Brucella NpeA is a secreted Type IV effector containing an N-WASP-binding short linear motif that promotes niche formation.

The modulation of actin polymerization is a common theme among microbial pathogens. Even though microorganisms show a wide repertoire of strategies to subvert the activity of actin, most of them converge in the ones that activate nucleating factors, such as the Arp2/3 complex. Brucella spp. are intracellular pathogens capable of establishing chronic infections in their hosts. The ability to subvert the host cell response is dependent on the capacity of the bacterium to attach, invade, avoid degradation in the phagocytic compartment, replicate in an endoplasmic reticulum-derived compartment and egress. Even though a significant number of mechanisms deployed by Brucella in these different phases have been identified and characterized, none of them have been described to target actin as a cellular component. In this manuscript, we describe the identification of a novel virulence factor (NpeA) that promotes niche formation. NpeA harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP and stabilizes the autoinhibited state. Our results show that NpeA is secreted in a Type IV secretion system-dependent manner and that deletion of the gene diminishes the intracellular replication capacity of the bacterium. In vitro and ex vivo experiments demonstrate that NpeA binds N-WASP and that the short linear motif is required for the biological activity of the protein.IMPORTANCEThe modulation of actin-binding effectors that regulate the activity of this fundamental cellular protein is a common theme among bacterial pathogens. The neural Wiskott-Aldrich syndrome protein (N-WASP) is a protein that several pathogens target to hijack actin dynamics. The highly adapted intracellular bacterium Brucella has evolved a wide repertoire of virulence factors that modulate many activities of the host cell to establish successful intracellular replication niches, but, to date, no effector proteins have been implicated in the modulation of actin dynamics. We present here the identification of a virulence factor that harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP stabilizing its autoinhibited state. We demonstrate that this protein is a Type IV secretion effector that targets N-WASP-promoting intracellular survival and niche formation.

Using Linear Motif Database Resources to Identify SH2 Domain Binders.

The SH2-binding phosphotyrosine class of short linear motifs (SLiMs) are key conditional regulatory elements, particularly in signaling protein complexes beneath the cell's plasma membrane. In addition to transmitting cellular signaling information, they can also play roles in cellular hijack by invasive pathogens. Researchers can take advantage of bioinformatics tools and resources to predict the motifs at conserved phosphotyrosine residues in regions of intrinsically disordered protein. A candidate SH2-binding motif can be established and assigned to one or more of the SH2 domain subgroups. It is, however, not so straightforward to predict which SH2 domains are capable of binding the given candidate. This is largely due to the cooperative nature of the binding amino acids which enables poorer binding residues to be tolerated when the other residues are optimal. High-throughput peptide arrays are powerful tools used to derive SH2 domain-binding specificity, but they are unable to capture these cooperative effects and also suffer from other shortcomings. Tissue and cell type expression can help to restrict the list of available interactors: for example, some well-studied SH2 domain proteins are only present in the immune cell lineages. In this article, we provide a table of motif patterns and four bioinformatics strategies that introduce a range of tools that can be used in motif hunting in cellular and pathogen proteins. Experimental followup is essential to determine which SH2 domain/motif-containing proteins are the actual functional partners.

The Eukaryotic Linear Motif resource: 2022 release.

Almost twenty years after its initial release, the Eukaryotic Linear Motif (ELM) resource remains an invaluable source of information for the study of motif-mediated protein-protein interactions. ELM provides a comprehensive, regularly updated and well-organised repository of manually curated, experimentally validated short linear motifs (SLiMs). An increasing number of SLiM-mediated interactions are discovered each year and keeping the resource up-to-date continues to be a great challenge. In the current update, 30 novel motif classes have been added and five existing classes have undergone major revisions. The update includes 411 new motif instances mostly focused on cell-cycle regulation, control of the actin cytoskeleton, membrane remodelling and vesicle trafficking pathways, liquid-liquid phase separation and integrin signalling. Many of the newly annotated motif-mediated interactions are targets of pathogenic motif mimicry by viral, bacterial or eukaryotic pathogens, providing invaluable insights into the molecular mechanisms underlying infectious diseases. The current ELM release includes 317 motif classes incorporating 3934 individual motif instances manually curated from 3867 scientific publications. ELM is available at: http://elm.eu.org.

Short linear motif candidates in the cell entry system used by SARS-CoV-2 and their potential therapeutic implications.

The first reported receptor for SARS-CoV-2 on host cells was the angiotensin-converting enzyme 2 (ACE2). However, the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. We examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling. These SLiM candidates are highly conserved in vertebrates and may interact with the µ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that they may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin ß3 and ACE2, suggesting that these proteins could directly recruit autophagy components. Our findings identify several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.

Mimicry of Short Linear Motifs by Bacterial Pathogens: A Drugging Opportunity.

