Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the ...complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones.
We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays.
Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration.
These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Identification of small open reading frames (smORFs) encoding small proteins (≤ 100 amino acids; SEPs) is a challenge in the fields of genome annotation and protein discovery. Here, by combining a ...novel bioinformatics tool (RanSEPs) with “‐omics” approaches, we were able to describe 109 bacterial small ORFomes. Predictions were first validated by performing an exhaustive search of SEPs present in Mycoplasma pneumoniae proteome via mass spectrometry, which illustrated the limitations of shotgun approaches. Then, RanSEPs predictions were validated and compared with other tools using proteomic datasets from different bacterial species and SEPs from the literature. We found that up to 16 ± 9% of proteins in an organism could be classified as SEPs. Integration of RanSEPs predictions with transcriptomics data showed that some annotated non‐coding RNAs could in fact encode for SEPs. A functional study of SEPs highlighted an enrichment in the membrane, translation, metabolism, and nucleotide‐binding categories. Additionally, 9.7% of the SEPs included a N‐terminus predicted signal peptide. We envision RanSEPs as a tool to unmask the hidden universe of small bacterial proteins.
Synopsis
RanSEPs is a random forest‐based computational approach capable of predicting small encoded proteins in a species‐specific context. Running this tool in 109 bacterial genomes indicated that up to 16 ± 9.5% of the proteins in a genome could be SEPs.
Integration of transcriptomics and proteomics from 12 bacterial species showed that high‐throughput experimental characterization of small proteins (SEPs) presents multiple limitations and false positive detections.
RanSEPs is a computational approach that assigns coding potential scores to SEP candidates in a species‐specific manner based on sequence features.
After running RanSEPs in 109 bacterial genomes, we determined that between 6 and 25% of the proteins of a bacterial genome could be SEPs.
Function prediction of RanSEPs‐predicted SEPs revealed an enrichment in translation, metabolism and nucleotide‐binding proteins.
RanSEPs is a random forest‐based computational approach capable of predicting small encoded proteins in a species‐specific context. Running this tool in 109 bacterial genomes indicated that up to 16 ± 9.5% of the proteins in a genome could be SEPs.
Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome‐reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat ...biofilm‐associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome‐reduced bacterium that can fight against clinically relevant biofilm‐associated bacterial infections.
Synopsis
A non‐pathogenic strain of Mycoplasma pneumoniae is engineered to express biofilm dispersing agents as well as bactericidal peptides against Staphylococcus aureus. The engineered strain efficiently dissolves S. aureus biofilms in vitro and in vivo.
Mycoplasma pneumoniae offers unique features that might be of interest for a bacterial‐based therapeutic vector.
Here, an attenuated version of this bacterium is generated after studying the role of different pathogenic factors.
Using strong synthetic promoters and native secretion signals, a genetic platform is designed coding for antibiofilm and bactericidal enzymes.
In vitro, ex vivo and in vivo studies confirmed the ability of the engineered M. pneumoniae strain to efficiently dissolve S. aureus biofilms.
A non‐pathogenic strain of Mycoplasma pneumoniae is engineered to express biofilm dispersing agents as well as bactericidal peptides against Staphylococcus aureus. The engineered strain efficiently dissolves S. aureus biofilms in vitro and in vivo.
Protein degradation is a crucial cellular process in all‐living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to ...maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP‐dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA‐seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half‐life measurements also suggest a role for Lon‐modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation.
SYNOPSIS
A minimal protein degradation machinery required for maintaining proteome homeostasis is defined in the genome‐reduced bacterium Mycoplasma pneumoniae. Genetic and high‐throughput analyses identify substrates and pathways regulated by degradation.
All except one of the proteases in M. pneumoniae are essential for cell survival.
Phenotypic characterization, protease substrate analysis, and epistasis studies reveal distinct functions for Lon and FtsH, the only two ATP‐dependent proteases in M. pneumoniae.
Lon degrades misfolded proteins and unassembled subunits of protein complexes, whereas FtsH regulates the quality control of membrane proteins.
A minimal protein degradation machinery required for maintaining proteome homeostasis is defined in the genome‐reduced bacterium M. pneumoniae. Genetic and high‐throughput analyses identify substrates and pathways regulated by degradation.
Abstract
Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of ...tRNAs, mRNAs, RNA half‐lives, proteins, and protein half‐lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium
Mycoplasma pneumoniae
(
Mpn
). We found that, although the initiation rate varied over 160‐fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100‐fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in
Mpn
.
