Publications by authors named "Partho Ghosh"

58 Publications

Nonimmune antibody interactions of Group A Streptococcus M and M-like proteins.

PLoS Pathog 2021 02 25;17(2):e1009248. Epub 2021 Feb 25.

Department of Chemistry & Biochemistry, La Jolla, California, United States of America.

M and M-like proteins are major virulence factors of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes. These proteins confer resistance against innate and adaptive immune responses by recruiting specific human proteins to the streptococcal surface. Nonimmune recruitment of immunoglobulins G (IgG) and A (IgA) through their fragment crystallizable (Fc) domains by M and M-like proteins was described almost 40 years ago, but its impact on virulence remains unresolved. These interactions have been suggested to be consequential under immune conditions at mucosal surfaces and in secretions but not in plasma, while other evidence suggests importance in evading phagocytic killing in nonimmune blood. Recently, an indirect effect of Fc-binding through ligand-induced stabilization of an M-like protein was shown to increase virulence. Nonimmune recruitment has also been seen to contribute to tissue damage in animal models of autoimmune diseases triggered by S. pyogenes infection. The damage was treatable by targeting Fc-binding. This and other potential therapeutic applications warrant renewed attention to Fc-binding by M and M-like proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1009248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906336PMC
February 2021

Determinants of adenine-mutagenesis in diversity-generating retroelements.

Nucleic Acids Res 2021 01;49(2):1033-1045

Department of Chemistry & Biochemistry, 9500 Gilman Drive, La Jolla, CA, 92093-0375, USA.

Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial 'dark matter'. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa1240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826257PMC
January 2021

Crystal structure of a Thermus aquaticus diversity-generating retroelement variable protein.

PLoS One 2019 10;14(1):e0205618. Epub 2019 Jan 10.

Department of Chemistry & Biochemistry, University of California San Diego, La Jolla, CA, United States of America.

Diversity-generating retroelements (DGRs) are widely distributed in bacteria, archaea, and microbial viruses, and bring about unparalleled levels of sequence variation in target proteins. While DGR variable proteins share low sequence identity, the structures of several such proteins have revealed the C-type lectin (CLec)-fold as a conserved scaffold for accommodating massive sequence variation. This conservation has led to the suggestion that the CLec-fold may be useful in molecular surface display applications. Thermostability is an attractive feature in such applications, and thus we studied the variable protein of a DGR encoded by a prophage of the thermophile Thermus aquaticus. We report here the 2.8 Å resolution crystal structure of the variable protein from the T. aquaticus DGR, called TaqVP, and confirm that it has a CLec-fold. Remarkably, its variable region is nearly identical in structure to those of several other CLec-fold DGR variable proteins despite low sequence identity among these. TaqVP was found to be thermostable, which appears to be a property shared by several CLec-fold DGR variable proteins. These results provide impetus for the pursuit of the DGR variable protein CLec-fold in molecular display applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205618PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328139PMC
September 2019

Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex.

Nucleic Acids Res 2018 10;46(18):9711-9725

Department of Chemistry & Biochemistry, University of California, San Diego, CA 92093, USA.

Diversity-generating retroelements (DGRs) create unparalleled levels of protein sequence variation through mutagenic retrohoming. Sequence information is transferred from an invariant template region (TR), through an RNA intermediate, to a protein-coding variable region. Selective infidelity at adenines during transfer is a hallmark of DGRs from disparate bacteria, archaea, and microbial viruses. We recapitulated selective infidelity in vitro for the prototypical Bordetella bacteriophage DGR. A complex of the DGR reverse transcriptase bRT and pentameric accessory variability determinant (Avd) protein along with DGR RNA were necessary and sufficient for synthesis of template-primed, covalently linked RNA-cDNA molecules, as observed in vivo. We identified RNA-cDNA molecules to be branched and most plausibly linked through 2'-5' phosphodiester bonds. Adenine-mutagenesis was intrinsic to the bRT-Avd complex, which displayed unprecedented promiscuity while reverse transcribing adenines of either DGR or non-DGR RNA templates. In contrast, bRT-Avd processivity was strictly dependent on the template, occurring only for the DGR RNA. This restriction was mainly due to a noncoding segment downstream of TR, which specifically bound Avd and created a privileged site for processive polymerization. Restriction to DGR RNA may protect the host genome from damage. These results define the early steps in a novel pathway for massive sequence diversification.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gky620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182149PMC
October 2018

Diversity-generating retroelements: natural variation, classification and evolution inferred from a large-scale genomic survey.