Bacterial pathogens have developed complex strategies to successfully survive and proliferate within their hosts. Throughout the infection cycle, direct interaction with host cells occurs. Many bacteria have been found to secrete proteins, such as effectors and toxins, directly into the host cell with the potential to interfere with cell regulatory processes, either enzymatically or through protein-protein interactions (PPIs). Short linear motifs (SLiMs) are abundant peptide modules in cell signaling proteins. Here, we cover the reported examples of eukaryotic-like SLiM mimicry being used by pathogenic bacteria to hijack host cell machinery and discuss how drugs targeting SLiM-regulated cell signaling networks are being evaluated for interference with bacterial infections. This emerging anti-infective opportunity may become an essential contributor to antibiotic replacement strategies.

ELM-the eukaryotic linear motif resource in 2020.

The eukaryotic linear motif (ELM) resource is a repository of manually curated experimentally validated short linear motifs (SLiMs). Since the initial release almost 20 years ago, ELM has become an indispensable resource for the molecular biology community for investigating functional regions in many proteins. In this update, we have added 21 novel motif classes, made major revisions to 12 motif classes and added >400 new instances mostly focused on DNA damage, the cytoskeleton, SH2-binding phosphotyrosine motifs and motif mimicry by pathogenic bacterial effector proteins. The current release of the ELM database contains 289 motif classes and 3523 individual protein motif instances manually curated from 3467 scientific publications. ELM is available at: http://elm.eu.org.

The eukaryotic linear motif resource - 2018 update.

Short linear motifs (SLiMs) are protein binding modules that play major roles in almost all cellular processes. SLiMs are short, often highly degenerate, difficult to characterize and hard to detect. The eukaryotic linear motif (ELM) resource (elm.eu.org) is dedicated to SLiMs, consisting of a manually curated database of over 275 motif classes and over 3000 motif instances, and a pipeline to discover candidate SLiMs in protein sequences. For 15 years, ELM has been one of the major resources for motif research. In this database update, we present the latest additions to the database including 32 new motif classes, and new features including Uniprot and Reactome integration. Finally, to help provide cellular context, we present some biological insights about SLiMs in the cell cycle, as targets for bacterial pathogenicity and their functionality in the human kinome.

Strict tropism for CD71+/CD234+ human reticulocytes limits the zoonotic potential of Plasmodium cynomolgi.

Two malaria parasites of Southeast Asian macaques, Plasmodium knowlesi and P cynomolgi, can infect humans experimentally. In Malaysia, where both species are common, zoonotic knowlesi malaria has recently become dominant, and cases are recorded throughout the region. By contrast, to date, only a single case of naturally acquired P cynomolgi has been found in humans. In this study, we show that whereas P cynomolgi merozoites invade monkey red blood cells indiscriminately in vitro, in humans, they are restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC or CD234). This likely contributes to the paucity of detectable zoonotic cynomolgi malaria. We further describe postinvasion morphologic and rheologic alterations in P cynomolgi-infected human reticulocytes that are strikingly similar to those observed for P vivax These observations stress the value of P cynomolgi as a model in the development of blood stage vaccines against vivax malaria.

Exploring Short Linear Motifs Using the ELM Database and Tools.

The Eukaryotic Linear Motif (ELM) resource is dedicated to the characterization and prediction of short linear motifs (SLiMs). SLiMs are compact, degenerate peptide segments found in many proteins and essential to almost all cellular processes. However, despite their abundance, SLiMs remain largely uncharacterized. The ELM database is a collection of manually annotated SLiM instances curated from experimental literature. In this article we illustrate how to browse and search the database for curated SLiM data, and cover the different types of data integrated in the resource. We also cover how to use this resource in order to predict SLiMs in known as well as novel proteins, and how to interpret the results generated by the ELM prediction pipeline. The ELM database is a very rich resource, and in the following protocols we give helpful examples to demonstrate how this knowledge can be used to improve your own research. © 2017 by John Wiley & Sons, Inc.

Lower IgG somatic hypermutation rates during acute dengue virus infection is compatible with a germinal center-independent B cell response.

