Identical Protein Research Articles

Review of respiratory therapies in patients with spinal muscular atrophy

The purpose of this article was to systematize available literary data and to provide general recommendations for respiratory therapy in patients with spinal muscular atrophy. Spinal muscular atrophy (SMA) is a severe neuromuscular disease with autosomal recessive inheritance with degeneration of alpha motor neurons in the anterior horns of the spinal cord, leading to progressive proximal muscle weakness and paralysis. SMN 1–2 genes potentially encode identical proteins, although most of the transcripts of the SMN1 genes are halfsized, whereas most transcripts of the SMN2 genes do not contain the seventh exon. Therefore, the SMN2 gene is only partially functional, and a low-level SMN protein is produced in SMA patients. Moreover, the number of copies of the SMN2 can not be considered an exact predictive factor for any particular patient. The main causes of mortality and deterioration in the quality of life are the development of secondary respiratory failure.
 Type 1 (a, b, c) is the heaviest: early onset and lack of motor abilities, usually patients with a disease of this type survive no more than 2 years.
 Type 2 – an intermediate type characterized by a later onset, the patient may take a sedentary position, survival may reach the adult height.
 Type 3 is the softest form that manifests itself at the age of 1 year, the patient can walk and stand. The forecast is more favorable.
 Type 4 “adult form” manifests itself at the age from 10 to 20 or from 20 to 30 years and has a favorable outlook.
 The main causes of respiratory failure in patients with neuromuscular diseases are weakness of the respiratory muscles, unproductive cough and sleep disturbances. The weakness of the respiratory muscles, defined as the inability of resting respiratory muscles in the state of rest to create a normal level of pressure and air flow velocity when entering and exhaling, is common. Patients with neuromuscular diseases are susceptible to sleep disruption, especially in the REM sleep phase, with the most frequent form of this disorder being hypoventilation. Over time, hypoventilation in a dream can become more prolonged, resulting in the development of a severe form of hypoxia, an increase in the level of carbon dioxide in the blood and the suppression of the activity of the respiratory center. Thus, as a result of the review of literary data, a strategy of respiratory support in patients with CMA was proposed.

Variant proteins stimulate more IgM+ GC B-cells revealing a mechanism of cross-reactive recognition by antibody memory.

Vaccines induce memory B-cells that provide high affinity secondary antibody responses to identical antigens. Memory B-cells can also re-instigate affinity maturation, but how this happens against antigenic variants is poorly understood despite its potential impact on driving broadly protective immunity against pathogens such as Influenza and Dengue. We immunised mice sequentially with identical or variant Dengue-virus envelope proteins and analysed antibody and germinal-centre (GC) responses. Variant protein boosts induced GCs with a higher proportion of IgM+ B cells. The most variant protein re-stimulated GCs with the highest proportion of IgM+ cells with the most diverse, least mutated V-genes and with a slower but efficient serum antibody response. Recombinant antibodies from GC B-cells showed a higher affinity for the variant antigen than antibodies from a primary response, confirming a memory origin. This reveals a new process of antibody memory, that IgM memory cells with fewer mutations participate in secondary responses to variant antigens, demonstrating how the hierarchical structure of B-cell memory is used and indicating the potential and limits of cross-reactive antibody based immunity.

Structural characteristics of the serum endonuclease Dnase1L3

Abstract Systemic Lupus Erythematosus (SLE) is a debilitating autoimmune disease with poly- and mono-genic etiologies. One cause of monogenic SLE is the disruption of Dnase1L3. Dnase1L3 is a serum endonuclease that degrades chromatin in apoptotic microparticles. Dnase1L3 could represent a novel therapy for some cases of SLE. However, the structure of Dnase1L3 is predicted by the 42% identical protein Dnase1. To better understand the structure-function of Dnase1L3, we set out to solve the crystal structure. To solve the crystal structure, we first generated a recombinant His6-Maltose Binding Protein-Dnase1L3 fusion protein. We developed a protocol for purifying Dnase1L3. We isolated Dnase1L3 from the cell lysate using Nickel-NTA beads in maltose buffer, removed purification tags on Dnase1L3 with Tobacco Etch Virus protease, and purified Dnase1L3 on a negatively charged ion exchange column. We then purified Dnase1L3 by size exclusion chromatography to 96.7% final purity. We verified purity at each step by Coomassie staining and, confirmed identity of the final product by Western blot. Recombinant Dnase1L3 robustly degraded plasmid DNA, with a specific activity of 1.83 x 104 U/mg. We found Dnase1L3 had a propensity for small mosaic crystals in a Tris and MgCl2 crystallization buffer. However, larger crystals were grown at 7 °C, compared to 22 °C. Overall, we have developed a new purification method for generating active Dnase1L3 useful for crystallography and other high-purity applications. These applications will further our understanding of Dnase1L3 structure and its function in SLE.