Nucleic Acids Res 2018 01;46(1):11-24

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.

Diversity-generating retroelements (DGRs) are novel genetic elements that use reverse transcription to generate vast numbers of sequence variants in specific target genes. Here, we present a detailed comparative bioinformatic analysis that depicts the landscape of DGR sequences in nature as represented by data in GenBank. Over 350 unique DGRs are identified, which together form a curated reference set of putatively functional DGRs. We classify target genes, variable repeats and DGR cassette architectures, and identify two new accessory genes. The great variability of target genes implies roles of DGRs in many undiscovered biological processes. There is much evidence for horizontal transfers of DGRs, and we identify lineages of DGRs that appear to have specialized properties. Because GenBank contains data from only 10% of described species, the compilation may not be wholly representative of DGRs present in nature. Indeed, many DGR subtypes are present only once in the set and DGRs of the candidate phylum radiation bacteria, and Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea archaea, are exceptionally diverse in sequence, with little information available about functions of their target genes. Nonetheless, this study provides a detailed framework for classifying and studying DGRs as they are uncovered and studied in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkx1150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758913PMC
January 2018

Variation, Indispensability, and Masking in the M protein.

Authors:
Partho Ghosh

Trends Microbiol 2018 02 31;26(2):132-144. Epub 2017 Aug 31.

Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

The M protein is the major surface-associated virulence factor of group A Streptococcus (GAS) and an antigenically variable target of host immunity. How selection pressures to escape immune recognition, maintain indispensable functions, and mask vulnerabilities have shaped the sequences of the >220M protein types is unclear. Recent experiments have shed light on this question by showing that, hidden within the antigenic variability of many M protein types, are sequence patterns conserved for recruiting human C4b-binding protein (C4BP). Other host factors may be recruited in a similar manner by conserved but hidden sequence patterns in the M protein. The identification of such patterns may be applicable to the development of a GAS vaccine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tim.2017.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5794653PMC
February 2018

Group A streptococcal M protein activates the NLRP3 inflammasome.

Nat Microbiol 2017 Oct 7;2(10):1425-1434. Epub 2017 Aug 7.

Department of Pediatrics, University of California, San Diego, La Jolla, CA, 92093, USA.

Group A Streptococcus (GAS) is among the top ten causes of infection-related mortality in humans. M protein is the most abundant GAS surface protein, and M1 serotype GAS strains are associated with invasive infections, including necrotizing fasciitis and toxic shock syndrome. Here, we report that released, soluble M1 protein triggers programmed cell death in macrophages (Mϕ). M1 served as a second signal for caspase-1-dependent NLRP3 inflammasome activation, inducing maturation and release of proinflammatory cytokine interleukin-1β (IL-1β) and macrophage pyroptosis. The structurally dynamic B-repeat domain of M1 was critical for inflammasome activation, which involved K efflux and M1 protein internalization by clathrin-mediated endocytosis. Mouse intraperitoneal challenge showed that soluble M1 was sufficient and specific for IL-1β activation, which may represent an early warning to activate host immunity against the pathogen. Conversely, in systemic infection, hyperinflammation associated with M1-mediated pyroptosis and IL-1β release could aggravate tissue injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-017-0005-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750061PMC
October 2017

Retroelement-guided protein diversification abounds in vast lineages of Bacteria and Archaea.

Nat Microbiol 2017 Apr 3;2:17045. Epub 2017 Apr 3.

Marine Science Institute, University of California, Santa Barbara, California 93106, USA.

