Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12

ABSTRACT Escherichia coli is one of the most common gram-negative bacteria that cause meningitis in neonates. Our previous studies have shown that outer membrane protein A (OmpA) of E. coli interacts with a 95-kDa human brain microvascular endothelial cell (HBMEC) glycoprotein, Ecgp, for invasion. Here, we report the identification of a gene that encodes Ecgp by screening of an HBMEC cDNA expression library as well as by 5′ rapid amplification of cDNA ends. The sequence of the Ecgp gene shows that it is highly similar to gp96, a tumor rejection antigen-1, and contains an endoplasmic reticulum retention signal, KDEL. Overexpression of either Ecgp or gp96 in both HBMECs and CHO cells increases E. coli binding and invasion. We further show that Ecgp gene-transfected HBMECs express Ecgp on the cell surface despite the presence of the KDEL motif. Northern blot analysis of total RNA from various eukaryotic cells indicates that Ecgp is significantly expressed in HBMECs. Recombinant His-tagged Ecgp blocked E. coli invasion efficiently by binding directly to the bacteria. These results suggest that OmpA of E. coli K1 interacts with a gp96-like molecule on HBMECs for invasion.

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Immunoprotective evaluation of Escherichia coli outer membrane protein A against the main pathogens of animal mastitis

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v19i1.23 ◽

2020 ◽

Vol 19 (1) ◽

pp. 155-162

Author(s):

Chen Chen ◽

Nana Wu ◽

Na Rong ◽

Chao Kang ◽

Chunlin Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein A ◽

Enzyme Linked Immunosorbent Assay ◽

Protective Function ◽

E Coli ◽

Ompa Protein ◽

Outer Membrane Protein A

Purpose: To evaluate prokaryotic expression of the Escherichia coli (E. coli) outer membrane protein A (OmpA) and its immunoprotective function against the main pathogens of animal mastitis.Methods: A molecular cloning method was used to develop a prokaryotic strain expressing OmpA protein, which was purified by Ni-affinity chromatography. Polyclonal antiserum was generated in mice immunized with OmpA protein. Enzyme-linked immunosorbent assay (ELISA) and western blotting were used to determine the titer and verify anti-OmpA serum specificity, respectively. Interaction between OmpA antiserum and main pathogens of animal mastitis was verified by ELISA and a pull-down method. The immune protective function of OmpA protein was evaluated in mice challenged with pathogens of animal mastitis. Optimal fermentation conditions to produce OmpA protein were determined by the L9(34) orthogonal test.Results: A prokaryotic strain expressing OmpA protein was developed, and purified OmpA was used to develop a mouse polyclonal antibody. The anti-OmpA serum exhibited high specificity and a titer of 1:1600. Anti-OmpA serum directly interacted with E. coli and Staphylococcus aureus (S. aureus). OmpA demonstrated a significant immune protective function of 58.33 % against E. coli and 46.15 % against S. aureus. The optimal conditions for expressing fermentation OmpA were a strain absorbance of 0.5 at a wavelength of 600 nm, IPTG final concentration of 0.3 mmol/L, induction time of 12 h, and induction temperature of 28 °C.Conclusion: OmpA possesses selective immunogenicity and a significant immune protective effect against the main pathogens of animal mastitis. The results suggest that OmpA may potentially be used as a vaccine for animal mastitis. Keywords: E. coli, OmpA protein, Immunoprotection, Animal mastitis, Protein fermentation

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Sequence of the regulatory region of ompT, the gene specifying major outer membrane protein a (3b) of Escherichia coli K-12: Implications for regulation and processing

MGG Molecular & General Genetics ◽

10.1007/bf00382077 ◽

1984 ◽

Vol 193 (3) ◽

pp. 414-421 ◽

Cited By ~ 3

Author(s):

Gerald Gordon ◽

Randall C. Gayda ◽

Alvin Markovitz

Keyword(s):

Escherichia Coli ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein A ◽

Regulatory Region ◽

Major Outer Membrane Protein ◽

Outer Membrane Protein A ◽

K 12

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Interaction of e. coli outer-membrane protein A with sugars on the receptors of the brain microvascular endothelial cells

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.10257 ◽

2002 ◽

Vol 50 (2) ◽

pp. 213-221 ◽

Cited By ~ 25

Author(s):

Deepshikha Datta ◽

Nagarajan Vaidehi ◽

Wely B. Floriano ◽

Kwang S. Kim ◽

Nemani V. Prasadarao ◽

...

Keyword(s):

Endothelial Cells ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein A ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Outer Membrane Protein A ◽

The Brain

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Entry and Intracellular Replication of Escherichia coli K1 in Macrophages Require Expression of Outer Membrane Protein A

Infection and Immunity ◽

10.1128/iai.71.10.5951-5961.2003 ◽

2003 ◽

Vol 71 (10) ◽

pp. 5951-5961 ◽

Cited By ~ 60

Author(s):

Sunil K. Sukumaran ◽

Hiroyuki Shimada ◽

Nemani V. Prasadarao

Keyword(s):

Escherichia Coli ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Human Peripheral Blood ◽

Protein A ◽

Raw 264.7 Cells ◽

Human Macrophage ◽

E Coli ◽

Outer Membrane Protein A

ABSTRACT Interactions between Escherichia coli K1, which causes meningitis in neonates, and macrophages have not been explored well. In this study we found that E. coli K1 was able to enter, survive, and replicate intracellularly in both murine and human macrophage cell lines, as well as in monocytes and macrophages of newborn rats. In addition, we demonstrated that OmpA + E. coli also enters and replicates in human peripheral blood monocytes in vitro. Outer membrane protein A (OmpA) expression on E. coli contributes to binding to macrophages, phagocytosis, and survival within macrophages. Opsonization with either complement proteins or antibody is not required for uptake and survival of the bacteria within the macrophages. Transmission electron microscopy and immunocytochemistry studies with the infected macrophages indicated that OmpA+ E. coli multiplies enormously in a single phagosome and bursts the cell. Internalization of OmpA+ E. coli by RAW 264.7 cells occurred by both actin- and microtubule-dependent processes, which are independent of RGD-mediated integrin receptors. Internalization and intracellular survival within phagocytic cells thus may play an important role in the development of bacteremia, which is crucial for E. coli crossing of the blood-brain barrier.

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