Evaluating lignin degradation under limited oxygen conditions by bacterial isolates from forest soil.

Lignin, a heterogeneous aromatic polymer present in plant biomass, is intertwined with cellulose and hemicellulose fibrils, posing challenges to its effective utilization due to its phenolic nature and recalcitrance to degradation. In this study, three lignin utilizing bacteria, Klebsiella sp. LEA1, Pseudomonas sp. LEA2, and Burkholderia sp. LEA3, were isolated from deciduous forest soil samples in Nan province, Thailand. These isolates were capable of growing on alkali lignin and various lignin-associated monomers at 40 °C under microaerobic conditions. The presence of Cu2+ significantly enhanced guaiacol oxidation in Klebsiella sp. LEA1 and Pseudomonas sp. LEA2. Lignin-related monomers and intermediates such as 2,6-dimethoxyphenol, 4-vinyl guaiacol, 4-hydroxybenzoic acid, benzoic acid, catechol, and succinic acid were detected mostly during the late stage of incubation of Klebsiella sp. LEA1 and Pseudomonas sp. LEA2 in lignin minimal salt media via GC-MS analysis. The intermediates identified from Klebsiella sp. LEA1 degradation suggested that conversion and utilization occurred through the ß-ketoadipate (ortho-cleavage) pathway under limited oxygen conditions. The ability of these bacteria to thrive on alkaline lignin and produce various lignin-related intermediates under limited oxygen conditions suggests their potential utility in oxygen-limited processes and the production of renewable chemicals from plant biomass.

Andrographolide Induces ROS-Mediated Cytotoxicity, Lipid Peroxidation, and Compromised Cell Integrity in Saccharomyces cerevisiae.

Andrographolide, a bioactive compound found in Andrographis paniculata, has gained significant attention for its potential therapeutic properties. Despite its promising benefits, the understanding of its side effects and underlying mechanisms remains limited. Here, we investigated the impact of andrographolide in Saccharomyces cerevisiae and observed that andrographolide induced cytotoxicity, particularly when oxidative phosphorylation was active. Furthermore, andrographolide affected various cellular processes, including vacuole fragmentation, endoplasmic reticulum stress, lipid droplet accumulation, reactive oxygen species levels, and compromised cell integrity. Moreover, we unexpectedly observed that andrographolide induced the precipitation of biomolecules secreted from yeast cells, adding an additional source of stress. Overall, this study provides insights into the cellular effects and potential mechanisms of andrographolide in yeast, shedding light on its side effects and underlying cytotoxicity pathways.

Receptor binding protein of prophage reversibly recognizes the low-molecular weight subunit of the surface-layer protein SlpA in Clostridioides difficile.

Receptor-binding proteins (RBPs) are located at the viral tail and mediate the initial recognition of phage to a specific bacterial host. Phage RBPs have co-evolved with numerous types of host receptors resulting in the formation of a diverse assortment of cognate pairs of RBP-receptors that function during the phage attachment step. Although several Clostridioides difficile bacteriophages have been discovered, their RBPs are poorly described. Using homology analysis, putative prophage-tail structure (pts) genes were identified from the prophage genome of the C. difficile HN10 strain. Competition and enzyme-linked immunosorbent assays, using recombinant PtsHN10M, demonstrated the interaction of this Pts to C. difficile cells, suggesting a role as a phage RBP. Gel filtration and cross-linking assay revealed the native form of this protein as a homotrimer. Moreover, truncated variants indicated that the C-terminal domain of PtsHN10M was important for binding to C. difficile cells. Interaction of PtsHN10M was also observed to the low-molecular weight subunit of surface-layer protein A (SlpA), located at the outermost surface of C. difficile cells. Altogether, our study highlights the function of PtsHN10M as an RBP and potentially paves the way toward phage engineering and phage therapy against C. difficile infection.

Insights into the Mechanism of Pre-mRNA Splicing of Tiny Introns from the Genome of a Giant Ciliate Stentor coeruleus.

