Publications by authors named "Tanya Myers-Morales"

16 Publications

  • Page 1 of 1

Burkholderia multivorans requires species-specific GltJK for entry of a contact-dependent growth inhibition system protein.

Mol Microbiol 2021 Sep 28;116(3):957-973. Epub 2021 Jul 28.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, USA.

Interbacterial antagonism and communication are driving forces behind microbial community development. In many Gram-negative bacteria, contact-dependent growth inhibition (CDI) systems contribute to these microbial interactions. CDI systems deliver the toxic C-terminus of a large surface exposed protein to the cytoplasm of neighboring bacteria upon cell-contact. Termed the BcpA-CT, import of this toxic effector domain is mediated by specific, yet largely unknown receptors on the recipient cell outer and inner membranes. In this study, we demonstrated that cytoplasmic membrane proteins GltJK, components of a predicted ABC-type transporter, are required for entry of CDI system protein BcpA-2 into Burkholderia multivorans recipient cells. Consistent with current CDI models, gltJK were also required for recipient cell susceptibility to a distinct BcpA-CT that shared sequences within the predicted "translocation domain" of BcpA-2. Strikingly, this translocation domain showed low sequence identity to the analogous region of an Escherichia coli GltJK-utilizing CDI system protein. Our results demonstrated that recipient bacteria expressing E. coli gltJK were resistant to BcpA-2-mediated interbacterial antagonism, suggesting that BcpA-2 specifically recognizes Burkholderia GltJK. Using a series of chimeric proteins, the specificity determinant was mapped to Burkholderia-specific sequences at the GltK C-terminus, providing insight into BcpA transport across the recipient cell cytoplasmic membrane.
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http://dx.doi.org/10.1111/mmi.14783DOI Listing
September 2021

Neurotropic Lineage III Strains of Listeria monocytogenes Disseminate to the Brain without Reaching High Titer in the Blood.

mSphere 2020 09 16;5(5). Epub 2020 Sep 16.

Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USA

is thought to colonize the brain using one of three mechanisms: direct invasion of the blood-brain barrier, transportation across the barrier by infected monocytes, and axonal migration to the brain stem. The first two pathways seem to occur following unrestricted bacterial growth in the blood and thus have been linked to immunocompromise. In contrast, cell-to-cell spread within nerves is thought to be mediated by a particular subset of neurotropic strains. In this study, we used a mouse model of foodborne transmission to evaluate the neurotropism of several isolates. Two strains preferentially colonized the brain stems of BALB/cByJ mice 5 days postinfection and were not detectable in blood at that time point. In contrast, infection with other strains resulted in robust systemic infection of the viscera but no dissemination to the brain. Both neurotropic strains (L2010-2198, a human rhombencephalitis isolate, and UKVDL9, a sheep brain isolate) typed as phylogenetic lineage III, the least characterized group of Neither of these strains encodes InlF, an internalin-like protein that was recently shown to promote invasion of the blood-brain barrier. Acute neurologic deficits were observed in mice infected with the neurotropic strains, and milder symptoms persisted for up to 16 days in some animals. These results demonstrate that neurotropic strains are not restricted to any one particular lineage and suggest that the foodborne mouse model of listeriosis can be used to investigate the pathogenic mechanisms that allow to invade the brain stem. Progress in understanding the two naturally occurring central nervous system (CNS) manifestations of listeriosis (meningitis/meningoencephalitis and rhombencephalitis) has been limited by the lack of small animal models that can readily distinguish between these distinct infections. We report here that certain neurotropic strains of can spread to the brains of young otherwise healthy mice and cause neurological deficits without causing a fatal bacteremia. The novel strains described here fall within phylogenetic lineage III, a small collection of isolates that have not been well characterized to date. The animal model reported here mimics many features of human rhombencephalitis and will be useful for studying the mechanisms that allow to disseminate to the brain stem following natural foodborne transmission.
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http://dx.doi.org/10.1128/mSphere.00871-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494839PMC
September 2020

Burkholderia cepacia Complex Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition.

