Role of TlyA in the Biology of Uncultivable Mycobacteria

TlyA proteins are related to distinct functions in a diverse spectrum of bacterial pathogens including mycobacterial spp. There are several annotated proteins function as hemolysin or pore forming molecules that play an important role in the virulence of pathogenic organisms. Many studies reported the dual activity of mycobacterial TlyA as ‘hemolysin’ and ‘S-adenosylmethionine dependent rRNA methylase’. To act as a hemolysin, a sequence must have a signal sequence and transmembrane segment which helps the protein to enter the extracellular environment. Interestingly, the mycobacterial tlyA has neither a traditional signal sequences of general/sec/tat pathways nor any transmembrane segments are present. Still it can reach the extracellular milieu with the help of non-classical signal mechanisms. Also, retention of tlyA in cultivable mycobacterial pathogens (such as Mycobacterium tuberculosis and M. marinum) as well as uncultivated mycobacterial pathogens despite their extreme reductive evolution (such as M. leprae, M. lepromatosis and M. uberis) suggests its crucial role in evolutionary biology of pathogenic mycobacteria. Numerous virulence factors have been characterised from the uncultivable mycobacteria but the information of TlyA protein is still limited in terms of molecular and structural characterisation. The genomic insights offered by comparative analysis of TlyA sequences and its conserved domains reveal its pore forming activity which further confirms its role as a virulence protein, particularly in uncultivable mycobacteria. Therefore, this review presents a comparative analysis of mycobacterial TlyA family by sequence homology and alignment to improve our understanding of this unconventional hemolysin and RNA methyltransferase TlyA of uncultivable mycobacteria.

USP10 regulates B cell response to SARS-CoV-2 or HIV-1 nanoparticle vaccines through deubiquitinating AID

Activation-induced cytidine deaminase (AID) initiates class-switch recombination and somatic hypermutation (SHM) in antibody genes. Protein expression and activity are tightly controlled by various mechanisms. However, it remains unknown whether a signal from the extracellular environment directly affects the AID activity in the nucleus where it works. Here, we demonstrated that a deubiquitinase USP10, which specifically stabilizes nuclear AID protein, can translocate into the nucleus after AKT-mediated phosphorylation at its T674 within the NLS domain. Interestingly, the signals from BCR and TLR1/2 synergistically promoted this phosphorylation. The deficiency of USP10 in B cells significantly decreased AID protein levels, subsequently reducing neutralizing antibody production after immunization with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or human immunodeficiency virus type 1 (HIV-1) nanoparticle vaccines. Collectively, we demonstrated that USP10 functions as an integrator for both BCR and TLR signals and directly regulates nuclear AID activity. Its manipulation could be used for the development of vaccines and adjuvants.

LAPped in Proof: LC3‐Associated Phagocytosis and the Arms Race Against Bacterial Pathogens

Cells of the innate immune system continuously patrol the extracellular environment for potential microbial threats that are to be neutralized by phagocytosis and delivery to lysosomes. In addition, phagocytes employ autophagy as an innate immune mechanism against pathogens that succeed to escape the phagolysosomal pathway and invade the cytosol. In recent years, LC3-associated phagocytosis (LAP) has emerged as an intermediate between phagocytosis and autophagy. During LAP, phagocytes target extracellular microbes while using parts of the autophagic machinery to label the cargo-containing phagosomes for lysosomal degradation. LAP contributes greatly to host immunity against a multitude of bacterial pathogens. In the pursuit of survival, bacteria have developed elaborate strategies to disarm or circumvent the LAP process. In this review, we will outline the nature of the LAP mechanism and discuss recent insights into its interplay with bacterial pathogens.

