Laminin-binding Protein Research Articles

Unveilling genetic profiles and correlations of biofilm-associated genes, quorum sensing, and antibiotic resistance in Staphylococcus aureus isolated from a Malaysian Teaching Hospital

BackgroundStaphylococcus aureus is a notorious multidrug resistant pathogen prevalent in healthcare facilities worldwide. Unveiling the mechanisms underlying biofilm formation, quorum sensing and antibiotic resistance can help in developing more effective therapy for S. aureus infection. There is a scarcity of literature addressing the genetic profiles and correlations of biofilm-associated genes, quorum sensing, and antibiotic resistance among S. aureus isolates from Malaysia.MethodsBiofilm and slime production of 68 methicillin-susceptible S. aureus (MSSA) and 54 methicillin-resistant (MRSA) isolates were determined using a a plate-based crystal violet assay and Congo Red agar method, respectively. The minimum inhibitory concentration values against 14 antibiotics were determined using VITEK® AST-GP67 cards and interpreted according to CLSI-M100 guidelines. Genetic profiling of 11 S. aureus biofilm-associated genes and agr/sar quorum sensing genes was performed using single or multiplex polymerase chain reaction (PCR) assays.ResultsIn this study, 75.9% (n = 41) of MRSA and 83.8% (n = 57) of MSSA isolates showed strong biofilm-forming capabilities. Intermediate slime production was detected in approximately 70% of the isolates. Compared to MSSA, significantly higher resistance of clindamycin, erythromycin, and fluoroquinolones was noted among the MRSA isolates. The presence of intracellular adhesion A (icaA) gene was detected in all S. aureus isolates. All MSSA isolates harbored the laminin-binding protein (eno) gene, while all MRSA isolates harbored intracellular adhesion D (icaD), clumping factors A and B (clfA and clfB) genes. The presence of agrI and elastin-binding protein (ebpS) genes was significantly associated with biofilm production in MSSA and MRSA isolates, respectively. In addition, agrI gene was also significantly correlated with oxacillin, cefoxitin, and fluoroquinolone resistance.ConclusionsThe high prevalence of biofilm and slime production among MSSA and MRSA isolates correlates well with the detection of a high prevalence of biofilm-associated genes and agr quorum sensing system. A significant association of agrI gene was found with cefoxitin, oxacillin, and fluoroquinolone resistance. A more focused approach targeting biofilm-associated and quorum sensing genes is important in developing new surveillance and treatment strategies against S. aureus biofilm infection.

Material matters: Degradation products affect regenerating Schwann cells

Devices to treat peripheral nerve injury (PNI) must balance many considerations to effectively guide regenerating nerves across a gap and achieve functional recovery. To enhance efficacy, design features like luminal fillers have been explored extensively. Material choice for PNI devices is also critical, as the determining factor of device mechanics, and degradation rate and has increasingly been found to directly impact biological response. This study investigated the ways in which synthetic polymer materials impact the differentiation state and myelination potential of Schwann cells, peripheral nerve glia. Microporous substrates of polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA) 85:15, or PLGA 50:50 were chosen, as materials already used in nerve repair devices, representing a wide range of mechanics and degradation profiles. Schwann cells co-cultured with dorsal root ganglion (DRG) neurons on the substrates expressed more mature myelination proteins (MPZ) on PLGA substrates compared to PCL. Changes to myelination and differentiation state of glia were reflected in adhesion proteins expressed by glia, including β-dystroglycan and integrin α6, both laminin binding proteins. Importantly, degradation products of the polymers affected glial expression independently of direct attachment. Fast degrading PLGA 50:50 substrates released measurable amounts of degradation products (lactic acid) within the culture period, which may push Schwann cells towards glycolytic metabolism, decreasing expression of early transcription factors like sox10. This study shows the importance of understanding not only material effects on attachment, but also on cellular metabolism which drives myelination responses.

Dual transgene amelioration of Lama2-null muscular dystrophy.

