scholarly journals Murine Vaginal Colonization Model for Investigating Asymptomatic Mucosal Carriage of Streptococcus pyogenes

2013 ◽  
Vol 81 (5) ◽  
pp. 1606-1617 ◽  
Author(s):  
Michael E. Watson ◽  
Hailyn V. Nielsen ◽  
Scott J. Hultgren ◽  
Michael G. Caparon
Keyword(s):  
Streptococcus Pyogenes ◽  
Critical Role ◽  
Protein A ◽  
Vaginal Fluid ◽  
Vaginal Mucosa ◽  
Lactate Oxidase ◽  
Content Type ◽  
Vaginal Colonization ◽  
Colonization Model

ABSTRACTWhile many virulence factors promotingStreptococcus pyogenesinvasive disease have been described, specific streptococcal factors and host properties influencing asymptomatic mucosal carriage remain uncertain. To address the need for a refined model of prolongedS. pyogenesasymptomatic mucosal colonization, we have adapted a preestrogenized murine vaginal colonization model forS. pyogenes. In this model, derivatives of strains HSC5, SF370, JRS4, NZ131, and MEW123 established a reproducible, asymptomatic colonization of the vaginal mucosa over a period of typically 3 to 4 weeks' duration at a relatively high colonization efficiency. Prior treatment with estradiol prolonged streptococcal colonization and was associated with reduced inflammation in the colonized vaginal epithelium as well as a decreased leukocyte presence in vaginal fluid compared to the levels of inflammation and leukocyte presence in non-estradiol-treated control mice. The utility of our model for investigatingS. pyogenesfactors contributing to mucosal carriage was verified, as a mutant with a mutation in the transcriptional regulator catabolite control protein A (CcpA) demonstrated significant impairment in vaginal colonization. An assessment ofin vivotranscriptional activity in the CcpA−strain for several known CcpA-regulated genes identified significantly elevated transcription of lactate oxidase (lctO) correlating with excessive generation of hydrogen peroxide to self-lethal levels. Deletion oflctOdid not impair colonization, but deletion oflctOin a CcpA−strain prolonged carriage, exceeding even that of the wild-type strain. Thus, while LctO is not essential for vaginal colonization, its dysregulation is deleterious, highlighting the critical role of CcpA in promoting mucosal colonization. The vaginal colonization model should prove effective for future analyses ofS. pyogenesmucosal colonization.

scholarly journals Transcriptomic Analysis ofStreptococcus pyogenesColonizing the Vaginal Mucosa IdentifieshupY, an MtsR-Regulated Adhesin Involved in Heme Utilization

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Laura C. C. Cook ◽  
Nilanjana Chatterjee ◽  
Yan Li ◽  
Jorge Andrade ◽  
Michael J. Federle ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Streptococcus Agalactiae ◽  
Iron Homeostasis ◽  
Vaginal Mucosa ◽  
Heme Binding ◽  
Human Pathogen ◽  
Intracellular Iron ◽  
Essential Information ◽  
Content Type ◽  
Vaginal Colonization

ABSTRACTStreptococcus pyogenes(group A streptococcus [GAS]) is a serious human pathogen with the ability to colonize mucosal surfaces such as the nasopharynx and vaginal tract, often leading to infections such as pharyngitis and vulvovaginitis. We present genome-wide transcriptome sequencing (RNASeq) data showing the transcriptomic changes GAS undergoes during vaginal colonization. These data reveal that the regulon controlled by MtsR, a master metal regulator, is activated during vaginal colonization. This regulon includes two genes highly expressed during vaginal colonization,hupYZ. Here we show that HupY binds hemein vitro, affects intracellular concentrations of iron, and is essential for proper growth of GAS using hemoglobin or serum as the sole iron source. HupY is also important for murine vaginal colonization of both GAS and the related vaginal colonizer and pathogenStreptococcus agalactiae(group B streptococcus [GBS]). These data provide essential information on the link between metal regulation and mucosal colonization in both GAS and GBS.IMPORTANCEColonization of the host requires the ability to adapt to an environment that is often low in essential nutrients such as iron. Here we present data showing that the transcriptome of the important human pathogenStreptococcus pyogenesshows extensive remodeling duringin vivogrowth, resulting in, among many other differentially expressed genes and pathways, a significant increase in genes involved in acquiring iron from host heme. Data show that HupY, previously characterized as an adhesin in bothS. pyogenesand the related pathogenStreptococcus agalactiae, binds heme and affects intracellular iron concentrations. HupY, a protein with no known heme binding domains, represents a novel heme binding protein playing an important role in bacterial iron homeostasis as well as vaginal colonization.

