Abstract
Escherichia coli O157:H7 is the most well-studied serotype of enterohemorrhagic E. coli (EHEC) class of E. coli intestinal pathogens, and is responsible for many outbreaks of serious food-borne illness worldwide each year. Adherence mechanisms are a critical component of its pathogenesis, persistence in natural reservoirs, and environmental contamination. E. coli O157:H7 has a highly effective virulence operon, the Locus of Enterocyte Effacement (LEE), and its encoded intimate adherence mechanism is well characterized. However, factors involved in the preceding initial attachment are not well understood. In this study, we propose a mechanism of initial adherence used by E. coli O157:H7 in vitro. We describe a bacterial protein not previously reported to be involved in adherence, Slp, and its interactions with the human host protein polymeric immunoglobulin receptor (pIgR). The human pIgR has previously been shown to act as an adherence receptor for some mucosal pathogens, and is highly expressed in the intestine. Following observation of significant colocalization between E. coli O157:H7 and pIgR location on Caco-2 cells, a co-immunoprecipitation (Co-IP) assay using a human recombinant Fc-tagged pIgR protein led to the identification of this protein. Disruption of Slp expression in E. coli O157:H7, through deletion of its encoding gene slp, produced a significant adherence deficiency to Caco-2 cells at early time points associated with initial adherence. Plasmid complementation of slp fully restored the wild-type phenotype. Furthermore, immunofluorescence microscopy revealed evidence that this interaction is specific to the pathogenic strains of E. coli tested, and not the nonpathogenic strain E. coli K12. Additionally, deletion of slp resulted in the absence of the corresponding protein band in further Co-IP assays, while the plasmid-encoded slp complementation of the deletion mutant strain restored the wild-type pattern. These data support the proposal that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor.
Author summary Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) are responsible for tens of thousands of cases of food-borne illness in the United States each year. E. coli O157:H7 has a particularly effective intimate adherence mechanism. However, the mechanisms of initial adherence, which facilitate attachment and virulence prior to the engagement of intimate adherence, are not well understood. In this study, we describe an initial adherence interaction between the E. coli O157:H7 Slp and the human polymeric immunoglobulin receptor (pIgR) expressed by the human colonic epithelial cell line Caco-2. The relationship was first demonstrated as a significant colocalization between the locations of E. coli O157:H7 bacterial cells and pIgR protein using immunofluorescence microscopy. The E. coli O157:H7 Slp protein was identified, and disruption of the slp gene resulted in a severe adherence deficiency to Caco-2 cells during initial adherence. This effect was reversed upon complementation of the Δslp strain with a plasmid-encoded slp gene, and the constitutive over-expression of slp resulted in hyper-adherence exceeding that of the wild-type E. coli O157:H7. These data support the proposition that Slp directly contributes to initial adherence, with the pIgR protein as its proposed receptor.
Introduction
It has been estimated that the most common enterohemorrhagic E. coli (EHEC) serotype, E. coli O157:H7, causes more than 70,000 cases of food-borne illnesses in the United States each year, with an additional 35,000 infections caused by non-O157 EHEC serotypes (1). EHEC infections typically result in abdominal cramping, watery or bloody diarrhea, and are also sometimes accompanied by fever, nausea, and vomiting (2). Among these cases, a subset develop a severe complication known as hemolytic uremic syndrome (HUS), which is primarily observed in infected children under the age of five years (3). In the U.S., E. coli O157:H7 outbreaks occurring between 1982 and 2002 had highly variable rates of HUS ranging from less than 1% to greater than 15% (4).
E. coli O157:H7 utilizes a fairly well-characterized set of virulence factors to colonize the human gastrointestinal (GI) tract during infection, including the robust intimate adherence mechanism carried out by factors encoded in the Locus of Enterocyte Effacement (LEE) pathogenicity island (5). The three main factors required for intimate adherence are (i) intimin, (ii) Tir, and (iii) type three secretion system (T3SS); which are the bacterial adhesin, translocated intimin receptor, and secretion apparatus, respectively (6). Upon infiltration of the mucin layer in the colon, the bacteria require an initial attachment in order to stay stably adjacent to the intestinal epithelial cell (IEC) for long enough to have the opportunity to engage its intimate adherence mechanism before being cleared from the digestive tract (7).