Multiple Pathways in Capsid Assembly.

For a three-dimensional structure to spontaneously self-assemble from many identical components, the steps on the pathway must be kinetically accessible. Many virus capsids are icosahedral and assembled from hundreds of identical proteins, but how they navigate the assembly process is poorly understood. Capsid assembly is thought to involve stepwise addition of subunits to a growing capsid fragment. Coarse-grained models suggest that the reaction occurs on a downhill energy landscape, so intermediates are expected to be fleeting. In this work, charge detection mass spectrometry (CDMS) has been used to track assembly of the hepatitis B virus (HBV) capsid in real time. The icosahedral T = 4 capsid of HBV is assembled from 120 capsid protein dimers. Our results indicate that there are multiple pathways for assembly. Under conditions that favor a modest association energy there is no accumulation of large intermediates, which indicates that available pathways include ones on a downhill energy surface. Under higher salt conditions, where subunit interactions are strengthened, around half of the products of the initial assembly reaction have masses close to the T = 4 capsid and the other half are stalled intermediates which emerge abruptly at around 90 dimers, indicating a bifurcation in the ensemble of assembly paths. When incubated at room temperature, the 90-dimer intermediates accumulate dimers and gradually shift to higher mass and merge with the capsid peak. Though free subunits are present in solution, the stalled intermediates indicate the presence of a local minima on the energy landscape. Some intermediates may result from hole closure, where the growing capsid distorts to close the hole due to the missing capsid proteins or from a species where subsequent additions are particularly labile.

Localizing Conformational Hinges by NMR: Where Do Hepatitis B Virus Core Proteins Adapt for Capsid Assembly?

The hepatitis B virus (HBV) icosahedral nucleocapsid is assembled from 240 chemically identical core protein molecules and, structurally, comprises four groups of symmetrically nonequivalent subunits. We show here that this asymmetry is reflected in solid-state NMR spectra of the capsids, in which peak splitting is observed for a subset of residues. We compare this information to dihedral angle variations from available 3D structures and also to computational predictions of "dynamic" domains and molecular hinges. We find that although, at the given resolution, dihedral angles variations directly obtained from the X-ray structures are not precise enough to be interpreted, the chemical-shift information from NMR correlates, and interestingly goes beyond, information from bioinformatics approaches. Our study reveals the high sensitivity with which NMR can detect the residues allowing the subtle conformational adaptations needed in lattice formation. Our findings are important for understanding the formation and modulation of protein assemblies in general.