Null mutations of the Lama2-gene cause a severe congenital muscular dystrophy and associated neuropathy. In the absence of laminin-α2 (Lmα2) there is a compensatory replacement by Lmα4, a subunit that lacks the polymerization and α-dystroglycan (αDG)-binding properties of Lmα2. The dystrophic phenotype in the dy3K/dy3K Lama2-/- mouse were evaluated with transgenes driving expression of two synthetic laminin-binding linker proteins. Transgenic muscle-specific expression of αLNNd, a chimeric protein that enables α4-laminin polymerization, and miniagrin (mag), a protein that increases laminin binding to the receptor αDG, separately improved median mouse survival two-fold. The double transgenes (DT) improved mean survival three-fold with increases in overall body weight, muscle size, and grip strength, but, given absence of neuronal expression, did not prevent hindlimb paresis. Muscle improvements included increased myofiber size and number and reduced fibrosis. Myofiber hypertrophy with increased mTOR and Akt phosphorylation were characteristics of mag-dy3K/dy3K and DT-dy3K/dy3K muscle. Elevations of matrix-bound α4-, β1 and γ1 laminin subunits were detected in muscle extracts and immunostained sections in response to DT expression. Collectively, these findings reveal a complimentary polymerization and αDG-binding benefit to Lama2-/- mouse muscle largely mediated through modified laminin-411.

Laminin-binding protein of Streptococcus suis serotype 2 influences zinc acquisition and cytokine responses

Streptococcus suis serotype 2 is an important bacterial pathogen of swine, responsible for substantial economic losses to the swine industry worldwide. The knowledge on the pathogenesis of the infection caused by S. suis is still poorly known. It has been previously described that S. suis possesses at least one lipoprotein with double laminin and zinc (Zn)-binding properties, which was described in the literature as either laminin-binding protein (Lmb, as in the current study), lipoprotein 103, CDS 0330 or AdcAII. In the present study, the role of the Lmb in the pathogenesis of the infection caused by S. suis serotype 2 was dissected. Using isogenic mutants, results showed that Lmb does not play an important role in the laminin-binding activity of S. suis, even when clearly exposed at the bacterial surface. In addition, the presence of this lipoprotein does not influence bacterial adhesion to and invasion of porcine respiratory epithelial and brain endothelial cells and it does not increase the susceptibility of S. suis to phagocytosis. On the other hand, the Lmb was shown to play an important role as cytokine activator when tested in vitro with dendritic cells. Finally, this lipoprotein plays a critical role in Zn acquisition from the host environment allowing bacteria to grow in vivo. The significant lower virulence of the Lmb defective mutant may be related to a combination of a lower bacterial survival due to the incapacity to acquire Zn from their surrounding milieu and a reduced cytokine activation.

Acanthamoeba Mannose and Laminin Binding Proteins Variation across Species and Genotypes.

Acanthamoeba is a ubiquitous free-living amoeba capable of being an opportunistic pathogen in humans and animals. A critical step in infection is the adhesion of the amoeba to host cells and tissues, and two major parasite adhesins, mannose-binding protein (MBP) and laminin-binding protein (LBP), are known to recognize the cell surface glycoproteins and those of the extracellular matrix, respectively. In this study, the available genomes of Acanthamoeba were analysed to recover the sequences of MBP and LBP using previously published genetic data. Genes for both proteins were successfully obtained from strains belonging to various genotypes (T4A, T4D, T4G, T4F, T2, T5, T10, T22, T7 and T18), resulting in a single gene for LBP but identifying two types of MBP, MBP1 and MBP2. Phylogenetic analysis based on deduced amino acid sequences shows that both MBP and LBP have a branching pattern that is consistent with that based on 18S rDNA, indicating that changes in both proteins occurred during diversification of Acanthamoeba lines. Notably, all MBPs possess a conserved motif, shared with some bacterial C-type lectins, which could be the recognition site for mannose binding.

Oxacillin (Methicillin) Resistant Staphylococci in Domestic Animals in the Czech Republic.

