Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface

BACKGROUND: Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein. RESULTS: In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking. CONCLUSION: Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53

LcrG secretion is not required for blocking of Yops secretion in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>LcrG, a negative regulator of the <it>Yersinia </it>type III secretion apparatus has been shown to be primarily a cytoplasmic protein, but is secreted at least in <it>Y. pestis</it>. LcrG secretion has not been functionally analyzed and the relevance of LcrG secretion on LcrG function is unknown.</p> <p>Results</p> <p>An LcrG-GAL4AD chimera, originally constructed for two-hybrid analyses to analyze LcrG protein interactions, appeared to be not secreted but the LcrG-GAL4AD chimera retained the ability to regulate Yops secretion. This result led to further investigation to determine the significance of LcrG secretion on LcrG function. Additional analyses including deletion and substitution mutations of amino acids 2–6 in the N-terminus of LcrG were constructed to analyze LcrG secretion and LcrG's ability to control secretion. Some changes to the N-terminus of LcrG were found to not affect LcrG's secretion or LcrG's secretion-controlling activity. However, substitution of poly-isoleucine in the N-terminus of LcrG did eliminate LcrG secretion but did not affect LcrG's secretion controlling activity.</p> <p>Conclusion</p> <p>These results indicate that secretion of LcrG, while observable and T3SS mediated, is not relevant for LcrG's ability to control secretion.</p

Immunization of mice with YscF provides protection from Yersinia pestis infections

BACKGROUND: Yersinia pestis, the causative agent of plague, is a pathogen with a tremendous ability to cause harm and panic in populations. Due to the severity of plague and its potential for use as a bioweapon, better preventatives and therapeutics for plague are desirable. Subunit vaccines directed against the F1 capsular antigen and the V antigen (also known as LcrV) of Y. pestis are under development. However, these new vaccine formulations have some possible limitations. The F1 antigen is not required for full virulence of Y. pestis and LcrV has a demonstrated immunosuppressive effect. These limitations could damper the ability of F1/LcrV based vaccines to protect against F1-minus Y. pestis strains and could lead to a high rate of undesired side effects in vaccinated populations. For these reasons, the use of other antigens in a plague vaccine formulation may be advantageous. RESULTS: Desired features in vaccine candidates would be antigens that are conserved, essential for virulence and accessible to circulating antibody. Several of the proteins required for the construction or function of the type III secretion system (TTSS) complex could be ideal contenders to meet the desired features of a vaccine candidate. Accordingly, the TTSS needle complex protein, YscF, was selected to investigate its potential as a protective antigen. In this study we describe the overexpression, purification and use of YscF as a protective antigen. YscF immunization triggers a robust antibody response to YscF and that antibody response is able to afford significant protection to immunized mice following challenge with Y. pestis. Additionally, evidence is presented that suggests antibody to YscF is likely not protective by blocking the activity of the TTSS. CONCLUSION: In this study we investigated YscF, a surface-expressed protein of the Yersinia pestis type III secretion complex, as a protective antigen against experimental plague infection. Immunization of mice with YscF resulted in a high anti-YscF titer and provided protection against i.v. challenge with Y. pestis. This is the first report to our knowledge utilizing a conserved protein from the type III secretion complex of a gram-negative pathogen as a candidate for vaccine development

Zika Virus-Specific IgY Results Are Therapeutic Following a Lethal Zika Virus Challenge without Inducing Antibody-Dependent Enhancement

The Zika virus (ZIKV) is a newly emerged pathogen in the Western hemisphere. It was declared a global health emergency by the World Health Organization in 2016. There have been 223,477 confirmed cases, including 3720 congenital syndrome cases since 2015. ZIKV infection symptoms range from asymptomatic to Gullain–Barré syndrome and extensive neuropathology in infected fetuses. Passive and active vaccines have been unsuccessful in the protection from or the treatment of flaviviral infections due to antibody-dependent enhancement (ADE). ADE causes an increased viral load due to an increased monocyte opsonization by non-neutralizing, low-avidity antibodies from a previous dengue virus (DENV) infection or from a previous exposure to ZIKV. We have previously demonstrated that polyclonal avian IgY generated against whole-killed DENV-2 ameliorates DENV infection in mice while not inducing ADE. This is likely due to the inability of the Fc portion of IgY to bind to mammalian Fc receptors. We have shown here that ZIKV oligoclonal IgY is able to neutralize the virus in vitro and in IFNAR−/− mice. The concentration of ZIKV-specific IgY yielding 50% neutralization (NT50) was 25 µg/mL. The exposure of the ZIKV, prior to culture with ZIKV-specific IgY or 4G2 flavivirus-enveloped IgG, demonstrated that the ZIKV-specific IgY does not induce ADE. ZIKV IgY was protective in vivo when administered following a lethal ZIKV challenge in 3-week-old IFNAR−/− mice. We propose polyclonal ZIKV-specific IgY may provide a viable passive immunotherapy for a ZIKV infection without inducing ADE