Heparan sulfate chain length modulates the cellular targeting of prions in vivo

Prion diseases are fatal neurodegenerative disorders caused by the misfolding of the physiologically expressed prion protein into an insoluble form that accumulates as amorphous aggregates (subfibrillar prions) or amyloid fibers (fibrillar prions). Identical prion protein sequences can misfold into different conformational variants or strains that affect specific neural cell populations, leading to a wide variety of clinical signs and brain regions targeted. The mechanisms underlying the cell targeting of prion strains are unknown. Heparan sulfate (HS) molecules are glycosaminoglycans abundantly expressed in the brain and that profoundly vary in length and sulfation depending on the cell type. HS co‐localize with prion plaques of Creutzfeldt–Jakob disease (CJD), Gerstmann‐Sträussler‐Scheinker (GSS) syndrome, and sheep scrapie, and promote prion replication and fibril formation in vitro. Paradoxically, HS have been used to slow or even prevent prion formation in CJD patients and scrapie‐infected rodents. We hypothesize that the interaction of HS with prions is a major determinant underlying the selective cell vulnerability in prion diseases. Here we characterized the HS length that most effectively modulates the in vitro prion conversion into the aggregated state. We also determined how manipulating the HS chain length shifts the cell tropism of prion strains in vivo. While longer HS chain lengths led to more efficient prion conversion for subfibrillar strains (RML and ME7), intermediate lengths were more effective for the fibrillar strain mCWD. Ext1+/‐ mice expressing shorter HS molecules only altered prion disease phenotype when inoculated with the fibrillar prion mCWD, affecting the disease progression and neuropathology. Together these results suggest that the specific length of the HS together with the prion aggregate conformation determine the binding affinity of prions to HS, and underlie the specific cellular targeting in the CNS. These results have implications for other misfolding proteins such as amyloid‐β and α‐synuclein, which also interact with HS.Support or Funding InformationNIH, NINDS R01 grant 069566 Creutzfeldt–Jakob disease Foundation Fundación Ramón ArecesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dynamics of a Protein Chain Motor Driving Helical Bacteria under Stress

The wall-less, helical bacterial genus Spiroplasma has a unique propulsion system; it is not driven by propeller-like flagella but by a membrane-bound, cytoplasmic, linear motor that consists of a contractile chain of identical proteins spanning the entire cell length. By a coordinated spread of conformational changes of the proteins, kinks propagate in pairs along the cell body. However, the mechanisms for the initiation or delay of kinks and their coordinated spread remain unclear. Here, we show how we manipulate the initiation of kinks, their propagation velocities, and the time between two kinks for a single cell trapped in an optical line potential. By interferometric three-dimensional shape tracking, we measured the cells’ deformations in response to various external stress situations. We observed a significant dependency of force generation on the cells’ local ligand concentrations (likely ATP) and ligand hydrolysis, which we altered in different ways. We developed a mechanistic, mathematical model based on Kramer’s rates, describing the subsequent cooperative and conformational switching of the chain’s proteins. The model reproduces our experimental observations and can explain deformation characteristics even when the motor is driven to its extreme. Nature has invented a set of minimalistic mechanical driving concepts. To understand or even rebuild them, it is essential to reveal the molecular mechanisms of such protein chain motors, which need only two components—coupled proteins and ligands—to function.

Human herpesvirus portal proteins: Structure, function, and antiviral prospects.

Herpesviruses (Herpesvirales) and tailed bacteriophages (Caudovirales) package their dsDNA genomes through an evolutionarily conserved mechanism. Much is known about the biochemistry and structural biology of phage portal proteins and the DNA encapsidation (viral genome cleavage and packaging) process. Although not at the same level of detail, studies on HSV-1, CMV, VZV, and HHV-8 have revealed important information on the function and structure of herpesvirus portal proteins. During dsDNA phage and herpesviral genome replication, concatamers of viral dsDNA are cleaved into single length units by a virus-encoded terminase and packaged into preformed procapsids through a channel located at a single capsid vertex (portal). Oligomeric portals are formed by the interaction of identical portal protein monomers. Comparing portal protein primary aa sequences between phage and herpesviruses reveals little to no sequence similarity. In contrast, the secondary and tertiary structures of known portals are remarkable. In all cases, function is highly conserved in that portals are essential for DNA packaging and also play a role in releasing viral genomic DNA during infection. Preclinical studies have described small molecules that target the HSV-1 and VZV portals and prevent viral replication by inhibiting encapsidation. This review summarizes what is known concerning the structure and function of herpesvirus portal proteins primarily based on their conserved bacteriophage counterparts and the potential to develop novel portal-specific DNA encapsidation inhibitors.

