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Farag, Salah
Publications (3 of 3) Show all publications
Costa, T., Francis, M. K., Farag, S., Edgren, T. & Francis, M. S. (2019). Measurement of Yersinia translocon pore formation in erythrocytes. In: Viveka Vadyvaloo and Matthew B. Lawrenz (Ed.), Pathogenic Yersinia: methods and protocols (pp. 211-229). New York, NY, U.S.A.: Humana Press
Open this publication in new window or tab >>Measurement of Yersinia translocon pore formation in erythrocytes
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2019 (English)In: Pathogenic Yersinia: methods and protocols / [ed] Viveka Vadyvaloo and Matthew B. Lawrenz, New York, NY, U.S.A.: Humana Press, 2019, p. 211-229Chapter in book (Refereed)
Abstract [en]

Many Gram-negative pathogens produce a type III secretion system capable of intoxicating eukaryotic cells with immune-modulating effector proteins. Fundamental to this injection process is the prior secretion of two translocator proteins destined for injectisome translocon pore assembly within the host cell plasma membrane. It is through this pore that effectors are believed to travel to gain access to the host cell interior. Yersinia species especially pathogenic to humans and animals assemble this translocon pore utilizing two hydrophobic translocator proteins-YopB and YopD. Although a full molecular understanding of the biogenesis, function and regulation of this translocon pore and subsequent effector delivery into host cells remains elusive, some of what we know about these processes can be attributed to studies of bacterial infections of erythrocytes. Herein we describe the methodology of erythrocyte infections by Yersinia, and how analysis of the resultant contact-dependent hemolysis can serve as a relative measurement of YopB- and YopD-dependent translocon pore formation.

Place, publisher, year, edition, pages
New York, NY, U.S.A.: Humana Press, 2019
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 2010
Keywords
Biogenesis, Contact-dependent hemolysis, Effector recognition and intracellular delivery, Function and regulation, Host immune response, Membrane integration, Type III translocon pore complex
National Category
Microbiology in the medical area Microbiology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Microbiology; Molecular Biology; Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-161385 (URN)10.1007/978-1-4939-9541-7_15 (DOI)31177441 (PubMedID)978-1-4939-9540-0 (ISBN)
Funder
Swedish Research Council, 2014-2105Swedish Research Council, 2009-5628
Available from: 2019-07-03 Created: 2019-07-03 Last updated: 2019-07-03Bibliographically approved
Amer, A., Costa, T., Farag, S., Avican, U., Forsberg, Å. & Francis, M. (2013). Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis. PLoS ONE, 8(10), Article ID e77767.
Open this publication in new window or tab >>Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 10, article id e77767Article in journal (Refereed) Published
Abstract [en]

Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2013
Keywords
secretion control, hierarchy, translocation, InvE family, ribosome slippage, virulence
National Category
Microbiology Biochemistry and Molecular Biology Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-81379 (URN)10.1371/journal.pone.0077767 (DOI)000325483600088 ()24098594 (PubMedID)
Funder
Swedish Research Council
Available from: 2013-10-08 Created: 2013-10-08 Last updated: 2018-06-08Bibliographically approved
Costa, T., Amer, A., Farag, S., Wolf-Watz, H., Fällman, M., Fahlgren, A., . . . Francis, M. (2013). Type III secretion translocon assemblies that attenuate Yersinia virulence. Cellular Microbiology, 15(7), 1088-1110
Open this publication in new window or tab >>Type III secretion translocon assemblies that attenuate Yersinia virulence
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2013 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, no 7, p. 1088-1110Article in journal (Refereed) Published
Abstract [en]

Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopD(I262P) and YopD(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopD(A263P) and YopD(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2013
Keywords
virulence, coiled-coil, effector delivery, pore formation, regulation, YopD
National Category
Microbiology Biochemistry and Molecular Biology Microbiology in the medical area
Research subject
Infectious Diseases; Microbiology; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-67117 (URN)10.1111/cmi.12100 (DOI)000320394800004 ()
Funder
Swedish Research Council
Note

This work, performed within the framework of the Umea Centre for Microbial Research-Linnaeus Program, was supported by grants from the Carl Tryggers Foundation for Scientific Research (M. S. F.), Swedish Research Council (H.W.-W., M. F., T. E., M. S. F.), Foundation for Medical Research at Umea University (M. S. F.) and J C Kempe Memorial Fund (T. R. C., A. A. A.).

Available from: 2013-03-13 Created: 2013-03-13 Last updated: 2018-06-08Bibliographically approved
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