A Superfamily of T6SS Antibacterial Effectors Displaying L,D-carboxypeptidase Activity Towards Peptidoglycan

SummaryType VI secretion systems (T6SSs) are contractile nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella SPI-6 T6SS antibacterial effector called Tae5STM (type VI amidase effector 5). Tae5STM is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tai5STM). Microscopy analysis revealed that cells expressing the effector stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovered similarities between Tae5STM and the catalytic domain of L,D-transpeptidase. Point mutations on conserved catalytic histidine and cysteine residues abrogated toxicity. Biochemical assays revealed that Tae5STM displays L,D-carboxypeptidase activity, cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and D-alanine4. Phylogenetic analysis showed that Tae5STM homologs constitutes a new superfamily of T6SS-associated amidase effectors distributed among α-, β- and γ-proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition.

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A family of T6SS antibacterial effectors related to L,D-transpeptidases targets the Peptidoglycan

Access Microbiology ◽

10.1099/acmi.ac2020.po0033 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Stephanie Sibinelli de Sousa ◽

Julia Takuno Hespanhol ◽

Bruno Matsuyama ◽

Stephane Mesnage ◽

Gianlucca Nicastro ◽

...

Keyword(s):

Cell Envelope ◽

Point Mutations ◽

Time Lapse ◽

Secretion Systems ◽

Bacterial Antagonism ◽

New Family ◽

Type Vi Secretion ◽

Altered Cell ◽

Bacterial Effectors ◽

Insight Into

Type VI secretion systems (T6SSs) are contractile nanomachines widely used by bacteria to intoxicate competitors. Salmonella Typhimurium encodes a T6SS within the Salmonella pathogenicity island 6 (SPI-6) that is used during competition against species of the gut microbiota. We characterized a new SPI-6 T6SS antibacterial effector named Tlde1 (type VI L,D-transpeptidase effector 1). Tlde1 is toxic in target-cell periplasm and its toxicity is neutralized by co-expression with immunity protein Tldi1 (type VI L,D-transpeptidase immunity 1). Time-lapse microscopy revealed that intoxicated cells display altered cell division and lose cell envelope integrity. Bioinformatics analysis showed that Tlde1 is evolutionarily related to L,D-transpeptidases. Point mutations on conserved histidine121 and cysteine131 residues eliminated toxicity. Co-incubation of purified recombinant Tlde1 and peptidoglycan tetrapeptides showed that Tlde1 displays both L,D-carboxypeptidase activity by cleaving GM-tetrapeptides between meso-diaminopimelic acid3 and D-alanine4, and L,D-transpeptidase exchange activity by replacing D-alanine4 for a non-canonical D-amino acid. Tlde1 constitutes a new family of T6SS effectors widespread in Proteobacteria. This work increases our knowledge about the bacterial effectors used in interbacterial competitions and provides molecular insight into a new mechanism of bacterial antagonism.

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Pathological ATX3 Expression Induces Cell Perturbations in E. coli as Revealed by Biochemical and Biophysical Investigations

International Journal of Molecular Sciences ◽

10.3390/ijms22020943 ◽

2021 ◽

Vol 22 (2) ◽

pp. 943

Author(s):

Diletta Ami ◽

Barbara Sciandrone ◽

Paolo Mereghetti ◽

Jacopo Falvo ◽

Tiziano Catelani ◽

...

Keyword(s):

Cell Envelope ◽

Spinocerebellar Ataxia Type ◽

Spinocerebellar Ataxia Type 3 ◽

Intact Cells ◽

E Coli ◽

Fourier Transform Infrared Microspectroscopy ◽

Electron Microscopy Analysis ◽

Microscopy Analysis ◽

The Fourier Transform ◽

Type 3

Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.

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Genome-wide and high-density CRISPR-Cas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance

10.1101/203224 ◽

2017 ◽

Author(s):

Stephen J. Pettitt ◽

Dragomir B. Krastev ◽

Inger Brandsma ◽

Amy Drean ◽

Feifei Song ◽

...