Assessment of Serum Level of Squamous Cell Carcinoma Antigen II in Patients with Lichen Planus

Lichen planus (LP) is a chronic mucocutaneous autoimmune inflammatory disorder of the stratified squamous epitheliumthat mostly affects middle-aged adults. No significant differences in the incidence for LP are noticed between male and femalepatients. Squamous cell carcinoma antigen (SCCA) is known as a tumor biomarker for various squamous cell tumors. It was firstidentified in the uterine cervix by Kato and Torigoe. Total SCCA comprises nearly identical proteins. Both SCCAI and II belongto the ovalbumin-serpin proteinase inhibitor family. The aim of this work was to assess the serum levels of SCCAII in patientswith LP and compare it to its level in normal group. This was a case-control study conducted over a period of six months fromSeptember 2018 to march 2019 included 100 participants, 80 patients suffering from LP. In addition to 20 apparently healthy.Patients were selected from the outpatient clinic of Dermatology and Andrology Department of Benha University Hospitals andKafr Shokr central hospital. We found that Serum levels of SCCAII were significantly higher in patients with LP compared tohealthy control subjects. In conclusion, significant elevation of SSCCAII is associated with LP. Serum SCCAII may become anovel and useful biomarker for evaluating disease activity.

Breaking Symmetry in Viral Icosahedral Capsids as Seen through the Lenses of X-ray Crystallography and Cryo-Electron Microscopy.

The majority of viruses on Earth form capsids built by multiple copies of one or more types of a coat protein arranged with 532 symmetry, generating an icosahedral shell. This highly repetitive structure is ideal to closely pack identical protein subunits and to enclose the nucleic acid genomes. However, the icosahedral capsid is not merely a passive cage but undergoes dynamic events to promote packaging, maturation and the transfer of the viral genome into the host. These essential processes are often mediated by proteinaceous complexes that interrupt the shell’s icosahedral symmetry, providing a gateway through the capsid. In this review, we take an inventory of molecular structures observed either internally, or at the 5-fold vertices of icosahedral DNA viruses that infect bacteria, archea and eukaryotes. Taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of individual components, we review the design principles of non-icosahedral structural components that interrupt icosahedral symmetry and discuss how these macromolecules play vital roles in genome packaging, ejection and host receptor-binding.

Measuring Structural Changes as a Function of Protein Environment using Infrared Spectroscopy

The formulation of biopharmaceutical protein drugs is based on the ability of physiochemical measurement techniques to characterize the structure, stability and dynamics of proteins in different chemical environments. The need to formulate these drugs at high concentrations and use them at much lower concentrations makes it important to have techniques that can compare properties between different concentrations. Infrared spectroscopy has long been used to deduce concentration and structural descriptions of proteins. We are developing new infrared methods to compare the structures, dynamics and functions of nearly identical biopharmaceutical protein samples. We will present our results describing structural changes in model polymers, proteins and the NIST reference monoclonal antibody across different pH, ionic strength and concentration conditions.

Correction: Specialized ribosomes and specific ribosomal protein paralogs control translation of mitochondrial proteins.

Genome duplication in eukaryotes created paralog pairs of ribosomal proteins (RPs) that show high sequence similarity/identity. However, individual paralogs can confer vastly different effects upon cellular processes, e.g., specific yeast paralogs regulate actin organization, bud site selection, and mRNA localization, although how specificity is conferred is unknown. Changes in the RP composition of ribosomes might allow for specialized translation of different subsets of mRNAs, yet it is unclear whether specialized ribosomes exist and if paralog specificity controls translation. Using translatome analyses, we show that the translation of mitochondrial proteins is highly down-regulated in yeast lacking RP paralogs required for normal mitochondrial function (e.g., RPL1b). Although RPL1a and RPL1b encode identical proteins, Rpl1b-containing ribosomes confer more efficient translation of respiration-related proteins. Thus, ribosomes varying in RP composition may confer specialized functions, and RP paralog specificity defines a novel means of translational control.

