979SVL03 b 21 copy - moodle2.units.it

MOBILOME, II

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Comparison of two explanations for unexpected phylogenetic distribution

The presence of a gene with characteristics that are typical for an unrelated group can be due to horizontal gene transfer (HGT, arrow)

An alternative explanation is an ancient gene duplication (*) followed by differential gene loss (x). The more sister lineages have only the typical gene, the more independent gene-loss events must be postulated under this scenario

Lamarck introduced tree-like binary schemes for taxonomic classification1 and Charles Darwin described the evolution of species as the tree of life

Darwin also noted that the 'coral of life' might be a more appropriate metaphor, because only the outermost layer in the tree of life is actually alive, resting on a base of dead branches

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Mobilome importance, II• Understanding evolution driven by HGT—> positive/negative/neutral

✴ Only genes involved in key informational and metabolic pathways are subject to strong selection

• Insight into microbial behavior and its dynamic change due to mobilome

• MGEs can confer profoundly different phenotypes on different strains of the same species

• Human activities and behaviors provide selective pressures that shape mobile gene pools, and that acquisition of mobile genes is important for colonizing specific human populations (gut microbiome)

• Focus on human health: microbial virulence and microbial defense

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• Hyperparasitic defense in prokaryotes is presented by the phage-inducible chromosomal islands (PICIs)

• Phage-derived satellite elements integrated within host chromosome but reactivated and excised on phage infection

• PICIs do not encode any virion components but abrogate phage reproduction and spread by hijacking the virions —> modulating packaging specificity machinery and inducing the formation of small capsids —> enough space for the encapsidation of PICI DNA but not of phage genome

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• Addiction complexes come with genes coding for a toxin and an antitoxin

• Antitoxin < stable toxin, and is broken down at a faster rate

• As the addiction genes are on the plasmid, if plasmid is lost through segregation —> toxin will remain in cell (as it breaks down at a slower rate), but without antitoxin —> toxin kills the cell

• The evolution of microbial genomes is generally interpreted in terms of the interplay between three factors:

(i) gene gain, via horizontal gene transfer (HGT) and gene duplication; (ii) gene loss, via deletion; (iii) natural selection that affects gene fixation and maintenance

• The intrinsic bias toward DNA deletion (and hence gene loss) that characterizes mutational processes in prokaryotes results in nonadaptive genome reduction, whereas selection contributes to maintaining slightly beneficial genes

Disentangling the effects of selection and loss bias on gene dynamics

Iranzo et al., 2017

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C, energy production and conversion; D, cell division; E, amino acid metabolism and transport; F, nucleotide metabolism and transport; G, carbohydrate metabolism and transport; H, coenzyme metabolism; I, lipid metabolism; J, translation; K, transcription; L, replication and repair; M, membrane and cell wall structure and biogenesis; N, secretion and motility; O, posttranslational modification, protein turnover, and chaperone functions; P, inorganic ion transport and metabolism; Q, biosynthesis, transport, and catabolism of secondary metabolites; R, general functional prediction only (typically, prediction of biochemical activity); S, function unknown; T, signal transduction; U, intracellular trafficking and secretion; V, defense mechanisms; Tr, transposon; Pl, conjugative plasmid; and Ph, prophage or phage-related

8Iranzo et al., 2017

Effective loss bias and mean abundances of gene families from different functional categories

Free-living (FL)facultative host-associated (FHA)obligate intracellular parasitic (OP) microbes

Among the genetic elements that are typically considered parasitic, prophages show the highest fitness cost, followed by conjugative plasmids and transposons, which are only weakly deleterious in the long termGenes involved in antiparasite defense do not seem to provide long-term benefits on average but rather are slightly deleterious, almost to the same extent as transposons

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Positive and negative contribution to the microbial fitness

Strong genomic degeneration

Stronger selection for gene retention

Shuttling of components between MGEs and cellular organisms

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The functions of the defense systems and their components in cells and mobile genetic elements (MGEs)

a| A generic depiction of a class 1 CRISPR–Cas locus. The block arrows show cas genes (cas1–cas8) and the yellow rectangles show the repeat array. b | Restriction–modification systems. Note that restriction–modification systems and their components operate in bothprokaryotes and eukaryotes as well as their viruses. c | Toxin–antitoxin (TA) systems and their role in cellular organisms and MGEs. d| The locus encoding the contractile tail of bacteriophage P2, with genes encoding tail proteins, identified by roman letters, shown in red and other genes shown in grey. The tails of bacteriophages were recruited to produce various structures involvedin intercellular conflicts, including tailocins and type VI secretion systems(T6SS). e| Capture of apoptotic factors by large viruses of eukaryotes. BCL-2, B cell lymphoma 2; FLIP, FLICE-like inhibitory protein; MTase, methyltransferase; PCD, programmed cell death; PICI, phage-inducible chromosomalisland; REase, restriction endonuclease; SOD, superoxide dismutase; TNFR,tumour necrosis factor receptor; (v), (viral).