Several adhesins putatively involved in initial adherence have been reported, though the research has not yet reached a consensus. For example, long polar fimbriae (LPF) have been described as initial adhesins in E. coli O157:H7, but with some conflicting reports. LPF is a fimbrial adhesin with two loci, lpf1 and lpf2, with homologs in Salmonella (8). Recently, LPF has been described in the role of translocation of EHEC across human M cells in in vitro human organ culture simulated conditions, but lpf deletion mutant strains of E. coli O157:H7 did not show any significant effects on adherence to Caco-2 cells after three hours post-infection (9). One study showed that a non-fimbriated E. coli K12 strain expressing the lpf operon demonstrated some increased adherence to MDBK and HeLa cells after six hours of infection, and single deletion mutations in lpf1 or lpf2 showed a modest reduction in adherence under the same conditions (8). However, expression of lpf2 in E. coli K12 was shown to result in reduced adherence to Caco-2 cells, so the exact role of LPF in initial adherence remains unclear (10). The EHEC factor for Adherence (Efa) has also been investigated as an initial adhesin in enteropathogenic (EPEC) strains, and E. coli O157:H7 contains a truncated version of efa (11). Insertion mutations disrupting the expression of efa1 in EHEC showed reduced agglutination with human erythrocytes and autoagglutination with other EHEC cells, in addition to a significant reduction in adherence to Chinese hamster ovary (CHO) cells and a HeLa-derived cell line. However, the coinciding reduction in the secretion of the LEE-encoded effector EspA suggests that this effect may be more involved in intimate adherence than initial attachment (12) (13). Similarly, the effector ToxB found on the E. coli O157:H7 pO157 plasmid shares some sequence homology with Efa and has been shown to contribute to adherence. Its mechanism is likely to be involved with promotion of the LEE-encoded effector proteins EspA, EspB, and Tir and therefore is also unlikely to be involved in initial adherence (14). Flagella (H antigens) are well characterized for their role in motility, but have also been implicated in adherence in the bovine host. Flagella in E. coli O157:H7 were able to bind bovine intestinal mucus, while H negative mutant strains showed reduced adherence to bovine intestinal tissue explants; though other studies have reported H negative strains as being able to colonize young calves (6) (15). Although flagella have been shown to impact EPEC adherence in vitro, any role in EHEC or with human IECs remains unknown (16). Many other proteins have been identified as being of interest in the study of adherence, but roles in initial adherence are either unclear, not studied in human cells, or do not have enough data to be conclusive (7).
Under normal conditions, the human polymeric immunoglobulin receptor (pIgR) system plays a critical role in innate mucosal immunity (17). The human pIgR is a glycoprotein varying in size from 80–120 kDa depending on the level of glycosylation, and primarily functions as a transport for dimeric immunoglobulin A (dIgA) into the intestinal lumen (18). In the lumen, the transported pIgR-dIgA complex is cleaved from the IEC surface and termed secretory IgA (sIgA), where it plays an important role in maintaining the balance between immune function and microflora homeostasis (19) (20). In addition, pIgR lacking dIgA can also be transported. The unbound pIgR is also cleaved and released (free secretory component or SC), which also serves as part of innate immunity (21) (22). In spite of its importance in immunity, the human pIgR is also known to be hijacked by pathogens and used as a route for adherence and invasion. For example, the human pIgR is known to contribute to the adherence and invasion of Streptococcus pneumoniae, and the invasion and intercellular spread of Epstein-Barr virus during infection (17) (23). Although S. pneumoniae is a Gram-positive pathogen, it is a pathogen that targets mucosal epithelial cells, as does EHEC (24) (25) (26). Additionally, a study in 2007 found that pigr expression at six hours post-infection in bovine hosts was upregulated when experimentally infected with E. coli O157:H7 and a non-O157 EHEC strain (1.93 and 3.51 fold-change (FC), respectively); but not in response to non-colonizing control strains (27). Although this effect on gene expression may have been due to the bovine host immune response to EHEC colonization, as a mucosal epithelium colonizing pathogen EHEC also has the potential to use the pIgR during adherence to IECs during infection.
In this study, we describe the interaction between the E. coli O157:H7 outer membrane protein Slp (carbon starvation-inducible lipoprotein) and the pIgR, and its role in initial adherence to Caco-2 cells in vitro. Utilizing immunofluorescent microscopy to investigate the possibility, we demonstrated that during initial adherence, E. coli O157:H7 had a significant correlation with its location and the location of pIgR protein on the Caco-2 cell surface. The colocalization of E. coli cells with pIgR protein was statistically significant with E. coli O157:H7 (O157:H7-WT), but not with the nonpathogenic E. coli strain K12. Additionally, several other non-O157 pathogenic E. coli strains showed significant colocalization patterns. These observations were further investigated using a commercially available recombinant Fc-tagged human pIgR protein (pIgR-Fc) in a co-immunoprecipitation (Co-IP) assay, leading to the identification of Slp as the interacting protein involved in the colocalization phenotype observed in the pathogenic strains of E. coli tested. A deletion mutation of E. coli O157:H7 lacking the slp gene (O157:H7-Δslp) resulted in an elimination of the colocalization patterns observed with immunofluorescent microscopy, the absence of the ∼20 kDa band corresponding to Slp following Co-IP, and a significant adherence deficiency to Caco-2 cells. Furthermore, the O157:H7-Δslp strain was complemented in trans with the slp gene (O157:H7-p::slp), which restored the wild-type phenotypes seen in colocalization, Co-IP, and adherence to Caco-2 cells in vitro. Taken together, these results support the direct interaction between the E. coli O157:H7 Slp and the host pIgR and its contribution to initial adherence.
