Putative Lysine Research Articles

A Clostridioides difficile endolysin modulates toxin secretion without cell lysis

The Clostridia produce and secrete Large Clostridial Glucosylating Toxins (LCGTs) responsible for disease symptoms, but the secretion mechanism is largely unknown. Recently, a holin-like protein was shown to be essential for toxin secretion. Holins, typically bacteriophage-specific proteins, are part of the holin-endo(lysin) system that releases phage progeny. To determine if the clostridia also use a lysin, we investigated two conserved putative lysins, M7404_01910 and M7404_02200, in the release of the LCGTs TcdA and TcdB from a Clostridioides difficile ribotype 027 strain, M7404. Sequence analysis and structural modelling indicates that both proteins are related to N-acetylmuramoyl-l-alanine amidases, similar to CD27L, a lysin from the C. difficile phage ΦCD27. Disruption of these genes reveal that only M7404_02200 contributes to toxin secretion and does so in a non-lytic fashion. Peptidoglycan hydrolysis assays show that recombinant M7404_02200 is an active peptidoglycan amidase, confirming its role in TcdA and TcdB secretion in C. difficile M7404.

Discovery of Antimicrobial Lysins from the "Dark Matter" of Uncharacterized Phages Using Artificial Intelligence.

The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential antibacterial agents, experimental screening methods for novel lysins pose significant challenges due to the enormous workload. Here, the first unified software package, namely DeepLysin, is developed to employ artificial intelligence for mining the vast genome reservoirs ("dark matter") for novel antibacterial phage lysins. Putative lysins are computationally screened from uncharacterized Staphylococcus aureus phages and 17 novel lysins are randomly selected for experimental validation. Seven candidates exhibit excellent in vitro antibacterial activity, with LLysSA9 exceeding that of the best-in-class alternative. The efficacy of LLysSA9 is further demonstrated in mouse bloodstream and wound infection models. Therefore, this study demonstrates the potential of integrating computational and experimental approaches to expedite the discovery of new antibacterial proteins for combating increasing antimicrobial resistance.

Preclinical characterization andin silico safety assessment of three virulent bacteriophages targeting carbapenem-resistant uropathogenic Escherichia coli.

Phage therapy has recently been revitalized in the West with many successful applications against multi-drug-resistant bacterial infections. However, the lack of geographically diverse bacteriophage (phage) genomes has constrained our understanding of phage diversity and its genetics underpinning host specificity, lytic capability, and phage-bacteria co-evolution. This study aims to locally isolate virulent phages against uropathogenic Escherichia coli (E. coli) and study its phenotypic and genomic features. Three obligately virulent Escherichia phages (øEc_Makalu_001, øEc_Makalu_002, and øEc_Makalu_003) that could infect uropathogenic E. coli were isolated and characterized. All three phages belonged to Krischvirus genus. One-step growth curve showed that the latent period of the phages ranged from 15 to 20min, the outbreak period ~ 50min, and the burst size ranged between 74 and 127 PFU/bacterium. Moreover, the phages could tolerate a pH range of 6 to 9 and a temperature range of 25-37°C for up to 180min without significant loss of phage viability. All phages showed a broad host spectrum and could lyse up to 30% of the 35 tested E. coli isolates. Genomes of all phages were approximately ~ 163kb with a gene density of 1.73 gene/kbp and an average gene length of ~ 951bp. The coding density in all phages was approximately 95%. Putative lysin, holin, endolysin, and spanin genes were found in the genomes of all three phages. All phages were strictly virulent with functional lysis modules and lacked any known virulence or toxin genes and antimicrobial resistance genes. Pre-clinical experimental and genomic analysis suggest these phages may be suitable candidates for therapeutic applications.

Lysosomal membrane transporter purification and reconstitution for functional studies.