Major radiations of enigmatic Bacteria and Archaea with large inventories of uncharacterized proteins are a striking feature of the Tree of Life. The processes that led to functional diversity in these lineages, which may contribute to a host-dependent lifestyle, are poorly understood. Here, we show that diversity-generating retroelements (DGRs), which guide site-specific protein hypervariability, are prominent features of genomically reduced organisms from the bacterial candidate phyla radiation (CPR) and as yet uncultivated phyla belonging to the DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota and Nanohaloarchaea) archaeal superphylum. From reconstructed genomes we have defined monophyletic bacterial and archaeal DGR lineages that expand the known DGR range by 120% and reveal a history of horizontal retroelement transfer. Retroelement-guided diversification is further shown to be active in current CPR and DPANN populations, with an assortment of protein targets potentially involved in attachment, defence and regulation. Based on observations of DGR abundance, function and evolutionary history, we find that targeted protein diversification is a pronounced trait of CPR and DPANN phyla compared to other bacterial and archaeal phyla. This diversification mechanism may provide CPR and DPANN organisms with a versatile tool that could be used for adaptation to a dynamic, host-dependent existence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmicrobiol.2017.45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436926PMC
April 2017

Group A Streptococcal M1 Protein Provides Resistance against the Antimicrobial Activity of Histones.

Sci Rep 2017 02 21;7:43039. Epub 2017 Feb 21.

Department of Pediatrics, University of California San Diego, La Jolla, California, United States of America.

Histones are essential elements of chromatin structure and gene regulation in eukaryotes. An unexpected attribute of these nuclear proteins is their antimicrobial activity. A framework for histone release and function in host defense in vivo was revealed with the discovery of neutrophil extracellular traps, a specialized cell death process in which DNA-based structures containing histones are extruded to ensnare and kill bacteria. Investigating the susceptibility of various Gram-positive pathogens to histones, we found high-level resistance by one leading human pathogen, group A Streptococcus (GAS). A screen of isogenic mutants revealed that the highly surface-expressed M1 protein, a classical GAS virulence factor, was required for high-level histone resistance. Biochemical and microscopic analyses revealed that the N-terminal domain of M1 protein binds and inactivates histones before they reach their cell wall target of action. This finding illustrates a new pathogenic function for this classic GAS virulence factor, and highlights a potential innate immune evasion strategy that may be employed by other bacterial pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep43039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5318940PMC
February 2017

Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein.

Nat Microbiol 2016 Sep 5;1(11):16155. Epub 2016 Sep 5.

Department of Chemistry &Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.

No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ∼90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant 'reading head' in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M-C4BP interaction, and also inform a path towards vaccine design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmicrobiol.2016.155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014329PMC
September 2016

Conservation of the C-type lectin fold for accommodating massive sequence variation in archaeal diversity-generating retroelements.

BMC Struct Biol 2016 08 31;16(1):13. Epub 2016 Aug 31.

Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, CA, 92093, USA.

Background: Diversity-generating retroelements (DGRs) provide organisms with a unique means for adaptation to a dynamic environment through massive protein sequence variation. The potential scope of this variation exceeds that of the vertebrate adaptive immune system. DGRs were known to exist only in viruses and bacteria until their recent discovery in archaea belonging to the 'microbial dark matter', specifically in organisms closely related to Nanoarchaeota. However, Nanoarchaeota DGR variable proteins were unassignable to known protein folds and apparently unrelated to characterized DGR variable proteins.

Results: To address the issue of how Nanoarchaeota DGR variable proteins accommodate massive sequence variation, we determined the 2.52 Å resolution limit crystal structure of one such protein, AvpA, which revealed a C-type lectin (CLec)-fold that organizes a putative ligand-binding site that is capable of accommodating 10(13) sequences. This fold is surprisingly reminiscent of the CLec-folds of viral and bacterial DGR variable protein, but differs sufficiently to define a new CLec-fold subclass, which is consistent with early divergence between bacterial and archaeal DGRs. The structure also enabled identification of a group of AvpA-like proteins in multiple putative DGRs from uncultivated archaea. These variable proteins may aid Nanoarchaeota and these uncultivated archaea in symbiotic relationships.