The aim of this study was to describe the prevalence of different Staphylococcus species isolated from pathological processes and lesions in domestic animals in the Czech Republic and to detect and describe oxacillin (methicillin)-resistant strains (MRS). During the years 2019–2020, a total of 5218 veterinary clinical samples from the Czech Republic were tested. Testing was performed by culture methods and typing by molecular phenotypic methods MALDI-TOF MS and PCR. Antimicrobial susceptibility testing of the strains was performed by the disk diffusion method. A total of 854 staphylococci strains were identified (16.37% prevalence), out of which 43 strains of 6 species of staphylococci were MRS (n = 43; 0.82% prevalence). Of the MRS strains, the most prevalent species were Staphylococcus pseudintermedius (n = 24; 0.46% prevalence) and Staphylococcus aureus (n = 7; 0.13% prevalence). Susceptibility testing showed resistance to beta-lactam antibiotics and, depending on the species, also to trimethoprim/sulfamethoxazole, gentamicin, tetracycline, erythromycin, clindamycin, and enrofloxacin. For further characterization of MRS, PCR assay for virulence factor genes was performed. Seven of the 14 target genes were observed only in S. aureus, except for the eno gene encoding laminin-binding protein, which was also detected in other staphylococci. It is necessary to emphasize the issue of correct using of antimicrobials in practice and antibiotic policy in university teaching and to create stricter legislation that would prevent the widespread use of antimicrobials in veterinary medicine, especially in livestock to reduce the emergence and spread of antimicrobial resistance.

In silico screening and epitope mapping of leptospiral outer membrane protein—Lsa46

Leptospirosis is one of the neglected diseases caused by the spirochete, Leptospira interrogans. Leptospiral surface adhesion (Lsa) proteins are surface exposed outer membrane proteins present in the pathogen. It acts as laminin and plasminogen binding proteins which enable them to infect host cells. The major target for the development of vaccine in the current era focuses on surface exposed outer membrane proteins, as they can induce strong and fast immune response in hosts. Therefore, the present study mapped the potential epitopes of the Leptospiral outer membrane proteins, mainly the surface adhesion proteins. Protein sequence analysis of Lsa proteins was done by in silico methods. The primary protein sequence analysis revealed Lsa46 as a suitable target which can be a potent Leptospiral vaccine candidate. Its structure was modelled by threading based method in I-TASSER server and validated by Ramachandran plot. The predicted epitope’s interactions with human IgG, IgM(Fab) and T-cell receptor TCR(αβ) were performed by molecular docking studies using Biovia Discovery studio 2018. One of the predicted B-cell epitopes and the IgG showed desirable binding interactions, while four of the predicted B-cell epitopes and T-cell epitopes showed desirable binding interactions with IgM and TCR respectively. The molecular dynamic simulation studies carried out with the molecular docked complexes gave minimized energies indicating stable interactions. The structural analysis of the entire simulated complex showed a stable nature except for one of the Epitope-IgM complex. Further the binding free energy calculation of eight receptor-ligand complex predicted them energetically stable. The results of the study help in elucidating the structural and functional characterization of Lsa46 for epitope-based vaccine design. Communicated by Ramaswamy H. Sarma.

First Report of Antimicrobial Susceptibility and Virulence Gene Characterization Associated with Staphylococcus aureus Carriage in Healthy Camels from Tunisia

A total of 318 nasal and rectal swabs were collected from 159 apparently healthy camels (Camelus dromedarius) randomly selected from five regions in southern and central Tunisia and screened for Staphylococcus aureus carriage. Staphylococcus spp. were recovered from 152 of 159 camels studied (95.6%) and in total 258 swabs (81%) were positive. Among these isolates, 16 were coagulase positive Staphylococcus (CoPS) (6.2%) and were characterized by biochemical and molecular tests as S. aureus. These were isolated from 14 camels (8.8%) with co-carriage in nasal and rectal mucosa by two camels. All S. aureus isolates recovered were methicillin-susceptible Staphylococcus aureus (MSSA) and were characterized by spa typing and PFGE. Three different spa types were recovered: t729, t4013 and a spa type newly registered as t19687, which was the most common. PFGE analysis revealed seven different patterns and these were characterized by MLST, which revealed five different sequence types (ST6, ST88, ST3583 and two new sequences, ST6504 and ST6506). All isolates harbored different virulence genes, including hld, encoding delta hemolysin; lukE–lukD, encoding bicomponent leukotoxin LukE–LukD; the clfB gene, encoding clumping factor B; the laminin gene, encoding laminin-binding protein; and cap8, encoding capsule type 8. Fifteen isolates harbored hemolysin beta (hlb) and fourteen encoded hemolysin alpha (hla) and hemolysin G2 (hlgv). Adhesin factors, including clfA and fnbB, were detected in five and four isolates respectively. Binding proteins, including collagen (cbp) and elastin-binding protein (ebp), were detected in two S. aureus isolates while fibrinogen-binding protein (fib) was identified in four isolates. This study provides the first set of genotyping data on the population structure and presence of toxin genes of S. aureus strains in Tunisian camels.

