Clostridioides difficile strain-dependent and strain-independent adaptations to a microaerobic environment

Weiss, Amy L.; Lopez, Christopher A.; Beavers, William N.; Rodriguez, J. I.; Skaar, Eric P.

doi:10.1099/mgen.0.000738

Cited by 14 publications

(12 citation statements)

References 65 publications

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“…We mined published C. difficile RNA-sequencing data ( 50 , 51 ) and observed expression from all intron and IS605 elements, though precise mapping to predicted ωRNA regions was confounded by their highly repetitive nature and the sequencing method ( fig. S4 ).…”

Section: Resultsmentioning

confidence: 99%

Antagonistic conflict between transposon-encoded introns and guide RNAs

Žedaveinytė,

Meers,

et al. 2023

Preprint

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TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life, presumably due to the critical roles they play in transposon proliferation. IS605-family TnpB homologs function in bacteria as programmable homing endonucleases by exploiting transposon-encoded guide RNAs to cleave vacant genomic sites, thereby driving transposon maintenance through DSB-stimulated homologous recombination. Whether this pathway is conserved in other genetic contexts, and in association with other transposases, is unknown. Here we uncover molecular mechanisms of transposition and RNA-guided DNA cleavage by IS607-family elements that, remarkably, also encode catalytic, self-splicing group I introns. After reconstituting and systematically investigating each of these biochemical activities for a candidate ‘IStron’ derived fromClostridium botulinum, we discovered sequence and structural features of the transposon-encoded RNA that satisfy molecular requirements of a group I intron and TnpB guide RNA, while still retaining the ability to be faithfully mobilized at the DNA level by the TnpA transposase. Strikingly, intron splicing was strongly repressed not only by TnpB, but also by the secondary structure of ωRNA alone, allowing the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts have evolved a sensitive equilibrium to balance competing and mutually exclusive activities that promote transposon maintenance while limiting adverse fitness costs on the host. Collectively, this work explains how diverse enzymatic activities emerged during the selfish spread of IS607-family elements and highlights molecular innovation in the multi-functional utility of transposon-encoded noncoding RNAs.

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Section: Resultsmentioning

confidence: 99%

Antagonistic conflict between transposon-encoded introns and guide RNAs

Žedaveinytė,

Meers,

et al. 2023

Preprint

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“…Previous work has shown that C. difficile fhuB was upregulated during hamster infection ( 4 ) and growth under microaerophilic conditions (33) suggesting the FhuDBGC transporter may be important during colonization. To determine the impact of the ΔfhuDBGC mutation on colonization, we used the cefoperazone mouse model of C. difficile infection (CDI) (17).…”

Section: Resultsmentioning

confidence: 99%

Clostridioides difficileutilizes siderophores as an iron source and FhuDBGC contributes to ferrichrome uptake

Hastie,

Carmichael,

Werner

et al. 2023

Preprint

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Clostridioides difficileremains a public health threat commonly observed following antibiotic use. Due to the importance of iron in many cell processes, most bacteria, includingC. difficile, have multiple mechanisms of acquiring iron. Previous studies have examined ferrous iron uptake inC. difficile, here we focus on the role of siderophores. In a growth assay, we show thatC. difficilecan use a variety of siderophores as the sole iron source. InC. difficile,two ABC transporters induced under low iron conditions are predicted siderophore importers: FhuDBGC and YclNOPQ. We hypothesized that these transporters are responsible for the uptake of the siderophores we tested. To investigate the specificity of these transporters, we purified the substrate binding proteins and examined siderophore binding using thermal shift. We demonstrate increased stability between one siderophore binding protein, FhuD, and the siderophore ferrichrome, suggesting a binding interaction. This specificity correlates with the inability of anΔfhuDBGCmutant to grow efficiently under iron limiting conditions in the presence of ferrichrome. WhileC. difficileused additional siderophores in our growth experiments, we did not observe increased thermal stability between the receptor proteins and any of the other siderophores tested, suggesting these siderophores do not bind these receptors and other siderophore import mechanisms remain to be elucidated. Redundancy in iron acquisition is a microbial survival adaptation to cope with the constant battle for iron within a host. Greater knowledge about howC. difficileacquires iron will provide insight about howC. difficilecolonizes and persists in the colon.IMPORTANCEThis study is the first example ofC. difficilegrowing with siderophores as the sole iron source and describes the characterization of the ferric hydroxamate uptake ABC transporter (FhuDBGC). This transporter shows specificity to the siderophore ferrichrome. While not required for pathogenesis, this transporter highlights the redundancy in iron acquisition mechanisms whichC. difficileuses to compete for iron during an infection.

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“…The WT and complemented strains likely succumb to the effects of superoxide before the stress response genes are activated. However, sodA in C. difficile has been associated only with spore coat formation or maturation ( 39 ) and not with aerobic stress management ( 48 , 49 ). Instead σ B -associated desulfoferrodoxin, reverse rubrerythrins, and NADH-rubredoxin reductases have been described as remedial agents ( 17 ).…”

Section: Discussionmentioning

confidence: 99%