- Email: [email protected]
YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop
Accepted Manuscript YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop Rima Tapader, Dipro Bose, Amit Pal PII:
S0882-4010(16)30799-9
DOI:
10.1016/j.micpath.2017.02.020
Reference:
YMPAT 2129
To appear in:
Microbial Pathogenesis
Received Date: 20 November 2016 Revised Date:
9 February 2017
Accepted Date: 13 February 2017
Please cite this article as: Tapader R, Bose D, Pal A, YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.02.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Title Page
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Title: YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop
5
Authors
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Rima Tapader1, Dipro Bose2, Amit Pal1 Affiliations 1
SC
7 8 9 10
RI PT
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Division of Pathophysiology, National Institute of Cholera and Enteric Diseases (NICED),
Kolkata-10, India, 2Centre for Infectious Diseases & Control, School of Biosciences and
12
Technology, VIT University, Vellore-632014, India.
13 14
Address for correspondence
15
Amit Pal,
16
Mailing address: Division of Pathophysiology, National Institute of Cholera and Enteric
17
Diseases, P33, CIT Road, Scheme XM, Beliaghata, Kolkata, 700010
18
Phone: +91-33-2353-7470; ext: 3094. Fax: +91-33-2370-5066
19
Email: [email protected]
20
Running Title
21
YghJ induces hemorrhagic fluid accumulation in mouse ileum
22
Keywords
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YghJ (SsLE); mouse ileal loop; hemorrhagic fluid response; Intestinal pathogenic E. coli; Neonatal septicemic E. coli
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Abstract
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YghJ, also known as SsLE (Secreted and surface associated lipoprotein) is a cell surface
29
associated and secreted lipoprotein harbouring M60 metalloprotease domain. Though the
30
gene is known to be conserved among both pathogenic and commensal E. coli isolates, the
31
expression and secretion of YghJ was found to be higher among diverse Escherichia coli
32
pathotypes. YghJ, secreted from intestinal pathogens such as enterotoxigenic E. coli (ETEC)
33
and enteropathogenic E. coli (EPEC) has been demonstrated to possess mucinase activity and
34
hence facilitates colonization of these enteric pathogens to intestinal epithelial cells.
35
Importantly, YghJ is also reported to be secreted from extraintestinal pathogenic E. coli
36
isolates. In our previous study we have shown that YghJ, purified from a neonatal septicemic
37
E. coli isolate could trigger induction of various proinflammatory cytokines in vitro. This led
38
us to investigate the role of YghJ in causing in vivo tissue hemorrhage. In the present study,
39
we validate the earlier in vitro finding and have showed that YghJ can cause extensive tissue
40
damage in mouse ileum and is also able to induce significant fluid accumulation in a dose
41
dependent manner in a mouse ileal loop (MIL) assay. Hence, our present study not only
42
confirms the pathogenic potential of YghJ in sepsis pathophysiology but also indicates the
43
enterotoxic ability of YghJ which makes it an important virulence determinant of intestinal
44
pathogenic E. coli.
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Introduction
52
YghJ belongs to a large and diverse family of proteins containing a putative metalloprotease
53
domain named M60-like pfam 13402 which is defined as a novel sub-family of extracellular
54
zinc (Zn)-metallopeptidases. This novel domain was found to be conserved among diverse
55
prokaryotic and eukaryotic microbes including commensals as well as pathogens of
56
invertebrates and vertebrates [1]. YghJ was identified as a conserved, secreted molecule in an
57
immunoproteome [2] and transcriptome analysis of enterotoxigenic Escherichia coli (ETEC)
58
[3]. It has been shown to possess mucinase activity in a similar manner like its closest
59
homolog V. cholerae colonization factor AcfD [4]. Though the gene is reported to be
60
conserved also among the commensal E. coli, the expression and secretion of YghJ among
61
pathogenic E. coli isolates was found to be significantly higher than the commensal E. coli
62
isolates [4,5]. In another recent study, YghJ was shown to be expressed during the course of
63
ETEC infection and was suggested to be one of such proteins that trigger immune response to
64
ETEC infection [6]. YghJ also known as SsLE (Secreted and surface associated lipoprotein)
65
was found to be secreted from other intestinal E. coli pathotypes such as enteropathogenic E.