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a | A generic depiction of a class 1 CRISPR–Cas locus. The block arrows show cas genes (cas1–cas8) and the yellow rectangles show the repeat array.

b | Restriction–modification systems. Note that restriction–modification systems and their components operate in both prokaryotes and eukaryotes as well as their viruses.

MTase, methyltransferase; PICI, phage-inducible chromosomal island; REase, restriction endonucleaseKoo

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c | Toxin–antitoxin (TA) systems and their role in cellular organisms and MGEs. d | The locus encoding the contractile tail of bacteriophage P2, with genes encoding tail proteins, identified by roman letters, shown in red and other genes shown in grey. The tails of bacteriophages were recruited to produce various structures involved in intercellular conflicts, including tailocins and type VI secretion systems (T6SS). e | Capture of apoptotic factors by large viruses of eukaryotes. BCL-2, B cell lymphoma 2; FLIP, FLICE-like inhibitory protein; PCD, programmed cell death; SOD, superoxide dismutase; TNFR, tumour necrosis factor receptor; (v), (viral)

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Targeted and Whole Metagenomic Technologies for Extracting MGEs

Carr et al., 202013


Carr et al., 202014

Targeted metagenomic methods currently include purifying MGEs prior to shotgun sequencing

Free phage particles, along with other virus-like particles (VLPs), are purified in several stages of physical and/or enzymatic treatments

Nucleic acids extracted from VLPs are then sequenced and assembled into contiguous sequences for further annotation

According to sample type the protocol is not reproducible in terms of efficiency, feacal sample

Size-fractionation is an alternative technique involving enrichment of extracted DNA for novel viral particles by filtering the samples through a size-exclusion membrane that has been applied to the cow rumen virome


Carr et al., 202015

Circular plasmids are isolated using high-throughput transposon-aided capture (TRACA) from metagenomic DNA, which are then typically transformed into Escherichia coli for cloning, followed by shotgun sequencing and PCR-based approaches to close gaps in sequences.

For plasmids, TRACA enriches metagenomic DNA for circular plasmids by using a DNase that selectively removes linear DNA

Plasmids are subsequently ‘captured’ by inserting a transposon (in an in vitro transposition reaction) with an origin of replication and selection marker before transforming them into (typically) E. coli for cloning

This is followed by shotgun sequencing, with additional PCR to close gaps in sequences

However, TRACA has a bias towards capturing smaller plasmids (between 3 and 10 kb), excludes linearised plasmids, and potentially inactivates plasmid genes as a result of transposon insertion


Carr et al., 202016

Alternatively, inverse-PCR together with multiple displacement amplification (another DNA amplification technique) has also been applied to identify small circular plasmids in metagenomic samples

Targeted metagenomic approach, using PCR amplification, can be used to identify transposable elements by targeting the repeat regions.

Metagenomic DNA is amplified by PCR primers targeting transposable elements, purified and ligated into plasmid vectors, then transformed into host strains. After clonal expansion, the plasmids are isolated, sequenced, and annotated for transposable elements


Carr et al., 202017

Genome structure reflects bacterial lifestyle

Genome reduction is common in intracellular bacteria (obligate intracellular pathogens, endosymbionts) contributes to evolution of strictly host-dependent bacterial variants — as bacteria rely on host cell to compensate for gene function lost

Gene acquisition by horizontal transfer between different species is common in extracellular bacteria (facultative pathogens, symbionts), which involves mobile genetic elements, such as plasmids, genomic islands (GEIs) and bacteriophages, increases the versatility and adaptability of the recipient — allows bacteria to adapt to a new or changing environment

Point mutations and genetic rearrangements constantly contribute to evolution of new gene variants in all types of bacteria

Dobrindt et al., 2004

Evolution of bacterial variants by acquisition and loss of genetic information

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• Major bacterial enteropathogens, antibiotic resistance reservoirs, and pathogenesis in human gut• Major enteric bacterial species and common reservoirs for proliferation and resistance exchange• A close-up view of the human gut, representing various pathobiont species and pathogenic tendencies. C. diff:

Clostridioides difficile, VRE: Vancomycin-resistant enterococcus, BFG: Bacteroides fragilis group. *Indicates E. coli can assume multiple pathogenic manifestations within the gut

