scholarly journals Molecular analyses identifies new domains and structural differences among Streptococcus pneumoniae immune evasion proteins PspC and Hic

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shanshan Du ◽  
Cláudia Vilhena ◽  
Samantha King ◽  
Alfredo Sahagún-Ruiz ◽  
Sven Hammerschmidt ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Functional Characterization ◽  
Surface Proteins ◽  
Genomic Diversity ◽  
Functional Evaluation ◽  
Structural Differences ◽  
Long Time ◽  
Modular Domain ◽  
Novel Strategy ◽  
Domain Level

AbstractThe PspC and Hic proteins of Streptococcuspneumoniae are some of the most variable microbial immune evasion proteins identified to date. Due to structural similarities and conserved binding profiles, it was assumed for a long time that these pneumococcal surface proteins represent a protein family comprised of eleven subgroups. Recently, however, the evaluation of more proteins revealed a greater diversity of individual proteins. In contrast to previous assumptions a pattern evaluation of six PspC and five Hic variants, each representing one of the previously defined subgroups, revealed distinct structural and likely functionally regions of the proteins, and identified nine new domains and new domain alternates. Several domains are unique to PspC and Hic variants, while other domains are also present in other virulence factors encoded by pneumococci and other bacterial pathogens. This knowledge improved pattern evaluation at the level of full-length proteins, allowed a sequence comparison at the domain level and identified domains with a modular composition. This novel strategy increased understanding of individual proteins variability and modular domain composition, enabled a structural and functional characterization at the domain level and furthermore revealed substantial structural differences between PspC and Hic proteins. Given the exceptional genomic diversity of the multifunctional PspC and Hic proteins a detailed structural and functional evaluation need to be performed at the strain level. Such knowledge will also be useful for molecular strain typing and characterizing PspC and Hic proteins from new clinical S. pneumoniae strains.

scholarly journals Same, Same, but Different: Molecular Analyses of Streptococcus pneumoniae Immune Evasion Proteins Identifies new Domains and Reveals Structural Differences between PspC and Hic Variants

2020 ◽  
Author(s):  
Shanshan Du ◽  
Claudia Vilhena ◽  
Samantha King ◽  
Alfredo Sahagun ◽  
Sven Hammerschmidt ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Immune Evasion ◽  
Antigenic Variation ◽  
Immune Escape ◽  
Functional Characterization ◽  
Surface Proteins ◽  
Surface Distribution ◽  
Domain Composition ◽  
Structural Differences ◽  
Domain Level

AbstractPspC and Hic proteins of Streptococcus pneumoniae are some of the most variable microbial immune evasion proteins identified to date. Due to structural similarities and conserved binding profiles it was assumed over a long time that these pneumococcal surface proteins represent a protein family, comprising eleven subgroups. Recently, however, by evaluating more proteins larger diversity of individual proteins became apparent. In contrast to previous assumptions a pattern evaluation of six PspC and five Hic variants, each representing one of the previously defined subgroups, revealed distinct structural and likely functionally regions of the proteins, and identified nine new domains and new domain alternates. Several domains are unique to PspC and Hic variants, while other domains are shared with other S. pneumoniae and bacterial virulent determinants. This understanding improved pattern evaluation on the level of full-length proteins, allowed a sequence comparison on the domain level and furthermore identified domains with a modular composition. This novel concept allows a better characterization of variability, and modular domain composition of individual proteins, enables a structural and functional characterization at the domain level and furthermore shows substantial structural differences between PspC and Hic proteins. Such knowledge will also be useful for molecular strain typing, characterizing PspC and Hic proteins from new clinical S. pneumoniae strains, including those derived from patients who present with pneumococcal hemolytic uremic syndrome. Furthermore this analysis explains the role of multifaceted intact PspC and Hic proteins in pathogen host interactions. and can provide a basis for rational vaccine design.Author SummaryThe human pathobiont Streptococcus pneumoniae expresses highly polymorphic PspC or Hic proteins, which bind a repertoire of host immune regulators and combine antigenic variation with conserved immune evasion features. Understanding domain composition of each protein encoded by more than 60 000 pspC or hic genes deposited in the data banks defines their diversity, a role in immune escape and can furthermore delineate structure function approach for single protein domains. PspC and Hic proteins show variable domain composition and sequence diversity, which explain differences in binding of human regulators and likely in immune escape. The results of our analyses provide insights in the domain composition of these diverse immune evasion proteins, identifies new domains, defines domains which are unique to PspC or Hic variants, and identifies domains which are shared with other bacterial immune evasion proteins. These data have implication on cell wall attachment, surface distribution and in immune escape.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

scholarly journals The Brilliance of Borrelia: Mechanisms of Host Immune Evasion by Lyme Disease-Causing Spirochetes

