Multiple Regulatory Mechanisms Control the Production of CmrRST, an Atypical Signal Transduction System in Clostridioides difficile

Garrett, Elizabeth M.; Mehra, Anchal; Sekulović, Ognjen; Tamayo, Rita

doi:10.1128/mbio.02969-21

Cited by 12 publications

(15 citation statements)

References 47 publications

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“…In this case, heterogeneity is controlled by a specific DNA recombination event mediated by RecV (38) and the Rho factor (39). In addition, C. difficile colony morphology is also subjected to phenotypic heterogeneity resulting in changes in bacterial physiology and pathogenesis and this occurs through phase variation of the CmrRST signal transduction system expression (40,41).…”

Section: Discussionmentioning

confidence: 99%

The cell wall lipoprotein CD1687 acts as a DNA binding protein during deoxycholate-induced biofilm formation inClostridioides difficile

Auria¹,

Hunault

England

et al. 2022

Preprint

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The ability of bacterial pathogens to establish recurrent and persistent infections is frequently associated with their ability to form biofilms. Clostridioides difficile infections have a high rate of recurrence and relapses and it is hypothesised that biofilms are involved in its pathogenicity and persistence. Biofilm formation by C. difficile is still poorly understood. It has been shown that specific molecules such as deoxycholate (DCA) or metronidazole induce biofilm formation, but the mechanisms involved remain elusive. In this study, we describe the role of the C. difficile lipoprotein CD1687 during DCA-induced biofilm formation. We showed that the expression of CD1687, which is part of an operon within the CD1685-CD1689 gene cluster, is controlled by multiple transcription starting sites and some are induced in response to DCA. Only CD1687 is required for biofilm formation and the overexpression of CD1687 is sufficient to induce biofilm formation. Using RNAseq analysis, we showed that CD1687 affects the expression of transporters and metabolic pathways and we identified several potential binding partners by pull-down assay, including transport-associated extracellular proteins. We then demonstrated that CD1687 is surface exposed in C. difficile, and that this localization is required for DCA-induced biofilm formation. Given this localization and the fact that C. difficile forms eDNA-rich biofilms, we confirmed that CD1687 binds DNA in a non-specific manner. We thus hypothesize that CD1687 is a component of the downstream response to DCA leading to biofilm formation by promoting interaction between the cells and the biofilm matrix by binding eDNA.

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Section: Discussionmentioning

confidence: 99%

The cell wall lipoprotein CD1687 acts as a DNA binding protein during deoxycholate-induced biofilm formation inClostridioides difficile

Auria¹,

Hunault

England

et al. 2022

Preprint

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“…Sekulovic et al identified the nucleotide within the inverted repeats where recombination occurs for each switch [10]. Deleting this nucleotide and a nucleotide on each side was recently demonstrated to prevent site-specific recombination and inversion of the flg and cmr switches [16,39]. We used this strategy to create strains with the equivalent 3-nucleotide deletion in the right inverted repeat (RIR) of the pdcB switch, one in which the switch is in the reference genome orientation, and the other with the switch in the inverse orientation (Fig 2C).…”

Section: Pdcb Switch Orientation Modulates Pdcb Expressionmentioning

confidence: 99%

“…FN545816) and are listed in S2 Table . To create the plasmids with pdcB (Cdi2) and pdcC (Cdi3) switch fusions to phoZ, the switch regions were PCR amplified from C. difficile R20291 genomic DNA. The PCR products were cloned into pMC123::phoZ (pRT1343) [16] and transformed into DH5α. Transformants were selected on LB-Cm agar and desired clones were identified by PCR and confirmed by sequencing.…”

Section: Dna Manipulation and Strain Constructionmentioning

confidence: 99%

“…Accordingly, the production of the glucosylating toxins required for virulence is indirectly subject to phase variation via the flagellar (flg) switch [2,12]. Phase variation via the cwpV and flg switches occurs through RNA-mediated mechanisms post-transcription initiation, while a promoter within the cmr switch mediates phase variation of CmrRST [1,2,13,16].…”