66
coli (EPEC) and has been shown to aid in the colonization of this pathogen to the intestinal
67
epithelial cells [7]. Interestingly, in a study by Serino et al., YghJ was identified as one of the
68
most potential vaccine candidates for ExPEC isolates by a “substractive reverse vaccinology”
69
approach [8].
70
In our earlier study we have identified YghJ in the culture supernatant of a clinical neonatal
71
septicemic Escherichia coli (NSEC) isolate [5]. We reported that recombinant YghJ (rYghJ)
72
could induce production of several proinflammatory cytokines that are essentially implicated
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3
ACCEPTED MANUSCRIPT in sepsis pathophysiology such as IL-8, TNF-α, IL-1α, IL-1β and also down-regulated
74
production of major anti-inflammatory cytokine IL-10 in intestinal epithelial cells and mouse
75
macrophages [5]. This gave us a hint to further explore the effect of YghJ in causing in vivo
76
tissue hemorrhage. In our present study, we substantiate the role of YghJ in promoting
77
virulence of pathogenic E. coli isolates in a mouse ileal loop (MIL) assay and delineate that
78
YghJ could cause substantial tissue haemorrhage in mouse ileum. This study strongly
79
supports our previous finding and is the first to report the role of YghJ in triggering
80
hemorrhagic fluid accumulation in mouse ileal loop (MIL) assay.
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Materials and Methods
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Purification of recombinant YghJ (rYghJ)
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rYghJ was purified as described previously [5]. Briefly, the recombinant clone pTYghJ7 was
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induced with 0.02% L-arabinose for 5 hrs for optimum expression of YghJ. rYghJ was
85
purified by Ni-NTA column chromatography and successive Gel–filtration chromatography
86
(Sephadex G-200). Protease activity was confirmed using Methoxysuccinyl (MeOSuc)-Ala-
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Ala-Pro-Met-p-nitroanilide (AAPM pNA) (Calbiochem) oligopeptide substrate.
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Mouse intestinal fluid accumulation
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The diarrheagenic potential of rYghJ and its ability to cause in vivo tissue damage was
90
assessed by MIL assay as described previously [9]. Briefly, 4-5 weeks old adult BALB/c
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mice were anesthesized with ketamine and rYghJ was injected at various doses from 20
92
µg/ml (120 nM) to 60 µg/ml (360 nM) into ligated mouse ileum of about 5 cm with PBS as
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control. After 6 h of incubation, mice were sacrificed and loops were excised to determine the
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fluid accumulation (FA) ratio. FA as expressed by fluid accumulated in loop (g) to the length
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of the loop (cm) was estimated to evaluate the enterotoxic activity of rYghJ. Values were
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expressed as mean ± standard deviation (SD) of the mean (n=5 mice per group).
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Histopathological analysis of mouse ileal tissues
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For histopathological analysis, mouse ileal tissues were collected and fixed in 10% neutral
100
buffered formalin solution. Three-four micrometer tissue sections were cut in a microtomy
101
rotor (Leica, Germany), embedded in paraffin and further processed using standard protocol.
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The sections were stained with hematoxylin and eosin and observed under light microscope.
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Images were taken with Leica DMLB microscope (Solms, Germany) under a magnification
104
of X40.
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Statistical analysis
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All experiments were performed in triplicates and values were expressed as mean ± standard
107
deviation of the mean. Statistical significance was determined using student’s t-test and the
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Bonferroni test with one asterick indicating p value between 0.01 to 0.05 and two astericks
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indicating p value between 0.01 to 0.001.