Antisense Oligonucleotide-Mediated Terminal Intron Retention of the SMN2 Transcript

The severe childhood disease spinal muscular atrophy (SMA) arises from the homozygous loss of the survival motor neuron 1 gene (SMN1). A homologous gene potentially encoding an identical protein, SMN2 can partially compensate for the loss of SMN1; however, the exclusion of a critical exon in the coding region during mRNA maturation results in insufficient levels of functional protein. The rate of transcription is known to influence the alternative splicing of gene transcripts, with a fast transcription rate correlating to an increase in alternative splicing. Conversely, a slower transcription rate is more likely to result in the inclusion of all exons in the transcript. Targeting SMN2 with antisense oligonucleotides to influence the processing of terminal exon 8 could be a way to slow transcription and induce the inclusion of exon 7. Interestingly, following oligomer treatment of SMA patient fibroblasts, we observed the inclusion of exon 7, as well as intron 7, in the transcript. Because the normal termination codon is located in exon 7, this exon/intron 7-SMN2 transcript should encode the normal protein and only carry a longer 3′ UTR. Further studies showed the extra 3′ UTR length contained a number of regulatory motifs that modify transcript and protein regulation, leading to translational repression of SMN. Although unlikely to provide therapeutic benefit for SMA patients, this novel technique for gene regulation could provide another avenue for the repression of undesirable gene expression in a variety of other diseases.

Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding.

Human nucleic acid-binding protein 1 and 2 (hNABP1 and hNABP2, also known as hSSB2 and hSSB1 respectively) form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single-stranded (ss) DNA- and RNA-binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In the present study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution; however, C9ORF80 exhibits anomalous behavior on SDS–PAGE and gel filtration because of 48% random coil present in the protein. Using electrophoretic mobility shift assay (EMSA), INTS3 displays higher affinity toward ssRNA than ssDNA, and C9ORF80 binds ssDNA but not ssRNA. Neither of them binds dsDNA, dsRNA, or RNA : DNA hybrid. INTS3 requires minimum of 30 nucleotides, whereas C9OFR80 requires 20 nucleotides for its binding, which increased with the increasing length of ssDNA. Interestingly, our GST pulldown results suggest that the N-terminus of INTS3 is involved in protein–protein interaction, while EMSA implies that the C-terminus is required for nucleic acid binding. Furthermore, we purified the INTS3–hNABP1/2–C9ORF80 heterotrimeric complex. It exhibits weaker binding compared with the individual hNABP1/2; interestingly, the hNABP1 complex prefers ssDNA, whereas hNABP2 complex prefers ssRNA. Using reconstituted heterotrimeric complex from individual proteins, EMSA demonstrates that INTS3, but not C9ORF80, affects the nucleic acid-binding ability of hNABP1 and hNABP2, indicating that INTS3 might regulate hNABP1/2's biological function, while the role of C9ORF80 remains unknown.

Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels.

Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (Mn = 700 g mol−1) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.

In Vitro-Reassembled Plant Virus-Like Particles of Hibiscus Chlorotic Ringspot Virus (HCRSV) as Nano-Protein Cages for Drugs.

Spherical shaped plant viruses require a precise quantity, size, and shape of their coat protein subunits to assemble into virions of identical dimensions. The capsid of spherical plant virus particles typically consists of a precisely shaped protein cage, which in many cases is assembled from identical coat protein subunits. In addition to packaging the viral genome, such protein cages may have the capacity to load foreign compounds, either large molecules (e.g., polymers) or small molecules (e.g., anticancer chemotherapy drugs). Therefore, reassembled protein cages of suitable viruses can serve as carriers for cargo loading, which is what makes them an attractive platform for drug delivery. Here we describe methods to reassemble plant virus-like particles of hibiscus chlorotic ringspot virus (HCRSV) as nano-protein cages including the techniques to purify coat protein, prepare virus-like particles, and load them with foreign compounds.

