scholarly journals Developmental Growth Control Exerted via the Protein A Kinase Tpk2 in Ashbya gossypii

2015 ◽  
Vol 14 (6) ◽  
pp. 593-601 ◽  
Author(s):  
Lisa Wasserstrom ◽  
Klaus Lengeler ◽  
Andrea Walther ◽  
Jürgen Wendland
Keyword(s):  
Protein A ◽  
Growth Control ◽  
Spore Wall ◽  
Ashbya Gossypii ◽  
Content Type ◽  
Developmental Growth ◽  
Late Step ◽  
Transcript Profiles ◽  
Wall Assembly ◽  
Developmental Switch

ABSTRACT Sporulation in Ashbya gossypii is induced by nutrient-limited conditions and leads to the formation of haploid spores. Using RNA-seq, we have determined a gene set induced upon sporulation, which bears considerable overlap with that of Saccharomyces cerevisiae but also contains A. gossypii -specific genes. Addition of cyclic AMP (cAMP) to nutrient-limited media blocks sporulation and represses the induction of sporulation specific genes. Deletion of the protein kinase A (PKA) catalytic subunits encoded by TPK1 and TPK2 showed reduced growth in tpk1 but enhanced growth in the tpk2 strain; however, both mutants sporulated well. Sporulation can be blocked by cAMP in tpk1 but not in tpk2 strains. Similarly, TPK2 acts at a second developmental switch promoting the break in spore dormancy. In S. cerevisiae , PKA phosphorylates and inhibits Msn2/4. The transcript profiles of the tpk1 and msn2/4 mutants were very similar to that of the wild type under sporulation conditions. However, deletion of the single A. gossypii MSN2/4 homolog generated a specific sporulation defect. We identified a set of genes involved in spore wall assembly that was downregulated in the msn2/4 mutant, particularly DIT2 , suggesting that poor spore viability may be due to lysis of spores. Our results reveal specific functional differences between the two catalytic PKA subunits in A. gossypii and identified Tpk2 as the key A kinase that transduces developmental decisions of growth. Our data also suggest that Msn2/4 is involved only at a late step of sporulation in A. gossypii and is not a major regulator of IME1 .

scholarly journals Clustering of Nuclei in Multinucleated Hyphae Is Prevented by Dynein-Driven Bidirectional Nuclear Movements and Microtubule Growth Control in Ashbya gossypii

2011 ◽  
Vol 10 (7) ◽  
pp. 902-915 ◽  
Author(s):  
Sandrine Grava ◽  
Miyako Keller ◽  
Sylvia Voegeli ◽  
Shanon Seger ◽  
Claudia Lang ◽  
...  
Keyword(s):  
Growth Control ◽  
Accessory Proteins ◽  
Nuclear Density ◽  
Ashbya Gossypii ◽  
Cytoplasmic Microtubule ◽  
Content Type ◽  
Microtubule Motor ◽  
Nuclear Clusters ◽  
Microtubule Growth ◽  
Nuclear Clustering

ABSTRACT During filamentous fungus development, multinucleated hyphae employ a system for long-range nuclear migration to maintain an equal nuclear density. A decade ago the microtubule motor dynein was shown to play a central role in this process. Previous studies with Ashbya gossypii revealed extensive bidirectional movements and bypassings of nuclei, an autonomous cytoplasmic microtubule (cMT) cytoskeleton emanating from each nucleus, and pulling of nuclei by sliding of cMTs along the cortex. Here, we show that dynein is the sole motor for bidirectional movements and bypassing because these movements are concomitantly decreased in mutants carrying truncations of the dynein heavy-chain DYN1 promoter. The dynactin component Jnm1, the accessory proteins Dyn2 and Ndl1, and the potential dynein cortical anchor Num1 are also involved in the dynamic distribution of nuclei. In their absence, nuclei aggregate to different degrees, whereby the mutants with dense nuclear clusters grow extremely long cMTs. As in budding yeast, we found that dynein is delivered to cMT plus ends, and its activity or processivity is probably controlled by dynactin and Num1. Together with its role in powering nuclear movements, we propose that dynein also plays (directly or indirectly) a role in the control of cMT length. Those combined dynein actions prevent nuclear clustering in A. gossypii and thus reveal a novel cellular role for dynein.