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Results
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Purification of rYghJ
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rYghJ showed presence of single band in NATIVE and SDS-PAGE (data not shown).
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rYghJ induced significant hemorrhagic fluid accumulation in MIL
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rYghJ showed a dose-dependent hemorrhagic fluid response in MIL (Fig.1A). At 120 nM FA
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ratio was insignificant and no hemorrhage could be detected. At 150 nM fluid accumulation
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without hemorrhage was observed (FA ratio, 0.122±0.022). With increasing concentration of
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ACCEPTED MANUSCRIPT rYghJ upto 360 nM, significant increase in hemorrhagic fluid accumulation was found as
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evidenced by remarkably higher FA ratios (FA ratio, 0.245±0.03) (Fig. 1). YghJ was also
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incubated with Polymyxin B (PMB) at a dose of 50 µg/ml for 1 hr before injection. YghJ
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even in the presence of PMB showed significant hemorrhagic fluid accumulation (FA ratio
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without PMB, 0.17±0.025 vs FA ratio with PMB, 0.155±0.03) (Fig. 1). Furthermore, on
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incubation of YghJ with metalloprotease inhibitors EDTA (10 mM) and 1,10 Phenanthroline
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(10 mM), the FA ratio was remarkably reduced.
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rYghJ caused extensive damage to mouse ileum
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The results of MIL were further substantiated by histopathological analysis of the ileal
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tissues. Histopathological analysis showed normal villi with mucosal and submucosal
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structure in PBS treated controls (Fig. 2A). At a dose of 180 nM of YghJ, the villous
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architechture was grossly ruptured, dilated and accumulation of RBCs and inflammatory cells
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were observed in the mucosa (Fig. 2B). At the dose of 360 nM of YghJ, the villous structure
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was completely distorted with accumulation of RBCs and inflammatory cells at the lamina
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propia and the epithelium of the mucosa as well as in the submucosa. The crypt epithelium
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was also found to be remarkably damaged (Fig. 2C). On treatment with EDTA and 1,10-
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Phenanthroline, all the layers of the ileal tissue exhibited less damage and retained almost
134
normal villi architechture. The mucosa and submucosa showed significantly reduced
135
hemorrhage (Fig. 2D).
136
Discussion
137
Secreted microbial proteases are one of the most critical and essential weapons of pathogens
138
to exacerbate the virulence of a particular pathogen in the progression of a disease [10]. YghJ,
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the extracellular metalloprotease is reported to be secreted from diverse E. coli pathotypes
140
including both intestinal [4,7] and extraintestinal pathogenic E. coli [5] and is proposed to be
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ACCEPTED MANUSCRIPT one of the most potent vaccine candidates for different E. coli pathotypes [2,8]. However, the
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pathogenic role of YghJ in promoting virulence of its producer organisms is still not explored
143
completely. YghJ produced from ETEC and EPEC has been shown to possess mucinase
144
activity and hence is suggested to have role in colonization of intestinal pathogenic E. coli
145
[4,7]. In our recent study, we reported that YghJ cloned from a neonatal septicemic E. coli
146
can alter cellular morphology of various cell lines and can trigger induction of several
147
proinflammatory cytokines which are attributed as one of the key mediators in the
148
pathogenesis of sepsis [5]. However, the role of YghJ in causing in vivo tissue hemorrhage
149
has not been studied till date. In this study we found that purified rYghJ caused extensive
150
hemorrhage to mouse ileum in a dose dependent manner which strikingly validates our
151
previous findings. In addition, the ability of YghJ to induce hemorrhagic fluid accumulation
152
in mouse ileum essentially reflects the enterotoxicity of YghJ and is also supportive to earlier
153
reports where YghJ has been demonstrated to be highly prevalent also among intestinal
154
pathogenic E. coli (IPECs). This clearly indicates that YghJ promotes virulence in IPECs not
155
only by accelerating colonization but also enhances the diarrheagenic potential of ETEC and
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EPEC. The fluid accumulation of purified rYghJ could be inhibited using specific Zn++
157
containing metalloprotease inhibitor 1,10 Phenanthroline which undoubtedly establishes the
158
involvement of M60 metalloprotease domain in the enterotoxicity of YghJ. Further studies
159
are being done to construct yghJ deletion mutant to substantiate its role in hemorrhagic fluid
160
response. One common limitation of expressing recombinant His-Tag protein in E. coli is the
161
contamination with LPS [11], known to induce proinflammatory cytokines [12]. Polymyxin
162
B (PMB), the Bacillus polymyxa-derived antibiotic binds with a very strong affinity to the
163
Lipid A moiety of LPS and hence can block the LPS-mediated proinflammation [13,14].