Conclusions: Our results have uncovered the widespread conservation of the CLec-fold in viruses, bacteria, and archaea for accommodating massive sequence variation. In addition, to our knowledge, this is the first report of an archaeal CLec-fold protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12900-016-0064-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006420PMC
August 2016

Coiled-coil destabilizing residues in the group A Streptococcus M1 protein are required for functional interaction.

Proc Natl Acad Sci U S A 2016 08 10;113(34):9515-20. Epub 2016 Aug 10.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093;

The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A "capture-and-collapse" model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1606160113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003295PMC
August 2016

Group A Streptococcal M1 Protein Sequesters Cathelicidin to Evade Innate Immune Killing.

Cell Host Microbe 2015 Oct;18(4):471-7

Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093, USA; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

The antimicrobial peptide LL-37 is generated upon proteolytic cleavage of cathelicidin and limits invading pathogens by directly targeting microbial membranes as well as stimulating innate immune cell function. However, some microbes evade LL-37-mediated defense. Notably, group A Streptococcus (GAS) strains belonging to the hypervirulent M1T1 serogroup are more resistant to human LL-37 than other GAS serogroups. We show that the GAS surface-associated M1 protein sequesters and neutralizes LL-37 antimicrobial activity through its N-terminal domain. M1 protein also binds the cathelicidin precursor hCAP-18, preventing its proteolytic maturation into antimicrobial forms. Exogenous M1 protein rescues M1-deficient GAS from killing by neutrophils and within neutrophil extracellular traps and neutralizes LL-37 chemotactic properties. M1 also binds murine cathelicidin, and its virulence contribution in a murine model of necrotizing skin infection is largely driven by its ability to neutralize this host defense peptide. Thus, cathelicidin resistance is essential for the pathogenesis of hyperinvasive M1T1 GAS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2015.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636435PMC
October 2015

Diversity-generating Retroelements in Phage and Bacterial Genomes.

Microbiol Spectr 2014 Dec;2(6)

Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095.

Diversity-generating retroelements (DGRs) are DNA diversification machines found in diverse bacterial and bacteriophage genomes that accelerate the evolution of ligand-receptor interactions. Diversification results from a unidirectional transfer of sequence information from an invariant template repeat (TR) to a variable repeat (VR) located in a protein-encoding gene. Information transfer is coupled to site-specific mutagenesis in a process called mutagenic homing, which occurs through an RNA intermediate and is catalyzed by a unique, DGR-encoded reverse transcriptase that converts adenine residues in the TR into random nucleotides in the VR. In the prototype DGR found in the Bordetella bacteriophage BPP-1, the variable protein Mtd is responsible for phage receptor recognition. VR diversification enables progeny phage to switch tropism, accelerating their adaptation to changes in sequence or availability of host cell-surface molecules for infection. Since their discovery, hundreds of DGRs have been identified, and their functions are just beginning to be understood. VR-encoded residues of many DGR-diversified proteins are displayed in the context of a C-type lectin fold, although other scaffolds, including the immunoglobulin fold, may also be used. DGR homing is postulated to occur through a specialized target DNA-primed reverse transcription mechanism that allows repeated rounds of diversification and selection, and the ability to engineer DGRs to target heterologous genes suggests applications for bioengineering. This chapter provides a comprehensive review of our current understanding of this newly discovered family of beneficial retroelements.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/microbiolspec.MDNA3-0029-2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498404PMC
December 2014

Targeted diversity generation by intraterrestrial archaea and archaeal viruses.

Nat Commun 2015 Mar 23;6:6585. Epub 2015 Mar 23.

1] Marine Science Institute, University of California, Santa Barbara, California 93106, USA [2] Department of Earth Science, University of California Santa Barbara, Santa Barbara, California 93106 USA.