Lysosomes achieve their function through numerous transporters that import or export nutrients across their membrane. However, technical challenges in membrane protein overexpression, purification, and reconstitution hinder our understanding of lysosome transporter function. Here, we developed a platform to overexpress and purify the putative lysine transporter Ypq1 using a constitutive overexpression system in protease- and ubiquitination-deficient yeast vacuoles. Using this method, we purified and reconstituted Ypq1 into proteoliposomes and showed lysine transport function, supporting its role as a basic amino acid transporter on the vacuole membrane. We also found that the absence of lysine destabilizes purified Ypq1 and causes it to aggregate, consistent with its propensity to be downregulated in vivo upon lysine starvation. Our approach may be useful for the biochemical characterization of many transporters and membrane proteins to understand organellar transport and regulation.

Characterization of a lytic Pseudomonas aeruginosa phage vB_PaeP_ASP23 and functional analysis of its lysin LysASP and holin HolASP.

In this study, we isolated a lytic Pseudomonas aeruginosa phage (vB_PaeP_ASP23) from the sewage of a mink farm, characterized its complete genome and analyzed the function of its putative lysin and holin. Morphological characterization and genome annotation showed that phage ASP23 belonged to the Krylovirinae family genus Phikmvvirus, and it had a latent period of 10 min and a burst size of 140 pfu/infected cell. In minks challenged with P. aeruginosa, phage ASP23 significantly reduced bacterial counts in the liver, lung, and blood. The whole-genome sequencing showed that its genome was a 42,735-bp linear and double-stranded DNA (dsDNA), with a G + C content of 62.15%. Its genome contained 54 predicted open reading frames (ORFs), 25 of which had known functions. The lysin of phage ASP23 (LysASP), in combination with EDTA, showed high lytic activity against P. aeruginosa L64. The holin of phage ASP23 was synthesized by M13 phage display technology, to produce recombinant phages (HolASP). Though HolASP exhibited a narrow lytic spectrum, it was effective against Staphylococcus aureus and Bacillus subtilis. However, these two bacteria were insensitive to LysASP. The findings highlight the potential of phage ASP23 to be used in the development of new antibacterial agents.

Enterococcal bacteriophage: A survey of the tail associated lysin landscape

Bacteriophages are viruses that exclusively infect bacteria which require local degradation of cell barriers. This degradation is accomplished by various lysins located mainly within the phage tail structure. In this paper we surveyed and analysed the genomes of 506 isolated bacteriophage and prophage infecting or harboured within the genomes of the medically important Enterococcus faecalis and faecium. We highlight and characterise the major features of the genomes of phage in the morphological groups podovirus, siphovirus and myovirus, and explore their categorisation according to the new ICTV classifications, with a focus on putative extracellular lysins chiefly within tail modules. Our analysis reveals a range of potential cell-wall targeting enzyme domains that are part of tail, tape measure or other predicted base structures of these phages or prophages. These largely fall into protein domains targeting pentapeptide or glycosidic linkages within peptidoglycan but also potentially the enterococcal polysaccharide antigen (EPA) and wall teichoic acids of these species (i.e., Pectinesterases and Phosphodiesterases). Notably, there is a great variety of domain architectures that reveal the diversity of evolutionary solutions to attack the Enterococcus cell wall. Despite this variety, most phage and prophage possess a putative endopeptidase (70%), reflecting the ubiquity of this cell surface barrier. We also identified a predicted lytic transglycosylase domain belonging to the glycosyl hydrolase (GH) family 23 and present exclusively within tape measure proteins. Our data also reveal distinct features of the genomes of podo-, sipho- and myo-type viruses that most likely relate to their size and complexity. Overall, we lay a foundation for expression of recombinant TAL proteins and engineering of enterococcal and other phage that will be invaluable for researchers in this field.

Cancer Stem Cell and Aggressiveness Traits Are Promoted by Stable Endothelin-Converting Enzyme-1c in Glioblastoma Cells.

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM.