Keyword(s):

Catalytic Domain ◽

Synthetic Lethality ◽

Parp Inhibitor ◽

Point Mutations ◽

Underlying Mechanism ◽

Parp Inhibitors ◽

High Density ◽

Single Amino Acid ◽

Genome Wide ◽

Amino Acid Mutations

AbstractPARP inhibitors (PARPi) target homologous recombination defective tumour cells via synthetic lethality. Genome-wide and high-density CRISPR-Cas9 “tag, mutate and enrich” mutagenesis screens identified single amino acid mutations in PARP1 that cause profound PARPi-resistance. These included PARP1 mutations outside of the DNA interacting regions of the protein, such as mutations in solvent exposed regions of the catalytic domain and clusters of mutations around points of contact between ZnF, WGR and HD domains. These mutations altered PARP1 trapping, as did a mutation found in a clinical case of PARPi resistance. These genetic studies reinforce the importance of trapped PARP1 as a key cytotoxic DNA lesion and suggest that interactions between non-DNA binding domains of PARP1 influence cytotoxicity. Finally, different mechanisms of PARPi resistance (BRCA1 reversion, PARP1, 53BP1, REV7 mutation) had differing effects on chemotherapy sensitivity, suggesting that the underlying mechanism of PARPi resistance likely influences the success of subsequent therapies.

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The Impact of Acquired Genetic Abnormalities on the Clinical Translation of Human Pluripotent Stem Cells

Cells ◽

10.3390/cells10113246 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3246

Author(s):

Alexander Keller ◽

Claudia Spits

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Chromosomal Abnormalities ◽

Current Knowledge ◽

Point Mutations ◽

Human Pluripotent Stem Cells ◽

Clinical Translation ◽

Copy Number Changes ◽

Clinical Potential ◽

The Impact

Human pluripotent stem cells (hPSC) are known to acquire chromosomal abnormalities, which range from point mutations to large copy number changes, including full chromosome aneuploidy. These aberrations have a wide-ranging influence on the state of cells, in both the undifferentiated and differentiated state. Currently, very little is known on how these abnormalities will impact the clinical translation of hPSC, and particularly their potential to prime cells for oncogenic transformation. A further complication is that many of these abnormalities exist in a mosaic state in culture, which complicates their detection with conventional karyotyping methods. In this review we discuss current knowledge on how these aberrations influence the cell state and how this may impact the future of research and the cells’ clinical potential.

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Molecular Mechanisms of FVII Deficiency: Expression of Mutations Clustered in the IVS7 Donor Splice Site of Factor VII Gene

Blood ◽

10.1182/blood.v92.5.1646 ◽

1998 ◽

Vol 92 (5) ◽

pp. 1646-1651 ◽

Cited By ~ 28

Author(s):

M. Pinotti ◽

R. Toso ◽

R. Redaelli ◽

M. Berrettini ◽

G. Marchetti ◽

...

Keyword(s):

Splice Site ◽

Molecular Mechanisms ◽

Catalytic Domain ◽

Point Mutations ◽

Factor Vii ◽

Donor Splice Site ◽

Donor Splice ◽

Expression Studies ◽

Fvii Deficiency ◽

American Society

Abstract In three Italian patients, two point mutations and a short deletion were found in the intron 7 of factor VII gene, clustered in the donor splice site and located in the first of several repeats. The mutation 9726+5G→A, the most frequent cause of symptomatic factor VII deficiency in Italy, as well as the deletion (9729del4) gave rise in expression studies to abnormally spliced transcripts, which were exclusively produced from the cryptic site in the second repeat. The insertion in the mature mRNA of the first intronic repeat caused (9726+5G→A) a reading frameshift, abolishing most of the factor VII catalytic domain, or produced (9729del4), an altered factor with 11 additional residues, the activity of which was not detectable in the cell medium after mutagenesis and expression studies. Studies of factor VII ectopic mRNA from leukocytes and expression studies indicated that the deleted gene produced 30% of normally spliced transcript. Differently, the 9726+5G→A mutation permitted a very low level (0.2% to 1%) of correct splicing to occur, which could be of great importance to prevent the onset, in the homozygous patients, of most of the life-threatening bleeding symptoms. The 9726+7A→G mutation was found to be a rare and functionally silent polymorphism. These findings, which provide further evidence of the interplay of sequence and position in the 5′ splice site selection, throw light on the heterogeneous molecular bases and clinical phenotypes of FVII deficiency. © 1998 by The American Society of Hematology.