Transcriptomic analysis of human brain microvascular endothelial cells exposed to laminin binding protein (adhesion lipoprotein) and Streptococcus pneumoniae

Streptococcus pneumoniae invades the CNS and triggers a strong cellular response. To date, signaling events that occur in the human brain microvascular endothelial cells (hBMECs), in response to pneumococci or its surface adhesins are not mapped comprehensively. We evaluated the response of hBMECs to the adhesion lipoprotein (a laminin binding protein—Lbp) or live pneumococci. Lbp is a surface adhesin recently identified as a potential ligand, which binds to the hBMECs. Transcriptomic analysis was performed by RNA-seq of three independent biological replicates and validated with qRT-PCR using 11 genes. In total 350 differentially expressed genes (DEGs) were identified after infection with S. pneumoniae, whereas 443 DEGs when challenged with Lbp. Total 231 DEGs were common in both treatments. Integrative functional analysis revealed participation of DEGs in cytokine, chemokine, TNF signaling pathways and phagosome formation. Moreover, Lbp induced cell senescence and breakdown, and remodeling of ECM. This is the first report which maps complete picture of cell signaling events in the hBMECs triggered against S. pneumoniae and Lbp. The data obtained here could contribute in a better understanding of the invasion of pneumococci across BBB and underscores role of Lbp adhesin in evoking the gene expression in neurovascular unit.

Biofilm-Formation Ability and the Presence of Adhesion Genes in Coagulase-Negative Staphylococci Isolates from Chicken Broilers.

Simple SummaryBacteria of the genus Staphylococcus are universally present on the mucous membranes and skin of warm-blooded animals. They are divided into two groups on the basis of their ability to clot blood plasma: the coagulase-positive (CoPS) and coagulase-negative staphylococci (CoNS). Some species can cause opportunistic infections in poultry. Identification and characterization of strains of the genus Staphylococcus isolated from farm animals are crucial in epidemiological research and for developing effective methods to treat infections and food poisoning induced by these bacteria. The main virulence factor of coagulase-negative staphylococci is considered to be their ability to form complex biofilm structures on the surfaces of damaged tissues. Biofilms increase the invasive properties of CoNS and their ability to cause infection. The purpose of this study was to determine the biofilm-forming potential of coagulase-negative Staphylococcus strains isolated from poultry. The frequency of selected genes potentially playing a role in the biofilm formation process was also determined. The results of the study indicate that the majority (79.3%) of CoNS isolated from broiler chickens in this study were capable of producing a biofilm.The aim of the study was to analyze the biofilm-production capacity of 87 coagulase-negative Staphylococcus strains (CoNS) isolated from broiler chickens and to determine the occurrence of biofilm-associated genes. The biofilm production capacity of staphylococci was assessed using the microtiter plate method (MTP), and the frequency of genes was determined by PCR. The ability to form a biofilm in vitro was shown in 79.3% of examined strains. Strong biofilm capacity was demonstrated in 26.4% of strains, moderate capacity in 25.3%, weak capacity in 27.6%, and a complete lack of biofilm production capacity in 20.7% of strains. The icaAB gene responsible for the production of extracellular polysaccharide adhesins was detected in 6.9% of strains. The other four genes, i.e., bap (encoding biofilm-associated protein), atlE (encoding cell surface protein exhibiting vitronectin-binding activity), fbe (encoding fibrinogen-binding protein), and eno (encoding laminin-binding protein) were detected in 5.7%, 19.5%, 8%, and 70.1% of strains, respectively. Demonstration of genes that play a role in bacterial biofilm formation may serve as a genetic basis to distinguish between symbiotic and potentially invasive coagulase-negative staphylococcal strains.