scholarly journals Sporulation in Ashbya gossypii

2020 ◽  
Vol 6 (3) ◽  
pp. 157
Author(s):  
Jürgen Wendland
Keyword(s):  
Protein A ◽  
Spore Wall ◽  
Regulatory Genes ◽  
Ashbya Gossypii ◽  
Filamentous Ascomycete ◽  
Open Questions ◽  
Type Gene ◽  
Strong Positive Signal ◽  
Pka Pathway

Ashbya gossypii is a filamentous ascomycete belonging to the yeast family of Saccharomycetaceae. At the end of its growth phase Ashbya generates abundant amounts of riboflavin and spores that form within sporangia derived from fragmented cellular compartments of hyphae. The length of spores differs within species of the genus. Needle-shaped Ashbya spores aggregate via terminal filaments. A. gossypii is a homothallic fungus which may possess a and α mating types. However, the solo-MATa type strain is self-fertile and sporulates abundantly apparently without the need of prior mating. The central components required for the regulation of sporulation, encoded by IME1, IME2, IME4, KAR4, are conserved with Saccharomyces cerevisiae. Nutrient depletion generates a strong positive signal for sporulation via the cAMP-PKA pathway and SOK2, which is also essential for sporulation. Strong inhibitors of sporulation besides mutations in the central regulatory genes are the addition of exogenous cAMP or the overexpression of the mating type gene MATα2. Sporulation has been dissected using gene-function analyses and global RNA-seq transcriptomics. This revealed a role of Msn2/4, another potential PKA-target, for spore wall formation and a key dual role of the protein A kinase Tpk2 at the onset of sporulation as well as for breaking the dormancy of spores to initiate germination. Recent work has provided an overview of ascus development, regulation of sporulation and spore maturation. This will be summarized in the current review with a focus on the central regulatory genes. Current research and open questions will also be discussed.

scholarly journals CD4 T Cell Antigens from Staphylococcus aureus Newman Strain Identified following Immunization with Heat-Killed Bacteria

2012 ◽  
Vol 19 (4) ◽  
pp. 477-489 ◽  
Author(s):  
Paulraj K. Lawrence ◽  
Bachra Rokbi ◽  
Nadège Arnaud-Barbe ◽  
Eric L. Sutten ◽  
Junzo Norimine ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
T Cell ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Protein A ◽  
Cd4 T Cell ◽  
Content Type ◽  
Intramammary Infections ◽  
T Cell Antigens ◽  
Ifn Γ

ABSTRACTStaphylococcus aureusis a commensal bacterium associated with the skin and mucosal surfaces of humans and animals that can also cause chronic infection. The emergence of antibiotic-resistant strains such as methicillin-resistantS. aureus(MRSA) and strains causing chronic intramammary infections (IMI) in cows results in severe human and livestock infections. Conventional approaches to vaccine development have yielded only a few noneffective vaccines against MRSA or IMI strains, so there is a need for improved vaccine development. CD4 T lymphocytes are required for promoting gamma interferon (IFN-γ) mediated immunoglobulin isotype switching in B lymphocytes to produce high-affinity IgG antibodies and IFN-γ-mediated phagocyte activation for an effective resolution of bacterial infection. However, the lack of known CD4 T cell antigens fromS. aureushas made it difficult to design effective vaccines. The goal of this study was to identifyS. aureusproteins recognized by immune CD4 T cells. Using a reverse genetics approach, 43 antigens were selected from theS. aureusNewman strain. These included lipoproteins, proteases, transcription regulators, an alkaline shock protein, conserved-domain proteins, hemolysins, fibrinogen-binding protein, staphylokinase, exotoxin, enterotoxin, sortase, and protein A. Screening of expressed proteins for recall T cell responses in outbred, immune calves identified 13 proteins that share over 80% sequence identity among MRSA or IMI strains. These may be useful for inclusion in a broadly protective multiantigen vaccine against MRSA or IMI.

scholarly journals Anaplasma phagocytophilum Outer Membrane Protein A Interacts with Sialylated Glycoproteins To Promote Infection of Mammalian Host Cells

2012 ◽  
Vol 80 (11) ◽  
pp. 3748-3760 ◽  
Author(s):  
Nore Ojogun ◽  
Amandeep Kahlon ◽  
Stephanie A. Ragland ◽  
Matthew J. Troese ◽  
Juliana E. Mastronunzio ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Anaplasma Phagocytophilum ◽  
Protein A ◽  
Host Cells ◽  
Mammalian Host ◽  
Content Type ◽  
Outer Membrane Protein A ◽  
Sialylated Glycoproteins