Results
Initial adherence timeline of E. coli O157:H7 during adherence to Caco-2 cells
In order to effectively study the roles of different factors during attachment, a specific timeline was required to define initial adherence in this model. Using the expression of LEE-encoded genes as a benchmark for the beginning of the intimate adherence stage of attachment, significant LEE upregulation has been cited as taking place between two and six hours post-infection in vitro. When adhered to Caco-2 cells, E. coli O157:H7 Sakai demonstrated increased production of EspA, EspB, and Tir protein production after 4.5 hours post-infection (28). During adherence to HeLa cells, LEE1 in E. coli O157:H7 Sakai has been shown to be activated by four hours post-infection (29) (30); while eae expression in an undefined E. coli O157:H7 strain demonstrated upregulation as early as two hours after infection (31). Other studies of EPEC have shown LEE promoter or LEE-encoded gene upregulation between four and six hours in several different epithelial cell lines, but the differences in the LEE operon between EPEC and EHEC strains make the relevance to E. coli O157:H7 unclear (32) (5). Following previous work, under the conditions of this study the intimin-encoding eae gene was used as a benchmark for the onset of intimate adherence. Compared to a non-adhered control culture to isolate the gene expression profiles of only initially attached bacterial cells, quantitative PCR (qPCR) was used to calculate FC of eae expression in adhered E. coli O157:H7 bacteria was calculated using the relative expression ratio method for comparative Ct analysis (33). The expression of eae surpassed the threshold for significance (FC ≥ 2) after four hours and increased thereafter, consistent with previous literature (Fig 1). Thus, initial adherence in this model was defined as taking place from zero to three hours post-infection, when eae was not significantly upregulated.
Colocalization and covariance of pIgR with E. coli
To investigate the possibility of a pIgR adherence mechanism in E. coli O157:H7 initial adherence, immunofluorescence was used to assess colocalization between the location of pIgR protein and locations of adhered E. coli bacteria and quantify any correlation using covariance analysis (Fig 2). The covariance, shown as R, attested that there was no significant correlation between the locations of E. coli K12 bacteria and the pIgR protein; while O157:H7-WT did exhibit a significant correlation (Fig 2B-C). This relationship is further detailed when observed over time, where O157:H7-WT showed an initial peak of covariance (R > 0.7) at one hour post-infection, and decreased over two and three hours (R > 0.5 and 0.4, respectively). This pattern of early peak followed by steady decrease was consistent with the predicted pattern of an adhesin active during initial adherence, as its activity would be less necessary as the intimate adherence factors assumed adherence function at later timepoints. No such pattern was observed in E. coli K12. Several other extra-intestinal (ExPEC) pathogenic strains of E. coli were also tested and showed significant covariance at two hours post-infection, indicating the possibility that an adherence mechanism is not limited to only E. coli O157:H7 (Fig 2A-B).
Protein-protein interactions between E. coli O157:H7 and the pIgR
To demonstrate a direct protein-protein interaction between E. coli O157:H7 and the pIgR, a co-immunoprecipitation (Co-IP) assay was done using a C-terminal Fc tagged human recombinant pIgR protein (pIgR-Fc) and O157:H7-WT proteins (whole-cell lysate was used to avoid the accidental exclusion of any proteins). Recovered proteins were run in a reducing SDS-PAGE gel (Fig 3), and bands of interest were later identified through LC MS/MS analysis (Table 1).
Lane D shows the presence of a concentrated band of approximately 20 kDa (noted as }) in the treated sample, not visible in the untreated control sample (Lane C), indicating the likelihood of a direct protein-protein interaction with the pIgR-Fc protein (Fig 3). There were several other observable bands in the treated sample between 26–43 kDa, but they corresponded in size to the most concentrated bands observed in the control, making it likely that they were non-specific and not indicative of protein binding.
A direct relationship between the O157:H7-WT protein Slp and the pIgR-Fc protein was demonstrated through disruption of the slp gene. A deletion mutation of the slp gene (O157:H7-Δslp) eliminated the band of interest recovered with the wild-type strain (O157:H7-WT) (Lane E), and the subsequent complementation of a plasmid-encoded slp gene (O157:H7-p::slp) restored the wild-type phenotype (Lane F) (Fig 3).