Characterization of KMSP1, a newly isolated virulent bacteriophage infecting Staphylococcus aureus, and its application to dairy products

Staphylococcus aureus is one of the major pathogens causing foodborne outbreaks and severe infections worldwide. Generally, various physical and chemical treatments have been applied to control S. aureus in the food industry. However, conventional treatments usually affected food quality and often produced toxic compounds. Therefore, bacteriophage (phage), a natural antimicrobial agent, has been suggested as an alternative strategy to control foodborne pathogens including S. aureus. In this study, KMSP1, a bacteriophage infecting S. aureus was isolated from a raw milk sample and characterized. Transmission electron microscopy (TEM) analysis revealed that phage KMSP1 belongs to the Myoviridae family. Phage KMSP1 efficiently inhibited bacterial growth for >28 h post-infection. In addition, phage KMSP1 could infect a broad spectrum of S. aureus strains, including methicillin-resistant S. aureus (MRSA) strains. Whole-genome sequence analysis showed that KMSP1 is a lytic phage with the absence of genes related to lysogen formation, toxin production, and antibiotics resistance, respectively. In the genome of KMSP1, the presence of putative tail lysin containing a cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain could be one of the reasons for the effective antimicrobial activity of KMSP1. Furthermore, high stability of phage KMSP1 at temperature ranging from 4 to 55 °C and pH ranging from 5 to 11, suggested its potential use in various food systems. Receptor analysis revealed that KMSP1 utilized cell wall teichoic acid (WTA), one of the major virulence factors of S. aureus, as a host receptor. Application of phage KMSP1 at an MOI of 104 achieved a significant reduction of log 8.8 CFU/mL of viable cell number in pasteurized milk and log 4.3 CFU/cm2 in sliced cheddar cheese after 24 h. Taken together, the strong antimicrobial activity of phage KMSP1 suggested that it could be developed as a biocontrol agent in dairy products to control S. aureus contamination.

HDAC8 Disrupts DNA Damage Response By Deacetylation of U2AF1 in Inv(16) Acute Myeloid Leukemia

Genomic instability due to defective DNA damage response (DDR) and DNA repair increases the probability of acquiring mutations, which may contribute to the pathogenesis of acute myeloid leukemia (AML). Inv(16) AML [inv(16)(p13q22) or t(16;16)(p13.1;q22)] creates a fusion gene CBFb-MYH11 (CM) by fusing core binding factor b (CBFb) with MYH11 which encodes smooth muscle myosin heavy chain (SMMHC). Our previous studies using a conditional Cbfb-MYH11 knock-in (KI) mouse model showed that preleukemic progenitors are prone to leukemia initiation, however, the underlying mechanism is not clear. Here, we show increased DNA double-strand breaks measured by comet assay and γH2AX staining in CM preleukemic progenitors compared to control counterparts. RNA-seq using sorted LSK (Lin-Sca1+Kit+) cells isolated from BM of CM KI mice (n = 5) and control (n = 4) identified a total of 1019 genes differentially expressed (526 up; 493 down; > 1.5-fold change; p < 0.01). Gene set enrichment analysis revealed that G2/M checkpoint, E2F targets, and mitotic spindle pathways were differentially downregulated in CM LSK, suggesting a possible contribution of cell cycle checkpoint dysregulation. To further explore the mechanism underlying CM-induced DNA damage, we expressed CM in 32D cells and subjected them to irradiation (IR; 3.5 Gy) followed by γH2AX and RAD51 staining. Significantly more γH2AX foci per nucleus were observed in CM cells (CM 35.03 vs Ctrl 24.71 at 0.5h, p<0.0001; CM 14.79 vs Ctrl 11.67 at 3h, p=0.0186), while fewer RAD51 foci per nucleus were seen in CM cells (CM 4.932 vs Ctrl 9.6 at 3h, p<0.0001), suggesting that CM may impair homologous recombination (HR)-mediated DNA repair. We reported that CM interacts with HDAC8 and enhances its activity, thus we examined whether HDAC8 may mediate the impact on DDR and HR activity. HDAC8 overexpression (OE) in 32D cells resulted in significantly more γH2AX foci per nucleus after IR (3.5 Gy) (HDAC8-OE 42.20 vs Ctrl 32.17 at 0.5h, p<0.0001; HDAC8-OE 15.45 vs Ctrl 10.63 at 3h, p<0.0001). Similar to CM cells, fewer RAD51 foci per nucleus were seen in HDAC8-OE cells (HDAC8-OE 11.63 vs Ctrl 17.39 at 3h, p<0.0001). To verify HDAC8 function in HR repair, we used the I-SceI and DR-GFP (homology-directed repair) or EJ5-GFP (end-joining-directed repair) DNA repair reporter (Gunn A et al, Methods Mol Biol, 2012). The frequency of repaired GFP+ population in the HR-directed repair reporter was reduced in HDAC8-OE cells compared to control (HDAC8-OE 7.807% vs Ctrl 11.34%, p=0.0169), while no difference was observed in the NHEJ-directed repair reporter. These results indicate that high HDAC8 expression disturbs HR DNA repair. The U2 small nuclear RNA auxiliary factor 1 (U2AF1) has been reported as a component of BRCA1-mRNA splicing machinery that regulates pre-mRNA splicing of critical genes involved in HR DNA repair. Here, we show that U2AF1 is subjected to post-translational acetylation and that U2AF1 interacts with HDAC8. We show that U2AF1 acetylation and its interaction with HDAC8 were increased following DNA damage and that HDAC8 is critical for deacetylating U2AF1. Site-directed mutagenesis for reported putative acetylation lysine sites (K15, K23, K175) (Elia AE et al, Mol Cell, 2015) revealed that the acetylation level of U2AF1-K23R decreased after IR and corresponded to reduced interaction with HDAC8 and the BRCA1/BCLAF1 complex. Notably, the interaction with BRCA1/BCLAF1 complex was rescued by mutating lysine to the acetylation mimic glutamine (Q). These results indicate that the acetylation state of U2AF1 is essential for the assembly of BRCA1-mRNA splicing complex and suggest that HDAC8 may regulate HR DNA repair by modulating the U2AF1 acetylation state. Similar to HDAC8 OE, CM expression reduced U2AF1 acetylation and impaired its interaction with BRCA1/BCLAF1 complex. Collectively, our results suggest that the increased DNA damage induced by CM may be due to the reduced acetylation of U2AF1 resulting from high HDAC8 activity. These results highlight a direct impact of inv(16) fusion protein and high HDAC8 activity on increased genomic instability in AML.

