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Microbiology Spectrum Jun 2024infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and pathogenesis studies are eagerly required for CDI therapy. The major...
infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in pathogenesis; however, a lack of isogenic mutants has greatly hampered analysis of SlpA functions. In this study, the whole gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of in resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCE infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic mutants. Surprisingly, the first deletion strain was generated in this study via CRISPR-Cas9, further negating the previous thought about being essential. Results in this study will provide direct proof for roles of SlpA in pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.
Topics: Clostridioides difficile; Bacterial Proteins; Humans; Gene Deletion; Clostridium Infections; Biofilms; Anti-Bacterial Agents; Virulence; CRISPR-Cas Systems; Bacterial Adhesion; Membrane Glycoproteins
PubMed: 38709045
DOI: 10.1128/spectrum.04005-23 -
Frontiers in Microbiology 2024Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The...
INTRODUCTION
Pholiota nameko is a widely consumed edible fungus. This study focuses on two crucial developmental stages of Pholiota nameko, namely, mycelium and ascospores. The objectives of this research were to investigate changes in microbial diversity and community structure during the growth of Pholiota nameko and to analyze the adaptability of the dominant strains to their respective habitats through metabolic.
METHODS
Specifically, we conducted second-generation sequencing of the 16S rRNA gene (Illumina) on samples obtained from these stages. In addition, we isolated and characterized endophytes present in Pholiota nameko, focusing on examining the impact of dominant endophyte genera on autolysis. We also conducted a metabolic pathway analysis.
RESULTS AND DISCUSSION
The results unveiled 578,414 valid sequences of Pholiota nameko endophytic fungi. At the phylum level, the dominant taxa were Basidiomycota, Ascomycota, Zoopagomycota, and Mucoromycota. At the genus level, the dominant taxa observed were Pholiota, Inocybe, Fusarium, and Hortiboletus. For endophytic bacteria, we obtained 458,475 valid sequences. The dominant phyla were Proteobacteria, TM6, Firmicutes, and Bacteroidetes, while the dominant genera were Edaphobacter, Xanthomonas, Burkholderia, and Pseudomonas. Moreover, we identified the isolated strains in Pholiota nameko using 16S rDNA, and most of them were found to belong to the genus Pseudomonas, with Pseudomonas putida being the most prevalent strain. The findings revealed that the Pseudomonas putida strain has the ability to slow down the breakdown of soluble proteins and partially suppress the metabolic processes that generate superoxide anion radicals in Pholiota nameko, thereby reducing autolysis. Additionally, our results demonstrated that molybdenum enzyme-mediated anaerobic oxidative phosphorylation reactions were the primary energy metabolism pathway in the Pseudomonas putida strain. This suggests that the molybdenum cofactor synthesis pathway might be the main mechanism through which Pholiota nameko adapts to its complex and diverse habitats.
PubMed: 38690362
DOI: 10.3389/fmicb.2024.1319886 -
Synthetic and Systems Biotechnology Sep 2024Microbial cell factories utilize renewable raw materials for industrial chemical production, providing a promising path for sustainable development. is widely used in...
Microbial cell factories utilize renewable raw materials for industrial chemical production, providing a promising path for sustainable development. is widely used in industry for its food safety properties, but challenges remain in the limitations of microbial fermentation. This study proposes a novel strategy based on lifespan engineering to design robust chassis cells to supplement traditional metabolic modification strategies that can alleviate cell autolysis, tolerate toxic substrates, and get a higher mass transfer efficiency. The modified chassis cells could produce high levels of l-glutaminase, and tolerate hydroquinone to produce -arbutin efficiently. In a 5 L bioreactor, the l-glutaminase enzyme activity of the final strain CRE15TG was increased to 2817.4 ± 21.7 U mL, about 1.98-fold compared with that of the wild type. The -arbutin yield of strain CRE15A was increased to 134.7 g L, about 1.34-fold compared with that of the WT. To our knowledge, both of the products in this study performed the highest yields reported so far. The chassis modification strategy described in this study can Improve the utilization efficiency of chassis cells, mitigate the possible adverse effects caused by excessive metabolic modification of engineered strains, and provide a new idea for the future design of microbial cell factories.
PubMed: 38634000
DOI: 10.1016/j.synbio.2024.04.004 -
Infection and Drug Resistance 2024(MRSA) is a cause of staph infection that is difficult to treat because of resistance to some antibiotics. A recent study indicated that diarylurea is a novel...
BACKGROUND
(MRSA) is a cause of staph infection that is difficult to treat because of resistance to some antibiotics. A recent study indicated that diarylurea is a novel antibacterial agent against multi-drug resistant . In this work, we refined the bactericidal mechanism of as a peptidoglycan (PG) hydrolase by affecting AtlA-mediated PG homeostasis.
METHODS
A wild-type strain (WT) and a mutant strain (Δ) were used to investigate the effects of on the cell wall, PG, and autolysin regulatory system by antimicrobial susceptibility testing, hemolytic toxin assay, microanalysis, autolysis assay, qRT-PCR, ELISA and mouse model of pneumonia.