Slp and initial adherence in vitro
Relative expression of the slp gene showed differing patterns between E. coli K12, O157:H7-WT, O157:H7-Δslp, and O157:H7-p::slp as measured by qPCR (Fig 4A). The slp gene is reported to be transcribed at a low baseline level in most conditions and upregulated during entry of bacteria into stationary-phase (34). Over 16 hours of growth, O157:H7-WT slp expression showed a transient peak at 14 hours, corresponding to the entry of the culture into stationary-phase growth. E. coli K12 slp expression remained relatively stable and low throughout growth, and did not show any peaks or changes at any time point. The O157:H7-Δslp strain did not show any detectable levels of gene expression, while the O157:H7-p::slp strain showed high levels of constitutive over-expression of slp at high levels at all timepoints, ranging from 10 to over 100 magnitude differences in FC as compared to E. coli K12 and O157:H7-WT.
When measuring initial adherence to Caco-2 cells by quantifying the number of attached bacteria over time, the O157:H7-WT presented a pattern showing a steady increase of adhered bacterial cells over three hours (Fig 4B). These results were consistent with O157:H7-WT bacterial cells as they were steadily coming into contact with and attaching to the Caco-2 cells over time, as the adherence increased two- to three-fold per hour (approximately 20%, 55%, and 100% at 1, 2, and 3 hours post-infection, respectively). When compared to the wild-type, deletion of the slp gene resulted in a significant adherence deficiency to Caco-2 cells, with O157:H7-Δslp adherence not reaching more than 10% at any time point. The O157:H7-Δslp adherence increased very slightly over three hours (from <5% to <10% at 1 and 3 hours post-infection, respectively). Conversely, the O157:H7-p::slp showed a hyper-adherent pattern, surpassing wild-type levels at all timepoints. The O157:H7-p::slp adherence followed roughly the same pattern as seen in the wild-type, with adherence increasing two- to three-fold per hour (approximately 60%, 100%, and 225% at 1, 2, and 3 hours post-infection, respectively). At three hours, the number of adhered O157:H7-p::slp cells was more than double the wild-type.
Colocalization and covariance were measured in the O157:H7-Δslp and O157:H7-p::slp strains over three hours of adherence to Caco-2 cells (Fig 4C-D). As in Fig 2, E. coli K12 did not show any statistically significant colocalization or any changes over time (R = 0.25, 0.28, and 0.22 at 1, 2, and 3 hours post-infection). The O157:H7-WT cells showed a significant colocalization at all timepoints, with the highest correlation (R = ∼0.7) seen at one hour post-infection, with a decrease to R = 0.55 and 0.50 at two and three hours respectively. The O157:H7-Δslp strain demonstrated adherence levels comparable to E. coli K12, with significantly diminished colocalization and no changes over time (R = approximately 0.2 at all time points). The O157:H7-p::slp strain restored colocalization comparable to that of the wild-type strain, but colocalization did not decrease over time in the same manner (R = 0.65, 0.70, and 0.63 at 1, 2, and 3 hours post-infection, respectively).
Discussion
Initial adherence of EHEC bacteria is a known step in pathogenesis- a loose attachment between the bacterium and the epithelial cell is required in order to have the opportunity to make an intimate attachment using T3SS effectors (35). The initial adherence in EHEC strains is not yet fully understood, and it is becoming clear that mechanisms vary between strains, hosts, and environmental signaling factors present (7). Studies examining initial adhesins have used a diverse array of host species as models with a variety of EHEC strains, which have shown that adherence mechanisms are not conserved. Even among EHEC strains with very similar virulence profiles, putative adhesins have inconsistent behavior between host type (7) (36) (37) (14). For example, in E. coli O157:H7, the flagellar antigen H7 has been implicated in bovine cell adherence using bovine intestinal tissue explants (38). Flagella in E. coli O157:H7 were able to bind bovine intestinal mucus, while H negative mutant strains showed reduced adherence to bovine intestinal tissue explants (6). However, purified H7 protein did not show any ability to bind to HeLa cells when other purified H proteins were able to bind in the same conditions (39). Here, using human colonic epithelial cells, we demonstrated a novel EHEC initial adherence mechanism in vitro, involving the E. coli O157:H7 outer membrane protein Slp and the human pIgR protein.