In the evolutionary arms race between microbes, their parasites, and their neighbours, the capacity for rapid protein diversification is a potent weapon. Diversity-generating retroelements (DGRs) use mutagenic reverse transcription and retrohoming to generate myriad variants of a target gene. Originally discovered in pathogens, these retroelements have been identified in bacteria and their viruses, but never in archaea. Here we report the discovery of intact DGRs in two distinct intraterrestrial archaeal systems: a novel virus that appears to infect archaea in the marine subsurface, and, separately, two uncultivated nanoarchaea from the terrestrial subsurface. The viral DGR system targets putative tail fibre ligand-binding domains, potentially generating >10(18) protein variants. The two single-cell nanoarchaeal genomes each possess ≥4 distinct DGRs. Against an expected background of low genome-wide mutation rates, these results demonstrate a previously unsuspected potential for rapid, targeted sequence diversification in intraterrestrial archaea and their viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms7585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372165PMC
March 2015

Structure of the essential Plasmodium host cell traversal protein SPECT1.

PLoS One 2014 5;9(12):e114685. Epub 2014 Dec 5.

Department of Chemistry & Biochemistry, University of California San Diego, La Jolla, California, United States of America.

Host cell traversal by Plasmodium, the protozoan cause of malaria, is an essential part of this parasite's virulence. In this process, the parasite enters a host cell through a parasite-induced pore, traverses the host cell, and then exits the host cell. Two P. berghei proteins, SPECT1 and SPECT2, are required for host cell traversal by the sporozoite form of the parasite. In the absence of either, no pore formation is observed. While SPECT2 has sequence homology to pore-forming proteins, SPECT1 has no homology to proteins of known structure or function. Here we present the 2.75 Å resolution structure of a slightly truncated version of P. berghei SPECT1. The structure reveals that the protein forms a four-helix bundle, with the rare feature of having all of these helices in parallel or antiparallel alignment. Also notable is the presence of a large, conserved, hydrophobic internal cavity in the protein, which may constitute a ligand-binding site or be indicative of partial instability in SPECT1, or both. The structure of SPECT1 will make possible targeted mutagenesis experiments aimed at understanding its mechanism of action in host cell traversal.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114685PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257719PMC
April 2016

Mutual exclusivity of hyaluronan and hyaluronidase in invasive group A Streptococcus.

J Biol Chem 2014 Nov 29;289(46):32303-32315. Epub 2014 Sep 29.

Department of Pediatrics, University of California San Diego, La Jolla, California 92093; School of Chemistry and Molecular Biosciences and The University of Queensland, St. Lucia, Queensland 4072, Australia; Australian Infectious Diseases Research Centre, The University of Queensland, St. Lucia, Queensland 4072, Australia,. Electronic address:

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.602847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231703PMC
November 2014

The fibrinogen-binding M1 protein reduces pharyngeal cell adherence and colonization phenotypes of M1T1 group A Streptococcus.

J Biol Chem 2014 Feb 19;289(6):3539-46. Epub 2013 Dec 19.

From the Departments of Pediatrics and.

Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis ("strep throat") to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulent WT M1T1 GAS strain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surface M1 protein cleavage by the GAS cysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreased GAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.529537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3916555PMC
February 2014

Functionally essential interaction between Yersinia YscO and the T3S4 domain of YscP.

J Bacteriol 2013 Oct 9;195(20):4631-8. Epub 2013 Aug 9.

Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, California, USA.

The type III secretion (T3S) system is essential to the virulence of a large number of Gram-negative bacterial pathogens, including Yersinia. YscO is required for T3S in Yersinia and is known to interact with several other T3S proteins, including the chaperone SycD and the needle length regulator YscP. To define which interactions of YscO are required for T3S, we pursued model-guided mutagenesis: three conserved and surface-exposed regions of modeled YscO were targeted for multiple alanine substitutions. Most of the mutations abrogated T3S and did so in a recessive manner, consistent with a loss of function. Both functional and nonfunctional YscO mutant proteins interacted with SycD, indicating that the mutations had not affected protein stability. Likewise, both functional and nonfunctional versions of YscO were exclusively intrabacterial. Functional and nonfunctional versions of YscO were, however, distinguishable with respect to interaction with YscP. This interaction was observed only for wild-type YscO and a T3S-proficient mutant of YscO but not for the several T3S-deficient mutants of YscO. Evidence is presented that the YscO-YscP interaction is direct and that the type III secretion substrate specificity switch (T3S4) domain of YscP is sufficient for this interaction. These results provide evidence that the interaction of YscO with YscP, and in particular the T3S4 domain of YscP, is essential to type III secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.00876-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807450PMC
October 2013

Surface display of a massively variable lipoprotein by a Legionella diversity-generating retroelement.