RESULTS
The results revealed that down-regulated the expression of genes related to peptidoglycan hydrolase (PGH) (, and ), and reduced the levels of PG, muramyl dipeptide (MDP), cytokines, and hemolytic toxin, while Δ interfered with the genes regulation and PG homeostasis. In the mouse MRSA pneumonia model, the same trend was observed in the nucleotide oligomerization domain protein 2 (NOD2) and relative proinflammatory factors.
CONCLUSION
may act as a novel inhibitor of PG hydrolyse, disrupting autolysin-mediated PG homeostasis, and reducing inflammation by down-regulating the MDP-NOD2 pathway.
PubMed: 38560705
DOI: 10.2147/IDR.S455339 -
MSystems Apr 2024Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance is...
UNLABELLED
Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment.
IMPORTANCE
The emergence of multidrug-resistant (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism.
Topics: Humans; Staphylococcus aureus; Vancomycin; Ribonuclease III; Methicillin-Resistant Staphylococcus aureus; RNA, Bacterial; RNA, Small Untranslated; Anti-Bacterial Agents; Staphylococcal Infections; RNA, Messenger; Bacteria; Carbon
PubMed: 38534138
DOI: 10.1128/msystems.00971-23 -
Veterinary Microbiology May 2024Chlamydia pecorum is a widespread veterinary chlamydial species causing endemic infections in livestock, such as ruminants and pigs, globally. However, there is limited...
Chlamydia pecorum is a widespread veterinary chlamydial species causing endemic infections in livestock, such as ruminants and pigs, globally. However, there is limited contemporary knowledge on infecting strain diversity in various hosts. This study aimed to evaluate the genetic diversity of C. pecorum strains infecting Swiss livestock through C. pecorum genotyping and phylogenetic analyses in comparison to the global population, while also assessing chlamydial strains for plasmid carriage. A total of 263 C. pecorum positive samples from clinically healthy ruminant and pig herds (Bovines = 216, sheep = 25, pigs = 14) as well as placentae from eight C. pecorum positive ruminant abortion cases from other Swiss herds were investigated. The ompA and Multi-Locus sequence typing revealed novel C. pecorum genotypes, and bovine strains exhibited considerable genetic diversity, contrasting with lower diversity in sheep and pig strains. C. pecorum plasmid was detected in 100.0% of sheep (41/41) and pig (255/255) samples, and in 69.4% of bovine samples (150/216). In contrast, no plasmid was detected in the eight C. pecorum-positive ruminant abortion cases either representing plasmid-less strains or possibly escaping PCR detection due to autolysis of the placenta. This study supports the genetic diversity of C. pecorum strains, particularly in bovines, and identifies novel sequence types in Swiss livestock.
Topics: Animals; Sheep; Cattle; Swine; Chlamydia Infections; Livestock; Switzerland; Multilocus Sequence Typing; Phylogeny; Genetic Variation; Chlamydia; Ruminants; Cattle Diseases; Swine Diseases
PubMed: 38502978
DOI: 10.1016/j.vetmic.2024.110057 -
Heliyon Mar 2024Fish protein hydrolysates were obtained from cultured rainbow trout (Oncorhynchus mykiss) viscera using commercial and endogenous enzymes. Two methods were employed for...
Fish protein hydrolysates were obtained from cultured rainbow trout (Oncorhynchus mykiss) viscera using commercial and endogenous enzymes. Two methods were employed for hydrolysis: acid autolysis (also known as silage) at room temperature for 10 days in acidic conditions, until total solubilisation, and enzymatic hydrolysis using Alcalase 2.4 LFG, Protana Prime, and the endogenous enzymes in the viscera. The effectiveness of both methods in releasing free amino acids (FAA) was assessed. After evaluating the results, the most effective enzymatic hydrolysis was optimized. The findings indicated that enzymatic hydrolysis with Alcalase, Protana Prime and endogenous enzymes combined for 7 h at a dose of 1% of protein, and a 7-day acid autolysis yielded the highest degree of hydrolysis (83.8% and 75.8%), a yield of FAA from viscera of 5.9% and 3.2%, and a yield of FAA from total protein of 71.3% and 52.5%, respectively. In conclusion, the use of commercial enzymes was more efficient in releasing amino acids, but endogenous enzymes showed a strong proteolytic capacity during acid autolysis, suggesting it also as a promising method to produce FAA-rich hydrolysates.
PubMed: 38468971
DOI: 10.1016/j.heliyon.2024.e27030 -
BMC Microbiology Mar 2024Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an...
BACKGROUND
Autolysis by cellular peptidoglycan hydrolases (PGH) is a well-known phenomenon in bacteria. During food fermentation, autolysis of starter cultures can exert an accelerating effect, as described in many studies on cheese ripening. In contrast, very little is known about autolysis of starter cultures used in other fermentations. Staphylococcus (S.) carnosus is often used in raw sausage fermentations, contributing to nitrate reduction and flavor formation. In this study, we analyzed the influence of PGHs of the strains S. carnosus TMW 2.146 and S. carnosus TMW 2.2525 on their autolytic behavior. The staphylococcal major autolysin (Atl), a bifunctional enzyme with an N-acetylmuramoyl-L-alanine amidase and a glucosaminidase as an active site, is assumed to be the enzyme by which autolysis is mainly mediated.