The role of the pIgR in pathogenesis is a known mechanism for other pathogens, but it had not been demonstrated in E. coli adherence. The pIgR is a major component of innate mucosal immunity, and its regulation is influenced by a variety of factors activated as part of the general inflammatory response to pathogens during colonization and infection (21). It was speculated that the pIgR behavior observed could have been affected by an immune response to the presence of EHEC; however, the effects of EHEC on the pIgR system do not explain the results reported. Like many enterics, E. coli is a Gram-negative organism, and contains LPS in its membrane (40). The LPS molecule is a potent activator (and the only known ligand) of Toll-like receptor 4 (TLR4), which causes an increased expression of the transcription factor NF-κB when activated (41). NF-κB is a highly effective activator of multiple proinflammatory signals, and among other effects it induces transcription of the pigr gene (42). Shiga toxins (Stx) are also capable of inducing expression of pigr through an induction of proinflammatory cytokine IL-8 during ribotoxic stress response in vitro (43). However, in order to upregulate pigr adequately to effect attachment as early as zero to three hours post-infection, exposure of the host cell to LPS or Stx would have to be reasonably prompt, prolonged and stable; which is unlikely to occur in the variable intestinal environment (41). Additionally, there is no evidence to suggest that the pIgR has any mechanism of location specificity, and inflammatory signals only affect the rate at which pIgR reaches the membrane without any effect on its location (19) (44) (45).
In this study, we hypothesized that the pIgR may be involved in EHEC adherence, and determined the bacterial protein binding to the human pIgR protein using a Co-IP assay. The Co-Escherichia coli through interactionsIP produced a distinct band of approximately 20 kDa that was significantly more concentrated when E. coli O157:H7 proteins were incubated with pIgR-Fc than with E. coli O157:H7 proteins alone. Identification using LC MS/MS led to the E. coli O157:H7 outer membrane protein Slp, a 22 kDa lipoprotein primarily known to be expressed in E. coli during entry into stationary-phase and during carbon starvation (34). Its encoding gene (slp) expression is extremely low at every other growth stage, only being upregulated significantly during entry into stationary-phase growth or during carbon starvation. Its function is not completely clear, though it has generally been accepted that one of Slp’s probable functions is related to membrane stability during stationary-phase growth (34) (46). The O157:H7-Δslp strain was observed to be slightly more easily lysed in water than the wild-type strain, which supports these findings. Bacterial lipoproteins are a broad range of proteins and have been known to be involved with many virulence functions, including adherence (47). Other genes with low levels of sequence homology have been identified in other Gram-negative bacteria or enteric pathogens, which suggests the possibility of a similar role among some limited classes of bacteria (46). The slp sequence is located on a genomic acid fitness island (AFI) along with several other defined and undefined genes and operons (48). The slp gene and AFI are present in all E. coli, but there are significant differences between strains. In E. coli K12, the AFI is approximately 14 kb and contains 12 protein-encoding genes, whereas the E. coli O157:H7 AFI is 23 kb and contains 21 protein-encoding genes. Due to the insertion of an O-island sequence encoding a heme transport locus, the E. coli O157:H7 AFI contains nine extra genes involved in heme uptake and utilization (chuA genes) (48) (49) (50). The slp is located in a small operon and is transcribed along with the gene yhiF (which encodes a putative LuxR family regulator protein), but of the AFI proteins, Slp and YhiF are not well characterized (49) (51). Several studies have outlined different potential functions of these proteins and how they may function together or separately, but the data are not yet conclusive. A 2007 study proposed that Slp and YhiF function together to protect the E. coli cell from its own toxic metabolic products during organic acid metabolism (52). Another study demonstrated the antimicrobial effects of cranberry concentrate in ground beef via bacterial membrane damage, and reported the differential expression of several outer membrane protein-encoding genes, including slp. The slp gene was modestly downregulated in conditions that showed anti-microbial activity, indicating a possibility of membrane weakening through lack of Slp’s function in membrane stabilization (53). There has also been evidence of increased slp gene expression during biofilm formation, though the significance of these findings is unclear (54). It is notable that Slp contributes to initial adherence and is involved in acid stress response and resistance, because pH is a potent signaling factor in E. coli O157:H7, and resistance to acid stress is a major component of E. coli O157:H7 virulence (55). Acid stress resistance in E. coli O157:H7 is crucial to survival in the low pH environment of the stomach, and the most significant AR mechanism used is the glutamate-decarboxylase (Gad) system, which is also found in the AFI (49). The regulation of adherence and virulence genes affected by pH is notoriously complex and not well understood; particularly with the Gad system (56). However, it has been shown that low pH results in a gene expression profile fit for initial adherence (55). In response to low pH, Gad-related regulators will downregulate the expression of LEE-encoded effectors not required at that stage of early attachment (i.e. eae and other intimate adherence related genes), and upregulate genes related to adherence and motility (55) (57) (49) (58). GadE, the Gad system master regulator, is known to affect the expression of genes outside the AFI both directly and indirectly (50). An acid- or Gad system-influenced mechanism of expression would be beneficial for initial adherence factors, as it is a consistent and robust signal present prior to the environment where initial adhesins would be required for effective colonization (58). With many virulence factors converging to be influenced or controlled by these acid-regulated pathways, regulation of adherence factor expression in the same manner would be more reliable and efficient than