Stentor coeruleus is a ciliate known for its regenerative ability. Recent genome sequencing reveals that its spliceosomal introns are exceptionally small. We wondered whether the multimegadalton spliceosome has any unique characteristics for removal of the tiny introns. First, we analyzed intron features and identified spliceosomal RNA/protein components. We found that all snRNAs are present, whereas many proteins are conserved but slightly reduced in size. Some regulators, such as Serine/Arginine-rich proteins, are noticeably undetected. Interestingly, while most parts of spliceosomal proteins, including Prp8's positively charged catalytic cavity, are conserved, regions of branching factors projecting to the active site are not. We conjecture that steric-clash avoidance between spliceosomal proteins and a sharply looped lariat might occur, and splicing regulation may differ from other species.

Potential Role of the Host-Derived Cell-Wall Binding Domain of Endolysin CD16/50L as a Molecular Anchor in Preservation of Uninfected Clostridioides difficile for New Rounds of Phage Infection.

Endolysin is a phage-encoded cell-wall hydrolase which degrades the peptidoglycan layer of the bacterial cell wall. The enzyme is often expressed at the late stage of the phage lytic cycle and is required for progeny escape. Endolysins of bacteriophage that infect Gram-positive bacteria often comprises two domains: a peptidoglycan hydrolase and a cell-wall binding domain (CBD). Although the catalytic domain of endolysin is relatively well-studied, the precise role of CBD is ambiguous and remains controversial. Here, we focus on the function of endolysin CBD from a recently isolated Clostridioides difficile phage. We found that the CBD is not required for lytic activity, which is strongly prevented by the surface layer of C. difficile. Intriguingly, hidden Markov model analysis suggested that the endolysin CBD is likely derived from the CWB2 motif of C. difficile cell-wall proteins but possesses a higher binding affinity to bacterial cell-wall polysaccharides. Moreover, the CBD forms a homodimer, formation of which is necessary for interaction with the surface saccharides. Importantly, endolysin diffusion and sequential cytolytic assays showed that CBD of endolysin is required for the enzyme to be anchored to post-lytic cell-wall remnants, suggesting its physiological roles in limiting diffusion of the enzyme, preserving neighboring host cells, and thereby enabling the phage progeny to initiate new rounds of infection. Taken together, this study provides an insight into regulation of endolysin through CBD and can potentially be applied for endolysin treatment against C. difficile infection. IMPORTANCE Endolysin is a peptidoglycan hydrolase encoded in a phage genome. The enzyme is attractive due to its potential use as antibacterial treatment. To utilize endolysin for the therapeutic propose, understanding of the fundamental role of endolysin becomes important. Here, we investigate the function of cell-wall binding domain (CBD) of an endolysin from a C. difficile phage. The domain is homologous to a cell-wall associating module of bacterial cell-wall proteins, likely acquired during phage-host coevolution. The interaction of CBD to bacterial cell walls reduces enzyme diffusion and thereby limits cell lysis of the neighboring bacteria. Our findings indicate that the endolysin is trapped to the cell-wall residuals through CBD and might serve as an advantage for phage replication. Thus, employing a CBD-less endolysin might be a feasible strategy for using endolysin for the treatment of C. difficile infection.

Frontiers in antibiotic alternatives for Clostridioides difficile infection.

Clostridioides difficile (C. difficile) is a gram-positive, anaerobic spore-forming bacterium and a major cause of antibiotic-associated diarrhea. Humans are naturally resistant to C. difficile infection (CDI) owing to the protection provided by healthy gut microbiota. When the gut microbiota is disturbed, C. difficile can colonize, produce toxins, and manifest clinical symptoms, ranging from asymptomatic diarrhea and colitis to death. Despite the steady-if not rising-prevalence of CDI, it will certainly become more problematic in a world of antibiotic overuse and the post-antibiotic era. C. difficile is naturally resistant to most of the currently used antibiotics as it uses multiple resistance mechanisms. Therefore, current CDI treatment regimens are extremely limited to only a few antibiotics, which include vancomycin, fidaxomicin, and metronidazole. Therefore, one of the main challenges experienced by the scientific community is the development of alternative approaches to control and treat CDI. In this Frontier article, we collectively summarize recent advances in alternative treatment approaches for CDI. Over the past few years, several studies have reported on natural product-derived compounds, drug repurposing, high-throughput library screening, phage therapy, and fecal microbiota transplantation. We also include an update on vaccine development, pre- and pro-biotics for CDI, and toxin antidote approaches. These measures tackle CDI at every stage of disease pathology via multiple mechanisms. We also discuss the gaps and concerns in these developments. The next epidemic of CDI is not a matter of if but a matter of when. Therefore, being well-equipped with a collection of alternative therapeutics is necessary and should be prioritized.