scholarly journals The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes

2016 ◽  
Vol 85 (3) ◽  
Author(s):  
Luis A. Vega ◽  
Kayla M. Valdes ◽  
Ganesh S. Sundar ◽  
Ashton T. Belew ◽  
Emrul Islam ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Immune Evasion ◽  
Immune Cell ◽  
Group A Streptococcus ◽  
Transcriptional Regulator ◽  
Innate Immune ◽  
Content Type ◽  
Human Host

ABSTRACTAs an exclusively human pathogen,Streptococcus pyogenes(the group A streptococcus [GAS]) has specifically adapted to evade host innate immunity and survive in multiple tissue niches, including blood. GAS can overcome the metabolic constraints of the blood environment and expresses various immunomodulatory factors necessary for survival and immune cell resistance. Here we present our investigation of one such factor, the predicted LysR family transcriptional regulator CpsY. The encoding gene,cpsY, was initially identified as being required for GAS survival in a transposon-site hybridization (TraSH) screen in whole human blood. CpsY is homologous with transcriptional regulators ofStreptococcus mutans(MetR),Streptococcus iniae(CpsY), andStreptococcus agalactiae(MtaR) that regulate methionine transport, amino acid metabolism, resistance to neutrophil-mediated killing, and survivalin vivo. Our investigation indicated that CpsY is involved in GAS resistance to innate immune cells of its human host. However, GAS CpsY does not manifest thein vitrophenotypes of its homologs in other streptococcal species. GAS CpsY appears to regulate a small set of genes that is markedly different from the regulons of its homologs. The differential expression of these genes depends on the growth medium, and CpsY modestly influences their expression. The GAS CpsY regulon includes known virulence factors (mntE,speB,spd,nga[spn],prtS[SpyCEP], andsse) and cell surface-associated factors of GAS (emm1,mur1.2,sibA[cdhA], andM5005_Spy0500). Intriguingly, the loss of CpsY in GAS does not result in virulence defects in murine models of infection, suggesting that CpsY function in immune evasion is specific to the human host.

scholarly journals Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

2016 ◽  
Vol 60 (4) ◽  
pp. 2435-2442 ◽  
Author(s):  
Tecla Ciociola ◽  
Thelma A. Pertinhez ◽  
Laura Giovati ◽  
Martina Sperindè ◽  
Walter Magliani ◽  
...  
Keyword(s):  
Self Assembly ◽  
Galleria Mellonella ◽  
Critical Role ◽  
Yeast Cells ◽  
Therapeutic Effects ◽  
Constant Region ◽  
Content Type ◽  
Complementarity Determining Regions

ABSTRACTSynthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

scholarly journals Combined Stimulation of Toll-Like Receptor 5 and NOD1 Strongly Potentiates Activity of NF-κB, Resulting in Enhanced Innate Immune Reactions and Resistance to Salmonella enterica Serovar Typhimurium Infection

2013 ◽  
Vol 81 (10) ◽  
pp. 3855-3864 ◽  
Author(s):  
Amir I. Tukhvatulin ◽  
Ilya I. Gitlin ◽  
Dmitry V. Shcheblyakov ◽  
Natalia M. Artemicheva ◽  
Lyudmila G. Burdelya ◽  
...  
Keyword(s):  
Critical Role ◽  
Immune Defense ◽  
Innate Immune ◽  
Microbial Infection ◽  
Immune Reactions ◽  
Content Type ◽  
Essential Components ◽  
Stimulation Of

