Conservation of C4BP-binding sequence patterns in Streptococcus pyogenes M and Enn proteins.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Conservation of C4BP-binding Sequence Patterns in Streptococcus pyogenes M and Enn Proteins.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP-binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP, and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to identification of amino acids that are crucial for C4BP-binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most populated among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.

Structural Requirements for Reverse Transcription by a Diversity-generating Retroelement.

Diversity-generating retroelements (DGRs), which are pervasive among microbes, create massive protein sequence variation through reverse transcription of a protein-coding RNA template coupled to frequent misincorporation at template adenines. For cDNA synthesis, the template must be surrounded by up- and downstream sequences. Cryo-EM revealed that this longer RNA formed an integral ribonucleoprotein (RNP) with the DGR reverse transcriptase bRT and associated protein Avd. The downstream, noncoding (nc) RNA formed stem-loops enveloping bRT and laying over barrel-shaped Avd, and duplexes with the upstream and template RNA. These RNA structural elements were required for reverse transcription, and several were conserved in DGRs from distant taxa. Multiple RNP conformations were visualized, and no large structural rearrangements occurred when adenine replaced guanine as the template base, suggesting energetics govern misincorporation at adenines. Our results explain how the downstream ncRNA primes cDNA synthesis, promotes processivity, terminates polymerization, and stringently limits mutagenesis to DGR variable proteins.

Structural and photophysical characterization of the small ultra-red fluorescent protein.

The small Ultra-Red Fluorescent Protein (smURFP) represents a new class of fluorescent protein with exceptional photostability and brightness derived from allophycocyanin in a previous directed evolution. Here, we report the smURFP crystal structure to better understand properties and enable further engineering of improved variants. We compare this structure to the structures of allophycocyanin and smURFP mutants to identify the structural origins of the molecular brightness. We then use a structure-guided approach to develop monomeric smURFP variants that fluoresce with phycocyanobilin but not biliverdin. Furthermore, we measure smURFP photophysical properties necessary for advanced imaging modalities, such as those relevant for two-photon, fluorescence lifetime, and single-molecule imaging. We observe that smURFP has the largest two-photon cross-section measured for a fluorescent protein, and that it produces more photons than organic dyes. Altogether, this study expands our understanding of the smURFP, which will inform future engineering toward optimal FPs compatible with whole organism studies.

A conserved 3D pattern in a Streptococcus pyogenes M protein immunogen elicits M-type crossreactivity.

Coiled coil-forming M proteins of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes (strep A) are immunodominant targets of opsonizing antibodies. However, antigenic sequence variability of M proteins into >220 M types, as defined by their hypervariable regions (HVRs), is considered to limit M proteins as vaccine immunogens because of type specificity in the antibody response. Surprisingly, a multi-HVR immunogen in clinical vaccine trials was shown to elicit M-type crossreactivity. The basis for this crossreactivity is unknown but may be due in part to antibody recognition of a 3D pattern conserved in many M protein HVRs that confers binding to human complement C4b-binding protein (C4BP). To test this hypothesis, we investigated whether a single M protein immunogen carrying the 3D pattern would elicit crossreactivity against other M types carrying the 3D pattern. We found that a 34-amino acid sequence of S. pyogenes M2 protein bearing the 3D pattern retained full C4BP-binding capacity when fused to a coiled coil-stabilizing sequence from the protein GCN4. We show that this immunogen, called M2G, elicited cross-reactive antibodies against a number of M types that carry the 3D pattern but not against those that lack the 3D pattern. We further show that the M2G antiserum-recognized M proteins displayed natively on the strep A surface and promoted the opsonophagocytic killing of strep A strains expressing these M proteins. As C4BP binding is a conserved virulence trait of strep A, we propose that targeting the 3D pattern may prove advantageous in vaccine design.

Structural and biochemical characterization of Leptospira interrogans Lsa45 reveals a penicillin-binding protein with esterase activity.