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Nonulosonic acids contribute to the pathogenicity of the oral bacterium Tannerella forsythia

Interface Focus ◽

10.1098/rsfs.2018.0064 ◽

2019 ◽

Vol 9 (2) ◽

pp. 20180064 ◽

Cited By ~ 6

Author(s):

Susanne Bloch ◽

Markus B. Tomek ◽

Valentin Friedrich ◽

Paul Messner ◽

Christina Schäffer

Keyword(s):

Cell Surface ◽

Current Knowledge ◽

Cell Envelope ◽

Treatment Strategies ◽

Periodontal Pathogen ◽

Tannerella Forsythia ◽

Oral Bacterium ◽

Complex Protein ◽

Unique Cell ◽

Systemic Health

Periodontitis is a polymicrobial, biofilm-caused, inflammatory disease affecting the tooth-supporting tissues. It is not only the leading cause of tooth loss worldwide, but can also impact systemic health. The development of effective treatment strategies is hampered by the complicated disease pathogenesis which is best described by a polymicrobial synergy and dysbiosis model. This model classifies the Gram-negative anaerobe Tannerella forsythia as a periodontal pathogen, making it a prime candidate for interference with the disease. Tannerella forsythia employs a protein O -glycosylation system that enables high-density display of nonulosonic acids via the bacterium's two-dimensional crystalline cell surface layer. Nonulosonic acids are sialic acid-like sugars which are well known for their pivotal biological roles. This review summarizes the current knowledge of T. forsythia' s unique cell envelope with a focus on composition, biosynthesis and functional implications of the cell surface O -glycan. We have obtained evidence that glycobiology affects the bacterium's immunogenicity and capability to establish itself in the polymicrobial oral biofilm. Analysis of the genomes of different T. forsythia isolates revealed that complex protein O -glycosylation involving nonulosonic acids is a hallmark of pathogenic T. forsythia strains and, thus, constitutes a valuable target for the design of novel anti-infective strategies to combat periodontitis.

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The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011817 ◽

2020 ◽

Vol 295 (15) ◽

pp. 5110-5123 ◽

Cited By ~ 4

Author(s):

Lin Shen ◽

Albertus Viljoen ◽

Sydney Villaume ◽

Maju Joe ◽

Iman Halloum ◽

...

Keyword(s):

Cell Wall ◽

Structural Changes ◽

Cell Envelope ◽

Wall Structure ◽

Tubercle Bacillus ◽

Bioinformatics Analyses ◽

The Past ◽

Biochemical Assays ◽

Mycobacterial Protein

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.

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Cloning and characterisation of the gene encoding HMG-CoA reductase from Taxus media and its functional identification in yeast

Functional Plant Biology ◽

10.1071/fp03153 ◽

2004 ◽

Vol 31 (1) ◽

pp. 73 ◽

Cited By ~ 22

Author(s):

Zhihua Liao ◽

Qiumin Tan ◽

Yourong Chai ◽

Kaijing Zuo ◽

Min Chen ◽

...