ABSTRACTPathogen recognition receptors (PRRs) are essential components of host innate immune systems that detect specific conserved pathogen-associated molecular patterns (PAMPs) presented by microorganisms. Members of two families of PRRs, transmembrane Toll-like receptors (TLRs 1, 2, 4, 5, and 6) and cytosolic NOD receptors (NOD1 and NOD2), are stimulated upon recognition of various bacterial PAMPs. Such stimulation leads to induction of a number of immune defense reactions, mainly triggered via activation of the transcription factor NF-κB. While coordination of responses initiated via different PRRs sensing multiple PAMPS present during an infection makes clear biological sense for the host, such interactions have not been fully characterized. Here, we demonstrate that combined stimulation of NOD1 and TLR5 (as well as other NOD and TLR family members) strongly potentiates activity of NF-κB and induces enhanced levels of innate immune reactions (e.g., cytokine production) bothin vitroandin vivo. Moreover, we show that an increased level of NF-κB activity plays a critical role in formation of downstream responses. In live mice, synergy between these receptors resulting in potentiation of NF-κB activity was organ specific, being most prominent in the gastrointestinal tract. Coordinated activity of NOD1 and TLR5 significantly increased protection of mice against enteroinvasiveSalmonellainfection. Obtained results suggest that cooperation of NOD and TLR receptors is important for effective responses to microbial infectionin vivo.

scholarly journals Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Germán E. Piñas ◽  
John S. Parkinson
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Kinase Activity ◽  
Critical Role ◽  
Active State ◽  
Dynamics Simulation ◽  
Cross Linking ◽  
Atp Binding ◽  
Content Type

ABSTRACT Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, “dipped” and “undipped,” for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units. IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called “dipped” CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

scholarly journals Development of a Novel Virus-Like Particle Vaccine Platform That Mimics the Immature Form of Alphavirus

2017 ◽  
Vol 24 (7) ◽  
Author(s):  
Akane Urakami ◽  
Atsuko Sakurai ◽  
Momoko Ishikawa ◽  
Moh Lan Yap ◽  
Yevel Flores-Garcia ◽  
...  
Keyword(s):  
Immune Responses ◽  
Malaria Infection ◽  
Protein A ◽  
Structural Proteins ◽  
Vaccine Platform ◽  
Content Type ◽  
Virus Like Particle ◽  
Immature Form ◽  
Novel Virus

ABSTRACT Virus-like particles (VLPs) are noninfectious multiprotein structures that are engineered to self-assemble from viral structural proteins. Here, we developed a novel VLP-based vaccine platform utilizing VLPs from the chikungunya virus. We identified two regions within the envelope protein, a structural component of chikungunya, where foreign antigens can be inserted without compromising VLP structure. Our VLP displays 480 copious copies of an inserted antigen on the VLP surface in a highly symmetric manner and is thus capable of inducing strong immune responses against any inserted antigen. Furthermore, by mimicking the structure of the immature form of the virus, we altered our VLP's in vivo dynamics and enhanced its immunogenicity. We used the circumsporozoite protein (CSP) of the Plasmodium falciparum malaria parasite as an antigen and demonstrated that our VLP-based vaccine elicits strong immune responses against CSP in animals. The sera from immunized monkeys protected mice from malaria infection. Likewise, mice vaccinated with P. yoelii CSP-containing VLPs were protected from an infectious sporozoite challenge. Hence, our uniquely engineered VLP platform can serve as a blueprint for the development of vaccines against other pathogens and diseases.

scholarly journals The Pseudomonas aeruginosa PrrF Small RNAs Regulate Iron Homeostasis during Acute Murine Lung Infection

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Alexandria A. Reinhart ◽  
Angela T. Nguyen ◽  
Luke K. Brewer ◽  
Justin Bevere ◽  
Jace W. Jones ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Small Rnas ◽  
Iron Homeostasis ◽  
Critical Role ◽  
Opportunistic Pathogen ◽  
Lung Infection ◽  
Content Type ◽  
The Individual

ABSTRACT Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrHIG sequence). Previous studies showed that the prrF locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrHIG sequence [prrF(ΔHIG)]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa. Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo. Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.

scholarly journals Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells.

