INTRODUCTION
Bacterial adherence to surfaces is an important survival mechanism. Attachment to host cells or extracellular matrix can provide access to specific niches and promote colonization of host tissues. Adhesins can also mediate biofilm formation through bacterium-bacterium associations and improve survival in the environment. Bacterial adhesins include hair-like appendages (fimbriae or pili) as well as other molecules, including proteins or polysaccharides, which are displayed on the cell surface (
1). Many types of fimbriae (pili) in Gram-negative bacteria are assembled by the chaperone/usher pathway (CUP) (
2,
3). In
Escherichia coli, numerous types of CUP fimbriae have been identified and characterized. Pathogenic
E. coli often can produce multiple types of fimbrial adhesins, and genomic analyses indicate that some strains may contain 10 or more fimbrial gene clusters (
3,
4). The ability to produce a variety of adhesins can provide a fitness advantage by expanding potential host receptor targets or promoting adherence to environmental substrates.
Two main types of fimbriae, type 1 and P fimbriae, from
E. coli have been extensively investigated to determine aspects of their roles in disease, particularly urinary tract infections (UTIs) (
5–7), as well as molecular aspects of biogenesis and assembly of these structures (
8). Type 1 fimbriae mediate adherence to mannose-containing receptors and have been shown to be critical for virulence of extraintestinal pathogenic
E. coli (ExPEC), including
E. coli strains causing urinary tract infections (
9) and neonatal meningitis (
10). P fimbriae were first described in uropathogenic
E. coli (UPEC) and were named based on receptor affinities for P blood group oligosaccharides, and they were also described as pyelonephritis-associated pili (Pap), since these fimbriae were more associated with
E. coli strains from cases of pyelonephritis (
11). P fimbriae have also been identified in other ExPEC isolates, including
E. coli associated with systemic infections in swine (
12) and some strains of avian pathogenic
E. coli (APEC) (
13–16). The P fimbrial gene clusters are commonly located on horizontally acquired chromosomal regions, which have been termed pathogenicity islands (
14,
17,
18).
The P fimbrial gene cluster comprises 11 genes, including regulatory genes (
papI and
papB) and genes dedicated to fimbrial assembly and structure (
papAHCDKJEFG). The
papA gene encodes the major fimbrial subunit and has been used to class P fimbriae into serological variants (F7
1 and F7
2 through F16) (
19). The adhesin specificity of P fimbriae is mediated by the
papG gene product. The G adhesins of P fimbriae were grouped into 3 major classes based on sequence differences and receptor specificity to different Gal(α1-4)Gal-containing glycolipids (
20,
21). PapG class I (PapGI) adhesins recognize globotriaosylceramide (Gb3), PapGII adhesins recognize globotetraosylceramide (Gb4), and PapGIII or PrsGIII adhesins recognize galactosylgloboside (Gb5). These glycolipid receptors are usually found on the surface of red blood cells and on human bladder and kidney cells. A distinct variant allele,
papGBF31, which was termed class IV, was also reported, although receptor specificity for this fimbrial adhesin was not described (
22).
P fimbriae are the archetype representatives of the π fimbrial family (
2), which includes a number of other types of
E. coli fimbriae, including Pix fimbriae present in some UPEC strains (
23,
24) and Sfp fimbriae encoded on plasmids in some lineages of enterohemorrhagic
E. coli (EHEC) (
25,
26). This report describes a new type of
E. coli fimbriae from the π group that we have named P-like (PL) fimbriae, since they share sequence similarity and genetic organization with P fimbriae. The PL fimbriae are distinct from other known members of the π fimbriae and are encoded on IncFIB plasmids containing numerous other virulence genes associated with ExPEC and APEC strains. As with P fimbriae (
20), PL fimbriae have also diversified into a number of different G adhesin classes and major subunit variants, suggesting adaptive potential for host specificity and tissue tropism. Herein, we characterized two different types of PL fimbria genes encoding distinct G adhesins that were cloned from avian pathogenic
E. coli strain QT598 and a urinary tract pathogenic
E. coli (UPEC) strain, UMEA3703-1, and demonstrate these fimbriae can mediate adherence to host epithelial cells.
DISCUSSION
A novel plasmid-carried fimbrial gene cluster was identified on the large colicin V plasmid of avian pathogenic
E. coli strain QT598 (serotype O1:K1, sequence type ST1385). Strains from this and related sequence types such as ST91 are commonly associated with extraintestinal infections in poultry and urinary tract infections in humans (
27) (
http://enterobase.warwick.ac.uk/). The
plf genes were shown to be adjacent to the RepFIB, and
intM genes on plasmid pEC598. The is a common integration site for a diversity of genes on F and related plasmids, and collectively this region has been named the cargo gene region (
28). The cargo gene region has been found to encode a diversity of accessory genes, insertion sequences, and integrons known to carry genes for resistance to antimicrobials and metals, microcins, and virulence genes (
28,
32). It is therefore likely that the
plf fimbrial gene cluster along with other genes was inherited by certain
E. coli strains through a recombination/integration event and that it has since disseminated or been transferred into a diversity of
E. coli isolates associated with different host or environmental sources (highlighted in Tables S1 and S2 in the supplemental material). As with the P fimbriae, PL fimbriae have also diversified considerably, and there has been notable divergence in the PlfG adhesin-encoding sequences into 5 distinct PlfG adhesin classes (
Fig. 4). Such changes may have occurred to promote adherence and colonization to a variety of surfaces or host cell receptors in different niches or environments.