Putative SET-domain methyltransferases in Cryptosporidium parvum and histone methylation during infection

ABSTRACT Cryptosporidium parvum is a leading cause of diarrhoeal illness worldwide being a significant threat to young children and immunocompromised patients, but the pathogenesis caused by this parasite remains poorly understood. C. parvum was recently linked with oncogenesis. Notably, the mechanisms of gene expression regulation are unexplored in Cryptosporidium and little is known about how the parasite impact host genome regulation. Here, we investigated potential histone lysine methylation, a dynamic epigenetic modification, during the life cycle of the parasite. We identified SET-domain containing proteins, putative lysine methyltransferases (KMTs), in the C. parvum genome and classified them phylogenetically into distinct subfamilies (namely CpSET1, CpSET2, CpSET8, CpKMTox and CpAKMT). Our structural analysis further characterized CpSET1, CpSET2 and CpSET8 as histone lysine methyltransferases (HKMTs). The expression of the CpSET genes varies considerably during the parasite life cycle and specific methyl-lysine antibodies showed dynamic changes in parasite histone methylation during development (CpSET1:H3K4; CpSET2:H3K36; CpSET8:H4K20). We investigated the impact of C. parvum infection on the host histone lysine methylation. Remarkably, parasite infection led to a considerable decrease in host H3K36me3 and H3K27me3 levels, highlighting the potential of the parasite to exploit the host epigenetic regulation to its advantage. This is the first study to describe epigenetic mechanisms occurring throughout the parasite life cycle and during the host–parasite interaction. A better understanding of histone methylation in both parasite and host genomes may highlight novel infection control strategies.

Developing a Riboswitch-Mediated Regulatory System for Metabolic Flux Control in Thermophilic Bacillus methanolicus.