Repurposing a platelet aggregation inhibitor ticagrelor as an antimicrobial against Clostridioides difficile.

Drug resistance in Clostridioides difficile becomes a public health concern worldwide, especially as the hypervirulent strains show decreased susceptibility to the first-line antibiotics for C. difficile treatment. Therefore, the simultaneous discovery and development of new compounds to fight this pathogen are urgently needed. In order to determinate new drugs active against C. difficile, we identified ticagrelor, utilized for the prevention of thrombotic events, as exhibiting potent growth-inhibitory activity against C. difficile. Whole-cell growth inhibition assays were performed and compared to vancomycin and metronidazole, followed by determining time-kill kinetics against C. difficile. Activities against biofilm formation and spore germination were also evaluated. Leakage analyses and electron microscopy were applied to confirm the disruption of membrane structure. Finally, ticagrelor's ability to synergize with vancomycin and metronidazole was determined using checkerboard assays. Our data showed that ticagrelor exerted activity with a MIC range of 20-40 µg/mL against C. difficile. This compound also exhibited an inhibitory effect on biofilm formation and spore germination. Additionally, ticagrelor did not interact with vancomycin nor metronidazole. Our findings revealed for the first time that ticagrelor could be further developed as a new antimicrobial agent for fighting against C. difficile.

Characterization of Bacteriophages Infecting Clinical Isolates of Clostridium difficile.

Clostridium difficile is recognized as a problematic pathogen, causing severe enteric diseases including antibiotic-associated diarrhea and pseudomembranous colitis. The emergence of antibiotic resistant C. difficile has driven a search for alternative anti-infection modalities. A promising strategy for controlling bacterial infection includes the use of bacteriophages and their gene products. Currently, knowledge of phages active against C. difficile is still relatively limited by the fact that the isolation of phages for this organism is a technically demanding method since bacterial host themselves are difficult to culture. To isolate and characterize phages specific to C. difficile, a genotoxic agent, mitomycin C, was used to induce temperate phages from 12 clinical isolates of C. difficile. Five temperate phages consisting of ΦHR24, ΦHN10, ΦHN16-1, ΦHN16-2, and ΦHN50 were successfully induced and isolated. Spotting assays were performed against a panel of 92 C. difficile isolates to screen for susceptible bacterial hosts. The results revealed that all the C. difficile phages obtained in this work displayed a relatively narrow host range of 0-6.5% of the tested isolates. Electron microscopic characterization revealed that all isolated phages contained an icosahedral head connected to a long contractile tail, suggesting that they belonged to the Myoviridae family. Restriction enzyme analysis indicated that these phages possess unique double-stranded DNA genome. Further electron microscopic characterization revealed that the ΦHN10 absorbed to the bacterial surface via attachment to cell wall, potentially interacting with S-layer protein. Bacteriophages isolated from this study could lead to development of novel therapeutic agents and detection strategies for C. difficile.

The Developmental Process of the Growing Motile Ciliary Tip Region.

Eukaryotic motile cilia/flagella play vital roles in various physiological processes in mammals and some protists. Defects in cilia formation underlie multiple human disorders, known as ciliopathies. The detailed processes of cilia growth and development are still far from clear despite extensive studies. In this study, we characterized the process of cilium formation (ciliogenesis) by investigating the newly developed motile cilia of deciliated protists using complementary techniques in electron microscopy and image analysis. Our results demonstrated that the distal tip region of motile cilia exhibit progressive morphological changes as cilia develop. This developmental process is time-dependent and continues after growing cilia reach their full lengths. The structural analysis of growing ciliary tips revealed that B-tubules of axonemal microtubule doublets terminate far away from the tip end, which is led by the flagellar tip complex (FTC), demonstrating that the FTC might not directly mediate the fast turnover of intraflagellar transport (IFT).