Histone demethylase UTX regulates glioblastoma progression through affecting periostin expression

The histone H3K27 demethylase UTX participates in regulating multiple cancer types. However, less is known about the UTX function in glioblastoma (GBM). This study aims to define the effect of UTX on GBM. GEPIA2 database analysis showed that UTX expression was significantly increased in GBM and inversely correlated with survival. Knockdown UTX inhibited GBM cell proliferation, migration, and invasion while promoting apoptosis. Moreover, knockdown UTX also hampered tumor growth in the heterotopic xenograft model. RNA-seq combined with qRT-PCR and ChIP-qPCR were used to identify the target genes. The results showed that the UTX-mediated genes were strongly associated with tumor progression and the extracellular environment. Protein-protein interaction analysis suggested that periostin (POSTN) interacted with most of the other UTX-mediated genes. POSTN supplement abolished the effect of UTX knockdown in GBM cells. Furthermore, silencing UTX exhibited similar antitumor effect in patient-derived glioblastoma stem-like cells, while UTX functions were partially restored after exposing POSTN. Our findings may reveal a new insight into the onset of gliomagenesis and progression, providing a promising therapeutic strategy for GBM treatment.

The Multifaceted Role of Connexins in Tumor Microenvironment Initiation and Maintaining

The modern paradigm of studying the processes of carcinogenesis and vital activity of tumor tissues implies increased attention to constituents of tumor microenvironment (TME) and their interactions. These interactions between the cells in TME can be mediated via protein junctions of different types. Connexins (Cnxs) are one of the major contributors to intercellular communication. They form gap junctions responsible for the transfer of ions, metabolites, peptides, miRNA, etc. between neighboring tumor cells as well as between tumor and stromal cells. Cnx hemichannels mediate purinergic signaling and bidirectional molecular transport with the extracellular environment. Additionally, Cnxs were reported to localize in tumor-derived exosomes and facilitate the release of their cargo. A large body of evidence implies that the role of connexins in cancer is multifaceted. Pro- or anti-tumorigenic properties of connexins are determined by their abundance, localization and functionality as well as channel assembly and non-channel functions. In this review we have summarized the data on the Cnxs contribution in TME and to the cancer initiation and progression.

Secretion Systems in Gram-Negative Bacterial Fish Pathogens

Bacterial fish pathogens are one of the key challenges in the aquaculture industry, one of the fast-growing industries worldwide. These pathogens rely on arsenal of virulence factors such as toxins, adhesins, effectors and enzymes to promote colonization and infection. Translocation of virulence factors across the membrane to either the extracellular environment or directly into the host cells is performed by single or multiple dedicated secretion systems. These secretion systems are often key to the infection process. They can range from simple single-protein systems to complex injection needles made from dozens of subunits. Here, we review the different types of secretion systems in Gram-negative bacterial fish pathogens and describe their putative roles in pathogenicity. We find that the available information is fragmented and often descriptive, and hope that our overview will help researchers to more systematically learn from the similarities and differences between the virulence factors and secretion systems of the fish-pathogenic species described here.

The role of cell-matrix interactions in connective tissue mechanics

Living tissue is able to withstand large stresses in everyday life, yet it also actively adapts to dynamic loads. This remarkable mechanical behaviour emerges from the interplay between living cells and their non-living extracellular environment. Here we review recent insights into the biophysical mechanisms involved in the reciprocal interplay between cells and the extracellular matrix and how this interplay determines tissue mechanics, with a focus on connective tissues. We first describe the roles of the main macromolecular components of the extracellular matrix in regards to tissue mechanics. We then proceed to highlight the main routes via which cells sense and respond to their biochemical and mechanical extracellular environment. Next we introduce the three main routes via which cells can modify their extracellular environment: exertion of contractile forces, secretion and deposition of matrix components, and matrix degradation. Finally we discuss how recent insights in the mechanobiology of cell-matrix interactions are furthering our understanding of the pathophysiology of connective tissue diseases and cancer, and facilitating the design of novel strategies for tissue engineering.

Investigating the Mechanism Behind the Release of Microcystins in Freshwater Cyanobacteria