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Induction of hemorrhagic fluid accumulation even in the presence of PMB clearly ascertains
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the enterotoxic role of YghJ which is completely independent of LPS.
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ACCEPTED MANUSCRIPT In conclusion, we report for the first time that YghJ causes extensive hemorrhage in mouse
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ileum in a dose dependent manner and the metalloprotease domain of YghJ is primarily
168
attributable to this hemorrhagic response of YghJ. This finding strongly supports our
169
previous report that YghJ can induce secretion of proinflammatory cytokines in vitro.
170
Furthermore, by establishing the ability of YghJ to induce hemorrhagic fluid accumulation in
171
mouse ileum suggests that YghJ could be one of such virulence factors of those enteric
172
pathogens associated with hemorrhagic diarrhea. Future studies are required to establish the
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enterotoxicity of YghJ in intestinal pathogenic E. coli causing hemorrhagic diarrhea and also
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among other enteric pathogens implicated in bloody diarhhea.
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Ethical approval
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Animal experiments were performed with necessary permission from the Institutional
178
Animal
179
Ethical Committee (IAEC) (Approval≠64/IAEC/NICED/2009).
180
Conflicts of interest
181
All authors declare that they have no conflicts of interest.
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Funding
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This work was supported by intramural funding (Indian Council of Medical Research). RT
184
was supported by a fellowship from University Grants Commission (UGC), India.
185
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9. A. Mondal, R. Tapader, N.S. Chatterjee, A. Ghosh, R. Sinha, H. Koley, et al., Cytotoxic and inflammatory responses induced by Outer Membrane Vesicles
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14. D. Van der Kleij, A.H. Van den Biggelaar, Y.C. Kruize, K. Retra, Y. Fillie, et al., Responses to Toll-like receptor ligands in children living in areas where
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schistosome infections are endemic, J. Infect. Dis. 189 (2004) 1044-1051.
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Figure Legends
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Figure 1. YghJ induces hemorrhagic fluid accumulation in mouse ileal loop assay.
246
Images of ligated mouse ileal loops treated with different doses of rYghJ (A) clearly
247
indicates hemorrhagic fluid accumulation in a dose dependent manner even in presence of
248
PMB, compared to the PBS treated negative control. Significant reduction in fluid
249
accumulation is seen on incubation of YghJ with EDTA and 1,10 Phe (A). The FA ratio (gm
250
of fluid accumulated to the length of loop in cm) for each treatment is represented in the
251
histogram (B) as mean ± SD (n=5 mice per treatment). Significance was determined using
252
student’s t-test and the Bonferroni test with one asterick indicating p value between 0.01 to
253
0.05 and two astericks indicating p value between 0.01 to 0.001.
254
Figure 2. Histopathological analysis of mouse ileum at a magnification of X40. A) PBS
255
treated control shows normal villi with mucosal and submucosal structure, B) YghJ, at a dose
256
of 180 nM causes rupture and dilation in the villi with accumulation of RBCs and in the
257
mucosa, C) At a dose of 360 nM, villi pattern are found to be completely distorted with
258
accumulation of RBCs and inflammatory cells in both mucosa and submucosa which reduces
259
remarkably on incubation of YghJ with EDTA and 1,10 phenanthroline (D).