Genome-wide transcriptomic data obtained in RNA-seq experiments can serve as a reliable source for identification of novel regulatory elements such as riboswitches and promoters. Riboswitches are parts of the 5′ untranslated region of mRNA molecules that can specifically bind various metabolites and control gene expression. For that reason, they have become an attractive tool for engineering biological systems, especially for the regulation of metabolic fluxes in industrial microorganisms. Promoters in the genomes of prokaryotes are located upstream of transcription start sites and their sequences are easily identifiable based on the primary transcriptome data. Bacillus methanolicus MGA3 is a candidate for use as an industrial workhorse in methanol-based bioprocesses and its metabolism has been studied in systems biology approaches in recent years, including transcriptome characterization through RNA-seq. Here, we identify a putative lysine riboswitch in B. methanolicus, and test and characterize it. We also select and experimentally verify 10 putative B. methanolicus-derived promoters differing in their predicted strength and present their functionality in combination with the lysine riboswitch. We further explore the potential of a B. subtilis-derived purine riboswitch for regulation of gene expression in the thermophilic B. methanolicus, establishing a novel tool for inducible gene expression in this bacterium.

Computational Analysis Indicates That PARP1 Acts as a Histone Deacetylases Interactor Sharing Common Lysine Residues for Acetylation, Ubiquitination, and SUMOylation in Alzheimer's and Parkinson's Disease.

Aim/Hypothesis: Lysine residues are known for the post-translational modifications (PTMs) such as acetylation, ubiquitination, and SUMOylation. In acetylation, histone deacetylase (HDAC) and its interactors cause transcriptional deregulation and cause mitochondrial dysfunction, apoptosis, inflammatory response, and cell-cycle impairment that cause brain homeostasis and neuronal cell death. Other regulatory PTMs involved in the pathogenesis of neurodegenerative diseases (NDDs) are ubiquitination and SUMOylation for the degradation of the misfolded proteins. Thus, we aim to investigate the potential acetylation/ubiquitination/SUMOylation crosstalk sites in the HDAC interactors, which cause NDDs. Furthermore, we aim to identify the influence of PTMs on the structural features of proteins and the impact of putative lysine mutation on disease susceptibility. Last, we aim to examine the impact of the putative mutation on acetylated lysine for ubiquitination and SUMOylation. Results: Herein, we integrate 1455 genes, 3094 genes, and 1940 genes related to HDAC interactors, Alzheimer’s disease (AD), and Parkinson’s disease (PD), respectively. Furthermore, the protein–protein interaction and PTM integrations from different databases identified 32 proteins that are associated with HDAC, AD, and PD with 1489 potential lysine-modified sites. HDAC interactors poly(ADP-ribose) polymerase 1 (PARP1), nucleophosmin (NPM1), and cyclin-dependent kinase 1 (CDK1) involved in the progression of NDDs and 64 and 75% of PTM sites in PARP1, NPM1, and CDK1 fall into coiled and ordered regions, respectively. Moreover, 15 putative lysine sites have been found in the crosstalk and K148, K249, K528, K637, K700, and K796 of PARP1 are crosstalk hotspots. Conclusion: The loss of acetylated hotspot sites results in the loss of ubiquitination and SUMOylation function on nearby sites, which is relatively higher when compared to the gain of function.

Structural and functional analysis of the Francisella lysine decarboxylase as a key actor in oxidative stress resistance

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.

Bacteriophages that infect Gram-negative bacteria as source of signal-arrest-release motif lysins

Treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria is challenging, a potential solution for which is the use of bacteriophage-derived lytic enzymes. However, the exogenous action of bacteriophage lysins against Gram-negative bacteria is hindered due to the presence of an impermeable outer membrane in these bacteria. Nevertheless, recent research has demonstrated that some lysins are capable of permeating the outer membrane of Gram-negative bacteria with the help of signal peptides. In the present study, we investigated the genomes of 309 bacteriophages that infect Gram-negative pathogens of clinical interest in order to determine the evolutionary markers of signal peptide-containing lysins. Complete genomes displayed 265 putative lysins, of which 17 (6.41%) contained signal-arrest-release motifs and 41 (15.47%) contained cleavable signal peptides. There was no apparent relationship between host specificity and lysin diversity. Nevertheless, the evolution of lysin genes might not be independent of the rest of the bacteriophage genome once pan-genome clustering and lysin diversity appear to be correlated. In addition, signal peptide- and signal-arrest-release-containing lysins were monophyletically distributed in the protein cladogram, suggesting that the natural selection of holin-independent lysins is divergent. Our study screened 58 (21.89%) out of 265 potential candidates for in vitro experimentation against MDR bacteria.