having multiple independent mechanisms during pathogenesis (59). Additionally, elaborate regulatory mechanisms also provide insight into possible reasons why E. coli K12, despite having the slp gene and a similar AFI, does not show a colocalization phenotype with pIgR. While no work has yet been done specifically regarding adherence and AFI-encoded gene regulation in response to acid stress in E. coli K12, the differences in slp expression in culture over time provide evidence that slp expression is managed by a different mechanism in E. coli K12 than in E. coli O157:H7. A study from 2011 highlighted some significant differences between E. coli K12 and E. coli O157:H7 AFI gene expression-namely, that E. coli O157:H7 carries a mutation causing the hdeB gene from being expressed (48). HdeA and HdeB are AR chaperone proteins that play a critical role in acid tolerance in E. coli K12. Deletion mutations of hdeA, hdeB, or hdeAB will significantly hinder the ability of E. coli K12 to survive acid stress, but E. coli O157:H7 was almost completely unaffected by the deletion of these genes (48). These findings illustrate the presence of different AFI gene expression regulation mechanisms and implies that these difference may explain why an AFI-containing E. coli strain does not appear to utilize the Slp during adherence. Given the complexity and sensitivity of acid-responsive regulatory mechanisms, and the current gap in knowledge regarding the exact roles of Gad, AFI, and other regulatory genes during infection of the human host; further study is required to fully understand these systems and how their gene products, such as Slp, affect pathogenesis in vivo.
E. coli O157:H7 and other EHEC strains pose a large public health burden worldwide, and with the most serious complications and outcomes affecting children under the age of five years, the need for effective treatments or interventions is urgent (3). Interventions are aimed at preventing the transmission from the colonized bovine host at either the pre- or post-harvest stage, by treating or preventing EHEC colonization before slaughter, or reducing or eliminating contamination post-harvest (60). Results of interventions such as vaccines and post-harvest sanitation have produced limited effects (61) (62) (60) (63). Many of the EHEC virulence and adherence related genes are significantly affected by an acidic environment, and this convergence of regulatory systems involving adherence and virulence provides a potential opportunity for interventions or therapies to target these pathways and attenuate virulence or adherence. The identification of an outer membrane protein that is conserved among E. coli and some Shigella species may also provide potential vaccine and intervention targets to mitigate disease caused by these pathogens. Additionally, this approach may contribute to the discovery of other potential initial adherence factors based on the pursuit of pH-influenced genes that may have a higher probability of involvement in initial adherence or other closely related functions of EHEC. In S. pneumoniae, the cell signaling pathway responsible for internalization is also used by other pathogens of diverse natures such as Staphylococcus aureus, Neisseria meningitidis, and Listeria monocytogenes(Agarwal et al., 2010), suggesting the possibility that this adherence mechanism might be more widespread than previously known.
Materials and Methods
Bacterial and mammalian growth and culture
All bacterial strains and plasmids are described in Table 2.
All bacterial cultures were inoculated from single colonies into LB broth with appropriate antibiotics when applicable and grown shaking at 37°C with 5% CO2 overnight. Caco-2 cells (human colonic adenocarcinoma cells; American Type Culture Collection: ATCC HTB-37) were grown at 37°C with 5% CO2, in Eagle’s Minimum Essential Medium (EMEM) (ATCC) plus 20% Fetal Bovine Serum (FBS) (Atlanta Biologicals) unless otherwise indicated. Caco-2 cells were seeded into tissue culture treated six well plates (Corning Life Sciences), containing glass cover slips if used for microscopy, and grown to confluency. pUC18 Complementation cloning plasmid
Immunofluorescent microscopy
Mammalian cells were grown as described, rinsed once with sterile phosphate buffered saline (PBS) and fresh media applied 2 to 4 hours prior to infection. Bacterial strains were inoculated at an MOI of 20, allowed to equilibrate to 37°C for five minutes, and incubated for 0 to 6 hours. After incubation, cell culture media was removed, and cells were washed gently (by tilting and swirling) with sterile PBS three times to remove unadhered bacteria. After infection, samples were fixed using 10% neutral buffered formalin (Azer Scientific) for ten minutes at room temperature and rinsed with PBS three times. Fluorescent stains were applied as listed below, with two PBS rinses in between each stain; slides were stored in glycerol-based mounting media and away from light. The pIgR protein was stained using an unconjugated PIGR rabbit IgG polyclonal antibody (#PA5–22096, Thermo Fisher Scientific) at a1:750 dilution in PBS for 60 minutes, followed by a DyLight 488-conjugated secondary goat anti-Rabbit IgG (H+L) antibody (#35552, Thermo Fisher Scientific), stained at 1:1,000 dilution in PBS for 60 minutes. E. coli were stained using an unconjugated E. coli goat IgG polyclonal antibody (#PA1–73032, Thermo Fisher Scientific), stained in 1:1,000 dilution for 60 minutes, followed by a DyLight 594-conjugated secondary donkey anti-goat IgG (H+L) Secondary Antibody (DyLight 594 #SA5–10088, Thermo Fisher Scientific), stained at 1:1,000 dilution in PBS for 60 minutes. Caco-2 cell nuclei were stained using Hoechst 33342 (#62249, Thermo Fisher Scientific), stained at 1:1,000 dilution in PBS for 5 minutes. Slides were washed three times in sterile PBS and mounted using glycerol based mounting media and stored away from light. Immunofluorescent images were obtained using a Keyence BZ-9000 Fluorescence Microscope (Keyence) with 40X objective lenses (Nikon) with filters for DAPI, GFP, Texas-Red, and phase contrast. Images were taken as z-stacks at 0.5 µM intervals. Covariance was measured using ImageJ (U. S. National Institutes of Health) and the JaCOP plugin (64). Statistical analysis was performed using XLSTAT 2017: Data Analysis and Statistical Solution for Microsoft Excel (Addinsoft). P-values were calculated using the mean PCC (R value) for the Student’s T-test and p ≤0.05 was considered significant (65).