Proc Natl Acad Sci U S A 2013 May 30;110(20):8212-7. Epub 2013 Apr 30.

Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by facilitating localized DNA sequence evolution through a specialized error-prone reverse transcription process. We characterized a DGR in Legionella pneumophila, an opportunistic human pathogen that causes Legionnaires disease. The L. pneumophila DGR is found within a horizontally acquired genomic island, and it can theoretically generate 10(26) unique nucleotide sequences in its target gene, legionella determinent target A (ldtA), creating a repertoire of 10(19) distinct proteins. Expression of the L. pneumophila DGR resulted in transfer of DNA sequence information from a template repeat to a variable repeat (VR) accompanied by adenine-specific mutagenesis of progeny VRs at the 3'end of ldtA. ldtA encodes a twin-arginine translocated lipoprotein that is anchored in the outer leaflet of the outer membrane, with its C-terminal variable region surface exposed. Related DGRs were identified in L. pneumophila clinical isolates that encode unique target proteins with homologous VRs, demonstrating the adaptability of DGR components. This work characterizes a DGR that diversifies a bacterial protein and confirms the hypothesis that DGR-mediated mutagenic homing occurs through a conserved mechanism. Comparative bioinformatics predicts that surface display of massively variable proteins is a defining feature of a subset of bacterial DGRs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1301366110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3657778PMC
May 2013

Coiled-coil irregularities of the M1 protein structure promote M1-fibrinogen interaction and influence group A Streptococcus host cell interactions and virulence.

J Mol Med (Berl) 2013 Jul 27;91(7):861-9. Epub 2013 Feb 27.

Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Rämistr 100, 8091 Zürich, Switzerland.

Group A Streptococcus (GAS) is a human pathogen causing a wide range of mild to severe and life-threatening diseases. The GAS M1 protein is a major virulence factor promoting GAS invasiveness and resistance to host innate immune clearance. M1 displays an irregular coiled-coil structure, including the B-repeats that bind fibrinogen. Previously, we found that B-repeat stabilisation generates an idealised version of M1 (M1) characterised by decreased fibrinogen binding in vitro. To extend these findings based on a soluble truncated version of M1, we now studied the importance of the B-repeat coiled-coil irregularities in full length M1 and M1 expressed in live GAS and tested whether the modulation of M1-fibrinogen interactions would open up novel therapeutic approaches. We found that altering either the M1 structure on the GAS cell surface or removing its target host protein fibrinogen blunted GAS virulence. GAS expressing M1 showed an impaired ability to adhere to and to invade human endothelial cells, was more readily killed by whole blood or neutrophils and most importantly was less virulent in a murine necrotising fasciitis model. M1-mediated virulence of wild-type GAS was strictly dependent on the presence and concentration of fibrinogen complementing our finding that M1-fibrinogen interactions are crucial for GAS virulence. Consistently blocking M1-fibrinogen interactions by fragment D reduced GAS virulence in vitro and in vivo. This supports our conclusion that M1-fibrinogen interactions are crucial for GAS virulence and that interference may open up novel complementary treatment options for GAS infections caused by the leading invasive GAS strain M1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00109-013-1012-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695690PMC
July 2013

Structure of the essential diversity-generating retroelement protein bAvd and its functionally important interaction with reverse transcriptase.

Structure 2013 Feb 27;21(2):266-76. Epub 2012 Dec 27.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.

Diversity-generating retroelements (DGRs) are the only known source of massive protein sequence variation in prokaryotes. These elements transfer coding information from a template region (TR) through an RNA intermediate to a protein-encoding variable region. This retrohoming process is accompanied by unique adenine-specific mutagenesis and, in the prototypical BPP-1 DGR, requires a reverse transcriptase (bRT) and an accessory variability determinant (bAvd) protein. To understand the role of bAvd, we determined its 2.69 Å resolution structure, which revealed a highly positively charged pentameric barrel. In accordance with its charge, bAvd bound both DNA and RNA, albeit without a discernable sequence preference. We found that the coding sequence of bAvd functioned as part of TR but identified means to mutate bAvd without affecting TR. This mutational analysis revealed a strict correspondence between retrohoming and interaction of bAvd with bRT, suggesting that the bRT-bAvd complex is important for DGR retrohoming.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2012.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3570691PMC
February 2013

Structure and glycolipid binding properties of the nematicidal protein Cry5B.