The PL fimbriae are new members of the π fimbrial family, which contains P-fimbria-like operons present in some
Betaproteobacteria and
Gammaproteobacteria (
2). More specifically, based on comparison of the fimbrial usher proteins, the PL fimbriae are part of a subgroup which includes true P fimbriae, as well as closely related Sfp and Pix fimbriae (
2) (
Fig. 2), all of which have been shown to mediate mannose-resistant hemagglutination (MRHA) of erythrocytes from humans in addition to some distinct MRHA profiles for erythrocytes from other species. Pix fimbriae, which have been identified in some uropathogenic
E. coli strains, were shown to agglutinate human erythrocytes, but not sheep or goat erythrocytes, and do not recognize the Gal-Gal sugars recognized by P fimbriae (
23). Sfp fimbriae also mediate MRHA of human erythrocytes, which was dependent on the
sfpG gene (
26). However, to our knowledge, no tests for MRHA for erythrocytes from other species have been reported. Interestingly, the G adhesin proteins from Pix and Sfp fimbriae share amino acid homology between them (63% identity and 81% similarity), suggesting these G adhesin proteins are more closely related to each other than to PlfG or PapG adhesins, which share no more than 25% amino acid identity. Herein, we demonstrated that PL fimbriae producing the class I adhesin mediated MRHA for erythrocytes from different species, including equine, ovine, bovine, rabbit, and human erythrocytes, whereas PL fimbriae producing the class II adhesin mediated MRHA only to human and turkey erythrocytes (
Fig. 6). Taken together, this subgroup of π fimbriae (true P fimbriae, Sfp, Pix, and PL fimbriae) have developed important differences in adhesin protein sequences that have expanded the capacity to adhere to a variety of receptors on erythrocytes and host cells from different species. It will be of interest to more specifically determine the lectin receptor specificity of this family of fimbriae.
The genetic organization of the
plf gene cluster includes 9 predicted fimbrial subunit genes, which is the number of predicted structural genes encoding P fimbriae (
Fig. 3). In contrast, both the Sfp and Pix fimbrial gene clusters comprise 7 structural genes and lack the genes corresponding to the
papK and
papE genes predicted to encode an adaptor and a minor fimbrial subunit (
Fig. 3). From this standpoint, overall, PL fimbriae are most similar to true P fimbriae.
To further demonstrate potential complementarity between P and PL fimbriae, we also generated hybrid fimbrial gene clusters, wherein the plfGQT598 gene was replaced by PapG adhesin-encoding genes belonging to class I, class II, or class III adhesins. Each of these clones was able to produce a functional fimbrial structure that also increased adherence to human urinary tract epithelial cells. This also further indicates that the PL fimbriae, despite having adhesins that are quite distinct in amino acid sequence from P fimbriae, also produce mannose-resistant hemagglutinins that can mediate adherence to human bladder and kidney cells and that the bioassembly of these fimbriae is compatible with that of P fimbrial G adhesins. It is interesting, however, that the production of the hybrid fimbriae from bacterial cells was substantially reduced compared to that of the PL fimbrial clones, suggesting that efficiency of biogenesis of the hybrid fimbriae is reduced.
As with the
plf gene cluster, the location of the
sfp genes is also on IncF plasmids, in close proximity to RepFIB of the pSFO157 plasmid (
26). However, it is flanked on both sides by insertion sequences that are distinct from the region adjacent to
plf genes on pEC598. The Sfp fimbriae were initially found not to be expressed by EHEC strains under normal laboratory conditions, and properties of these fimbriae were first determined using cloned fimbrial genes in
E. coli K-12 (
26). The
sfp genes encoding a fimbrial system with mannose-resistant hemagglutinin activity have been identified on a subgroup of sorbitol-fermenting EHEC/Shiga-toxigenic
E. coli (STEC) strains and some EHEC O165:H25/NM strains from humans and cattle, but are absent from most other types of
E. coli (
25,
26). This suggests that the
sfp genes were likely acquired independently by horizontal transfer to both a nonmotile sorbitol O157 strain and independently to an O165:H25/NM strain and have since remained in these branches of EHEC (
25). This is clearly in contrast to the
plf gene cluster, which is present in a diversity of
E. coli strains from multiple sources and has likely been transferred either through multiple conjugation and/or recombination events and has also diversified, since distinct G adhesin classes have emerged among strains.