<p>The frequency and distribution of toxic cyanobacterial blooms are increasing globally, creating the need for a better understanding of the processes involved in toxic secondary metabolite production. Microcystins (MCs) are potent hepatotoxins produced by a wide range of bloom-forming cyanobacteria genera such as Microcystis and Planktothrix. Although the release of MCs to the extracellular environment has long been considered a by-product of cell lysis and death, several studies suggest the presence of a mechanism that actively transports these toxins outside the cell membrane. The aim of the present study was to find evidence for a link between cell lysis and concentrations of extracellular MCs. A dual-fluorescence cell viability assay using the nucleic acid stain SYTOX Green was optimised for use on Microcystis and Planktothrix. A SYTOX Green concentration of 1 µM, and an incubation time of 30 minutes, yielded a bright and even fluorescent signal that readily identified lysed cells. The improved staining technique, in conjunction with liquid chromatography-mass spectrometry analyses, was employed in a culturing experiment to track the transfer of MCs to the extracellular environment in relation to the amount of cell lysis. For Microcystis, there was a strong and significant positive relationship between cell lysis and the concentration of extracellular MC. When the extracellular MC was predicted according to cell lysis levels and the MC content per cell, lysed cells were a major contributor of MCs to the extracellular environment, although the model overestimated the concentrations. Relationships for Planktothrix were significant but weaker, possibly due to reduced accuracy in the cell enumeration step, which would have altered the calculated MC content per cell. Whilst these findings support the hypothesis that cell lysis is the main contributor of extracellular MCs, the results do not exclude a role of MCs as signalling molecules. The recent finding that programmed cell death may occur in Microcystis under various environmental conditions may explain the commonly observed increase in extracellular MCs. Understanding the mechanisms involved in the transfer of MCs to the extracellular environment will provide further clarification on the function of these secondary metabolites and lead to the improvement of water quality management strategies.</p>

Investigating the Mechanism Behind the Release of Microcystins in Freshwater Cyanobacteria

<p>The frequency and distribution of toxic cyanobacterial blooms are increasing globally, creating the need for a better understanding of the processes involved in toxic secondary metabolite production. Microcystins (MCs) are potent hepatotoxins produced by a wide range of bloom-forming cyanobacteria genera such as Microcystis and Planktothrix. Although the release of MCs to the extracellular environment has long been considered a by-product of cell lysis and death, several studies suggest the presence of a mechanism that actively transports these toxins outside the cell membrane. The aim of the present study was to find evidence for a link between cell lysis and concentrations of extracellular MCs. A dual-fluorescence cell viability assay using the nucleic acid stain SYTOX Green was optimised for use on Microcystis and Planktothrix. A SYTOX Green concentration of 1 µM, and an incubation time of 30 minutes, yielded a bright and even fluorescent signal that readily identified lysed cells. The improved staining technique, in conjunction with liquid chromatography-mass spectrometry analyses, was employed in a culturing experiment to track the transfer of MCs to the extracellular environment in relation to the amount of cell lysis. For Microcystis, there was a strong and significant positive relationship between cell lysis and the concentration of extracellular MC. When the extracellular MC was predicted according to cell lysis levels and the MC content per cell, lysed cells were a major contributor of MCs to the extracellular environment, although the model overestimated the concentrations. Relationships for Planktothrix were significant but weaker, possibly due to reduced accuracy in the cell enumeration step, which would have altered the calculated MC content per cell. Whilst these findings support the hypothesis that cell lysis is the main contributor of extracellular MCs, the results do not exclude a role of MCs as signalling molecules. The recent finding that programmed cell death may occur in Microcystis under various environmental conditions may explain the commonly observed increase in extracellular MCs. Understanding the mechanisms involved in the transfer of MCs to the extracellular environment will provide further clarification on the function of these secondary metabolites and lead to the improvement of water quality management strategies.</p>

Chemical, molecular, and single cell analysis reveal chondroitin sulfate proteoglycan aberrancy in fibrolamellar carcinoma

Fibrolamellar carcinoma (FLC) is an aggressive liver cancer with no effective therapeutic options. The extracellular environment of FLC tumors is poorly characterized and may contribute to cancer growth and/or metastasis. To bridge this knowledge gap, we assessed pathways relevant to proteoglycans, a major component of the extracellular matrix. We first analyzed gene expression data from FLC and non-malignant liver tissue to identify changes in glycosaminoglycan (GAG) biosynthesis pathways. We then implemented a novel LC-MS/MS based method to quantify the abundance of different types of GAGs in patient tumors, followed by measurement of the levels of different GAG-associated proteins. Finally, we performed the first single-cell assay for transposon-accessible chromatin-sequencing on FLC tumors, to identify which cell types are linked to the most dominant GAG-associated protein in FLC. Our results reveal a pathologic aberrancy in chondroitin (but not heparan) sulfate proteoglycans in FLC and highlight a potential role for activated stellate cells.