J Bacteriol 2019 06 22;201(12). Epub 2019 May 22.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky, USA

species, including opportunistic pathogens in the complex (Bcc), have genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a phenomenon in which Gram-negative bacteria use the toxic C terminus of a polymorphic surface-exposed exoprotein, BcpA, to inhibit the growth of susceptible bacteria upon direct cell-cell contact. Production of a small immunity protein, BcpI, prevents autoinhibition. Although CDI systems appear widespread in Gram-negative bacteria, their function has been primarily examined in several model species. Here we demonstrate that genes encoding predicted CDI systems in Bcc species exhibit considerable diversity. We also show that , which causes pulmonary infections in patients with cystic fibrosis, expresses genes that encode two CDI systems, both of which appear distinct from the typical -type CDI system. Each system can mediate intrastrain interbacterial competition and contributes to bacterial adherence. Surprisingly, the immunity-protein-encoding gene of CDI system 1 could be mutated without obvious deleterious effects. We also show that nonpathogenic uses CDI to control growth during coculture, providing one of the first examples of interspecies CDI and suggesting that CDI systems could be manipulated to develop therapeutic strategies targeting Bcc pathogens. Competition among bacteria affects microbial colonization of environmental niches and host organisms, particularly during polymicrobial infections. The Bcc is a group of environmental bacteria that can cause life-threatening opportunistic infections in patients who have cystic fibrosis or are immunocompromised. Understanding the mechanisms used by these bacterial pathogens to compete with one another may lead to the development of more effective therapies. Findings presented here demonstrate that a Bcc species, , produces functional CDI system proteins and that growth of this pathogen can be controlled by CDI system proteins produced by neighboring cells.
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http://dx.doi.org/10.1128/JB.00012-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531619PMC
June 2019

Type I IFN Does Not Promote Susceptibility to Foodborne Listeria monocytogenes.

J Immunol 2016 Apr 19;196(7):3109-16. Epub 2016 Feb 19.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY 40536

Type I IFN (IFN-α/β) is thought to enhance growth of the foodborne intracellular pathogen Listeria monocytogenes by promoting mechanisms that dampen innate immunity to infection. However, the type I IFN response has been studied primarily using methods that bypass the stomach and, therefore, fail to replicate the natural course of L. monocytogenes infection. In this study, we compared i.v. and foodborne transmission of L. monocytogenes in mice lacking the common type I IFN receptor (IFNAR1(-/-)). Contrary to what was observed using i.v. infection, IFNAR1(-/-) and wild-type mice had similar bacterial burdens in the liver and spleen following foodborne infection. Splenocytes from wild-type mice infected i.v. produced significantly more IFN-β than did those infected by the foodborne route. Consequently, the immunosuppressive effects of type I IFN signaling, which included T cell death, increased IL-10 secretion, and repression of neutrophil recruitment to the spleen, were all observed following i.v. but not foodborne transmission of L. monocytogenes. Type I IFN was also previously shown to cause a loss of responsiveness to IFN-γ through downregulation of the IFN-γ receptor α-chain on macrophages and dendritic cells. However, we detected a decrease in surface expression of IFN-γ receptor α-chain even in the absence of IFN-α/β signaling, suggesting that in vivo, this infection-induced phenotype is not type I IFN-dependent. These results highlight the importance of using the natural route of infection for studies of host-pathogen interactions and suggest that the detrimental effects of IFN-α/β signaling on the innate immune response to L. monocytogenes may be an artifact of the i.v. infection model.
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http://dx.doi.org/10.4049/jimmunol.1502192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4799772PMC
April 2016

Intracellular Listeria monocytogenes comprises a minimal but vital fraction of the intestinal burden following foodborne infection.

Infect Immun 2015 Aug 26;83(8):3146-56. Epub 2015 May 26.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USA

Listeria monocytogenes is a highly adaptive bacterium that replicates as a free-living saprophyte in the environment as well as a facultative intracellular pathogen that causes invasive foodborne infections. The intracellular life cycle of L. monocytogenes is considered to be its primary virulence determinant during mammalian infection; however, the proportion of L. monocytogenes that is intracellular in vivo has not been studied extensively. In this report, we demonstrate that the majority of wild-type (strain EGDe) and mouse-adapted (InlA(m)-expressing) L. monocytogenes recovered from the mesenteric lymph nodes (MLN) was extracellular within the first few days after foodborne infection. In addition, significantly lower burdens of L. monocytogenes were recovered from the colon, spleen, and liver of gentamicin-treated mice than of control mice. This led us to investigate whether intracellular replication of L. monocytogenes was essential during the intestinal phase of infection. We found that lipoate protein ligase-deficient L. monocytogenes (ΔlplA1) mutants, which display impaired intracellular growth, were able to colonize the colon but did not persist efficiently and had a significant defect in spreading to the MLN, spleen, and liver. Together, these data indicate that the majority of the L. monocytogenes burden in the gastrointestinal tract is extracellular, but the small proportion of intracellular L. monocytogenes is essential for dissemination to the MLN and systemic organs.
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http://dx.doi.org/10.1128/IAI.00503-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496611PMC
August 2015