1995 ◽  
Vol 15 (7) ◽  
pp. 3857-3863 ◽  
Author(s):  
D A Fruman ◽  
S Y Pai ◽  
S J Burakoff ◽  
B E Bierer
Keyword(s):  
Aspartic Acid ◽  
Phorbol Esters ◽  
Critical Role ◽  
Protein A ◽  
Calcium Ionophore ◽  
Lymphoma Cells ◽  
Calcium Dependent ◽  
Calcineurin A ◽  
Alpha Gene

The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.

scholarly journals Formalin-Inactivated Coxiella burnetii Phase I Vaccine-Induced Protection Depends on B Cells To Produce Protective IgM and IgG

2013 ◽  
Vol 81 (6) ◽  
pp. 2112-2122 ◽  
Author(s):  
Guoquan Zhang ◽  
Ying Peng ◽  
Laura Schoenlaub ◽  
Alexandra Elliott ◽  
William Mitchell ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Phase I ◽  
B Cell ◽  
Coxiella Burnetii ◽  
Critical Role ◽  
Partial Protection ◽  
Content Type ◽  
Deficient Mice

ABSTRACTTo further understand the mechanisms of formalin-inactivatedCoxiella burnetiiphase I (PI) vaccine (PIV)-induced protection, we examined if B cell, T cell, CD4+T cell, or CD8+T cell deficiency in mice significantly affects the ability of PIV to confer protection against aC. burnetiiinfection. Interestingly, compared to wild-type (WT) mice, PIV conferred comparable levels of protection in CD4+T cell- or CD8+T cell-deficient mice and partial protection in T cell-deficient mice but did not provide measurable protection in B cell-deficient mice. These results suggest that PIV-induced protection depends on B cells. In addition, anti-PI-specific IgM was the major detectable antibody (Ab) in immune sera from PIV-vaccinated CD4+T cell-deficient mice, and passive transfer of immune sera from PIV-vaccinated CD4+T cell-deficient mice conferred significant protection. These results suggest that T cell-independent anti-PI-specific IgM may contribute to PIV-induced protection. Our results also suggested that PIV-induced protection may not depend on complement activation and Fc receptor-mediated effector functions. Furthermore, our results demonstrated that both IgM and IgG from PIV-vaccinated WT mouse sera were able to inhibitC. burnetiiinfectionin vivo, but only IgM from PIV-vaccinated CD4+T cell-deficient mouse sera inhibitedC. burnetiiinfection. Collectively, these findings suggest that PIV-induced protection depends on B cells to produce protective IgM and IgG and that T cell-independent anti-PI-specific IgM may play a critical role in PIV-induced protection againstC. burnetiiinfection.

scholarly journals Streptococcus pyogenes Arginine and Citrulline Catabolism Promotes Infection and Modulates Innate Immunity

2013 ◽  
Vol 82 (1) ◽  
pp. 233-242 ◽  
Author(s):  
Zachary T. Cusumano ◽  
Michael E. Watson ◽  
Michael G. Caparon
Keyword(s):  
Nitric Oxide ◽  
Innate Immunity ◽  
Streptococcus Pyogenes ◽  
Murine Model ◽  
Acid Stress ◽  
Arginine Deiminase ◽  
Content Type ◽  
Cutaneous Tissue

ABSTRACTA bacterium's ability to acquire nutrients from its host during infection is an essential component of pathogenesis. For the Gram-positive pathogenStreptococcus pyogenes, catabolism of the amino acid arginine via the arginine deiminase (ADI) pathway supplements energy production and provides protection against acid stressin vitro. Its expression is enhanced in murine models of infection, suggesting an important rolein vivo. To gain insight into the function of the ADI pathway in pathogenesis, the virulence of mutants defective in each of its enzymes was examined. Mutants unable to use arginine (ΔArcA) or citrulline (ΔArcB) were attenuated for carriage in a murine model of asymptomatic mucosal colonization. However, in a murine model of inflammatory infection of cutaneous tissue, the ΔArcA mutant was attenuated but the ΔArcB mutant was hyperattenuated, revealing an unexpected tissue-specific role for citrulline metabolism in pathogenesis. When mice defective for the arginine-dependent production of nitric oxide (iNOS−/−) were infected with the ΔArcA mutant, cutaneous virulence was rescued, demonstrating that the ability ofS. pyogenesto utilize arginine was dispensable in the absence of nitric oxide-mediated innate immunity. This work demonstrates the importance of arginine and citrulline catabolism and suggests a novel mechanism of virulence by whichS. pyogenesuses its metabolism to modulate innate immunity through depletion of an essential host nutrient.

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