Ail Binding to Fibronectin Facilitates Yersinia pestis Binding to Host Cells and Yop Delivery

ABSTRACT Yersinia pestis, the causative agent of plague, evades host immune responses and rapidly causes disease. The Y. pestis adhesin Ail mediates host cell binding and is critical for Yop delivery. To identify the Ail receptor(s), Ail was purified following overexpression in Escherichia coli. Ail bound specifically to fibronectin, an extracellular matrix protein with the potential to act as a bridge between Ail and host cells. Ail expressed by E. coli also mediated binding to purified fibronectin, and Ail-mediated E. coli adhesion to host cells was dependent on fibronectin. Ail expressed by Y. pestis bound purified fibronectin, as did the Y. pestis adhesin plasminogen activator (Pla). However, a KIM5 Δail mutant had decreased binding to host cells, while a KIM5 Δpla mutant had no significant defect in adhesion. Furthermore, treatment with antifibronectin antibodies decreased Ail-mediated adhesion by KIM5 and the KIM5 Δpla mutant, indicating that the Ail-fibronectin interaction was important for cell binding. Finally, antifibronectin antibodies inhibited the KIM5-mediated cytotoxicity of host cells in an Ail-dependent fashion. These data indicate that Ail is a key adhesin that mediates binding to host cells through interaction with fibronectin on the surface of host cells, and this interaction is important for Yop delivery by Y. pestis.

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The extracellular matrix protein mindin as a novel adjuvant elicits stronger immune responses for rBAG1, rSRS4 and rSRS9 antigens of Toxoplasma gondiiin BALB/c mice

BMC Infectious Diseases ◽

10.1186/1471-2334-14-429 ◽

2014 ◽

Vol 14 (1) ◽

Cited By ~ 10

Author(s):

Xiaojing Sun ◽

Mei Mei ◽

Xu Zhang ◽

Fusong Han ◽

Boyin Jia ◽

...

Keyword(s):

Extracellular Matrix ◽

Immune Responses ◽

Matrix Protein ◽

Extracellular Matrix Protein

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1280: Increased Susceptibility to Genitovaginal Prolapse Associated with a Polymorphism in the Promoter of the Extracellular Matrix Protein LAMC1

The Journal of Urology ◽

10.1016/s0022-5347(18)31494-0 ◽

2007 ◽

Vol 177 (4S) ◽

pp. 421-422

Author(s):

Ganka Nikolova ◽

Christian O. Twiss ◽

Hane Lee ◽

Nelson Stanley ◽

Janet Sinsheimer ◽

...

Keyword(s):

Extracellular Matrix ◽

Matrix Protein ◽

Extracellular Matrix Protein ◽

Increased Susceptibility

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Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.64.12.4862-4869.1998 ◽

1998 ◽

Vol 64 (12) ◽

pp. 4862-4869 ◽

Cited By ~ 39

Author(s):

Jörg F. Rippmann ◽

Michaela Klein ◽

Christian Hoischen ◽

Bodo Brocks ◽

Wolfgang J. Rettig ◽

...

Keyword(s):

Escherichia Coli ◽

Proteus Mirabilis ◽

Binding Activity ◽

Host Cells ◽

Heterologous Proteins ◽

Single Chain Variable Fragment ◽

Single Chain ◽

E Coli ◽

Active Product ◽

Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

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Homogenous overexpression of the extracellular matrix protein Netrin-1 in a hollow fiber bioreactor

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11438-0 ◽

2021 ◽

Author(s):

Aniel Moya-Torres ◽

Monika Gupta ◽

Fabian Heide ◽

Natalie Krahn ◽

Scott Legare ◽

...

Keyword(s):

Extracellular Matrix ◽

Hollow Fiber ◽

Mammalian Cells ◽

Matrix Protein ◽

Continuous Production ◽

Extracellular Matrix Protein ◽

Close Monitoring ◽

High Quality ◽

Biolayer Interferometry ◽

Hollow Fiber Bioreactor

Abstract The production of recombinant proteins for functional and biophysical studies, especially in the field of structural determination, still represents a challenge as high quality and quantities are needed to adequately perform experiments. This is in part solved by optimizing protein constructs and expression conditions to maximize the yields in regular flask expression systems. Still, work flow and effort can be substantial with no guarantee to obtain improvements. This study presents a combination of workflows that can be used to dramatically increase protein production and improve processing results, specifically for the extracellular matrix protein Netrin-1. This proteoglycan is an axon guidance cue which interacts with various receptors to initiate downstream signaling cascades affecting cell differentiation, proliferation, metabolism, and survival. We were able to produce large glycoprotein quantities in mammalian cells, which were engineered for protein overexpression and secretion into the media using the controlled environment provided by a hollow fiber bioreactor. Close monitoring of the internal bioreactor conditions allowed for stable production over an extended period of time. In addition to this, Netrin-1 concentrations were monitored in expression media through biolayer interferometry which allowed us to increase Netrin-1 media concentrations tenfold over our current flask systems while preserving excellent protein quality and in solution behavior. Our particular combination of genetic engineering, cell culture system, protein purification, and biophysical characterization permitted us to establish an efficient and continuous production of high-quality protein suitable for structural biology studies that can be translated to various biological systems. Key points • Hollow fiber bioreactor produces substantial yields of homogenous Netrin-1 • Biolayer interferometry allows target protein quantitation in expression media • High production yields in the bioreactor do not impair Netrin-1 proteoglycan quality Graphical abstract

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