Biochemistry 2012 Dec 26;51(49):9911-21. Epub 2012 Nov 26.

Department of Chemistry and Biochemistry, 9500 Gilman Drive, University of California, San Diego, La Jolla, CA 92093-0375, USA.

Crystal (Cry) proteins are globally used in agriculture as proteinaceous insecticides. They have also been recently recognized to have great potential as anthelmintic agents in targeting parasitic roundworms (e.g., hookworms). The most extensively characterized of the anthelmintic Cry proteins is Cry5B. We report here the 2.3 Å resolution structure of the proteolytically activated form of Cry5B. This structure, which is the first for a nematicidal Cry protein, shows the familiar three-domain arrangement seen in insecticidal Cry proteins. However, domain II is unusual in that it more closely resembles a banana lectin than it does other Cry proteins. This result is consistent with the fact that the receptor for Cry5B consists of a set of invertebrate-specific glycans (attached to lipids) and also suggests that domain II is important for receptor binding. We found that not only galactose but also N-acetylgalactosamine (GalNAc) is an efficient competitor for binding between Cry5B and glycolipids. GalNAc is one of the core arthroseries tetrasaccharides of the Cry5B receptor and galactose an antennary sugar that emanates from this core. These and prior data suggest that the minimal binding determinant for Cry5B consists of a core GalNAc and two antennary galactoses. Lastly, the protoxin form of Cry5B was found to bind nematode glycolipids with a specificity equal to that of activated Cry5B, but with lower affinity. This suggests that the initial binding of Cry5B protoxin to glycolipids can be stabilized at the nematode cell surface by proteolysis. These results lay the groundwork for the design of effective Cry5B-based anthelmintics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi301386qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567309PMC
December 2012

Structure and interactions of the cytoplasmic domain of the Yersinia type III secretion protein YscD.

J Bacteriol 2012 Nov 31;194(21):5949-58. Epub 2012 Aug 31.

Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, California, USA.

The virulence of a large number of Gram-negative bacterial pathogens depends on the type III secretion (T3S) system, which transports select bacterial proteins into host cells. An essential component of the Yersinia T3S system is YscD, a single-pass inner membrane protein. We report here the 2.52-Å resolution structure of the cytoplasmic domain of YscD, called YscDc. The structure confirms that YscDc consists of a forkhead-associated (FHA) fold, which in many but not all cases specifies binding to phosphothreonine. YscDc, however, lacks the structural properties associated with phosphothreonine binding and thus most likely interacts with partners in a phosphorylation-independent manner. Structural comparison highlighted two loop regions, L3 and L4, as potential sites of interactions. Alanine substitutions at L3 and L4 had no deleterious effects on protein structure or stability but abrogated T3S in a dominant negative manner. To gain insight into the function of L3 and L4, we identified proteins associated with YscD by affinity purification coupled to mass spectrometry. The lipoprotein YscJ was found associated with wild-type YscD, as was the effector YopH. Notably, the L3 and L4 substitution mutants interacted with more YopH than did wild-type YscD. These substitution mutants also interacted with SycH (the specific chaperone for YopH), the putative C-ring component YscQ, and the ruler component YscP, whereas wild-type YscD did not. These results suggest that substitutions in the L3 and L4 loops of YscD disrupted the dissociation of SycH from YopH, leading to the accumulation of a large protein complex that stalled the T3S apparatus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JB.00513-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486071PMC
November 2012

Two translation products of Yersinia yscQ assemble to form a complex essential to type III secretion.

Biochemistry 2012 Feb 15;51(8):1669-77. Epub 2012 Feb 15.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0375, United States.