Synopsis
image
Integration of the absolute quantifications of mRNAs, RNA half‐lives, proteins, protein half‐lives, tRNAs, with ribosome densities offers a comprehensive quantitative study of translation efficiency in the genome‐reduced bacterium
Mycoplasma pneumoniae
(
Mpn
).
Integration of the absolute quantifications of mRNAs, proteins, and protein half‐lives with ribosome densities allowed to derive the initiation and elongation rates for 475 genes (67% of all genes) in the genome‐reduced bacterium
Mpn
.
Translation initiation rate varies over 160‐fold among genes, and most of the known factors have little impact on translation efficiency.
Measured tRNA abundances vary over 100‐fold among tRNA isoacceptors, and this variation could not explain local codon elongation rates.
A comprehensive quantitative study of translation efficiency suggests the existence of unidentified mechanisms of translational regulation in
Mpn
.
The C‐terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this ...C‐terminal sequence and levels of protein expression remains unknown. Here, we identified C‐terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C‐terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels.
Synopsis
Large‐scale genomics analyses combined with high‐throughput experimental assays reveal that the C‐terminal amino acid composition has a strong influence on protein expression levels in bacteria.
C‐terminal amino acid biases are ubiquitous across bacterial taxonomy: positively charged residues (lysine, arginine) are enriched at the last position, while hydrophobic amino acids and threonine are depleted.
High‐throughput expression assays using a reporter gene library showed that protein expression varies up to 4‐fold, with C‐terminal positively and negatively charged residues increasing expression, and hydrophobic residues decreasing expression.
Modulation of protein degradation rate due to the identity of the C‐terminal residue could explain ˜ 85% of the variation in protein expression.
These results are relevant for the optimization of heterologous protein sequences, where the choice of C‐terminal residues could lead to increased expression levels.
Large‐scale genomics analyses combined with high‐throughput experimental assays reveal that the C‐terminal amino acid composition has a strong influence on protein expression levels in bacteria.
DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt ...defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems.
Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is active as a swapped domain dimer and is used in bacterial therapy of gut inflammation. IL-10 can be used as treatment of a wide range ...of pulmonary diseases. Here we have developed a non-pathogenic chassis (CV8) of the human lung bacterium Mycoplasma pneumoniae (MPN) to treat lung diseases. We find that IL-10 expression by MPN has a limited impact on the lung inflammatory response in mice. To solve these issues, we rationally designed a single-chain IL-10 (SC-IL10) with or without surface mutations, using our protein design software (ModelX and FoldX). As compared to the IL-10 WT, the designed SC-IL10 molecules increase the effective expression in MPN four-fold, and the activity in mouse and human cell lines between 10 and 60 times, depending on the cell line. The SC-IL10 molecules expressed in the mouse lung by CV8 in vivo have a powerful anti-inflammatory effect on Pseudomonas aeruginosa lung infection. This rational design strategy could be used to other molecules with immunomodulatory properties used in bacterial therapy.
Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome‐reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 ...residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non‐coding RNAs (ncRNAs) non‐overlapping with essential genes is 5% higher than of non‐transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non‐coding regions, thus changing the focus when aiming to define the minimal essential genome.
Synopsis
A genome essentiality analysis in the genome‐reduced bacterium Mycoplasma pneumoniae, reveals that protein essentiality should be considered at the domain level and that small proteins (< 100 aa) and ncRNAs are frequently essential genomic elements.
A genome essentiality analysis is performed using two mini‐transposon mutant libraries of M. pneumoniae.
The results indicate that ORF essentiality should be considered at the protein domain level.
Small ORFs are as essential as conventional ORFs and they can interact with DNA.
Some essential antisense ncRNAs are involved in the regulation of essential ORF expression.
A genome essentiality analysis in the genome‐reduced bacterium Mycoplasma pneumoniae, reveals that protein essentiality should be considered at the domain level and that small proteins (< 100 aa) and ncRNAs are frequently essential genomic elements.
Mycoplasma genomes exhibit an impressively low amount of genes involved in cell division and some species even lack the ftsZ gene, which is found widespread in the microbial world and is considered ...essential for cell division by binary fission. We constructed a Mycoplasma genitalium ftsZ null mutant by gene replacement to investigate the role of this gene and the presence of alternative cell division mechanisms in this minimal bacterium. Our results demonstrate that ftsZ is non-essential for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery. This is an alternative mechanism, completely independent of the FtsZ protein, to perform cell division by binary fission in a microorganism. We also propose that the mycoplasma cytoskeleton, a complex network of proteins involved in many aspects of the biology of these microorganisms, may have taken over the function of many genes involved in cell division, allowing their loss in the regressive evolution of the streamlined mycoplasma genomes.
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