ABSTRACTAnaplasma phagocytophilumis the tick-transmitted obligate intracellular bacterium that causes human granulocytic anaplasmosis (HGA).A. phagocytophilumbinding to sialyl Lewis x (sLex) and other sialylated glycans that decorate P selectin glycoprotein 1 (PSGL-1) and other glycoproteins is critical for infection of mammalian host cells. Here, we demonstrate the importance ofA. phagocytophilumouter membrane protein A (OmpA) APH_0338 in infection of mammalian host cells. OmpA is transcriptionally induced during transmission feeding ofA. phagocytophilum-infected ticks on mice and is upregulated during invasion of HL-60 cells. OmpA is presented on the pathogen's surface. Sera from HGA patients and experimentally infected mice recognize recombinant OmpA. Pretreatment ofA. phagocytophilumorganisms with OmpA antiserum reduces their abilities to infect HL-60 cells. The OmpA N-terminal region is predicted to contain the protein's extracellular domain. GlutathioneS-transferase (GST)-tagged versions of OmpA and OmpA amino acids 19 to 74 (OmpA19-74) but not OmpA75-205bind to, and competitively inhibitA. phagocytophiluminfection of, host cells. Pretreatment of host cells with sialidase or trypsin reduces or nearly eliminates, respectively, GST-OmpA adhesion. Therefore, OmpA interacts with sialylated glycoproteins. This study identifies the firstA. phagocytophilumadhesin-receptor pair and delineates the region of OmpA that is critical for infection.

scholarly journals A Novel, Multiple-Antigen Pneumococcal Vaccine Protects against LethalStreptococcus pneumoniaeChallenge

2018 ◽  
Vol 87 (3) ◽  
Author(s):  
Win-Yan Chan ◽  
Claire Entwisle ◽  
Giuseppe Ercoli ◽  
Elise Ramos-Sevillano ◽  
Ann McIlgorm ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein A ◽  
Surface Protein ◽  
Surface Proteins ◽  
Rabbit Serum ◽  
Protein Antigens ◽  
Content Type ◽  
Multiple Antigen ◽  
Bacterial Lysates ◽  
Chromatography Step

ABSTRACTCurrent vaccination againstStreptococcus pneumoniaeuses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared fromS. pneumoniaeTIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains ofS. pneumoniaewere opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologousS. pneumoniaestrains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection againstS. pneumoniae.

scholarly journals Structural Similarities and Differences between Two Functionally Distinct SecA Proteins, Mycobacterium tuberculosis SecA1 and SecA2

2015 ◽  
Vol 198 (4) ◽  
pp. 720-730 ◽  
Author(s):  
Stephanie Swanson ◽  
Thomas R. Ioerger ◽  
Nathan W. Rigel ◽  
Brittany K. Miller ◽  
Miriam Braunstein ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Signal Peptide ◽  
Protein A ◽  
Structural Difference ◽  
Structural Similarity ◽  
Structural Features ◽  
Dominant Negative ◽  
Content Type ◽  
Functional Regions ◽  
Suppressor Mutations

ABSTRACTWhile SecA is the ATPase component of the major bacterial secretory (Sec) system, mycobacteria and some Gram-positive pathogens have a second paralog, SecA2. In bacteria with two SecA paralogs, each SecA is functionally distinct, and they cannot compensate for one another. Compared to SecA1, SecA2 exports a distinct and smaller set of substrates, some of which have roles in virulence. In the mycobacterial system, some SecA2-dependent substrates lack a signal peptide, while others contain a signal peptide but possess features in the mature protein that necessitate a role for SecA2 in their export. It is unclear how SecA2 functions in protein export, and one open question is whether SecA2 works with the canonical SecYEG channel to export proteins. In this study, we report the structure ofMycobacterium tuberculosisSecA2 (MtbSecA2), which is the first structure of any SecA2 protein. A high level of structural similarity is observed between SecA2 and SecA1. The major structural difference is the absence of the helical wing domain, which is likely to play a role in howMtbSecA2 recognizes its unique substrates. Importantly, structural features critical to the interaction between SecA1 and SecYEG are preserved in SecA2. Furthermore, suppressor mutations of a dominant-negativesecA2mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG or the translocating polypeptide substrate. These results support a model in which the mycobacterial SecA2 works with SecYEG.IMPORTANCESecA2 is a paralog of SecA1, which is the ATPase of the canonical bacterial Sec secretion system. SecA2 has a nonredundant function with SecA1, and SecA2 exports a distinct and smaller set of substrates than SecA1. This work reports the crystal structure of SecA2 ofMycobacterium tuberculosis(the first SecA2 structure reported for any organism). Many of the structural features of SecA1 are conserved in the SecA2 structure, including putative contacts with the SecYEG channel. Several structural differences are also identified that could relate to the unique function and selectivity of SecA2. Suppressor mutations of asecA2mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG.