A One-Scale Model of Dynamical Supersymmetry Breaking

A model of gauge-mediated supersymmetry breaking is constructed in which the low-energy physics depends on a single dynamical scale. Strong coupling dynamics of gauge theories plays an important role, in particular through its effects on beta functions and through confinement. The model does not have distinct messenger and supersymmetry-breaking sectors. The scale of supersymmetry breaking is of order 10-100 \TeV, implying that the decay of the next-to-lightest superpartner into the gravitino is prompt. Superoblique corrections are enhanced. A Dirac fermion and one complex scalar, in a 10 or \bar{10} of (global) SU(5), are predicted to be relatively light and to satisfy certain mass relations with the standard model squarks and sleptons.Comment: 28 pages, uses revtex, h-physrev.bs

Non-Equilibrium Large N Yukawa Dynamics: marching through the Landau pole

The non-equilibrium dynamics of a Yukawa theory with N fermions coupled to a scalar field is studied in the large N limit with the goal of comparing the dynamics predicted from the renormalization group improved effective potential to that obtained including the fermionic backreaction. The effective potential is of the Coleman-Weinberg type. Its renormalization group improvement is unbounded from below and features a Landau pole. When viewed self-consistently, the initial time singularity does not arise. The different regimes of the dynamics of the fully renormalized theory are studied both analytically and numerically. Despite the existence of a Landau pole in the model, the dynamics of the mean field is smooth as it passes the location of the pole. This is a consequence of a remarkable cancellation between the effective potential and the dynamical chiral condensate. The asymptotic evolution is effectively described by a quartic upright effective potential. In all regimes, profuse particle production results in the formation of a dense fermionic plasma with occupation numbers nearly saturated up to a scale of the order of the mean field. This can be interpreted as a chemical potential. We discuss the implications of these results for cosmological preheating.Comment: 36 pages, 14 figures, LaTeX, submitted to Physical Review

Determination of Fundamental Supersymmetry Parameters from Chargino Production at Lepii

If accessible at LEP II, chargino production is likely to be one of the few available supersymmetric signals for many years. We consider the prospects for the determination of fundamental supersymmetry parameters in such a scenario. The study is complicated by the dependence of observables on a large number of these parameters. We propose a straightforward procedure for disentangling these dependences and demonstrate its effectiveness by presenting a number of case studies at representative points in parameter space. Working in the context of the minimal supersymmetric standard model, we find that chargino production by itself is a fairly sensitive probe of the supersymmetry-breaking sector. For significant regions of parameter space, it is possible to test the gaugino mass unification hypothesis and to measure the gaugino contents of the charginos and neutralinos, thereby testing the predictions of grand unification and the viability of the lightest supersymmetric particle as a dark matter candidate. For much of the parameter space, it is also possible to set limits on the mass of the electron sneutrino, which provide a valuable guide for future particle searches.Comment: 52pp, Revtex, 30 figures available upon request, SLAC-PUB-6497, RU-94-67 (text and figures available in ps form by anonymous ftp from preprint.slac.stanford.edu, directory pub/preprints/hep-ph/9408

Structure-Function Analysis of the C-Terminal Domain of LcrV from Yersinia pestis▿

LcrV, a multifunctional protein, acts as a positive regulator of effector protein secretion for the type III secretion system (T3SS) in Yersinia pestis by interaction with the negative regulator LcrG. In this study, LcrV was analyzed to identify regions required for LcrG interaction. Random-linker insertion mutagenesis, deletion analysis, and site-directed mutagenesis of hydrophobic amino acids between residues 290 and 311 allowed the isolation of an LcrV mutant (LcrV L291R F308R) defective for LcrG interaction. The new residues identified in LcrG interaction lie in helix 12 of LcrV; residues in helix 7 of LcrV are known to be involved in LcrG interaction. Helix 7 and helix 12 of LcrV interact to form an intramolecular coiled coil; these new results suggest that the intramolecular coiled coil in LcrV is required for LcrG interaction and activation of the T3SS

Type III Secretion Systems

Type III secretion system (TTSS) is believed to have originally evolved from the flagellar export system and is now dispersed among a number of both animal‐ and plant‐interacting gram‐negative bacteria. The aim of much current research on TTSS is to understand the mechanisms involved in effector secretion/injection and what the effectors are doing inside the host cell. Bacterial pathogens use several different protein secretion pathways to export virulence proteins from the bacterial cytoplasm to their site of action. Chaperones bind to effector proteins in the bacterial cytosol and remain cytosolic following export of their cognate substrate. Secreted proteins exhibiting significant structural similarities to YopB and YopD are present in all TTSSs of animal pathogens but not plant pathogens. Secretion of LcrQ upon cell contact depletes LcrQ from the cytoplasmic compartment and triggers increased transcription of type III genes. Secretion and polymerization of PrgI are required to complete the assembly of the needle complex and type III export apparatus. Two flagellar components, FlhB, an inner membrane protein with a substantial C‐terminal cytoplasmic domain, and FliK, the secreted hook‐length control protein, are proposed to be involved in switches substrate specificity process. An important question is how far knowledge of flagellar biogenesis can be extrapolated to understand the structure and function of the type III export apparatus. It is obvious that there will be features unique to each system; however, the basic process of transporting substrates across the bacterial membranes appears to be relatively well conserved