Caspase-3 mediates the pathogenic effect of Yersinia pestis YopM in liver of C57BL/6 mice and contributes to YopM's function in spleen.

PLoS One 2014 5;9(11):e110956. Epub 2014 Nov 5.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, United States of America.

The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G(+), F4/80(+), and iNOS(+) cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the ΔyopM-1 strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM's action and supports the hypothesis that in liver YopM's main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110956PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4220956PMC
June 2015

Cyclic di-GMP-dependent signaling pathways in the pathogenic Firmicute Listeria monocytogenes.

PLoS Pathog 2014 Aug 7;10(8):e1004301. Epub 2014 Aug 7.

Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, United States of America.

We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.
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http://dx.doi.org/10.1371/journal.ppat.1004301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125290PMC
August 2014

Fecal transplantation does not transfer either susceptibility or resistance to food borne listeriosis in C57BL/6 and BALB/c/By mice.

F1000Res 2013 20;2:177. Epub 2013 Aug 20.

Dept. of Microbiology, Immunology & Molecular Genetics, University of Kentucky, Lexington, KY, USA.

The composition of the intestinal microbiota has wide reaching effects on the health of an individual, including the development of protective innate immune responses.  In this report, a fecal transplantation approach was used to determine whether resistance to food borne listeriosis was dependent on the murine gut microbiota.  Transplantation of BALB/c/By feces did not increase the susceptibility of C57BL/6 mice to Listeria monocytogenes infection.   Likewise, transplantation of C57BL/6 fecal matter did not enhance the resistance of BALB/c/By mice.  Thus, intestinal microbiota composition is not a key factor that confers either susceptibility or resistance to food borne listeriosis in mice.
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http://dx.doi.org/10.12688/f1000research.2-177.v1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869484PMC
February 2014

A mouse model of foodborne Listeria monocytogenes infection.

Curr Protoc Microbiol 2013 Nov 5;31:9B.3.1-9B.3.16. Epub 2013 Nov 5.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky.

Listeria monocytogenes causes foodborne disease in humans that ranges in severity from mild, self-limiting gastroenteritis to life-threatening systemic infections of the blood, brain, or placenta. The most commonly used animal model of listeriosis is intravenous infection of mice. This systemic model is highly reproducible, and thus, useful for studying cell-mediated immune responses against an intracellular bacterial pathogen, but it completely bypasses the gastrointestinal phase of L. monocytogenes infection. Intragastric inoculation of L. monocytogenes produces more variable results and may cause direct bloodstream invasion in some animals. The foodborne transmission model described here does not require specialized skills to perform and results in infections that more closely mimic human disease. This natural feeding model can be used to study both the host- and pathogen-derived factors that govern susceptibility or resistance to orally acquired L. monocytogenes.
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http://dx.doi.org/10.1002/9780471729259.mc09b03s31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4191863PMC
November 2013

Temperature-dependence of yadBC phenotypes in Yersinia pestis.

Microbiology (Reading) 2014 Feb 12;160(Pt 2):396-405. Epub 2013 Nov 12.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY 40536-0298, USA.

