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Pathogens (Basel, Switzerland) May 2024pv. () is a significant phytopathogen causing black rot disease in crucifers. Its virulence relies heavily on the type III secretion system (T3SS), facilitating...
pv. () is a significant phytopathogen causing black rot disease in crucifers. Its virulence relies heavily on the type III secretion system (T3SS), facilitating effector translocation into plant cells. The type III effectors (T3Es) disrupt cellular processes, promoting pathogen proliferation. However, only a few T3Es from have been thoroughly characterized. In this study, we further investigated two effectors using the T3Es-deficient mutant and the protoplast system. XopE2 triggers immune responses via an unidentified activator of the salicylic acid (SA) signaling pathway, whereas XopL suppresses the expression of genes associated with patterns-triggered immunity (PTI) and the SA signaling pathway. These two effectors exert opposing effects on immune responses. Additionally, we examined the relationship between the specific domains and functions of these two effector proteins. Our findings demonstrate that the N-myristoylation motif and N-terminal domain are essential for the subcellular localization and virulence of XopE2 and XopL, respectively. These novel insights enhance our understanding of the pathogenic mechanisms of T3Es and contribute to developing effective strategies for controlling bacterial disease.
PubMed: 38921746
DOI: 10.3390/pathogens13060448 -
Marine Drugs Jun 2024Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal...
Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal protein A (SpA), to the peptidoglycan cell wall. Six pentacyclic polyketides (-) were isolated from the marine sponge sp., and their structures were elucidated using spectroscopic techniques and by comparing them to previously reported data. Among them, halenaquinol () was found to be the most potent SrtA inhibitor, with an IC of 13.94 μM (4.66 μg/mL). Semi-quantitative reverse transcription PCR data suggest that halenaquinol does not inhibit the transcription of and , while Western blot analysis and immunofluorescence microscopy images suggest that it blocks the cell wall surface anchoring of SpA by inhibiting the activity of SrtA. The onset and magnitude of the inhibition of SpA anchoring on the cell wall surface in that has been treated with halenaquinol at a value 8× that of the IC of SrtA are comparable to those for an -deletion mutant. These findings contribute to the understanding of the mechanism by which marine-derived pentacyclic polyketides inhibit SrtA, highlighting their potential as anti-infective agents targeting virulence.
Topics: Aminoacyltransferases; Cysteine Endopeptidases; Staphylococcus aureus; Cell Wall; Bacterial Proteins; Animals; Porifera; Anti-Bacterial Agents; Polyketides
PubMed: 38921577
DOI: 10.3390/md22060266 -
Marine Drugs Jun 2024Three new cyclic lipopeptides, olenamidonins A-C (-), in addition to two previously reported metabolites ( and ), were accumulated in the Δ deletion mutant of...
Three new cyclic lipopeptides, olenamidonins A-C (-), in addition to two previously reported metabolites ( and ), were accumulated in the Δ deletion mutant of deepsea-derived SCSIO 1071. The structures of these cyclic lipopeptides were determined by a combination of spectroscopic methods and circular dichroism (CD) measurement. The antibacterial assay results showed that compounds displayed different degrees of growth inhibition against multidrug-resistant (MDR) bacterial strains CCARM 5172 and CCARM 5203 with minimum inhibitory concentrations (MICs) of 1.56-6.25 μg/mL.
Topics: Streptomyces; Lipopeptides; Microbial Sensitivity Tests; Anti-Bacterial Agents; Enterococcus faecalis; Peptides, Cyclic; Enterococcus faecium; Drug Resistance, Multiple, Bacterial; Bacterial Proteins
PubMed: 38921573
DOI: 10.3390/md22060262 -
Journal of Fungi (Basel, Switzerland) May 2024Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating cell membrane fusion and intracellular...
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating cell membrane fusion and intracellular vesicular trafficking in eukaryotic cells, and have been well-documented to play critical roles in growth, development, and pathogenesis in the filamentous fungal plant pathogens. However, little is known about the contributions of SNAREs to the physiology and biocontrol potential in entomopathogenic filamentous fungi. Here, a genome-wide analysis of SNARE genes was performed taking advantage of the available whole genome sequence of , a classical entomopathogenic fungus. Based on the compared genomic method, 22 genes encoding putative SNAREs were identified from the whole genome of , and were classified into four groups (7 Qa-, 4 Qb-, 6 Qc-, and 5 R-SNAREs) according to the conserved structural features of their encoding proteins. An R-SNARE encoding gene was further functionally characterized by gene disruption and complementation. The null mutant showed a fluffy appearance in mycelial growth and an obvious lag in conidial germination. The null mutant also exhibited significantly increased sensitivity to oxidative stress and cell wall perturbing agents and reduced the yield of conidia production by 43.1% compared with the wild-type strain. Moreover, disruption of caused a significant decrease in conidial virulence to larvae. Overall, our results provide an overview of vesicle trafficking in and revealed that BbSec22 was a multifunctional protein associated with mycelial growth, sporulation, conidial germination, stress tolerance, and insecticidal virulence.