Comparison of the Prevalence of Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) among Staphylococcus aureus Isolates in a Burn Unit with Non-Burning Units.

Background:Staphylococcus aureus (S. aureus) is one of the most important pathogens in burn infections colonized in the nose and increase the risk of infections.Methods:Overall, 85 S. aureus isolates were isolated from clinical and nasal hospitalized patients and health care workers (HCWs) in a burn unit and non-burn units in Isfahan from June 2016 and September 2016. Genes encoding penicillin-binding protein 2a (mecA) and adhesive surface proteins, including fibronectin-binding proteins (fnbA,fnbB), fibrinogen binding protein (fib), laminin-binding protein(eno), collagen binding protein (cna), elastin binding protein (ebps), intracellular adhesion operon (icaA and icaD) were detected using PCR method.Results:The rate of methicillin-resistant S. aureus (MRSA) among burn and non-burn isolates were 62% (18/29) and 25% (14/56), respectively. The most prevalent MSCRAMMs genes in burn units were eno (86%) and fib (66%). The most common gene pattern in burn center was icaA+fib+eno. The frequency of icaD, fib and ebpS was higher in clinical samples than nasal samples. No relation was found between the MSCRAMMs genes in the burn unit and non-burn units.Conclusion:The high prevalence of MRSA in burn center can be a new challenge for clinicians. The higher frequency of icaD, fib and ebpS in clinical isolates than nasal isolates may reflect the important role of these genes in colonization and pathogenesis of S. aureus.

Feature of Adhesins Produced by Human Clinical Isolates of Mycobacterium intracellulare, Mycobacterium intracellulare subsp. chimaera and Closely Related Species.

The Mycobacterium avium complex includes two closely related species, Mycobacterium avium and Mycobacterium intracellulare. They are opportunistic pathogens in humans and responsible for severe disease in a wide variety of animals. Yet, little is known about factors involved in their pathogenicity. Here, we identified, purified and characterized adhesins belonging to the heparin-binding hemagglutinin (HBHA) and laminin-binding protein (LBP) family from M. intracellulare ATCC13950 and examined clinical isolates from patients with different pathologies associated with M. intracellulare infection for the presence and conservation of HBHA and LBP. Using a recombinant derivative strain of M. intracellulare ATCC13950 producing green fluorescent protein and luciferase, we found that the addition of heparin inhibited mycobacterial adherence to A549 cells, whereas the addition of laminin enhanced adherence. Both HBHA and LBP were purified by heparin-Sepharose chromatography and their methylation profiles were determined by mass spectrometry. Patients with M. intracellulare infection mounted strong antibody responses to both proteins. By using PCR and immunoblot analyses, we found that both proteins were highly conserved among all 17 examined clinical M. intracellulare isolates from patients with diverse disease manifestations, suggesting a conserved role of these adhesins in M. intracellulare virulence in humans and their potential use as a diagnostic tool.

PepO is a target of the two-component systems VicRK and CovR required for systemic virulence of Streptococcus mutans

ABSTRACT Streptococcus mutans , a cariogenic species, is often associated with cardiovascular infections. Systemic virulence of specific S. mutans serotypes has been associated with the expression of the collagen- and laminin-binding protein Cnm, which is transcriptionally regulated by VicRK and CovR. In this study, we characterized a VicRK- and CovR-regulated gene, pepO, coding for a conserved endopeptidase. Transcriptional and protein analyses revealed that pepO is highly expressed in S. mutans strains resistant to complement immunity (blood isolates) compared to oral isolates. Gel mobility assay, transcriptional, and Western blot analyses revealed that pepO is repressed by VicR and induced by CovR. Deletion of pepO in the Cnm+ strain OMZ175 (OMZpepO) or in the Cnm− UA159 (UApepO) led to an increased susceptibility to C3b deposition, and to low binding to complement proteins C1q and C4BP. Additionally, pepO mutants showed diminished ex vivo survival in human blood and impaired capacity to kill G. mellonella larvae. Inactivation of cnm in OMZ175 (OMZcnm) resulted in increased resistance to C3b deposition and unaltered blood survival, although both pepO and cnm mutants displayed attenuated virulence in G. mellonella. Unlike OMZcnm, OMZpepO could invade HCAEC endothelial cells. Supporting these phenotypes, recombinant proteins rPepO and rCnmA showed specific profiles of binding to C1q, C4BP, and to other plasma (plasminogen, fibronectin) and extracellular matrix proteins (type I collagen, laminin). Therefore this study identifies a novel VicRK/CovR-target required for immune evasion and host persistence, pepO, expanding the roles of VicRK and CovR in regulating S. mutans virulence.