YadB and YadC are putative trimeric autotransporters present only in the plague bacterium Yersinia pestis and its evolutionary predecessor, Yersinia pseudotuberculosis. Previously, yadBC was found to promote invasion of epithelioid cells by Y. pestis grown at 37 °C. In this study, we found that yadBC also promotes uptake of 37 °C-grown Y. pestis by mouse monocyte/macrophage cells. We tested whether yadBC might be required for lethality of the systemic stage of plague in which the bacteria would be pre-adapted to mammalian body temperature before colonizing internal organs and found no requirement for early colonization or growth over 3 days. We tested the hypothesis that YadB and YadC function on ambient temperature-grown Y. pestis in the flea vector or soon after infection of the dermis in bubonic plague. We found that yadBC did not promote uptake by monocyte/macrophage cells if the bacteria were grown at 28 °C, nor was there a role of yadBC in colonization of fleas by Y. pestis grown at 21 °C. However, the presence of yadBC did promote recoverability of the bacteria from infected skin for 28 °C-grown Y. pestis. Furthermore, the gene for the proinflammatory chemokine CXCL1 was upregulated in expression if the infecting Y. pestis lacked yadBC but not if yadBC was present. Also, yadBC was not required for recoverability if the bacteria were grown at 37 °C. These findings imply that thermally induced virulence properties dominate over effects of yadBC during plague but that yadBC has a unique function early after transmission of Y. pestis to skin.
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http://dx.doi.org/10.1099/mic.0.073205-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919538PMC
February 2014

Oral transmission of Listeria monocytogenes in mice via ingestion of contaminated food.

J Vis Exp 2013 May 6(75):e50381. Epub 2013 May 6.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, USA.

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.
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http://dx.doi.org/10.3791/50381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3676286PMC
May 2013

Toward a molecular pathogenic pathway for Yersinia pestis YopM.

Front Cell Infect Microbiol 2012 11;2:155. Epub 2012 Dec 11.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky Lexington, KY, USA.

YopM is one of the six "effector Yops" of the human-pathogenic Yersinia, but its mechanism has not been defined. After delivery to J774A.1 monocyte-like cells, YopM can rapidly bind and activate the serine/threonine kinases RSK1 and PRK2. However, in infected mice, effects of Y. pestis YopM have been seen only after 24-48 h post-infection (p.i.). To identify potential direct effects of YopM in-vivo we tested for effects of YopM at 1 h and 16-18 h p.i. in mice infected systemically with 10(6) bacteria. At 16 h p.i., there was a robust host response to both parent and ΔyopM-1 Y. pestis KIM5. Compared to cells from non-infected mice, CD11b(+) cells from spleens of infected mice produced more than 100-fold greater IFNγ. In the corresponding sera there were more than 100-fold greater amounts of IFNγ, G-CSF, and CXCL9, as well as more than 10-fold greater amounts of IL-6, CXCL10, and CXCL1. The only YopM-related differences were slightly lower CXCL10 and IL-6 in sera from mice infected 16 h with parent compared to ΔyopM-1 Y. pestis. Microarray analysis of the CD11b(+) cells did not identify consistent transcriptional differences of ≥4-fold at 18 h p.i. However, at 1 h p.i. mRNA for early growth response transcription factor 1 (Egr1) was decreased when YopM was present. Bone marrow-derived macrophages infected for 1 h also expressed lower Egr1 message when YopM was present. Infected J774A.1 cells showed greater expression of Egr1 at 1 h p.i. when YopM was present, but this pattern reversed at 3 h. At 6 h p.i., Cxcl10 mRNA was lower in parent-strain infected cells. We conclude that decreased Egr1 expression is a very early transcriptional effect of YopM and speculate that a pathway may exist from RSK1 through Egr1. These studies revealed novel early transcriptional effects of YopM but point to a time after 18 h of infection when critical transitional events lead to later major effects on cytokine gene transcription.
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http://dx.doi.org/10.3389/fcimb.2012.00155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518861PMC
December 2013

InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice.

PLoS Pathog 2012 15;8(11):e1003015. Epub 2012 Nov 15.

Department of Microbiology, Immunology, & Molecular Genetics, University of Kentucky, Lexington, Kentucky, United States of America.

Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA(m)) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA(m)), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA(m) significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.
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http://dx.doi.org/10.1371/journal.ppat.1003015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499570PMC
April 2013

yadBC of Yersinia pestis, a new virulence determinant for bubonic plague.

Infect Immun 2008 Feb 19;76(2):578-87. Epub 2007 Nov 19.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY 40536-0298, USA.

In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.
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http://dx.doi.org/10.1128/IAI.00219-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2223446PMC
February 2008

A surface-focused biotinylation procedure identifies the Yersinia pestis catalase KatY as a membrane-associated but non-surface-located protein.