Quantitative adherence
Infection of mammalian cells was done as described. After infection and incubation, samples were collected dispensing 100 µL sterile 10% triton X-100 (Sigma-Aldrich) in PBS and incubated at room temperature for 10 minutes until cells detached from the well plate. A 900 µL volume of sterile PBS was added to rinse the well and samples were collected using a sterile cell scraper for a total sample volume of 1 mL. Samples were resuspended and vortexed vigorously until no visible cell clumps remained, ten-fold serial dilutions were made by adding 100 µL of sample into 900 µL sterile PBS in succession. 100 µL samples of each dilution were plated and grown on LB plates overnight at 37°C before counting. All adherence assays were normalized by calculating colony forming units (CFU) per inoculum to ensure accuracy and performed in triplicate. To calculate adherence, bacterial counts were adjusted by dilution factor, averaged, and the percent adherence (to normalize across samples and experiments) was calculated from the number of adhered bacteria per the number of inoculated bacteria per well. The percent adherence per well was then compared to the maximum adherence count (wild-type count at three hours post-infection) to calculate relative adherence per sample.
Where (#A) = number of adhered bacteria; (#CFU) = number of colonies counted per plate; (PDF) = plate dilution factor; (#I) = number of inoculated bacteria per well (sample); (%AI) = percent adherence of inoculum (normalization between samples); (%AM) = maximum percent adherence (%AI) of wild-type bacteria (three hour samples); (%RA) = relative percent adherence (values shown in figures). P-values were calculated using the mean %RA for the Student’s T-test and p ≤ 0.05 was considered significant (65).
Quantitative PCR (qPCR)
Quantitative (real-time) PCR was done using the purified mRNA samples collected as described in Section 2.6 were reverse-transcribed to cDNA using the iScript cDNA Synthesis kit (BioRad). The cDNA samples were then concentrated and cleaned of reverse transcription reaction components using ethanol precipitation: to each sample, 10% (of total reverse transcription reaction volume) volume of 3M sodium acetate pH 5.2 was added; 2.5 volumes of 100% ethanol was added; sample was vortexed thoroughly and incubated at –20°C for a minimum of six hours to form a DNA precipitate. After precipitation, samples were centrifuged (in a standard microcentrifuge) at ˃10,000xg for 30 minutes at 4°C; pellets were rinsed once with ice-cold 70% ethanol; pellets were then air dried and resuspended in water and assessed for purity and concentration using AD 260/280 ratios. Using cDNA as PCR template, 20 µL qPCR reactions were made using 50–500 ng of template cDNA, 10 µL Applied Biosystems SYBR Green PCR Master Mix (Thermo Fisher Scientific), and molecular-grade water up to 20 uL total volume. The qPCR thermal cycling was done using the 7500/7500 Fast Real-Time PCR System (Applied Biosystems) using the default PCR cycling settings (hold stage: 10 minutes at 95°C; cycling stage (40 cycles): 15 seconds at 95°C + 1 minute at 60°C) and the primers listed in Table 3.
Relative expression was calculated using the relative expression ratio where efficiency (E) was calculated using the slope of a standard curve of ten-fold serial dilutions of DNA template, and fold-change (FC) was calculated using the relative expression ratio as described by Pfaffl (33). when E = efficiency of target or reference gene, ΔCt = (Ct value of control-Ct value of sample), target = the gene of interest, ref = endogenous control gene, control= untreated sample, and sample = experimental sample (33). A fold-change ≥ 2 was considered significant.