scholarly journals Curcumin Reduces the Motility of Salmonella enterica Serovar Typhimurium by Binding to the Flagella, Thereby Leading to Flagellar Fragility and Shedding

2016 ◽  
Vol 198 (13) ◽  
pp. 1798-1811 ◽  
Author(s):  
Sandhya Amol Marathe ◽  
Arjun Balakrishnan ◽  
Vidya Devi Negi ◽  
Deepika Sakorey ◽  
Nagasuma Chandra ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Binding Assay ◽  
Protein A ◽  
Intestinal Barrier ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Flagellar Filament ◽  
Fluorescence Binding ◽  
Fluorescence Binding Assay

ABSTRACTOne of the important virulence properties of the pathogen is its ability to travel to a favorable environment, cross the viscous mucus barrier (intestinal barrier for enteric pathogens), and reach the epithelia to initiate pathogenesis with the help of an appendage, like flagella. Nonetheless, flagella can act as an “Achilles heel,” revealing the pathogen's presence to the host through the stimulation of innate and adaptive immune responses. We assessed whether curcumin, a dietary polyphenol, could alter the motility ofSalmonella, a foodborne pathogen. It reduced the motility ofSalmonella entericaserovar Typhimurium by shortening the length of the flagellar filament (from ∼8 μm to ∼5 μm) and decreasing its density (4 or 5 flagella/bacterium instead of 8 or 9 flagella/bacterium). Upon curcumin treatment, the percentage of flagellated bacteria declined from ∼84% to 59%. However, no change was detected in the expression of the flagellin gene and protein. A fluorescence binding assay demonstrated binding of curcumin to the flagellar filament. This might make the filament fragile, breaking it into smaller fragments. Computational analysis predicted the binding of curcumin, its analogues, and its degraded products to a flagellin molecule at an interface between domains D1 and D2. Site-directed mutagenesis and a fluorescence binding assay confirmed the binding of curcumin to flagellin at residues ASN120, ASP123, ASN163, SER164, ASN173, and GLN175.IMPORTANCEThis work, to our knowledge the first report of its kind, examines how curcumin targets flagellar density and affects the pathogenesis of bacteria. We found that curcumin does not affect any of the flagellar synthesis genes. Instead, it binds to the flagellum and makes it fragile. It increases the torsional stress on the flagellar filament that then breaks, leaving fewer flagella around the bacteria. Flagella, which are crucial ligands for Toll-like receptor 5, are some of the most important appendages ofSalmonella. Curcumin is an important component of turmeric, which is a major spice used in Asian cooking. The loss of flagella can, in turn, change the pathogenesis of bacteria, making them more robust and fit in the host.

scholarly journals Response of Fatty Acid Synthesis Genes to the Binding of Human Salivary Amylase by Streptococcus gordonii

2012 ◽  
Vol 78 (6) ◽  
pp. 1865-1875 ◽  
Author(s):  
Anna E. Nikitkova ◽  
Elaine M. Haase ◽  
M. Margaret Vickerman ◽  
Steven R. Gill ◽  
Frank A. Scannapieco
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Protein A ◽  
Acid Synthesis ◽  
Differentially Expressed ◽  
Streptococcus Gordonii ◽  
Bacterial Survival ◽  
Salivary Amylase ◽  
Content Type

ABSTRACTStreptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding toS. gordoniimay be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes inS. gordoniistrain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated inS. gordoniiCH1 cells treated with native amylase relative to those treated with denatured amylase. AnabpA-deficient strain ofS. gordoniiexposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding toS. gordoniistrain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposedabpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response inS. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment.

scholarly journals CidR and CcpA Synergistically Regulate Staphylococcus aureus cidABC Expression