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 281
Author(s):  
Cassidy Anderson ◽  
Catherine A. Brissette
Keyword(s):  
Immune Response ◽  
Lyme Disease ◽  
Immune Evasion ◽  
Host Immune Response ◽  
Surface Proteins ◽  
Borrelia Burgdorferi Sensu Lato ◽  
Adaptive Responses ◽  
Tick Saliva ◽  
Vector Borne ◽  
Antimicrobial Peptide Resistance

Lyme disease (LD) has become the most common vector-borne illness in the northern hemisphere. The causative agent, Borrelia burgdorferi sensu lato, is capable of establishing a persistent infection within the host. This is despite the activation of both the innate and adaptive immune responses. B. burgdorferi utilizes several immune evasion tactics ranging from the regulation of surface proteins, tick saliva, antimicrobial peptide resistance, and the disabling of the germinal center. This review aims to cover the various methods by which B. burgdorferi evades detection and destruction by the host immune response, examining both the innate and adaptive responses. By understanding the methods employed by B. burgdorferi to evade the host immune response, we gain a deeper knowledge of B. burgdorferi pathogenesis and Lyme disease, and gain insight into how to create novel, effective treatments.

scholarly journals Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

2021 ◽  
Author(s):  
I. B. Trindade ◽  
G. Hernandez ◽  
E. Lebègue ◽  
F. Barrière ◽  
T. Cordeiro ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Paramagnetic Nmr ◽  
E Coli ◽  
The Past ◽  
Internal Cavities ◽  
Structural Differences ◽  
Bond Strengths ◽  
Redox Bohr Effect ◽  
Micromolar Range

AbstractIron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins. Graphic abstract

Abstract 050: Fine-mapping and Bioinformatics Characterization of QT Interval Loci in African Americans: The Population Architecture Using Genomics and Epidemiology (PAGE) Study

Circulation ◽  
2012 ◽  
Vol 125 (suppl_10) ◽  
Author(s):  
Christy L Avery ◽  
Praveen Sethupathy ◽  
Steven Buyske ◽  
Q. C He ◽  
Dan Y Lin ◽  
...  
Keyword(s):  
African American ◽  
African Americans ◽  
Fine Mapping ◽  
Qt Interval ◽  
Functional Characterization ◽  
Regulatory Elements ◽  
European Ancestry ◽  
Functional Evaluation ◽  
Human Cardiomyocytes

The QT interval (QT) is a heritable trait and its prolongation is an established risk factor for ventricular tachyarrhythmia and sudden cardiac death. Most genetic studies of QT have examined populations of European ancestry, although the increased genetic diversity in populations of African descent provides opportunity for fine-mapping, which can help narrow association signals and identify candidates for functional characterization. We examined whether eleven previously identified QT loci comprising 6,681 variants on the Illumina Metabochip array were associated with QT in 7,516 African American participants from the Atherosclerosis Risk in Communities study and Women’s Health Initiative clinical trial. Among associated loci, we used conditional analyses and queried bioinformatics databases to identify and functionally categorize signals. We identified nine of the eleven QT loci in African American populations ( P <0.0045 under an additive genetic model adjusting for ancestry and demographic characteristics: NOS1AP, ATP1B1, SCN5A, SLC35F1, KCNH2, KCNQ1, LITAF, NDRG4, and RFFL ). We also identified two independent secondary signals in NOS1AP and ATP1B1 ( P < 7.4x10 −6 ). Conditional analyses adjusting for published loci in European populations demonstrated that eight of these eleven SNPs (nine primary; two secondary) were independent of previously reported SNPs. We then performed the first bioinformatics-based functional characterization of QT loci using the eleven primary and secondary variants and SNPs in strong LD (r 2 > 0.5) among these African American participants. Only the SCN5A locus included a non-synonymous coding variant (rs1805124, H558R, r 2 = 0.7 with primary SNP rs9871385, P = 4.7x10 −4 ). The remaining ten loci harbored variants located exclusively within non-coding regions. Specifically, three contained SNPs within candidate long-range regulatory elements in human cardiomyocytes, five were in or near annotated promoter regions, and the remaining two were in un-annotated, but highly conserved non-coding elements. Several of the QT risk alleles at these SNPs significantly alter the predicted binding affinity for transcription factors, such as TBX5 and AhR, which have been previously implicated in cardiac formation and function. In summary, the findings provide compelling evidence that the same genes influence variation in QT across global populations and that additional, independent signals exist in African Americans. Moreover, of those SNPs identified as strong candidates for functional evaluation, the majority implicate gene regulatory dysfunction in QT prolongation.