PubMed: 38921379
DOI: 10.3390/jof10060393 -
Journal of Fungi (Basel, Switzerland) May 2024, a major postharvest pathogen, causes blue mold rot in citrus fruits through the deployment of various virulence factors. Recent studies highlight the role of the...
, a major postharvest pathogen, causes blue mold rot in citrus fruits through the deployment of various virulence factors. Recent studies highlight the role of the epigenetic reader, , in modulating the pathogenicity of phytopathogenic fungi. Our research revealed that the deletion of the gene in led to significant phenotypic alterations, including delayed mycelial growth, reduced spore production, and decreased utilization of sucrose. Additionally, the mutant strain exhibited increased sensitivity to pH fluctuations and elevated iron and calcium ion stress, culminating in reduced virulence on Gannan Novel oranges. Ultrastructural analyses disclosed notable disruptions in cell membrane integrity, disorganization within the cellular matrix, and signs of autophagy. Transcriptomic data further indicated a pronounced upregulation of hydrolytic enzymes, oxidoreductases, and transport proteins, suggesting a heightened energy demand. The observed phenomena were consistent with a carbon starvation response potentially triggering apoptotic pathways, including iron-dependent cell death. These findings collectively underscored the pivotal role of in maintaining the pathogenic traits of , proposing that targeting could offer a new avenue for controlling citrus fungal infections and subsequent fruit decay.
PubMed: 38921355
DOI: 10.3390/jof10060368 -
Cells Jun 2024Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinomas (PDACs) harbor activation mutations of...
Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinomas (PDACs) harbor activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilized proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. The PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, the inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. The concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (; ; ) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions, and the IGF-1 growth stimulation effect was AKT-dependent. The RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth, and the pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.
Topics: Proto-Oncogene Proteins c-akt; Pancreatic Neoplasms; Animals; Cell Line, Tumor; Mice; Humans; Down-Regulation; Proto-Oncogene Proteins p21(ras); Mutation; Cell Proliferation; Signal Transduction; Gene Expression Regulation, Neoplastic
PubMed: 38920688
DOI: 10.3390/cells13121061 -
Cells Jun 2024The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the...
The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Class IV (CIV) multi-dendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1) in vitro. Through targeted genetic manipulations, we confirm that an LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases the dendritic complexity in CIV neurons. Furthermore, both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus, we examine CIV dendritic development in disease conditions as well. The expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through the regulation of TORC1 in both health and disease.
Topics: Animals; Cullin Proteins; Drosophila Proteins; Dendrites; Drosophila melanogaster; Microtubules; Huntingtin Protein; Mechanistic Target of Rapamycin Complex 1; Signal Transduction; Sensory Receptor Cells; Larva; Transcription Factors
PubMed: 38920658
DOI: 10.3390/cells13121029 -
Cells Jun 2024causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival...
causes 6.4 million cases of tuberculosis and claims 1.6 million lives annually. Mycobacterial adhesion, invasion of host cells, and subsequent intracellular survival are crucial for the infection and dissemination process, yet the cellular mechanisms underlying these phenomena remain poorly understood. This study created a (BCG) transposon library using a MycomarT7 phage carrying a Himar1 Mariner transposon to identify genes related to mycobacteria adhesion and invasion. Using adhesion and invasion model screening, we found that the mutant strain B2909 lacked adhesion and invasion abilities because of an inactive gene, which encodes a fatty-acyl CoA ligase, although the specific function of this gene remains unclear. To investigate the role of FadD18, we constructed a complementary strain and observed that expression enhanced the colony size and promoted the formation of a stronger cord-like structure; FadD18 expression also inhibited BCG growth and reduced BCG intracellular survival in macrophages. Furthermore, FadD18 expression elevated levels of the proinflammatory cytokines IL-6, IL-1β, and TNF-α in infected macrophages by stimulating the NF-κB and MAPK signaling pathways. Overall, the FadD18 plays a key role in the adhesion and invasion abilities of mycobacteria while modulating the intracellular survival of BCG by influencing the production of proinflammatory cytokines.