Gene deletion and complementation
E. coli O157:H7 was used to make a genomic deletion lacking slp using the lambda Red recombinase system described by Datsenko and Wanner (66). Bacterial strains hosting plasmids were grown with antibiotics according to their respective resistance genes, and plasmids were purified using the Plasmid Midi Kit (Qiagen). PCR, using the mutation primers listed in Table 3, was performed using plasmid pKD3 as a template on an Eppendorf MasterCycler PCR System (Eppendorf). PCR reactions were done in 50 µL volumes: 0.25 µL Taq DNA Polymerase enzyme (5U/µL) (Omega Bio-Tek); 10X PCR buffer (#TQ2100–00, Omega Bio-Tek) at 5 µL; 1.0 µL dNTPs (10mM) (Lucigen); 1.0 µL each of forward and reverse primers (20 µM) (IDT), and water to 50 µL total volume. All PCR cycling conditions were done as follows, with adjusted annealing temperatures noted in Table 2.2: 95°C for 5 minutes; 35 cycles of 95°C for 5 minutes, (Annealing Tm)°C for 30 seconds, 72°C for 3e minutes; followed by a final extension step of 72°C for 5 minutes. PCR products were run in a 1.5% agarose gel in Tris-acetate-EDTA (TAE) buffer at 150 V for 1 to 2 hours to obtain clear band resolution, bands were excised from the gel, and purified using the MinElute Gel Extraction Kit (Qiagen). PCR products were transformed into electrocompetent E. coli O157:H7 containing plasmid pKD119, using volumes between 2 and 5 µL at concentrations between 100 and 500 ng/µL (as measured by Nanovue spectrophotometer). Transformed cells were incubated in 1 mL of pre-warmed 37°C SOC medium and incubated at 37°C for 1 hour, then plated on LB containing chloramphenicol. Colonies were selected for antibiotic resistance and screened by PCR. Transformants positive for antibiotic resistance and negative for screening PCR products were grown and made electrocompetent, transformed with plasmid pCP20 to excise the antibiotic resistance cassette, and screened for antibiotic resistance. Deletions were confirmed by sequencing the junction sequences flanking the deletion site, using The Pennsylvania State University’s Genomics Core Facility (University Park, PA). Plasmid complementation of the deletion strain was made using pUC18 and the primers listed in Table 3. PCR was done using E. coli O157:H7 genomic DNA as a template; and PCR reactions, PCR cycling conditions, PCR product purification, and transformations were done as described.
Co-immunoprecipitation (Co-IP) and Protein Identification
The Co-IP assay was done using the Invitrogen Dynabeads Protein A Immunoprecipitation kit (Invitrogen). Bacterial cultures were grown from starter culture diluted into 100 mL LB broth at an OD of 0.05 and grown with shaking to prevent sediment formation for approximately 14 hours at 37°C. Bacterial pellets were collected by centrifugation at 4°C at 4,000xg for 20 minutes and resuspended in 5mL water for sonication. Sonication was done using a hand-held sonicator (Thermo Fisher Scientific) on ice for a total of 20 minutes per sample, or until lysate turbidity was visibly reduced. A 5 mL volume of sonicated bacterial lysate was aliquoted into 1 mL samples. Untreated lysate was not incubated with protein; treated lysate was incubated with 10µg of Recombinant Human Polymeric Immunoglobulin Receptor/PIgR (C-Fc) CI09 protein purchased from Novoprotein (Novoprotein) containing an Fc tag at the C-terminus of the protein. Protein-lysate incubation was done overnight at 4°C for 16–18 hours on a rotator, and proteins collected using the Co-IP kit. Protein A coated beads from the kit were prepared for use and incubated with the lysate for 30 minutes at 4°C on a rotator. Beads were collected using a magnet stand and washed twice with kit wash solution. Beads pellet was then resuspended in 10 µL of kit elution solution and 10 µL of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer and incubated at 95°C for 5 minutes before loading into a 12% acrylamide SDS-PAGE gel. The SDS-PAGE gel was run for two hours at 100 V at room temperature and stained with Coomasie blue (BioRad). All protein identifications were done at protein facilities using liquid chromatography and tandem mass spectrometry (LC MS/MS) analysis. A total of three analyses were completed, at The Pennsylvania State Proteomics and Mass Spectrometry Core Facility (1) and the Protein Facility of the Iowa State University Office of Biotechnology (2 and 3). The protein bands were cut from the SDS-PAGE gel and processed at the respective facility. Peptide fragmentation patterns were compared to known databases of proteins of E. coli (PSU using SEQUEST and Uniprot; ISU using Mascot or Sequest HT). Raw data was analyzed using Thermo Scientific’s Proteome Discoverer Software.
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