Keyword(s):

Blot Analysis ◽

Catalytic Domain ◽

Bioinformatic Analysis ◽

Full Length ◽

Functional Identification ◽

Reading Frame ◽

Full Length Cdna ◽

Taxus Media ◽

Taxol Biosynthesis ◽

Coa Reductase

In plants, the first committed step in the pathway for biosynthesis of isoprenoids is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34). Here we report for the first time the cloning of a full-length cDNA encoding HMGR (Tm–HMGR) from a taxol-producing gymnosperm, Taxus media Rehder. The full-length cDNA of Tm–HMGR (GenBank accession number: AY277740) was 2307 base pairs (bp), with a 1791-bp open reading frame (ORF) encoding a 596-amino-acid polypeptide. Bioinformatic analysis revealed that Tm–HMGR contained two trans-membrane domains and a catalytic domain, and showed high homology to other plant HMGRs. Phylogenetic analysis indicated that Tm–HMGR was more ancient than other plant HMGRs. The structural modelling showed that Tm–HMGR had the typical spatial structure of HMGRs whose catalytic domains could be folded and divided into three spatial domains, L-domain, N-domain and S-domain. Southern blot analysis revealed that Tm–HMGR belonged to a small HMGR gene family. Northern blot analysis showed that Tm–HMGR was expressed in roots, stems and needles, with higher expression in stems and needles than in roots. Functional complementation of Tm–HMGR in a HMGR-deficient mutant yeast demonstrated that Tm–HMGR mediated the biosynthesis of mevalonate and provided the general precursor for taxol biosynthesis.

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Salmonella in Australia: understanding and controlling infection

Microbiology Australia ◽

10.1071/ma17044 ◽

2017 ◽

Vol 38 (3) ◽

pp. 112

Author(s):

Joshua PM Newson

Keyword(s):

Host Cell ◽

Cell Biology ◽

Protein Translocation ◽

Cell Signalling ◽

Effector Proteins ◽

Host Cells ◽

Secretion Systems ◽

Significant Burden ◽

Systemic Infections ◽

Bacterial Effectors

The bacterium Salmonella causes a spectrum of foodborne diseases ranging from acute gastroenteritis to systemic infections, and represents a significant burden of disease globally. In Australia, Salmonella is frequently associated with outbreaks and is a leading cause of foodborne illness, which results in a significant medical and economic burden. Salmonella infection involves colonisation of the small intestine, where the bacteria invades host cells and establishes an intracellular infection. To survive within host cells, Salmonella employs type-three secretion systems to deliver bacterial effector proteins into the cytoplasm of host cells. These bacterial effectors seek out and modify specific host proteins, disrupting host processes such as cell signalling, intracellular trafficking, and programmed cell death. This strategy of impairing host cells allows Salmonella to establish a replicative niche within the cell, where they can replicate to high numbers before escaping to infect neighbouring cells, or be transmitted to new hosts. While the importance of effector protein translocation to infection is well established, our understanding of many effector proteins remains incomplete. Many Salmonella effectors have unknown function and unknown roles during infection. A greater understanding of how Salmonella manipulates host cells during infection will lead to improved strategies to prevent, control, and eliminate disease. Further, studying effector proteins can be a useful means for exploring host cell biology and elucidating the details of host cell signalling.

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Connectomics at cellular precision

e-Neuroforum ◽

10.1515/s13295-016-0030-6 ◽

2016 ◽

Vol 22 (3) ◽

Author(s):

Moritz Helmstaedter

Keyword(s):

Electron Microscopy ◽

Current Knowledge ◽

Scientific Field ◽

The State ◽

Neuronal Circuits ◽

Dimensional Electron ◽

3 Dimensional ◽

Complete Mapping ◽

Electron Microscopy Analysis ◽

Microscopy Analysis

AbstractThe complete mapping of neuronal circuits in at least parts of brains has received substantial attention recently. Methodological breakthroughs have made the imaging of ever larger tissue blocks realistic using 3-dimensional electron microscopy. Analysis of such data, however, is still limiting the neuroscientific insights obtainable from cellular connectomics data. What is the state of this scientific field, which insights have been obtained, which are in reach? This brief overview summarizes the current knowledge in cellular connectomics.

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