Phage lysin that specifically eliminates Clostridium botulinum Group I cells

Clostridium botulinum poses a serious threat to food safety and public health by producing potent neurotoxin during its vegetative growth and causing life-threatening neuroparalysis, botulism. While high temperature can be utilized to eliminate C. botulinum spores and the neurotoxin, non-thermal elimination of newly germinated C. botulinum cells before onset of toxin production could provide an alternative or additional factor controlling the risk of botulism in some applications. Here we introduce a putative phage lysin that specifically lyses vegetative C. botulinum Group I cells. This lysin, called CBO1751, efficiently kills cells of C. botulinum Group I strains at the concentration of 5 µM, but shows little or no lytic activity against C. botulinum Group II or III or other Firmicutes strains. CBO1751 is active at pH from 6.5 to 10.5. The lytic activity of CBO1751 is tolerant to NaCl (200 mM), but highly susceptible to divalent cations Ca2+ and Mg2+ (50 mM). CBO1751 readily and effectively eliminates C. botulinum during spore germination, an early stage preceding vegetative growth and neurotoxin production. This is the first report of an antimicrobial lysin against C. botulinum, presenting high potential for developing a novel antibotulinal agent for non-thermal applications in food and agricultural industries.

SP_0916 Is an Arginine Decarboxylase That Catalyzes the Synthesis of Agmatine, Which Is Critical for Capsule Biosynthesis in Streptococcus pneumoniae

The global burden of invasive pneumococcal diseases, including pneumonia and sepsis, caused by Streptococcus pneumoniae, a Gram-positive bacterial pathogen, remains a major global health risk. The success of pneumococcus as a pathogen can be attributed to its ability to regulate the synthesis of capsular polysaccharide (CPS) during invasive disease. We previously reported that deletion of a putative lysine decarboxylase (LDC; ΔSP_0916) in pneumococcal serotype 4 (TIGR4) results in reduced CPS. SP_0916 locus is annotated as either an arginine or a LDC in pneumococcal genomes. In this study, by biochemical characterization of the recombinant SP_0916, we determined the substrate specificity of SP_0916 and show that it is an arginine decarboxylase (speA/ADC). We also show that deletion of the polyamine transporter (potABCD) predicted to import putrescine and spermidine results in reduced CPS, while deletion of spermidine synthase (speE) for the conversion of putrescine to spermidine had no impact on the capsule. Targeted metabolomics identified a correlation between reduced levels of agmatine and loss of capsule in ΔspeA and ΔpotABCD, while agmatine levels were comparable between the encapsulated TIGR4 and ΔspeE. Exogenous supplementation of agmatine restored CPS in both ΔpotABCD and ΔspeA. These results demonstrate that agmatine is critical for regulating the CPS, a predominant virulence factor in pneumococci.

Application of Bacteriophages to Control Vibrio alginolyticus Contamination in Oyster (Saccostrea glomerata) Larvae.

Mortalities of bivalve larvae and spat linked with Vibrio spp. infection have been described in hatcheries since 1959, causing potential development of resistant bacteria. A reliable and sustainable solution to this problem is yet to be developed. Potential treatment of bacterial infection with bacteriophages is gaining interest in aquaculture as a more sustainable option for managing Vibrio spp. infection. This study assessed the effectiveness of bacteriophages (Φ-5, Φ-6, and Φ-7) against pathogenic Vibrio isolates (USC-26004 and USC-26005). These phage isolates were found to belong to the Myoviridae viral family. A total of 212 ORFs of Φ-5 were identified and annotated. The genome of this phage contained putative thymidine kinase and lysin enzyme. During infections with phages, the OD values of the isolates USC-26005 and USC-26004 remained stable at a much lower reading compared to the control after 9 h of incubation. Mortality rate of oyster (Saccostrea glomerata) larvae was 28.2 ± 3.5% in the bacteriophage treatment group, compared to 77.9 ± 9.1% in the bacterial treatment group after 24 h incubation. Findings of this study indicate that lytic phages might be utilized as potential bio-control agents of luminescent bacterial disease in oyster hatcheries.