Leptospirosis is a bacterial disease that affects humans and animals and is caused by Leptospira. The recommended treatment for leptospirosis is antibiotic therapy, which should be given early in the course of the disease. Despite the use of these antibiotics, their role during the course of the disease is still not completely clear because of the lack of effective clinical trials, particularly for severe cases of the disease. Here, we present the characterization of L. interrogans Lsa45 protein by gel filtration, protein crystallography, SAXS, fluorescence and enzymatic assays. The oligomeric studies revealed that Lsa45 is monomeric in solution. The crystal structure of Lsa45 revealed the presence of two subdomains: a large α/ß subdomain and a small α-helical subdomain. The large subdomain contains the amino acids Ser122, Lys125, and Tyr217, which correspond to the catalytic triad that is essential for ß-lactamase or serine hydrolase activity in similar enzymes. Additionally, we also confirmed the bifunctional promiscuity of Lsa45, in hydrolyzing both the 4-nitrophenyl acetate (p-NPA) and nitrocefin ß-lactam antibiotic. Therefore, this study provides novel insights into the structure and function of enzymes from L. interrogans, which furthers our understanding of this bacterium and the development of new therapies for the prevention and treatment of leptospirosis.

Structural and biochemical characterization of Leptospira interrogans Lsa45 reveals a penicillin-binding protein with esterase activity

Leptospirosis is a bacterial disease that affects humans and animals and is caused by Leptospira. The recommended treatment for leptospirosis is antibiotic therapy, which should be given early in the course of the disease. Despite the use of these antibiotics, their role during the course of the disease is still not completely clear because of the lack of effective clinical trials, particularly for severe cases of the disease. Here, we present the characterization of L. interrogans Lsa45 protein by gel filtration, protein crystallography, SAXS, fluorescence and enzymatic assays. The oligomeric studies revealed that Lsa45 is monomeric in solution. The crystal structure of Lsa45 revealed the presence of two subdomains: a large α/β subdomain and a small α-helical subdomain. The large subdomain contains the amino acids Ser122, Lys125, and Tyr217, which correspond to the catalytic triad that is essential for β-lactamase or serine hydrolase activity in similar enzymes. Additionally, we also confirmed the bifunctional promiscuity of Lsa45, in hydrolyzing both the 4-nitrophenyl acetate (p-NPA) and nitrocefin β-lactam antibiotic. Therefore, this study provides novel insights into the structure and function of enzymes from L. interrogans, which furthers our understanding of this bacterium and the development of new therapies for the prevention and treatment of leptospirosis.

Strengthening of enterococcal biofilms by Esp.

Multidrug-resistant (MDR) Enterococcus faecalis are major causes of hospital-acquired infections. Numerous clinical strains of E. faecalis harbor a large pathogenicity island that encodes enterococcal surface protein (Esp), which is suggested to promote biofilm production and virulence, but this remains controversial. To resolve this issue, we characterized the Esp N-terminal region, the portion implicated in biofilm production. Small angle X-ray scattering indicated that the N-terminal region had a globular head, which consisted of two DEv-Ig domains as visualized by X-ray crystallography, followed by an extended tail. The N-terminal region was not required for biofilm production but instead significantly strengthened biofilms against mechanical or degradative disruption, greatly increasing retention of Enterococcus within biofilms. Biofilm strengthening required low pH, which resulted in Esp unfolding, aggregating, and forming amyloid-like structures. The pH threshold for biofilm strengthening depended on protein stability. A truncated fragment of the first DEv-Ig domain, plausibly generated by a host protease, was the least stable and sufficient to strengthen biofilms at pH ≤ 5.0, while the entire N-terminal region and intact Esp on the enterococcal surface was more stable and required a pH ≤ 4.3. These results suggested a virulence role of Esp in strengthening enterococcal biofilms in acidic abiotic or host environments.

An M protein coiled coil unfurls and exposes its hydrophobic core to capture LL-37.