Virulence Characteristics of mecA-Positive Multidrug-Resistant Clinical Coagulase-Negative Staphylococci.

Coagulase-negative staphylococci (CoNS) are an important group of opportunistic pathogenic microorganisms that cause infections in hospital settings and are generally resistant to many antimicrobial agents. We report on phenotypic and genotypic virulence characteristics of a select group of clinical, mecA-positive (encoding penicillin-binding protein 2a) CoNS isolates. All CoNS were resistant to two or more antimicrobials with S. epidermidis strain 214EP, showing resistance to fifteen of the sixteen antimicrobial agents tested. Aminoglycoside-resistance genes were the ones most commonly detected. The presence of megaplasmids containing both horizontal gene transfer and antimicrobial resistance genetic determinants indicates that CoNS may disseminate antibiotic resistance to other bacteria. Staphylococcus sciuri species produced six virulence enzymes, including a DNase, gelatinase, lipase, phosphatase, and protease that are suspected to degrade tissues into nutrients for bacterial growth and contribute to the pathogenicity of CoNS. The PCR assay for the detection of biofilm-associated genes found the eno (encoding laminin-binding protein) gene in all isolates. Measurement of their biofilm-forming ability and Spearman’s rank correlation coefficient analyses revealed that the results of crystal violet (CV) and extracellular polymeric substances (EPS) assays were significantly correlated (ρ = 0.9153, P = 3.612e-12). The presence of virulence factors, biofilm-formation capability, extracellular enzymes, multidrug resistance, and gene transfer markers in mecA-positive CoNS clinical strains used in this study makes them powerful opportunistic pathogens. The study also warrants a careful evaluation of nosocomial infections caused by CoNS and may be useful in studying the mechanism of virulence and factors associated with their pathogenicity in vivo and developing effective strategies for mitigation.

Effect of trans-Cinnamaldehyde on Methicillin-Resistant Staphylococcus aureus Biofilm Formation: Metabolic Activity Assessment and Analysis of the Biofilm-Associated Genes Expression.

The effects of trans-cinnamaldehyde (TC) on transcriptional profiles of biofilm-associated genes and the metabolic activity of two methicillin-resistant Staphylococcus aureus (MRSA) strains showing a different degree of adherence to polystyrene, were evaluated. Metabolic activity of S. aureus in biofilm was significantly decreased in the presence of TC at 1/2 minimum biofilm inhibition concentration (MBIC). Expression levels of the genes encoding laminin binding protein (eno), elastin binding protein (ebps) and fibrinogen binding protein (fib) in the presence of TC at 1/2 MBIC were lower than in untreated biofilm in both the weakly and strongly adhering strain. The highest decrease of expression level was observed in case of fib in the strongly adhering strain, in which the amount of fib transcript was 10-fold lower compared to biofilm without TC. In the presence of TC at 1/2 MBIC after 3, 6, 8 and 12 h, the expression level of icaA and icaD, that are involved in the biosynthesis of polysaccharide intercellular adhesin, was above half lower in the weakly adhering strain compared to biofilm without TC. In the strongly adhering strain the highest decrease in expression of these genes was observed after 3 and 6 h. This study showed that TC is a promising anti-biofilm agent for use in MRSA biofilm-related infections.