scholarly journals BspA and Pmp proteins ofTrichomonas vaginalismediate adherence to host cells

2018 ◽  
Author(s):  
Maria R. Handrich ◽  
Sriram G. Garg ◽  
Ewen W. Sommerville ◽  
Robert P. Hirt ◽  
Sven B. Gould
Keyword(s):  
Trichomonas Vaginalis ◽  
Functional Characterization ◽  
Surface Proteins ◽  
Host Cells ◽  
Protein Families ◽  
Sexually Transmitted ◽  
Trace Back ◽  
Oral Pathogens ◽  
Adhesion Performance

AbstractTrichomonas vaginalisis one of the most widespread, sexually transmitted pathogens. The infection involves a morphological switch from a free-swimming pyriform trophozoite to an amoeboid cell upon adhesion to host epithelial cells. While details on how the switch is induced and to what proteins of the host surface the parasite adheres remain poorly characterized, several surface proteins of the parasite itself have been identified as potential candidates. Among those are two expanded protein families that harbor domains that share similarity to functionally investigated surface proteins of prokaryotic oral pathogens; these are the BspA proteins of Bacteroidales and Spirochaetales, and the Pmp proteins of Chlamydiales. We sequenced the transcriptomes of five Trichomonads and screened for the presence of BspA and Pmp domain-containing proteins and tested the ability of individualT. vaginaliscandidates to mediate adhesion. Here we demonstrate that (i) BspA and Pmp domain-containing proteins are specifically expanded inT. vaginalisin comparison to other Trichomonads, and that (ii) individual proteins of both families have the ability to increase adhesion performance in a non-virulentT. vaginalisstrain andTetratrichomonas gallinarum, a parasite usually known to infect birds but not humans. Our results initiate the functional characterization of these two broadly distributed protein families, whose origin we trace back to the origin of Trichomonads themselves.

scholarly journals CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Jessica M. Bryant ◽  
Clément Regnault ◽  
Christine Scheidig-Benatar ◽  
Sebastian Baumgarten ◽  
Julien Guizetti ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Transcriptional Regulation ◽  
Gene Family ◽  
Genome Editing ◽  
Immune Evasion ◽  
Antigenic Variation ◽  
Surface Proteins ◽  
Functional Study ◽  
Monoallelic Expression ◽  
Immune Evasion Mechanism

ABSTRACT Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes—the intron—in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes—the intron—in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression.

scholarly journals A Yersinia ruckeri TIR Domain-Containing Protein (STIR-2) Mediates Immune Evasion by Targeting the MyD88 Adaptor

2019 ◽  
Vol 20 (18) ◽  
pp. 4409 ◽  
Author(s):  
Tao Liu ◽  
Wen-Yan Wei ◽  
Kai-Yu Wang ◽  
Er-Long Wang ◽  
Qian Yang
Keyword(s):  
Immune Evasion ◽  
Adaptor Protein ◽  
Innate Immune ◽  
Luciferase Reporter ◽  
Yersinia Ruckeri ◽  
Reporter System ◽  
Tir Domain ◽  
Novel Strategy

TIR domain-containing proteins are essential for bacterial pathogens to subvert host defenses. This study describes a fish pathogen, Yersinia ruckeri SC09 strain, with a novel TIR domain-containing protein (STIR-2) that affects Toll-like receptor (TLR) function. STIR-2 was identified in Y. ruckeri by bioinformatics analysis. The toxic effects of this gene on fish were determined by in vivo challenge experiments in knockout mutants and complement mutants of the stir-2 gene. In vitro, STIR-2 downregulated the expression and secretion of IL-6, IL-1β, and TNF-α. Furthermore, the results of NF-κB-dependent luciferase reporter system, co-immunoprecipitation, GST pull-down assays, and yeast two-hybrid assay indicated that STIR-2 inhibited the TLR signaling pathway by interacting with myeloid differentiation factor 88 (MyD88). In addition, STIR-2 promoted the intracellular survival of pathogenic Yersinia ruckeri SC09 strain by binding to the TIR adaptor protein MyD88 and inhibiting the pre-inflammatory signal of immune cells. These results showed that STIR-2 increased virulence in Y. ruckeri and suppressed the innate immune response by inhibiting TLR and MyD88-mediated signaling, serving as a novel strategy for innate immune evasion.

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