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Marat R. Sadykov ◽  
Ian H. Windham ◽  
Todd J. Widhelm ◽  
Vijaya Kumar Yajjala ◽  
Sean M. Watson ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Carbon Metabolism ◽  
Protein A ◽  
Transcriptional Regulators ◽  
Extracellular Dna ◽  
Regulatory Control ◽  
Pcr Analysis ◽  
Mobility Shift ◽  
Content Type ◽  
Biofilm Development

ABSTRACT The death and lysis of a subpopulation of Staphylococcus aureus cells during biofilm development benefit the whole bacterial population through the release of an important component of the biofilm matrix, extracellular DNA. Previously, we have demonstrated that these processes are affected by the gene products of the cidABC operon, the expression of which is controlled by the LysR-type transcriptional regulator, CidR. In this study, we characterized cis- and trans-acting elements essential for the induction of the cidABC operon. In addition to a CidR-binding site located within the cidABC promoter region, sequence analysis revealed the presence of a putative catabolite responsive element (cre box), suggestive of the involvement of the catabolite control protein A (CcpA) in the regulation of cidABC expression. This was confirmed using electrophoretic mobility shift assays and real-time reverse transcriptase PCR analysis demonstrating the direct positive control of cidABC transcription by the master regulator of carbon metabolism. Furthermore, the importance of CcpA and the identified cre site for the induction of the cidABC operon was demonstrated by examining the expression of PcidABC-lacZ reporter fusions in various mutant strains in which the genes involved in carbon metabolism and carbon catabolite repression were disrupted. Together the results of this study demonstrate the necessity of both transcriptional regulators, CidR and CcpA, for the induction of the cidABC operon and reveal the complexity of molecular interactions controlling its expression. IMPORTANCE This work focuses on the characterization of cis- and trans-acting elements essential for the induction of the cidABC operon in S. aureus. The results of this study are the first to demonstrate the synergistic control of cidABC expression by transcriptional regulators CidR and CcpA during carbohydrate metabolism. We established that the full induction of cidABC expression depends on the metabolic state of bacteria and requires both CidR and CcpA. Together, these findings delineate regulatory control of cidABC expression under different metabolic conditions and provide important new insights into our understanding of cell death mechanisms during biofilm development in S. aureus.

scholarly journals Interaction between SWP9 and Polar Tube Proteins of the Microsporidian Nosema bombycis and Function of SWP9 as a Scaffolding Protein Contribute to Polar Tube Tethering to the Spore Wall

2016 ◽  
Vol 85 (3) ◽  
Author(s):  
Donglin Yang ◽  
Lixia Pan ◽  
Pai Peng ◽  
Xiaoqun Dang ◽  
Chunfeng Li ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Spore Wall ◽  
Scaffolding Protein ◽  
Dependent Manner ◽  
Polar Tube ◽  
Content Type ◽  
Nosema Bombycis ◽  
Invasion Mechanism ◽  
In The Beginning ◽  
And Function

ABSTRACTAll microsporidia possess a unique, highly specialized invasion mechanism that involves the polar tube and spore wall. The interaction between spore wall proteins (SWPs) and polar tube proteins (PTPs) in the formation, arrangement, orderly orientation, and function of the polar tube and spore wall remains to be determined. This study was undertaken to examine the protein interactions ofNosema bombycisSWP7 (NbSWP7), NbSWP9, and PTPs. Coimmunoprecipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and yeast two-hybrid data demonstrated that NbSWP9, but not NbSWP7, interacts with NbPTP1 and NbPTP2. Furthermore, immunoelectron microscopy (IEM) showed that NbSWP9 was localized mainly in the developing polar tube of sporoblasts, while NbSWP7 was found randomly in the cytoplasm. However, both NbSWP9 and NbSWP7 were located in the polar tube and spore wall ofN. bombycismature spores. The reason why NbSWP7 was localized to the polar tube may be due to the interaction between NbSWP9 and NbSWP7. Interestingly, the majority of NbSWP9, but not NbSWP7, accumulated in the beginning part of the extruded polar tube and the ruptured spore wall called the anchoring disk (AD) when the mature spores germinated under weak-alkaline environmental stimulation. Additionally, anti-NbSWP9 antibody reduced spore germination in a dose-dependent manner. In conclusion, our study further confirmed that NbSWP9 is a scaffolding protein that not only anchors and holds the polar tube but also tethers the polar tube to the spore wall.

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