Linker Protein Repair of LAMA2 Dystrophic Neuromuscular Basement Membranes

An understanding of basement membrane (BM) assembly at a molecular level provides a foundation with which to develop repair strategies for diseases with defects of BM structure. As currently understood, laminins become anchored to cell surfaces through receptor-mediated interactions and polymerize. This provisional matrix binds to proteoglycans, nidogens and type IV collagen to form a mature BM. Identification of BM binding domains and their binding targets has enabled investigators to engineer proteins that link BM components to modify and improve their functions. This approach is illustrated by the development of two linker proteins to repair the LAMA2-deficient muscular dystrophy (LAMA2-MD). Dystrophy-causing mutations of the LAMA2 gene product (Lmα2) disrupt the BM molecular architecture, destabilizing it. In a mild ambulatory type of the dystrophy, α2LN mutations in laminin-211 prevents polymerization. In the more common and severe non-ambulatory type (MDC1A), an absent Lmα2 subunit is replaced by the naturally occurring Lmα4 subunit that is normally largely confined to the microvasculature. The compensatory laminin, however, is a poor substitute because it neither polymerizes nor binds adequately to the anchoring receptor α-dystroglycan. A chimeric laminin-binding protein called αLNNd enables laminins with defective or absent αLN domains to polymerize while another engineered protein, miniagrin (mag), promotes efficient α-dystroglycan receptor-binding in otherwise weakly adhesive laminins. Alone, αLNNd enables Lm211 with a self-assembly defect to polymerize and was used to ameliorate a mouse model of the ambulatory dystrophy. Together, these linker proteins alter Lm411 such that it both polymerizes and binds αDG such that it properly assembles. This combination was used to ameliorate a mouse model of the non-ambulatory dystrophy in which Lm411 replaced Lm211 as seen in the human disease. Collectively, these studies pave the way for the development of somatic gene delivery of repair proteins for treatment of LAMA2-MD. The studies further suggest a more general approach of linker-protein mediated repair in which a variety of existing BM protein domains can be combined together to stabilize BMs in other diseases.

A Novel Laminin-Binding Protein Mediates Microbial-Endothelial Cell Interactions and Facilitates Dissemination of Lyme Disease Pathogens.

Borrelia burgdorferi conserved gene products BB0406 and BB0405, members of a common B. burgdorferi paralogous gene family, share 59% similarity. Although both gene products can function as potential porins, only BB0405 is essential for infection. Here we show that, despite sequence homology and coexpression from the same operon, both proteins differ in their membrane localization attributes, antibody accessibility, and immunogenicity in mice. BB0406 is required for spirochete survival in mammalian hosts, particularly for the disseminated infection in distant organs. We identified that BB0406 interacts with laminin, one of the major constituents of the vascular basement membrane, and facilitates spirochete transmigration across host endothelial cell barriers. A better understanding of how B. burgdorferi transmigrates through dermal and tissue vascular barriers and establishes disseminated infections will contribute to the development of novel therapeutics to combat early infection.

The Laminin Interactome: A Multifactorial Laminin-Binding Strategy by Nontypeable Haemophilus influenzae for Effective Adherence and Colonization.

Laminin is a well-defined component of the airway basement membrane (BM). Efficient binding of laminin via multiple interactions is important for nontypeable Haemophilus influenzae (NTHi) colonization in the airway mucosa. In this study, we identified elongation factor thermo-unstable (EF-Tu), l-lactate dehydrogenase (LDH), protein D (PD), and peptidoglycan-associated lipoprotein P6 as novel laminin-binding proteins (Lbps) of NTHi. In parallel with other well-studied Lbps (protein 4 [P4], protein E [PE], protein F [PF], and Haemophilus adhesion and penetration protein [Hap]), EF-Tu, LDH, PD, and P6 exhibited interactions with laminin, and mediated NTHi laminin-dependent adherence to pulmonary epithelial cell lines. More importantly, the NTHi laminin interactome consisting of the well-studied and novel Lbps recognized laminin LG domains from the subunit α chains of laminin-111 and -332, the latter isoform of which is the main laminin in the airway BM. The NTHi interactome mainly targeted multiple heparin-binding domains of laminin. In conclusion, the NTHi interactome exhibited a high plasticity of interactions with different laminin isoforms via multiple heparin-binding sites.