BACKGROUND: The study of human B cell response to dengue virus (DENV) infection is critical to understand serotype-specific protection and the cross-reactive sub-neutralizing response. Whereas the first is beneficial and thus represents the ultimate goal of vaccination, the latter has been implicated in the development of severe disease, which occurs in a small, albeit significant, fraction of secondary DENV infections. Both primary and secondary infections are associated with the production of poly-reactive and cross-reactive IgG antibodies. METHODS: To gain insight into the effect of DENV infection on the B cell repertoire, we used VH region high-throughput cDNA sequencing of the peripheral blood IgG B cell compartment of 19 individuals during the acute phase of infection. For 11 individuals, a second sample obtained 6 months later was analyzed for comparison. Probabilities of sequencing antibody secreting cells or memory B cells were estimated using second-order Monte Carlo simulation. RESULTS: We found that in acute disease there is an increase in IgG B cell diversity and changes in the relative use of segments IGHV1-2, IGHV1-18, and IGHV1-69. Somewhat unexpectedly, an overall low proportion of somatic hypermutated antibody genes was observed during the acute phase plasmablasts, particularly in secondary infections and those cases with more severe disease. CONCLUSIONS: Our data are consistent with an innate-like antiviral recognition system mediated by B cells using defined germ-line coded B cell receptors, which could provide a rapid germinal center-independent antibody response during the early phase of infection. A model describing concurrent T-dependent and T-independent B cell responses in the context of DENV infection is proposed, which incorporates the selection of B cells using hypomutated IGHV segments and their potential role in poly/cross-reactivity. Its formal demonstration could lead to a definition of its potential implication in antibody-dependent enhancement, and may contribute to rational vaccine development efforts.

Longitudinal analysis of the peripheral B cell repertoire reveals unique effects of immunization with a new influenza virus strain.

BACKGROUND: Despite the potential to produce antibodies that can neutralize different virus (heterotypic neutralization), there is no knowledge of why vaccination against influenza induces protection predominantly against the utilized viral strains (homotypic response). Identification of structural patterns of the B cell repertoire associated to heterotypic neutralization may contribute to identify relevant epitopes for a universal vaccine against influenza. METHODS: Blood samples were collected from volunteers immunized with 2008/2009 trivalent inactivated vaccine (TIV), pandemic H1N1 (pdmH1N1) monovalent inactivated vaccine (MIV) and the 2014/2015 TIV. Neutralization was assessed by hemagglutination and microneutralization test. IgG V(H) amplicons derived from peripheral blood RNA from pre-immune and 7 days post vaccination were subjected to 454-Roche sequencing. Full reconstruction of the sampled repertoires was done with ImmunediveRsity. RESULTS: The TIV induced a predominantly homotypic neutralizing serologic response, while the 09 MIV induced a heterotypic neutralizing seroconversion in 17% of the individuals. Both the 08/09 and the 14/15 TIV were associated with a reduction in clonotypic diversity, whereas 09 MIV was the opposite. Moreover, TIV and MIV induced distinctive patterns of IGHV segment use that are consistent with B cell selection by conserved antigenic determinants shared by the pre-pandemic and the pandemic strains. However, low somatic hypermutation rates in IgG after 09 MIV immunization, but not after 08/09 and 14/15 TIV immunization were observed. Furthermore, no evidence of the original antigenic sin was found in the same individuals after vaccination with the three vaccines. CONCLUSIONS: Immunization with a new influenza virus strain (2009 pdmH1N1) induced unique effects in the peripheral B cell repertoire clonal structure, a stereotyped response involving distinctive IGHV segment use and low somatic hypermutation levels. These parameters were contrastingly different to those observed in response to pre-pandemic and post-pandemic vaccination, and may be the result of clonal selection of common antigenic determinants, as well as germinal center-independent responses that wane as the pandemic strain becomes seasonal. Our findings may contribute in the understanding of the structural and cellular basis required to develop a universal influenza vaccine.

Reconstructing and mining the B cell repertoire with ImmunediveRsity.

The B cell antigen receptor repertoire is highly diverse and constantly modified by clonal selection. High-throughput DNA sequencing (HTS) of the lymphocyte repertoire (Rep-Seq) represents a promising technology to explore such diversity ex-vivo and assist in the identification of antigen-specific antibodies based on molecular signatures of clonal selection. Therefore, integrative tools for repertoire reconstruction and analysis from antibody sequences are needed. We developed ImmunediveRity, a stand-alone pipeline primarily based in R programming for the integral analysis of B cell repertoire data generated by HTS. The pipeline integrates GNU software and in house scripts to perform quality filtering, sequencing noise correction and repertoire reconstruction based on V, D and J segment assignment, clonal origin and unique heavy chain identification. Post-analysis scripts generate a wealth of repertoire metrics that in conjunction with a rich graphical output facilitates sample comparison and repertoire mining. Its performance was tested with raw and curated human and mouse 454-Roche sequencing benchmarks providing good approximations of repertoire structure. Furthermore, ImmunediveRsity was used to mine the B cell repertoire of immunized mice with a model antigen, allowing the identification of previously validated antigen-specific antibodies, and revealing different and unexpected clonal diversity patterns in the post-immunization IgM and IgG compartments. Although ImmunediveRsity is similar to other recently developed tools, it offers significant advantages that facilitate repertoire analysis and repertoire mining. ImmunediveRsity is open source and free for academic purposes and it runs on 64 bit GNU/Linux and MacOS. Available at: https://bitbucket.org/ImmunediveRsity/immunediversity/.