluster of the ETT2 locus [Reference Ren6, Reference Chaudhuri23]. Here, the eip locus was only found in APEC strains that possess an intact ETT2 locus and, moreover, was located between the E. coli yicM and nlpA genes (data not shown), which is in the same position as that in EAEC 042 and NMEC CE10.
There was no correlation between the presence or distribution of other virulence genes and the ETT2 locus (Supplementary Table S1).
DISCUSSION
Pathogenic bacteria utilize several common strategies to overcome host defences and facilitate infections. T3SSs, which deliver effector proteins into host cells, are important virulence mechanisms in Gram-negative bacteria as they interfere with specific cellular and host immune responses to promote bacterial survival and enhance pathogenicity [Reference Galan1, Reference Raymond2]. Two different T3SSs have been identified in E. coli. The LEE-encoded T3SS (ETT1) is well-characterized in EHEC and EPEC strains [Reference Franzin and Sircili4], whereas ETT2 is present, in whole or part, in the majority of E. coli strains, including many commensal strains [Reference Ren6]. However, until now, no prevalence data for ETT2 in APEC isolates have been published.
The present study shows that over half (57·6%) of 245 APEC isolates harboured the ETT2 locus. We identified five different ETT2 isoforms in these isolates. Sequence analysis showed that there are gene deletions in most APEC strains (types B, C, D, E ETT2 isoforms), indicating that ETT2 has undergone widespread mutational attrition. Similar mutations were also found in other E. coli pathovars [Reference Cheng12, Reference Prager13]. A serotyping analysis indicated that ETT2 was present in the predominant APEC serotypes O78, O2 and O1 with over half (52·5%) of the isolates being O78. Further, all the ETT2 elements in isolates of this serotype were variants (not intact), which is consistent with previous studies [Reference Ideses10]. Indeed, an intact ETT2 locus was mostly present in serotypes O1 and O2 isolates, which also harboured the eip cluster. Further analysis indicated that an intact ETT2 gene cluster was predominant in phylogenetic groups D and B2, as reported previously [Reference Ren6], but by contrast, the distribution of ETT2 mutational isoforms varied.
Our results show that the APEC isolates studied harboured all of the ETT2 isoforms found in human ExPEC (UPEC and NMEC) with a similar frequency as reported for human diarrhoeagenic E. coli (65%), Shiga toxin-producing E. coli (STEC) (100%), as well as isolates from piglets (85·9%) and cows (47·4%), but was markedly higher than that in UPEC (3%) [Reference Zhou7, Reference Cheng12–Reference Osawa14]. Published studies suggest that ExPEC (APEC, NMEC, UPEC) strains possess a similar broad range of virulence factors and pathogenic mechanisms [Reference Tivendale16, Reference Ewers17]. More importantly, the predominant APEC serotypes O1, O2 and O78 have also been implicated in various human diseases, including newborn meningitis and urinary tract infections, indicating that poultry may be a vehicle or even a reservoir for human ExPEC (UPEC and NMEC) [Reference Tivendale16–Reference Johnson18, Reference Rodriguez-Siek24]. Bacteria could acquire a combination of mobile genetic elements, such as the ETT2 locus, through some mechanism of horizontal gene transfer, and become a highly adapted pathogen [Reference Brown and Finlay25]. The 1·3-kb IS transposase was found in type C isoform ETT2 strains, which might facilitate the transfer of ETT2 among E. coli strains.
ETT2 is reported to be associated primarily with the LEE, the high pathogenicity island, the locus of proteolytic activity or Stx2e in intestinal pathogenic E. coli (EHEC and STEC) strains [Reference Zhou7, Reference Prager13, Reference Schmidt26]. We investigated the association of ETT2 with the distribution of 15 other APEC virulence factors, but failed to find any correlation between them, which possibly indicates that the ETT2 cluster occurs independently of other virulence factors in these strains, perhaps due to different pathogenicity mechanisms and virulence factors.
Characterized T3SS needle complexes are composed of inner and outer membrane rings and a hollow needle. In most E. coli strains, ETT2 is highly variable and contains mutations, which leads to an incomplete and non-functional T3SS apparatus [Reference Ren6, Reference Cheng12, Reference Prager13]. Although degenerate, ETT2 still plays a crucial role in E. coli virulence. In EHEC O157:H7, ETT2 regulates virulence gene expression and protein secretion by the LEE [Reference Zhang8]. The EilA regulator of EAEC 042 coordinately activates the expression of effectors and influences bacterial adherence and biofilm formation, which are indirectly involved in virulence [Reference Sheikh9]. A degenerate ETT2 in APEC contributes to serum survival and virulence. However, it is not involved in the secretion of effectors. The inactivation of ETT2 affects bacterial surface properties, such as pellicle formation, which might decrease bacterial survival during an infection [Reference Ideses10].
Intact ETT2 has been shown to be crucial to bacterial virulence and plays a role in invasion, intracellular survival in HBMECs, and virulence of NMEC [Reference Yao11]. Moreover, in the latter study, the ETT2 deletion mutant exhibited the same levels of OmpA and K1 capsules as wild type, which affect the interaction of E. coli K1 with HBMECs, indicating that the ETT2 locus associated with NMEC CE10 has novel properties and mechanisms. It has been shown that the ETT2 T3SS not only secretes proteins that are encoded in the T3SS locus, but also those that are encoded outside of the locus [Reference Brown and Finlay25]. A previous study [Reference Ren6] and our results show that the eip locus, which encodes homologues of SPI-1 translocators and T3SS-related proteins, was present in all APEC strains harbouring an intact ETT2 cluster. The eip locus might cooperate with ETT2 to construct an active T3SS apparatus. Indeed, E. coli DH5α acquired the ability to secrete the EspB protein when complemented with ETT2 [Reference Makino27]. Additionally, putative T3SS effectors were discovered in EAEC 042, NMEC CE10 and APEC strains [Reference Yao11, Reference Chaudhuri23]. An intact ETT2 may deliver an unidentified effector(s) into host cells to interfere with host defence and facilitate bacterial infection. The exact function of ETT2 remains unknown, and whether and how an intact ETT2 locus encodes a functional secretion system should be investigated in the future to help us prevent poultry colibacillosis and potential human infections [Reference Lee and Kessler28].
SUPPLEMENTARY MATERIAL
For supplementary material accompanying this paper visit http://dx.doi.org/10.1017/S0950268816000820.
ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China (grant nos. 31572523, 31370045 and 31572546), the Chinese Special Fund for Agro-scientific Research in the Public Interest (grant no. 201303044) and the Shanghai Key Project on Agricultural Development through Science and Technology (grant no. 2015HNG1-9).
DECLARATION OF INTEREST
None.