Intramuscular Immunization of Streptococcus pyogenes SF370 protein extract and identification of multiple virulence factors through proteomic profiling in RHD induced Balb/c mice

Group A streptococcus (GAS) and autoimmunity are associated with heart related mitral valve damage, in adults. In this study Balb/c mice were intramuscularly immunized with S. pyogenes SF370 for 4 weeks. Prior to euthanization, physiological parameters like body weight and electrical signalling of the heart were recorded. After euthanization, the heart tissue homogenate was prepared and proteomic alterations were studied using SDS-PAGE and 2D electrophoresis. The expression levels of inflammatory genes like TNFα, IFNγ and TGF-β were quantified using real time PCR. Insilico analysis was performed to identify the functions of hypothetical proteins and virulence factors involved in the induction of rheumatic carditis. The results showed a reduction in body weight, ulceration, inflammation, cardiac lesions and prolonged PR interval in mice immunized with S. pyogenes SF370, as a result of RHD. The heart related proteins like α-actinin, fatty acid binding protein-heart, myosin light chain 3, hemoglobin subunit alpha, myoglobin regulatory light chain 2, (ventricular/cardiac muscle isoform), myosin-6, troponin-1 were found to be up-regulated when compared with the control. The functional annotation of S. pyogenes (SF370) was carried out by retrieving 1696 identified proteins and 653 hypothetical protein sequences in NCBI genome database. The conserved domain was identified for 505 proteins. The pfam database documented that the super families of 279 sequences and 40 signal peptides enabled the classification of proteins in different categories like biological (20%), cellular (22%) and molecular functions (36%). Putative transcription repair coupling factor and putative lysine aminopeptidase N terminal are the two virulence factors identified by VICMPRED in S. pyogenes SF370. The two identified virulence factors are involved in altering the mice heart proteome and thereby controlling the streptococcus pyogenes infection. Thus, the results of the present study reveals the role of immunogenic proteins in induction of rheumatic carditis and to elucidate the molecular mechanisms leading to autoimmune reactions in Balb/c mice.

Discordant Effects of Putative Lysine Acetyltransferase Inhibitors in Biochemical and Living Systems.

Lysine acetyltransferases (KATs) are exquisitely fine-tuned to target specific lysine residues on many proteins, including histones, with aberrant acetylation at distinct lysines implicated in different pathologies. However, researchers face a lack of molecular tools to probe the importance of site-specific acetylation events in vivo. Because of this, there can be a disconnect between the predicted in silico or in vitro effects of a drug and the actual observable in vivo response. We have previously reported on how an in vitro biochemical analysis of the site-specific effects of the compound C646 in combination with the KAT p300 can accurately predict changes in histone acetylation induced by the same compound in cells. Here, we build on this effort by further analyzing a number of reported p300 modulators, while also extending the analysis to correlate the effects of these drugs to developmental and phenotypical changes, utilizing cellular and zebrafish model systems. While this study demonstrates the utility of biochemical models as a starting point for predicting in vivo activity of multi-site targeting KATs, it also highlights the need for the development of new enzyme inhibitors that are more specific to the regulation of KAT activity in vivo.

Comparison of aminotransferases of three Bacillus strains Bacillus altitudinis W3, Bacillus velezensis SYBC H47, and Bacillus amyloliquefaciens YP6 via genome analysis and bioinformatics.

Aminotransferases have attracted considerable attention due to their extraordinary potential for the biosynthesis of chiral amines. Research on transaminase genes can facilitate their application to various fields. Herein, 89 putative aminotransferase genes potentially encoding useful biocatalysts were identified in three Bacillus strains genomes by genome annotation. Enzymes encoded by genes ota3, ota8, otae6, otae21, otaf1, otaf8, and otaf26 belong to pyridoxine 5'-phosphate-dependent enzyme class IV. These seven ω-aminotransferase genes are highly conserved according to phylogenetic tree and bioinformatics analyses, as are the putative lysine catalytic residues in the corresponding enzymes (ω-BPTA 1-7). The enzymes may possess similar activity to ω-aminotransferases from Arthrobacter sp. KNK 168. The potential application of these novel enzymes for the synthesis of medicinal amino compounds will be explored in future genetic engineering studies.