DNA sequence comparisons of gene clusters that are highly similar to the
plf fimbrial system of
E. coli QT598 from nucleotide databases provided a means to identify subgroups of PL fimbria genes encoding 5 distinct classes of PlfG adhesins (
Fig. 4). Since the PlfG class I- and class II-encoding alleles were predominant among isolates that notably included strains associated with human extraintestinal infections as well as infections from poultry, we focused our attention on functional characterization of one of each of the PL fimbriae belonging to these classes. It was also interesting to identify some variant alleles of the PlfG adhesin in other
E. coli strains as well as a subgroup that was identified in some strains of
Cronobacter sakazakii and other
Cronobacter spp. (
Fig. 4). Although
Cronobacter strains containing the
plf fimbrial clusters were sampled from spices,
C. sakazaki and related
Cronobacter spp. are important foodborne pathogens that can contaminate dehydrated milk and other products and cause serious extraintestinal infections, particularly in neonates (
33,
34).
The capacity of PL fimbriae to form biofilms at different temperatures was also investigated, and both the class I and class II PL fimbriae promoted biofilm formation, with PlfG class I producing more biofilm at 25 and 37°C, but not 42°C. In contrast, the PlfG class II adhesin produced very high levels of biofilm at all temperatures tested. The presence of the PlfG adhesin was important for high-level biofilm production for PlfG class II, since the absence of the plfG adhesin gene greatly reduced biofilm formation. Notably, after growth at 37°C, the level of biofilm produced by the ΔplfG clone was significantly higher than that with the empty vector and comparable to levels of biofilm produced by Pap reference clones and the PlfG class I clone. This suggests that other factors in addition to the G adhesin may also contribute to increased biofilm formation associated with expression of plf or pap fimbrial genes.
Since both types of PL fimbriae conveyed increased adherence to human epithelial bladder and kidney cells (
Fig. 7), we investigated the potential of these fimbriae to contribute to urinary tract colonization in a murine model. Deletion of the
plf genes from either
E. coli strain QT598 or strain UMEA-3703-1 did not have an appreciable effect on colonization of either the bladder or the kidneys. Furthermore, despite being isolated from a human UTI, strain UMEA-3703-1 was not a strong colonizer in the mouse UTI model.
The mouse UTI model may not be as representative of a human infection when using certain bacterial strain backgrounds or when investigating specific mechanisms of virulence such as adherence and fimbrial adhesins. P fimbriae have been shown to play a role in urinary infection, particularly for pyelonephritis in cynomolgus monkeys (
35), and these fimbriae alone can confer an asymptomatic
E. coli urinary strain the capacity to elicit strong regulatory modulation in humans by acting as an IRF-7 agonist and reprogramming the immune response in the urinary tract (
5). In the case of the murine model, it has been demonstrated that P fimbriae can reduce the immune response in the kidney by decreased production of polymeric Ig receptor and reduced secretion of IgA (
36). However, the role of P fimbriae in bacterial colonization in the UTI mouse model has been less evident. Initially,
pap genes cloned into an avirulent
E. coli K-12 strain or an intestinal commensal
E. coli strain were shown to increase colonization of the mouse kidney (
37,
38). In contrast, deletion of
pap from different UPEC strains did not alter colonization of the urinary tract in CBA/J mice (
39). Reasons why PL fimbriae as much as P fimbriae may not play as critical role in the mouse UTI model may be due to differences in lectin receptor target specificity present on murine cells and/or the potential redundancy of adherence mechanisms due to production of multiple fimbrial adhesins in UPEC strains. Since a slight increase in colonization of the plf mutant of strain UMEA-3703-1 in the bladder was observed (Fig. S4), it is also possible that loss of the
plf genes may alter expression of other types of fimbriae, since regulatory cross talk can occur and loss of one type of fimbriae can enhance expression of other types of fimbriae. The expression of
plf was also upregulated more than 40-fold by strain QT598 in the bladder and was increased by 5-fold in minimal medium compared to rich medium (
Fig. 9). This indicates that the expression of these fimbriae can be increased by cues during infection, which may include host factors or decreased nutrient availability. In the current study, we used an acute infection model with an endpoint of 48 h, and our detection of increased expression of
plf in the urinary tract was only determined at this time point. As such, these fimbriae may potentially play a more important role in more prolonged infections, and it may be of interest to investigate colonization at later time points using other models, such as catheter-associated infections.
Since the competitive coinfection model demonstrated a potential fitness advantage in the mouse kidneys, this nevertheless supports a potential advantage for production of these fimbriae by strain QT598 during UTI in the mouse model (
Fig. 10). The prevalence of
plf genes was associated with strains from human infections, as well as canine infections and from samples in poultry, including turkeys and chickens. In future studies, it will be of interest to determine whether PL fimbriae or specific PlfG adhesin classes may contribute to infection in other animal models such as poultry and to further investigate PL fimbria receptor specificity, potential role in modulation of host immune response and the regulation of production of this newly identified group of fimbriae.