Section: Introductionmentioning

confidence: 99%

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Coordinated modulation of multiple processes through phase variation of a c-di-GMP phosphodiesterase in Clostridioides difficile

et al. 2022

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The opportunistic nosocomial pathogen Clostridioides difficile exhibits phenotypic heterogeneity through phase variation, a stochastic, reversible process that modulates expression. In C. difficile, multiple sequences in the genome undergo inversion through site-specific recombination. Two such loci lie upstream of pdcB and pdcC, which encode phosphodiesterases (PDEs) that degrade the signaling molecule c-di-GMP. Numerous phenotypes are influenced by c-di-GMP in C. difficile including cell and colony morphology, motility, colonization, and virulence. In this study, we aimed to assess whether PdcB phase varies, identify the mechanism of regulation, and determine the effects on intracellular c-di-GMP levels and regulated phenotypes. We found that expression of pdcB is heterogeneous and the orientation of the invertible sequence, or ‘pdcB switch’, determines expression. The pdcB switch contains a promoter that when properly oriented promotes pdcB expression. Expression is augmented by an additional promoter upstream of the pdcB switch. Mutation of nucleotides at the site of recombination resulted in phase-locked strains with significant differences in pdcB expression. Characterization of these mutants showed that the pdcB locked-ON mutant has reduced intracellular c-di-GMP compared to the locked-OFF mutant, consistent with increased and decreased PdcB activity, respectively. These alterations in c-di-GMP had concomitant effects on multiple known c-di-GMP regulated processes, indicating that phase variation of PdcB allows C. difficile to coordinately diversify multiple phenotypes in the population to enhance survival.

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“…The genes encoding the CmrST TCS, are phase variable and consists of two RRs and a single HK. CmrRST regulates a variety of cell functions, including colony morphology and motility (Garrett et al, 2019, 2022). The agrACDB locus, homologous to the agr locus in S. aureus , modulates virulence by increasing production of flagella and TcdA (Ahmed et al, 2020; Choudhary et al, 2018; Martin et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

HexSDF are Required for Synthesis of a Novel Glycolipid that Mediates Daptomycin and Bacitracin Resistance inC. difficile

Pannullo

Guan

Goldfine

et al. 2022

Preprint

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Clostridioides difficileis a Gram-positive opportunistic pathogen that results in 250,000 infections, 12,000 deaths, and $1 billion in medical costs in the US each year. There has been recent interest in using a daptomycin analog, Surotomycin, to treatC. difficileinfections. Daptomycin interacts with both phosphatidylglycerol and Lipid II to disrupt the membrane and halt peptidoglycan synthesis.C. difficilehas an unusual lipid membrane composition as it has no phosphatidylserine or phosphatidylethanolamine, and ∼50% of its membrane is composed of glycolipids, including the uniqueC. difficilelipid aminohexosyl-hexosyldiradylglycerol (HNHDRG). We identified a two-component system (TCS) HexRK that is required forC. difficileresistance to daptomycin. Using RNAseq we found that HexRK regulates a three gene operon of unknown functionhexSDF. Based on bioinformatic predictions,hexSencodes a monogalactosyldiacylglycerol synthase,hexDencodes a polysaccharide deacetylase, andhexFencodes an MprF-like flippase. We find that deletion ofhexRKleads to a 4-fold decrease in daptomycin MIC, and that deletion ofhexSDFleads to an 8-16-fold decrease in daptomycin MIC. The ΔhexSDFmutant is also 4-fold less resistant to bacitracin but no other cell wall active antibiotics. Our data indicate that in the absence of HexSDF the phospholipid membrane composition is altered. In WTC. difficilethe unique glycolipid, HNHDRG makes up ∼17% of the lipids in the membrane. However, in a ΔhexSDFmutant, HNHDRG is completely absent. While it is unclear how HNHDRG contributes daptomycin resistance, the requirement for bacitracin resistance suggests it has a general role in cell membrane biogenesis.ImportanceClostridioides difficileis a major cause of hospital acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. Little is understood aboutC. difficilemembrane lipids, but a unique glycolipid, HNHDRG, has been previously identified inC. difficileand, currently, has not been identified in other organisms. Here we show that HexSDF and HexRK are required for synthesis of HNHDRG, and that production of HNHDRG impacts resistance to daptomycin and bacitracin.

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Multiple Regulatory Mechanisms Control the Production of CmrRST, an Atypical Signal Transduction System in Clostridioides difficile

Cited by 12 publications

References 47 publications

The cell wall lipoprotein CD1687 acts as a DNA binding protein during deoxycholate-induced biofilm formation inClostridioides difficile

The cell wall lipoprotein CD1687 acts as a DNA binding protein during deoxycholate-induced biofilm formation inClostridioides difficile

Coordinated modulation of multiple processes through phase variation of a c-di-GMP phosphodiesterase in Clostridioides difficile

HexSDF are Required for Synthesis of a Novel Glycolipid that Mediates Daptomycin and Bacitracin Resistance inC. difficile

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