The bacterial flagellar C-ring is composed of two essential proteins, FliM and FliN. The smaller protein, FliN, is similar to the C-terminus of the larger protein, FliM, both being composed of SpoA domains. While bacterial type III secretion (T3S) systems encode many proteins in common with the flagellum, they mostly have a single protein in place of FliM and FliN. This protein resembles FliM at its N-terminus and is as large as FliM but is more like FliN at its C-terminal SpoA domain. We have discovered that a FliN-sized cognate indeed exists in the Yersinia T3S system to accompany the FliM-sized cognate. The FliN-sized cognate, YscQ-C, is the product of an internal translation initiation site within the locus encoding the FliM-sized cognate YscQ. Both intact YscQ and YscQ-C were found to be required for T3S, indicating that the internal translation initiation site, which is conserved in some but not all YscQ orthologs, is crucial for function. The crystal structure of YscQ-C revealed a SpoA domain that forms a highly intertwined, domain-swapped homodimer, similar to those observed in FliN and the YscQ ortholog HrcQ(B). A single YscQ-C homodimer associated reversibly with a single molecule of intact YscQ, indicating conformational differences between the SpoA domains of intact YscQ and YscQ-C. A "snap-back" mechanism suggested by the structure can account for this. The 1:2 YscQ-YscQ-C complex is a close mimic of the 1:4 FliM-FliN complex and the likely building block of the putative Yersinia T3S system C-ring.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi201792pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3289748PMC
February 2012

Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement.

Proc Natl Acad Sci U S A 2011 Aug 22;108(35):14649-53. Epub 2011 Aug 22.

Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd (∼16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10(20) potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1105613108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3167528PMC
August 2011

The nonideal coiled coil of M protein and its multifarious functions in pathogenesis.

Authors:
Partho Ghosh

Adv Exp Med Biol 2011 ;715:197-211

Department of Chemistry and Biochemistry, University of California, San Diego, CA, 92093-0375, USA.

The M protein is a major virulence factor of Streptococcus pyogenes (group A Streptococcus, GAS). This gram-positive bacterial pathogen is responsible for mild infections, such as pharyngitis, and severe invasive disease, like streptococcal toxic shock syndrome. M protein contributes to GAS virulence in multifarious ways, including blocking deposition of antibodies and complement, helping formation of microcolonies, neutralizing antimicrobial peptides, and triggering a proinflammatory and procoagulatory state. These functions are specified by interactions between M protein and many host components, especially C4BP and fibrinogen. The former interaction is conserved among many antigenically variant M protein types but occurs in a strikingly sequence-independent manner, and the latter is associated in the M1 protein type with severe invasive disease. Remarkably for a protein of such diverse interactions, the M protein has a relatively simple but nonideal α-helical coiled coil sequence. This sequence nonideality is a crucial feature of M protein. Nonideal residues give rise to specific irregularities in its coiled-coil structure, which are essential for interactions with fibrinogen and establishment of a proinflammatory state. In addition, these structural irregularities are reminiscent of those in myosin and tropomyosin, which are targets for crossreactive antibodies in patients suffering from autoimmune sequelae of GAS infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-94-007-0940-9_12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5541941PMC
July 2011

Streptococcal M1 protein constructs a pathological host fibrinogen network.

Nature 2011 Apr;472(7341):64-8

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.

M1 protein, a major virulence factor of the leading invasive strain of group A Streptococcus, is sufficient to induce toxic-shock-like vascular leakage and tissue injury. These events are triggered by the formation of a complex between M1 and fibrinogen that, unlike M1 or fibrinogen alone, leads to neutrophil activation. Here we provide a structural explanation for the pathological properties of the complex formed between streptococcal M1 and human fibrinogen. A conformationally dynamic coiled-coil dimer of M1 was found to organize four fibrinogen molecules into a specific cross-like pattern. This pattern supported the construction of a supramolecular network that was required for neutrophil activation but was distinct from a fibrin clot. Disruption of this network into other supramolecular assemblies was not tolerated. These results have bearing on the pathophysiology of streptococcal toxic shock.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature09967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3268815PMC
April 2011
-->