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ACCEPTED MANUSCRIPT
E. coli recombinant YghJ causes significant fluid accumulation in mouse ileal loop.
•
YghJ can cause extensive tissue hemorrhage in mouse ileum.
•
Metalloprotease domain of YghJ is attributable to the hemorrhagic fluid response.
•
This study indicates that YghJ has enterotoxic ability.
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S0882-4010(16)30799-9
DOI:
10.1016/j.micpath.2017.02.020
Reference:
YMPAT 2129
To appear in:
Microbial Pathogenesis
Received Date: 20 November 2016 Revised Date:
9 February 2017
Accepted Date: 13 February 2017
Please cite this article as: Tapader R, Bose D, Pal A, YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.02.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Title Page
3 4
Title: YghJ, the secreted metalloprotease of pathogenic E. coli induces hemorrhagic fluid accumulation in mouse ileal loop
5
Authors
6
Rima Tapader1, Dipro Bose2, Amit Pal1 Affiliations 1
SC
7 8 9 10
RI PT
2
Division of Pathophysiology, National Institute of Cholera and Enteric Diseases (NICED),
Kolkata-10, India, 2Centre for Infectious Diseases & Control, School of Biosciences and
12
Technology, VIT University, Vellore-632014, India.
13 14
Address for correspondence
15
Amit Pal,
16
Mailing address: Division of Pathophysiology, National Institute of Cholera and Enteric
17
Diseases, P33, CIT Road, Scheme XM, Beliaghata, Kolkata, 700010
18
Phone: +91-33-2353-7470; ext: 3094. Fax: +91-33-2370-5066
19
Email: [email protected]
20
Running Title
21
YghJ induces hemorrhagic fluid accumulation in mouse ileum
22
Keywords
23 24
YghJ (SsLE); mouse ileal loop; hemorrhagic fluid response; Intestinal pathogenic E. coli; Neonatal septicemic E. coli
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1
ACCEPTED MANUSCRIPT 26
Abstract
28
YghJ, also known as SsLE (Secreted and surface associated lipoprotein) is a cell surface
29
associated and secreted lipoprotein harbouring M60 metalloprotease domain. Though the
30
gene is known to be conserved among both pathogenic and commensal E. coli isolates, the
31
expression and secretion of YghJ was found to be higher among diverse Escherichia coli
32
pathotypes. YghJ, secreted from intestinal pathogens such as enterotoxigenic E. coli (ETEC)
33
and enteropathogenic E. coli (EPEC) has been demonstrated to possess mucinase activity and
34
hence facilitates colonization of these enteric pathogens to intestinal epithelial cells.
35
Importantly, YghJ is also reported to be secreted from extraintestinal pathogenic E. coli
36
isolates. In our previous study we have shown that YghJ, purified from a neonatal septicemic
37
E. coli isolate could trigger induction of various proinflammatory cytokines in vitro. This led
38
us to investigate the role of YghJ in causing in vivo tissue hemorrhage. In the present study,
39
we validate the earlier in vitro finding and have showed that YghJ can cause extensive tissue
40
damage in mouse ileum and is also able to induce significant fluid accumulation in a dose
41
dependent manner in a mouse ileal loop (MIL) assay. Hence, our present study not only
42
confirms the pathogenic potential of YghJ in sepsis pathophysiology but also indicates the
43
enterotoxic ability of YghJ which makes it an important virulence determinant of intestinal
44
pathogenic E. coli.