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Mobilome’s connections at the ecosystem level for antibiotic resistance and pathogenesis

Gillings, 2013

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• Parvome: world of small bioactive molecules • Resistome: world of potential resistance determinants• The resistome comprises the genes that potentially encode resistance to antibiotics. • The mobilome comprises the mobile proportion of bacterial genomes. The mobilome and resistome

overlap, since many resistance genes are located on mobile elements. • Both the resistome and mobilome are a subset of the total coding capacity of prokaryotic cells, the

pangenome, which is expressed as the panproteome

Chinese box: connectivity among pangenome, mobilome, resitstome

• Gut microbiome harbors a ‘silent reservoir’ of antibiotic resistance (AR) genes—> contribute to emergence of multidrug-resistant pathogens through horizontal gene transfer (HGT)

• HGT higher in species than in genera

• Antibiotic treatment and inflammation are putative triggers for HGT, through production of reactive oxygen species and DNA damage

• Neutropenic (low neutrophils) patients undergoing hematopoietic stem cell transplantation, who receive multiple courses of antibiotics

Kent et al., 2020 22

HGT occurs frequently over a several-week period in humans

Role of resistance gene pollution in generating novel, complex DNA elements • Pollution of natural environments with antibiotics and disinfectants affects community structure

and leads to increased carriage of resistance genes in environmental organisms • Integrons from humans into prawns into humans —> acquiring new gene cassette• Resistance determinants released from human waste streams may interact with gene cassettes

and mobile DNA elements in aquatic ecosystems to generate new combinations of potential virulence genes in environmental bacteria

Gillings, 2014

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Miniature inverted repeat transposable elements (MITEs) of the order of tens of base pairs (bps) in length

• The principal function of CRISPR–Cas systems in archaea and bacteria is defense against mobile genetic elements (MGEs), including viruses, plasmids and transposons

• Several classes of MGE contributed to the origin and evolution of CRISPR–Cas

• CRISPR–Cas systems and their components were recruited by various MGEs for functions that remain largely uncharacterized

CRISPR- Cas

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2 CRISPR classes and 6 typesM

akarova et al., 2020

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Makarova et al., 2020

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Distribution of the six types of CRISPR–Cas system in the major archaeal and bacterial phyla


The CRISPR–Cas systems are non-uniformly distributed among bacterial and archaeal phyla 28

• CRISPR–cas loss or retention in prokaryotic genomes depends on the trade-off between the fitness cost, which is determined mostly by autoimmunity and the HGT curtailment, and the benefits of defense conferred by adaptive immunity

• Benefits most likely depend on the abundance and diversity of viruses in specific habitats, as well as on the biology of host–parasite interactions in specific groups of microorganisms

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High horizontal mobility of CRISPR–cas loci


Evolution within the continuum

Faure et al., 201930

• CRISPR–Cas systems and their components are encoded by a relatively small minority of MGEs

• Most of the CRISPR loci carried by MGEs are reduced to retain only part of the original functionality

• CRISPR–Cas components promote the propagation of the respective MGE by inhibiting host defences and gaining the upper hand in inter-MGE conflicts, or via other mechanisms, and thus are retained during MGE evolution

• The recruitment of CRISPR–Cas by MGEs neatly fits the ‘guns for hire’ concept under which defense systems are repeatedly recruited for offense activities by MGEs, and vice versa

• Recruitment of CRISPR–Cas system components by MGEs is part of a complex network of functional and evolutionary relationships that includes multiple contributions of different MGEs to the origin of CRISPR–Cas

• One of the key unifying themes is the mechanistic similarity between various nuclease reactions involved in CRISPR–Cas functions and in the life cycles of MGEs, which allows evolutionary shuttling of nucleases

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CRISPR-Cas - MGEs interactions

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Definitions, I

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Definitions, II

• MGEs have developed inhibitors of CRISPR–Cas function called anti-CRISPR (Acr) proteins

• The first acr genes were discovered in phages that inhibit the type I–F CRISPR–Cas system of Pseudomonas aeruginosa

1. Borges, A. L., Davidson, A. R. & Bondy-Denomy, J. The discovery, mechanisms, and evolutionary impact of anti-CRISPRs. Annu Rev. Virol. 4, 37–59 (2018)

2. Bondy-Denomy Pawluk, A., Maxwell, K. L. & Davidson, A. R. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432 (2013)

Multiple anti-CRISPRs, I

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• Acr proteins has revealed a diverse range of inhibitory activities:

• Interference with crRNA loading• Inhibition of target DNA recognition• Inhibition of DNA cleavage

• Low molecular weight

• Acrs lack conserved sequence and structural features —> rendering de novo prediction largely impractical with current methods