Topics: Mycobacterium tuberculosis; Cytokines; Macrophages; Mycobacterium bovis; Mice; Bacterial Proteins; Animals; Humans; NF-kappa B; Microbial Viability; Bacterial Adhesion
PubMed: 38920649
DOI: 10.3390/cells13121019 -
BMC Oral Health Jun 2024Streptococcus mutans (S. mutans) is an important pathogenic bacterium that causes dental caries, while Streptococcus gordonii (S. gordonii) is a non-cariogenic bacterium...
BACKGROUND
Streptococcus mutans (S. mutans) is an important pathogenic bacterium that causes dental caries, while Streptococcus gordonii (S. gordonii) is a non-cariogenic bacterium that inhibits the growth of S. mutans. The SepM protein can promote the inhibitory ability of S. mutans against S. gordonii by cleaving CSP-21 and activating the ComDE two-component system. This study was designed to explore sepM mutation in S. mutans clinical isolates and related function in the regulation of interactions with S. gordonii.
METHODS
The S. mutans clinical strains that can inhibit the growth of S. gordonii constitute the inhibitory group. 286 C-serotype S. mutans strains were categorized into S. gordonii inhibitory (n = 114) and non-inhibitory bacteria (n = 172). We detected sanger sequencing of sepM gene, the expression levels of related genes and proteins in clinical isolates, obtained prokaryotic expression and purification of mutated proteins, and analyzed the effect of the target mutations on the binding between SepM and CSP-21.
RESULTS
We found that C482T, G533A, and G661A missense mutations were presented at significantly higher frequency in the inhibitory group relative to the non-inhibitory group. There was no significant difference in the expression of the sepM gene between selected clinical isolates harboring the G533A mutation and the control group. The expression levels of SepM, phosphorylated ComD, and ComE in the mutation group were significantly higher than those in the control group. SepM_control and SepM_D221N (G661A at the gene level) were found to contain two residues close to the active center while SepM_G178D (G533A at the gene level) contained three residues close to the active center. At 25 °C and a pH of 5.5, SepM_D221N (G661A) exhibited higher affinity for CSP-21 (KD = 8.25 µM) than did the SepM control (KD = 33.1 µM), and at 25 °C and a pH of 7.5, SepM_G178D (G533A) exhibited higher affinity (KD = 3.02 µM) than the SepM control (KD = 15.9 µM). It means that it is pH dependent.
CONCLUSIONS
Our data suggest that increased cleavage of CSP-21 by the the mutant SepM may be a reason for the higher inhibitory effect of S. mutans on S. gordonii .
Topics: Streptococcus mutans; Bacterial Proteins; Streptococcus gordonii; Humans; Mutation; Mutation, Missense; Dental Caries
PubMed: 38918777
DOI: 10.1186/s12903-024-04436-x -
Communications Biology Jun 2024Myeloblastosis (MYB)-like proteins are a family of highly conserved transcription factors in animals, plants, and fungi and are involved in the regulation of mRNA...
Myeloblastosis (MYB)-like proteins are a family of highly conserved transcription factors in animals, plants, and fungi and are involved in the regulation of mRNA expression of genes. In this study, we identified and characterized one MYB-like protein in the model organism Aspergillus nidulans. We screened the mRNA levels of genes encoding MYB-like proteins containing two MYB repeats in conidia and found that the mRNA levels of four genes including flbD, cicD, and two uncharacterized genes, were high in conidia. To investigate the roles of two uncharacterized genes, AN4618 and AN10944, deletion mutants for each gene were generated. Our results revealed that AN4618 was required for fungal development. Therefore, we further investigated the role of AN4618, named as mylA, encoding the MYB-like protein containing two MYB repeats. Functional studies revealed that MylA was essential for normal fungal growth and development. Phenotypic and transcriptomic analyses demonstrated that deletion of mylA affected stress tolerance, cell wall integrity, and long-term viability in A. nidulans conidia. In addition, the germination rate of the mylA deletion mutant conidia was decreased compared with that of the wild-type conidia. Overall, this study suggests that MylA is critical for appropriate development, conidial maturation, dormancy, and germination in A. nidulans.
Topics: Aspergillus nidulans; Spores, Fungal; Fungal Proteins; Gene Expression Regulation, Fungal; Transcription Factors
PubMed: 38918572
DOI: 10.1038/s42003-024-05866-7