Surface-associated, coiled-coil M proteins of Streptococcus pyogenes (Strep A) disable human immunity through interaction with select proteins. However, coiled coils lack features typical of protein-protein interaction sites, and it is therefore challenging to understand how M proteins achieve specific binding, for example, with the human antimicrobial peptide LL-37, leading to its neutralization. The crystal structure of a complex of LL-37 with M87 protein, an antigenic M protein variant from a strain that is an emerging threat, revealed a novel interaction mode. The M87 coiled coil unfurled and asymmetrically exposed its hydrophobic core to capture LL-37. A single LL-37 molecule was bound by M87 in the crystal, but in solution additional LL-37 molecules were recruited, consistent with a 'protein trap' neutralization mechanism. The interaction mode visualized crystallographically was verified to contribute significantly to LL-37 resistance in an M87 Strep A strain and was identified to be conserved in a number of other M protein types that are prevalent in human populations. Our results provide specific detail for therapeutic inhibition of LL-37 neutralization by M proteins.

We share our environment with many different bacteria. Some are beneficial for our health, like gut bacteria, but others can cause severe disease if they infect and spread within the body's tissues. For example, the bacterium Streptococcus pyogenes can cause conditions ranging from skin infections to a rapidly spreading deep-tissue infection, giving it the nickname "flesh-eating bacterium". To prevent infection, our bodies have developed defence mechanisms that target disease-causing bacteria. These include antimicrobial molecules, such as LL-37, which is a small protein produced on the skin. LL-37 kills bacteria by puncturing their cell membrane (the bacterial equivalent of our skin); in other words, it acts like a tiny chemical dart that 'pops' the bacterial cell. However, some bacteria, including S. pyogenes, can disarm these defences. S. pyogenes captures LL-37 on its surface with so called M proteins, which prevent LL-37 from reaching and destroying the underlying membrane. However, it was unknown how exactly the two proteins interact, especially since LL-37 is a simple molecule that lacks the structural features that allow most proteins to bind to each other. Kolesinski et al. set out to determine how the M protein can 'grab' LL-37. A technique called X-ray crystallography allowed them to visualise the molecules atom by atom and to examine the configuration of the M protein after it had captured LL-37. The M protein selected for these experiments (M87) came from a strain associated with particularly severe disease, considered to be an emerging health threat. The results showed that M87 uncurled itself, thereby exposing specific parts that normally remain hidden. This way, it could capture LL-37, like a hand opening to grab an object. Kolesinski et al. have revealed a key molecular mechanism that enables a disease-causing bacterium to invade our immune defences. Identifying which regions of M87 are involved in capturing LL-37 may help design more effective therapies to combat S. pyogenes infections.

Contribution of Streptococcus pyogenes M87 protein to innate immune resistance and virulence.

Streptococcus pyogenes is a pre-eminent human pathogen, and classified by the hypervariable sequence of the emm gene encoding the cell surface M protein. Among a diversity of M/emm types, the prevalence of the M/emm87 strain has been steadily increasing in invasive S. pyogenes infections. Although M protein is the major virulence factor for globally disseminated M/emm1 strain, it is unclear if or how the corresponding M protein of M/emm87 strain (M87 protein) functions as a virulence factor. Here, we use targeted mutagenesis to show that the M87 protein contributes to bacterial resistance to neutrophil and whole blood killing and promotes the release of mature IL-1ß from macrophages. While deletion of emm87 did not influence epithelial cell adherence and nasal colonization, it significantly reduced S. pyogenes-induced mortality and bacterial loads in a murine systemic infection model. Our data suggest that emm87 is involved in pathogenesis by modulating the interaction between S. pyogenes and innate immune cells.

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

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.

Determinants of adenine-mutagenesis in diversity-generating retroelements.

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.

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

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.

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

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.

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

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.

Variation, Indispensability, and Masking in the M protein.

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.

Group A streptococcal M protein activates the NLRP3 inflammasome.

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-1ß (IL-1ß) 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-1ß 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-1ß release could aggravate tissue injury.

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

This corrects the article DOI: 10.1038/nmicrobiol.2016.155.

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

Major radiations of enigmatic Bacteria and Archaea with large inventories of uncharacterized proteins are a striking feature of the Tree of Life1-5. 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 hypervariability6-8, 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.

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

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.