• Acr genes tend to cluster within loci that encode more conserved Acr-associated (Aca) proteins

Multiple anti-CRISPRs, II

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Pinilla-Redondo et al., 2020

Phylogenetic diversity of Aca9

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Acr-associated (Aca) gene

• 50 % phage• 38% plasmids

• To uncover how MGEs within the Enterobacteriales order cope with the pressure of CRISPR–Cas immunity

• Bacteria that express acrs can tolerate “self-targeting” spacers that, in the absence of CRISPR–Cas inhibition, would otherwise cause lethal genomic cleavage

• The presence of these self-targeting spacers can therefore be used to identify bacterial genomes that likely encode anti-CRISPR proteins capable of inhibiting their endogenous CRISPR system

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Phylogenetic distribution of Aca5 homologs Pinilla-R

edondo et al., 2020

• Defense systems often colocalize within defense islands in bacterial genomes

• acr loci often cluster with antagonists of other host defense functions (e.g., anti-RM)

• Hypothesis MGEs organize counter defense strategies in “anti-defense islands”

Pinilla-Redondo et al., 2020

Anti-defense cluster region

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Bordenstein & Reznikoff, 200539

MGEs in intracellular bacteria genomes

obligate

facultative

• Categorized by reductive evolution • Genome streamlining• Genome stability • Counterintuitively MGEs• Genes that encode mobile-DNA functions

vary in their abundance and diversity• Transposable-element genes comprise the

largest fraction of mobile DNA per genome• Fractions of MGEs per genome significantly

increase with genome size among intracellular bacteria

• Among obligate intracellular species, the parasitic genera that host-switch (Wolbachia, Coxiella, Phytoplasma, Rickettsia, Chlamydia and Chlamydophila) tend to harbour mobile DNA in their genomes, whereas the stable mutualistic genera (Buchnera, Blochmannia and Wigglesworthia) do not

Escherichia coli typically colonizes the gastrointestinal tract of human infants within a few hours after birth

E. coli and its human host coexist in good health and with mutual benefit for decades

Commensal E. coli strains rarely cause disease except in immunocompromised hosts or where the normal gastrointestinal barriers are breached — as in peritonitis

Niche of commensal E. coli is the mucous layer of the mammalian colon

E. coli is a highly successful competitor at this crowded site

Most abundant facultative anaerobe of the human intestinal microflora

Artist: David S. Goodsell

Several highly adapted E. coli clones that have acquired specific virulence attributes, which confers an increased ability to adapt to new niches and allows them to cause a broad spectrum of disease

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MGEs additions, deletions and other genetic changes can give rise to pathogenic E. coli forms capable of causing:1 diarrhoea (EPEC, EHEC, EAEC DAEC),2 dysentery (EIEC)3 haemolytic uremic syndrome, hus (EHEC),3 urinary tract infections, uti (UPEC)4 meningitis (MNEC)

MGEs contribution to the evolution of pathogenic E. coli, I

Kasper et al., 2004

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E. coli virulence factors can be encoded by several mobile genetic elements:1. Transposons (Tn) (for example, heat

stable enterotoxin (ST) of ETEC),2. Plasmids (for example, heat-labile

enterotoxin (LT) of ETEC and invasion factors of EIEC),

3. Bacteriophage (for example, Shiga toxin of EHEC)

4. Pathogenicity islands (PAIs) — for example, the locus of enterocyte effacement (LEE) of EPEC/EHEC and PAIs I and II of UPEC

5. Commensal E. coli can also undergo deletions resulting in ‘black holes’, point mutations or other DNA rearrangements that can contribute to virulence

6. Plasmid important for resistome

MGEs contribution to the evolution of pathogenic E. coli, II

Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus

Durrant et al., 2019

• Certain species might rely more heavily on MGE movement as a mechanism of adaptation and evolution than others

• Recent work has demonstrated that individuals with very similar gut microbiomes can harbor very different MGE repertoires (Brito et al., 2016)

• Monitoring the MGE potential of individual bacterial species and microbiomes will inform our understanding of the extent of and consequences of MGE-derived genetic variation

• Among all MGEs, we define a transposable element (TE) as an element cluster found at more than three positions in the reference genome in sum across all analyzed isolates 516 elements were classified as TEs (10.3%))

• Differences across species suggest significant variability in MGE diversity and overall activity

MGEs fingerprint for species characterization

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New frontiers in Mobilomes

• MGEs fingerprint for uncultivable species characterization

• New mechanisms of MGEs control and interactions

• Trascriptome and proteome based mobilomes

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979SVL03 b 21 copy - moodle2.units.it