Biophysical Characterization of the Type III Secretion System Translocator Proteins and the Translocator Proteins Attached to Bacterium-Like Particles

Chen, Xiaotong; Choudhari, Shyamal P.; Kumar, Prashant; Toth, Ronald; Kim, Jae Hyun; Roosmalen, Maarten L. van; Leenhouts, Kees; Middaugh, C. Russell; Picking, Wendy L.; Picking, William D.

doi:10.1002/jps.24659

Cited by 9 publications

(10 citation statements)

References 22 publications

(27 reference statements)

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“…Possibly, the difficulty of purifying YopB in sufficient quantities prevented its evaluation in larger preclinical studies. The Picking laboratory was able to circumvent this challenge by coexpressing hydrophobic YopB with its chaperone, SycD, and then removing the chaperone with the detergent LDAO and retaining the protein in LDAO to maintain solubility (28).…”

Section: Discussionmentioning

confidence: 99%

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A Combined YopB and LcrV Subunit Vaccine Elicits Protective Immunity against Yersinia Infection in Adult and Infant Mice

Heine

Franco-Mahecha

Sears

et al. 2019

The Journal of Immunology

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Yersinia enterocolitica causes a severe enteric infection in infants and young children. There is no vaccine approved for use in humans. We investigated the immunogenicity and protective capacity of Yersinia YopB, a conserved type III secretion system protein, alone or combined with LcrV in adult mice immunized intranasally. YopB or LcrV (5 mg) administered with the Escherichia coli double mutant heat-labile toxin (dmLT) adjuvant afforded modest (10-30%) protection against lethal Y. enterocolitica oral infection. The combination of YopB and LcrV (5 mg each) dramatically improved vaccine efficacy (70-80%). Additionally, it afforded complete protection against Y. pestis pulmonary infection. Immunization with YopB/LcrV+dmLT resulted in Ag-specific serum IgG, systemic and mucosal Ab-secreting cells, as well as IFN-g, TNF-a, IL-2, IL-6, IL-17A, and KC production by spleen cells. Serum Abs elicited by YopB/LcrV+dmLT had enhanced bactericidal and opsonophagocytic killing activity. After Y. enterocolitica challenge, YopB/LcrV+dmLT-vaccinated mice exhibited intact intestinal tissue, active germinal centers in mesenteric lymph nodes, IgG + and IgA + plasmablasts in the lamina propria, and Abs in intestinal fluid. On the contrary, complete tissue destruction and abscesses were seen in placebo recipients that succumbed to infection. Mice immunized as infants with YopB+dmLT or LcrV+dmLT achieved 60% protection against lethal Y. enterocolitica infection, and vaccine efficacy increased to 90-100% when they received YopB/LcrV+dmLT. YopB+dmLT also afforded substantial (60%) protection when administered intradermally to infant mice. YopB/LcrV+dmLT is a promising subunit vaccine candidate with the potential to elicit broad protection against Yersinia spp.

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Section: Discussionmentioning

confidence: 99%

“…YopB was produced as previously described (28). Briefly, the YopB gene from Y. enterocolitica was amplified by PCR, cloned into pET15b, and used to transform E. coli NovaBlue.…”

Section: Vaccine Preparationmentioning

confidence: 99%

A Combined YopB and LcrV Subunit Vaccine Elicits Protective Immunity against Yersinia Infection in Adult and Infant Mice

Heine

Franco-Mahecha

Sears

et al. 2019

The Journal of Immunology

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“…Methods include the traditional size exclusion chromatography dynamic light scattering, differential scanning calorimetry, field-flow fractionation, atomic force microscopy, resonant mass measurement, sedimentation velocity analytical ultracentrifugation, Coulter counting, microflow imaging, and nanoparticle tracking analysis. [43][44][45][46][47][48] Size exclusion chromatography is most often the method of choice as it is relatively fast and cheap. Recently, methods such as bright-field differential dynamic microscopy have also been developed and used to quantify the dynamics of submicron particles in protein-rich liquid clusters.…”

Section: Product-related Impuritiesmentioning

confidence: 99%

Tools for the analysis and characterization of therapeutic protein species

Fuh¹,

Steffen²,

Schlüter³

2016

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A continuously increasing number of therapeutic proteins are being released into the market, including biosimilars. In contrast to small organic drugs, therapeutic proteins require an extensive analysis of their exact chemical composition because of their complexity and proof of the absence of contaminants, such as host cell proteins and nucleic acids. Especially challenging is the detection of low abundant species of therapeutic proteins because these species are usually very similar to the target therapeutic protein. However, the detection of these species is very important for the safety of patients because a very small change of the exact chemical composition may cause serious side effects. In this review, we give a brief overview about the most important analytical approaches for characterizing therapeutic protein species and their contaminants and focus on the progress in this field during the past 3 years. Top-down mass spectrometry of intact therapeutic proteins in the future may solve many of the current problems in their analysis.

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“…Both tetrameric and monomeric IpaB retain a strongly α-helical secondary structure (Dickenson et al, 2013b ), however, it appears that IpaB prepared in LDAO assumes a structure that is somewhat characteristic of a molten globular state (Chen et al, 2015 ). Interestingly, while IpaB prepared in either of these detergents associates with phospholipid membranes, only tetrameric IpaB causes the size-dependent release of fluorescent molecules trapped in liposomes (Dickenson et al, 2013b ).…”

Section: Introductionmentioning

confidence: 99%

The Many Faces of IpaB

Picking

2016

Front. Cell. Infect. Microbiol.

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The type III secretion system (T3SS) is Shigella's most important virulence factor. The T3SS apparatus (T3SA) is comprised of an envelope-spanning basal body and an external needle topped by a tip complex protein called IpaD. This nanomachine is used to deliver effector proteins into host cells to promote pathogen entry. A key component of the matured T3SS needle tip complex is the translocator protein IpaB. IpaB can exist in multiple states when prepared as a recombinant protein, however, it has also been described as having additional roles in Shigella pathogenesis. This mini-review will briefly describe some of the features of IpaB as a T3SS needle tip protein, as a pore-forming translocator protein and as an effector protein. Reflection on the potential importance of the different in vitro states of IpaB on its function and importance in serotype-independent vaccines is also provided.

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Biophysical Characterization of the Type III Secretion System Translocator Proteins and the Translocator Proteins Attached to Bacterium-Like Particles

Cited by 9 publications

References 22 publications

A Combined YopB and LcrV Subunit Vaccine Elicits Protective Immunity against Yersinia Infection in Adult and Infant Mice

A Combined YopB and LcrV Subunit Vaccine Elicits Protective Immunity against Yersinia Infection in Adult and Infant Mice

Tools for the analysis and characterization of therapeutic protein species

The Many Faces of IpaB

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