Bioinformatic Survey of S-Layer Proteins in Bifidobacteria

Surface layer (S-layer) proteins are one of the most commonly observed cell envelope components in both Archaea and Bacteria. It has versatile functions and holds considerable application potential in biotechnology. Bifidobacteria are representative probiotics conferring health promoting properties. However, there is little study of S-layer in bifidobacteria yet. The distribution and characteristics of S-layer in bifidobacteria are unknown. In this study, search for S-layer protein in the identical protein groups in NCBI yielded 49 hits belonging to bifidobacteria. These proteins were annotated as either “S-layer (domain) protein” or “putative S-layer (y) domain protein” that distributed among 26 species of Bifidobacterium genus. Multiple alignments suggest S-layer proteins are relatively conservative. Phylogenetic analysis of 24 S-layer (domain) protein sequences groups them into three distinct clusters, with the majority species in Cluster-2. S-layer (domain) protein has a universe motif DUF4381, though its function is unknown. Meanwhile, two other motifs CARDB and EphA2_TM involved in cell adhesion and cell signaling respectively, presented in most S-layer (domain) protein in bifidobacteria. All S-layer proteins have a typical N-terminal Sec-dependent signal peptide and a C-terminal trans-membrane region. Homological modeling of representative S-layer proteins from each cluster revealed a few unique structural features. All representative S-layer proteins have a plenty of β-meander motif that exclusively composed by β-barrel structural architectures linked together by hairpin loops.

A histidine-rich Pseudomonas metallothionein with a disordered tail displays higher binding capacity for cadmium than zinc.

Metallothioneins (MTs) are crucial players in metal-related physiological processes. They are characterized by a high cysteine content and unique metal binding properties resulting in specific metal-thiolate clusters formation. Here we present the first NMR solution structure of a Pseudomonas MT, PflQ2 MT, using the strain P. fluorescens Q2-87. It consists of a metal binding domain and an intrinsically disordered C-terminal tail, that was not observed in other MTs so far. While not influencing the structure or function of the metal binding domain, the tail contains a potential binding motif that might be important in so far undiscovered biological interactions. Unusual is the different metal binding capacity for three ZnIIversus four CdII ions that results in two novel metal-cluster topologies. Nevertheless, the affinity for the fourth CdII ion is reduced due to transient coordination. PflQ2 MT contains an unusually large number of four histidine residues, of which only one is involved in metal ion binding. The three non-coordinating histidine residues influence neither the protein fold nor the stability in vitro. We demonstrate that reinstatement of a second coordinating histidine residue, observed for cyanobacterial MTs, in place of a non-coordinating residue in Pseudomonas MTs, decreases the kinetic lability of the cluster, while preserving the overall metal ion binding stability and the protein fold. Since high thermodynamic stability combined with high kinetic lability of metal binding are mechanistic features critical for the function of MTs, the observed replacement might be advantageous for Pseudomonas MTs with respect to metal ion handling in vivo.

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing Leuconostoc pseudomesenteroides 607 Isolated from Persimmon.

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05Da and a partial N-terminal amino acid sequence of NH2-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.

Subtle variation within conserved effector operon gene products contributes to T6SS-mediated killing and immunity.

Type VI secretion systems (T6SS) function to deliver lethal payloads into target cells. Many studies have shown that protection against a single, lethal T6SS effector protein requires a cognate antidote immunity protein, both of which are often encoded together in a two-gene operon. The T6SS and an effector-immunity pair is sufficient for both killing and immunity. HereIn this paper we describe a T6SS effector operon that differs from conventional effector-immunity pairs in that eight genes are necessary for lethal effector function, yet can be countered by a single immunity protein. In this study, we investigated the role that the PefE T6SS immunity protein plays in recognition between two strains harboring nearly identical effector operons. Interestingly, despite containing seven of eight identical effector proteins, the less conserved immunity proteins only provided protection against their native effectors, suggesting that specificity and recognition could be dependent on variation within an immunity protein and one effector gene product. The variable effector gene product, PefD, is encoded upstream from pefE, and displays toxic activity that can be countered by PefE independent of T6SS-activity. Interestingly, while the entire pef operon was necessary to exert toxic activity via the T6SS in P. mirabilis, production of PefD and PefE alone was unable to exert this effector activity. Chimeric PefE proteins constructed from two P. mirabilis strains were used to localize immunity function to three amino acids. A promiscuous immunity protein was created using site-directed mutagenesis to change these residues from one variant to another. These findings support the notion that subtle differences between conserved effectors are sufficient for T6SS-mediated kin discrimination and that PefD requires additional factors to function as a T6SS-dependent effector.