Appl Environ Microbiol 2007 Sep 20;73(18):5750-9. Epub 2007 Jul 20.

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY 40536-0298, USA.

This study identified major surface proteins of the plague bacterium Yersinia pestis. We applied a novel surface biotinylation method, followed by NeutrAvidin (NA) bead capture, on-bead digestion, and identification by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pulldown and immunoblot analysis, this method determined whether a protein was accessible to the surface. We applied the method to test the hypothesis that the catalase KatY is a surface protein of the plague bacterium Y. pestis. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by Y. pestis cells grown at 37 degrees C. Similar findings by other groups had led to speculations that this protein might be exposed to the surface and might be a candidate for evaluation as a protective antigen for an improved plague vaccine. KatY was obtained only in the total membrane fraction, and stachyose greatly reduced its biotinylation as well as that of the periplasmic maltose binding protein, indicating that KatY is not on the bacterial surface. LC-MS-MS analysis of on-bead digests representing ca. 10(9) cells identified highly abundant species, including KatY, Pal, and OmpA, as well as the lipoprotein Pcp, all of which bound in a biotin-specific manner. Pla, Lpp, and OmpX (Ail) bound to the NA beads in a non-biotin-specific manner. There was no contamination from abundant cytoplasmic proteins. We hypothesize that OmpX and Pcp are highly abundant and likely to be important for the Y. pestis pathogenic process. We speculate that a portion of KatY associates with the outer membrane in intact cells but that it is located on the periplasmic side. Consistent with this idea, it did not protect C57BL/6 mice against bubonic plague.
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http://dx.doi.org/10.1128/AEM.02968-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2074897PMC
September 2007

Application of a Saccharomyces cerevisiae model to study requirements for trafficking of Yersinia pestis YopM in eucaryotic cells.

Infect Immun 2003 Feb;71(2):937-47

Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington 40536-0298, USA.

YopM is a leucine-rich repeat (LRR) virulence protein that is delivered into host cells when any of the three human-pathogenic species of Yersinia binds to mammalian cells. It exhibits heterogeneity of size and sequence among the yersiniae, but the functional consequences of this variability are not yet known. Yersinia pestis YopM was previously shown to accumulate in the nuclei of infected HeLa cells by a mechanism that requires vesicular trafficking. In this study, we characterized the trafficking of Y. pestis YopM in a Saccharomyces cerevisiae model previously found to support nuclear localization of YopM from an enteropathogenic Yersinia strain (C. F. Lesser and S. I. Miller, EMBO J. 20:1840-1849, 2001). Y. pestis YopM was N-terminally fused to the yeast enhanced green fluorescent protein (yEGFP) and inducibly expressed in the cytoplasm. yEGFP-YopM localized to the yeast nucleus, showing that this property is conserved for YopMs so far tested and that infection and the presence of other Yops are not required for its trafficking. When expressed in S. cerevisiae that is temperature sensitive for vesicular transport, YopM failed to accumulate in the nucleus at the nonpermissive temperature but did accumulate when the permissive temperature was restored. This shows that vesicular trafficking also is required in yeast for normal localization of YopM. YopM consists of a 71-residue leader sequence, 15 LRRs, and a 32-residue tail. Deletion analysis revealed that the leader sequence or tail is alone insufficient to direct YopM to the nucleus, showing that the LRR structure is required. Both the N-terminal and C-terminal halves of YopM localized to the nucleus, indicating the possible presence of two nuclear localization signals (NLSs) in YopM or domains in YopM where an NLS-containing protein might bind; this fits with the presence of two highly conserved regions among Yersinia YopMs. yEGFP-YopM lacking LRRs 4 to 7 or 7 to 10 accumulated in the nucleus in yeast, and YopM lacking these LRRs concentrated normally in the HeLa cell nucleus after delivery by Yersinia infection, showing that these LRRs are not essential for YopM trafficking in eucaryotic cells. However, because Y. pestis carrying either of these YopMs is strongly compromised in virulence in mice, these findings revealed that LRRs 4 to 10 map a region of YopM or support a conformation of YopM that is necessary for a pathogenic effect.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145383PMC
http://dx.doi.org/10.1128/IAI.71.2.937-947.2003DOI Listing
February 2003
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