Human MOSPD2: A bacterial Lmb mimicked auto-antigen is involved in immune infertility

Autoantibody production is one of the leading factors of immune infertility, an autoimmune disease of the male reproductive system. The potential involvement of MHC-class II derived self-peptides against bacterial proteins in the antisperm antibody (ASA) production has been reported previously. Apparently, Streptococcus agalactiae has been considered as an important pathogen to impart infection-induced infertility in a bacteriospermia associated leukocytospermia (LCS/BS) state. Hence, the present study attempts to confirm S. agalactiae specific Laminin binding protein (Lmb) derived self-peptide (‘KDSYTKKAKAFKKEA’) namely human Motile Sperm domain-containing protein 2 (MOSPD2) as an auto-antigen in LCS/BS condition. Semen samples were collected from infertile men with LCS/BS (n ​= ​17) and their fertile counterparts (n ​= ​10). Gram-positive bacteria were predominantly identified in the entire 17 LCS samples using culture method followed by 16S rDNA sequencing technique. TLRs 2 and 4 expression used as markers of immune response in spermatozoa and sperm dysfunction were elevated in the LCS/BS spermatozoa as compared to their fertile counterparts. A significant increase in oxidative stress indices i.e., protein carbonylation, lipid peroxidation and acridine orange test (AOT), was also observed in the LCS/BS spermatozoa. Spermatozoa lysate (both auto and heterologous), bacterial lysate (control) and synthesized MOSPD2 self-peptide were used to test their antigenicity against the autoantibodies by rocket immunoelectrophoresis (RIEP) assay. Seminal plasma from LCS/BS patients with S. agalactiae was used as the source of autoantibodies. Spermatozoa and bacteria lysate; and MOSPD2 self-peptide were able to bind autoantibodies in the seminal plasma. Besides, the self-peptide showed a dose dependent increase in the precipitation of antibody. T-cell epitope mapping of 48 Enterococcus faecalis and 91Staphylococcus aureus surface proteins confirmed MOSPD2 as a global auto-antigen. Thus, augmentation of TLR expression in LCS/BS spermatozoa inferred MOSPD2 to be a putative immunogen. Altogether, these findings will delineate the significance of MOSPD2 auto-antigen in a bacteria derived immune infertility condition.

Proteomic Study of a Parkinson's Disease Model of Undifferentiated SH-SY5Y Cells Induced by a Proteasome Inhibitor.

Parkinson's disease (PD) is one of the most common nervous system degenerative diseases. However, the etiology of this disease remains elusive. Here, a proteasome inhibitor (PSI)-induced undifferentiated SH-SY5Y PD model was established to analyze protein alterations through proteomic study. Methods: Cultured undifferentiated SH-SY5Y cells were divided into a control group and a group treated with 2.5 µM PSI (PSI-treated group). An methyl thiazolyl tetrazolium (MTT) assay was applied to detect cell viability. Acridine orange/ethidium bromide (AO/EB), α-synuclein immunofluorescence and hematoxylin and eosin (H&E) staining were applied to evaluate apoptosis and cytoplasmic inclusions, respectively. The protein spots that were significantly changed were separated, analyzed by 2D gel electrophoresis and DIGE De Cyder software, and subsequently identified by MALDI-TOF mass spectrometry and database searching. Results: The results of the MTT assay showed that there was a time and dose dependent change in cell viability following incubation with PSI. After 24 h incubation, PSI resulted in early apoptosis, and cytoplasmic inclusions were found in the PSI-treated group through H&E staining and α-synuclein immunofluorescence. Thus, undifferentiated SH-SY5Y cells could be used as PD model following PSI-induced inhibition of proteasomal function. In total, 18 proteins were differentially expressed between the groups, 7 of which were up-regulated and 11 of which were down-regulated. Among them, 5 protein spots were identified as being involved in the ubiquitin proteasome pathway-induced PD process. Conclusions: Mitochondrial heat shock protein 75 (MTHSP75), phosphoglycerate dehydrogenase (PHGDH), laminin binding protein (LBP), tyrosine 3/tryptophan 5-monooxygenase activation protein (14-3-3ε) and YWHAZ protein (14-3-3ζ) are involved in mitochondrial dysfunction, serine synthesis, amyloid clearance, apoptosis process and neuroprotection. These findings may provide new clues to deepen our understanding of PD pathogenesis.