SC
M AN U
TE D
EP
AC C
45
RI PT
27
46
47
48
2
ACCEPTED MANUSCRIPT 49
50
Introduction
52
YghJ belongs to a large and diverse family of proteins containing a putative metalloprotease
53
domain named M60-like pfam 13402 which is defined as a novel sub-family of extracellular
54
zinc (Zn)-metallopeptidases. This novel domain was found to be conserved among diverse
55
prokaryotic and eukaryotic microbes including commensals as well as pathogens of
56
invertebrates and vertebrates [1]. YghJ was identified as a conserved, secreted molecule in an
57
immunoproteome [2] and transcriptome analysis of enterotoxigenic Escherichia coli (ETEC)
58
[3]. It has been shown to possess mucinase activity in a similar manner like its closest
59
homolog V. cholerae colonization factor AcfD [4]. Though the gene is reported to be
60
conserved also among the commensal E. coli, the expression and secretion of YghJ among
61
pathogenic E. coli isolates was found to be significantly higher than the commensal E. coli
62
isolates [4,5]. In another recent study, YghJ was shown to be expressed during the course of
63
ETEC infection and was suggested to be one of such proteins that trigger immune response to
64
ETEC infection [6]. YghJ also known as SsLE (Secreted and surface associated lipoprotein)
65
was found to be secreted from other intestinal E. coli pathotypes such as enteropathogenic E.
66
coli (EPEC) and has been shown to aid in the colonization of this pathogen to the intestinal
67
epithelial cells [7]. Interestingly, in a study by Serino et al., YghJ was identified as one of the
68
most potential vaccine candidates for ExPEC isolates by a “substractive reverse vaccinology”
69
approach [8].
70
In our earlier study we have identified YghJ in the culture supernatant of a clinical neonatal
71
septicemic Escherichia coli (NSEC) isolate [5]. We reported that recombinant YghJ (rYghJ)
72
could induce production of several proinflammatory cytokines that are essentially implicated
AC C
EP
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M AN U
SC
RI PT
51
3
ACCEPTED MANUSCRIPT in sepsis pathophysiology such as IL-8, TNF-α, IL-1α, IL-1β and also down-regulated
74
production of major anti-inflammatory cytokine IL-10 in intestinal epithelial cells and mouse
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macrophages [5]. This gave us a hint to further explore the effect of YghJ in causing in vivo
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tissue hemorrhage. In our present study, we substantiate the role of YghJ in promoting
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virulence of pathogenic E. coli isolates in a mouse ileal loop (MIL) assay and delineate that
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YghJ could cause substantial tissue haemorrhage in mouse ileum. This study strongly
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supports our previous finding and is the first to report the role of YghJ in triggering
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hemorrhagic fluid accumulation in mouse ileal loop (MIL) assay.
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Materials and Methods
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Purification of recombinant YghJ (rYghJ)
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rYghJ was purified as described previously [5]. Briefly, the recombinant clone pTYghJ7 was
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induced with 0.02% L-arabinose for 5 hrs for optimum expression of YghJ. rYghJ was
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purified by Ni-NTA column chromatography and successive Gel–filtration chromatography
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(Sephadex G-200). Protease activity was confirmed using Methoxysuccinyl (MeOSuc)-Ala-
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Ala-Pro-Met-p-nitroanilide (AAPM pNA) (Calbiochem) oligopeptide substrate.
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Mouse intestinal fluid accumulation
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The diarrheagenic potential of rYghJ and its ability to cause in vivo tissue damage was
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assessed by MIL assay as described previously [9]. Briefly, 4-5 weeks old adult BALB/c
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mice were anesthesized with ketamine and rYghJ was injected at various doses from 20
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µg/ml (120 nM) to 60 µg/ml (360 nM) into ligated mouse ileum of about 5 cm with PBS as
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control. After 6 h of incubation, mice were sacrificed and loops were excised to determine the
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fluid accumulation (FA) ratio. FA as expressed by fluid accumulated in loop (g) to the length
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of the loop (cm) was estimated to evaluate the enterotoxic activity of rYghJ. Values were
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expressed as mean ± standard deviation (SD) of the mean (n=5 mice per group).
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Histopathological analysis of mouse ileal tissues
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For histopathological analysis, mouse ileal tissues were collected and fixed in 10% neutral
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buffered formalin solution. Three-four micrometer tissue sections were cut in a microtomy
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rotor (Leica, Germany), embedded in paraffin and further processed using standard protocol.