RESULTS
AtlC mutant strains showed impaired growth and almost no autolysis compared to their respective wild-type strains. Light microscopy and scanning electron microscopy showed that the mutants could no longer appropriately separate from each other during cell division, resulting in the formation of cell clusters. The surface of the mutants appeared rough with an irregular morphology compared to the smooth cell surfaces of the wild-types. Moreover, zymograms showed that eight lytic bands of S. carnosus, with a molecular mass between 140 and 35 kDa, are processed intermediates of AtlC. It was noticed that additional bands were found that had not been described in detail before and that the banding pattern changes over time. Some bands disappear entirely, while others become stronger or are newly formed. This suggests that AtlC is degraded into smaller fragments over time. A second knockout was generated for the gene encoding a N-acetylmuramoyl-L-alanine amidase domain-containing protein. Still, no phenotypic differences could be detected in this mutant compared to the wild-type, implying that the autolytic activity of S. carnosus is mediated by AtlC.
CONCLUSIONS
In this study, two knockout mutants of S. carnosus were generated. The atlC mutant showed a significantly altered phenotype compared to the wild-type, revealing AtlC as a key factor in staphylococcal autolysis. Furthermore, we show that Atl is degraded into smaller fragments, which are still cell wall lytic active.
Topics: N-Acetylmuramoyl-L-alanine Amidase; Staphylococcus
PubMed: 38459514
DOI: 10.1186/s12866-024-03231-6 -
Journal of Neuropathology and... Apr 2024Identification of central nervous system injury is a critical part of perinatal autopsies; however, injury is not always easily identifiable due to autolysis and...
Identification of central nervous system injury is a critical part of perinatal autopsies; however, injury is not always easily identifiable due to autolysis and immaturity of the developing brain. Here, the role of immunohistochemical stains in the identification of perinatal brain injury was investigated. Blinded semiquantitative scoring of injury was performed on sections of frontal lobe from 76 cases (51 liveborn and 25 stillborn) using H&E, GFAP, Iba-1, and β-APP stains. Digital image analysis was used to quantify GFAP and Iba-1 staining. Commonly observed pathologies included diffuse white matter gliosis (DWMG) and white matter necrosis (WMN). DWMG scores were very similar on H&E and GFAP stains for liveborn subjects. For stillborn subjects, DWMG scores were significantly higher on GFAP stain than H&E. β-APP was needed for identification of WMN in 71.4% of stillborn subjects compared to 15.4% of liveborn subjects. Diffuse staining for Iba-1 within cortex and white matter was positively correlated with subject age. Staining quantification on digital image analysis was highly correlated to semiquantitative scoring. Overall, GFAP and β-APP stains were most helpful in identifying white matter injury not seen on H&E in stillborn subjects. Immunostains may therefore be warranted as an integral part of stillborn brain autopsies.
Topics: Pregnancy; Female; Humans; Brain; Brain Injuries; White Matter; Frontal Lobe; Cerebral Cortex; Glial Fibrillary Acidic Protein
PubMed: 38441171
DOI: 10.1093/jnen/nlae019 -
Journal of Oral Microbiology 2024To analyze contributions to microbial ecology of Reactive Electrophile Species (RES), including methylglyoxal, generated during glycolysis.
OBJECTIVES
To analyze contributions to microbial ecology of Reactive Electrophile Species (RES), including methylglyoxal, generated during glycolysis.
METHODS
Genetic analyses were performed on the glyoxalase pathway in Streptococcus mutans (SM) and Streptococcus sanguinis (SS), followed by phenotypic assays and transcription analysis.
RESULTS
Deleting glyoxalase I (lguL) reduced RES tolerance to a far greater extent in SM than in SS, decreasing the competitiveness of SM against SS. Although SM displays a greater RES tolerance than SS, lguL-null mutants of either species showed similar tolerance; a finding consistent with the ability of methylglyoxal to induce the expression of lguL in SM, but not in SS. A novel paralogue of lguL (named gloA2) was identified in most streptococci. SM mutant ∆gloA2SM showed little change in methylglyoxal tolerance yet a significant growth defect and increased autolysis on fructose, a phenotype reversed by the addition of glutathione, or by the deletion of a fructose: phosphotransferase system (PTS) that generates fructose-1-phosphate (F-1-P).
CONCLUSIONS
Fructose contributes to RES generation in a PTS-specific manner, and GloA2 may be required to degrade certain RES derived from F-1-P. This study reveals the critical roles of RES in fitness and interbacterial competition and the effects of PTS in modulating RES metabolism.
PubMed: 38440286
DOI: 10.1080/20002297.2024.2322241