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The sections were stained with hematoxylin and eosin and observed under light microscope.
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Images were taken with Leica DMLB microscope (Solms, Germany) under a magnification
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of X40.
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Statistical analysis
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All experiments were performed in triplicates and values were expressed as mean ± standard
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deviation of the mean. Statistical significance was determined using student’s t-test and the
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Bonferroni test with one asterick indicating p value between 0.01 to 0.05 and two astericks
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indicating p value between 0.01 to 0.001.
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Results
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Purification of rYghJ
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rYghJ showed presence of single band in NATIVE and SDS-PAGE (data not shown).
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rYghJ induced significant hemorrhagic fluid accumulation in MIL
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rYghJ showed a dose-dependent hemorrhagic fluid response in MIL (Fig.1A). At 120 nM FA
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ratio was insignificant and no hemorrhage could be detected. At 150 nM fluid accumulation
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without hemorrhage was observed (FA ratio, 0.122±0.022). With increasing concentration of
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evidenced by remarkably higher FA ratios (FA ratio, 0.245±0.03) (Fig. 1). YghJ was also
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incubated with Polymyxin B (PMB) at a dose of 50 µg/ml for 1 hr before injection. YghJ
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even in the presence of PMB showed significant hemorrhagic fluid accumulation (FA ratio
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without PMB, 0.17±0.025 vs FA ratio with PMB, 0.155±0.03) (Fig. 1). Furthermore, on
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incubation of YghJ with metalloprotease inhibitors EDTA (10 mM) and 1,10 Phenanthroline
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(10 mM), the FA ratio was remarkably reduced.
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rYghJ caused extensive damage to mouse ileum
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The results of MIL were further substantiated by histopathological analysis of the ileal
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tissues. Histopathological analysis showed normal villi with mucosal and submucosal
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structure in PBS treated controls (Fig. 2A). At a dose of 180 nM of YghJ, the villous
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architechture was grossly ruptured, dilated and accumulation of RBCs and inflammatory cells
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were observed in the mucosa (Fig. 2B). At the dose of 360 nM of YghJ, the villous structure
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was completely distorted with accumulation of RBCs and inflammatory cells at the lamina
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propia and the epithelium of the mucosa as well as in the submucosa. The crypt epithelium
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was also found to be remarkably damaged (Fig. 2C). On treatment with EDTA and 1,10-
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Phenanthroline, all the layers of the ileal tissue exhibited less damage and retained almost
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normal villi architechture. The mucosa and submucosa showed significantly reduced
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hemorrhage (Fig. 2D).
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Discussion
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Secreted microbial proteases are one of the most critical and essential weapons of pathogens
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to exacerbate the virulence of a particular pathogen in the progression of a disease [10]. YghJ,
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the extracellular metalloprotease is reported to be secreted from diverse E. coli pathotypes
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including both intestinal [4,7] and extraintestinal pathogenic E. coli [5] and is proposed to be
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pathogenic role of YghJ in promoting virulence of its producer organisms is still not explored
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completely. YghJ produced from ETEC and EPEC has been shown to possess mucinase
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activity and hence is suggested to have role in colonization of intestinal pathogenic E. coli
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[4,7]. In our recent study, we reported that YghJ cloned from a neonatal septicemic E. coli
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can alter cellular morphology of various cell lines and can trigger induction of several
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proinflammatory cytokines which are attributed as one of the key mediators in the
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pathogenesis of sepsis [5]. However, the role of YghJ in causing in vivo tissue hemorrhage
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has not been studied till date. In this study we found that purified rYghJ caused extensive
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hemorrhage to mouse ileum in a dose dependent manner which strikingly validates our
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previous findings. In addition, the ability of YghJ to induce hemorrhagic fluid accumulation
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in mouse ileum essentially reflects the enterotoxicity of YghJ and is also supportive to earlier
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reports where YghJ has been demonstrated to be highly prevalent also among intestinal
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pathogenic E. coli (IPECs). This clearly indicates that YghJ promotes virulence in IPECs not
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only by accelerating colonization but also enhances the diarrheagenic potential of ETEC and
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EPEC. The fluid accumulation of purified rYghJ could be inhibited using specific Zn++
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containing metalloprotease inhibitor 1,10 Phenanthroline which undoubtedly establishes the
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involvement of M60 metalloprotease domain in the enterotoxicity of YghJ. Further studies
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are being done to construct yghJ deletion mutant to substantiate its role in hemorrhagic fluid
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response. One common limitation of expressing recombinant His-Tag protein in E. coli is the
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contamination with LPS [11], known to induce proinflammatory cytokines [12]. Polymyxin
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B (PMB), the Bacillus polymyxa-derived antibiotic binds with a very strong affinity to the
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Lipid A moiety of LPS and hence can block the LPS-mediated proinflammation [13,14].
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Induction of hemorrhagic fluid accumulation even in the presence of PMB clearly ascertains
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the enterotoxic role of YghJ which is completely independent of LPS.
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ACCEPTED MANUSCRIPT In conclusion, we report for the first time that YghJ causes extensive hemorrhage in mouse
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ileum in a dose dependent manner and the metalloprotease domain of YghJ is primarily
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attributable to this hemorrhagic response of YghJ. This finding strongly supports our
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previous report that YghJ can induce secretion of proinflammatory cytokines in vitro.
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Furthermore, by establishing the ability of YghJ to induce hemorrhagic fluid accumulation in
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mouse ileum suggests that YghJ could be one of such virulence factors of those enteric
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pathogens associated with hemorrhagic diarrhea. Future studies are required to establish the
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enterotoxicity of YghJ in intestinal pathogenic E. coli causing hemorrhagic diarrhea and also
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among other enteric pathogens implicated in bloody diarhhea.
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Ethical approval
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Animal experiments were performed with necessary permission from the Institutional
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Animal
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Ethical Committee (IAEC) (Approval≠64/IAEC/NICED/2009).
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Conflicts of interest
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All authors declare that they have no conflicts of interest.
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Funding
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This work was supported by intramural funding (Indian Council of Medical Research). RT
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was supported by a fellowship from University Grants Commission (UGC), India.
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Figure Legends
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Figure 1. YghJ induces hemorrhagic fluid accumulation in mouse ileal loop assay.
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Images of ligated mouse ileal loops treated with different doses of rYghJ (A) clearly
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indicates hemorrhagic fluid accumulation in a dose dependent manner even in presence of
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PMB, compared to the PBS treated negative control. Significant reduction in fluid
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accumulation is seen on incubation of YghJ with EDTA and 1,10 Phe (A). The FA ratio (gm
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of fluid accumulated to the length of loop in cm) for each treatment is represented in the
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histogram (B) as mean ± SD (n=5 mice per treatment). Significance was determined using
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student’s t-test and the Bonferroni test with one asterick indicating p value between 0.01 to
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0.05 and two astericks indicating p value between 0.01 to 0.001.
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Figure 2. Histopathological analysis of mouse ileum at a magnification of X40. A) PBS
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treated control shows normal villi with mucosal and submucosal structure, B) YghJ, at a dose
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of 180 nM causes rupture and dilation in the villi with accumulation of RBCs and in the
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mucosa, C) At a dose of 360 nM, villi pattern are found to be completely distorted with
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accumulation of RBCs and inflammatory cells in both mucosa and submucosa which reduces
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remarkably on incubation of YghJ with EDTA and 1,10 phenanthroline (D).
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E. coli recombinant YghJ causes significant fluid accumulation in mouse ileal loop.
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YghJ can cause extensive tissue hemorrhage in mouse ileum.
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Metalloprotease domain of YghJ is attributable to the hemorrhagic fluid response.
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This study indicates that YghJ has enterotoxic ability.
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