In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

García-García, Tránsito; Douché, Thibaut; Gianetto, Quentin Giai; Poncet, Sandrine; Omrani, Nesrine El; Smits, Wiep Klaas; Cuenot, Elodie; Matondo, Mariette; Martin‐Verstraete, Isabelle

doi:10.1016/j.mcpro.2022.100428

Cited by 7 publications

(12 citation statements)

References 101 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, identification of their phosphopeptides will depend on the sensitivity of the proteomic methods used. As previously suspected for different bacteria (Prust et al, 2021; Garcia-Garcia et al, 2022; Huemer et al, 2022), other non-Hanks-type kinases besides PASTA kinase must be involved to phosphorylate the broad substrate spectrum in S. pyogenes .…”

Section: Discussionmentioning

confidence: 66%

“…The above discussed phosphorylation events within the juxtamembrane region of SP-STK and at nine potentially cell cycle-related proteins occurred exclusively at threonine residues. A strong enrichment of phosphothreonine among peptides phosphorylated under the control of PASTA kinases and dephosphorylated under the control of their cognate phosphatases was recently reported for C. difficile (Garcia-Garcia et al, 2022) and is obvious from published lists of PASTA kinase substrates of various bacteria (Hirschfeld et al, 2020; Ulrych et al, 2021; Henry et al, 2019; Silvestroni et al, 2009; Ianetta et al, 2021; Hu et al, 2021; Prust et al, 2021). Thus, either the substrate specificities of these kinases are generally biased towards threonine or the regulation of cell cycle events by the PASTA kinases predominantly takes place at threonine while other functions of the kinase may target also serine residues.…”

Section: Discussionmentioning

confidence: 81%

“…Phosphorylation of the secretion motor ATPase SecA at T809 also showed comparable phosphorylation dynamics to the pT sites in SP-STK. SecA is a potential target of kinases in Staphylococcus aureus (Prust et al, 2021), S. suis (Hu et al, 2021) and Clostridioides difficile (Garcia-Garcia et al, 2022). Most interesting, in B. subtilis SecA is required for membrane targeting of DivIVA (Halbedel et al, 2014), and in L. monocytogenes an interaction between DivIVA and SecA2 has been described too (Halbedel et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

“…Early pioneering projects using this approach typically identified near one hundred phosphorylation sites in the phosphoproteomes of Bacillus subtilis (Macek et al, 2007), E. coli (Macek et al, 2008), Lactococcus lactis (Soufi et al, 2008) and other bacteria (reviewed by Yagüe et al, 2019). This number increased dramatically to several thousand with technological advances in qualitative and quantitative mass spectrometry, data management and phosphopeptide sample preparation (Lin et al, 2018; Prust et al, 2021; Garcia-Garcia et al, 2022). Despite these advances, there are major gaps in our knowledge of the functions of protein phosphorylation in bacteria.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Dynamic protein phosphorylation inStreptococcus pyogenesduring growth, stationary phase and starvation

Mikkat,

Kreutzer,

Patenge

2023

Preprint

View full text Add to dashboard Cite

BackgroundPhosphorylation of proteins at serine, threonine, and tyrosine residues plays an important role in physiological processes of bacteria such as cell cycle, metabolism, virulence, dormancy, sporulation, and stationary phase functions.Streptococcus pyogenespossesses a single known transmembrane serine/threonine kinase belonging to the class of penicillin-binding protein and serine/threonine-associated (PASTA) kinases. To gain initial insights into the targets and dynamics of protein phosphorylation inS. pyogenesserotype M49 strain 591, we performed a proteomics and phosphoproteomics workflow using cultures from different growth conditions, stationary phase and starvation.ResultsThe quantitative analysis of dynamic phosphorylation, which included a subset of 463 out of 815 identified phosphorylation sites, revealed two main types of phosphorylation events, distinguished by the growth phase in which they predominantly occur and their preference for either threonine or serine. A small group of phosphorylation events occurred almost exclusively at threonine residues of proteins related to the cell cycle and was enhanced in growing cells. Many of these phosphorylation sites are highly conserved targets of PASTA kinases in streptococci. The majority of phosphorylation events occurred during stationary phase or starvation, preferentially at serine residues. These phosphorylations may be important for regulatory processes in stationary phase or for persister cell formation, but their function and the kinases responsible for their formation need to be elucidated in further analyses. Moreover, our data indicate that the vast majority of proteins can be phosphorylated, but identification of their phosphopeptides depends on the sensitivity of the proteomic methods used.ConclusionsPASTA kinase-dependent cell cycle regulation processes found in related bacteria are conserved inS. pyogenes, but account for only a small part of all phosphorylation events. Most phosphorylation events take place during stationary growth phase and starvation. This phenomenon has also been described for some other bacteria and may therefore be a general feature of bacterial protein phosphorylation.

show abstract

Section: Discussionmentioning

confidence: 66%

Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Dynamic protein phosphorylation inStreptococcus pyogenesduring growth, stationary phase and starvation

Mikkat,

Kreutzer,

Patenge

2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Interestingly, CD1595.1 is less expressed in a sigF and a sigG mutant in transcriptome [71] and transcribed from a promoter recognized by s G [46], sharing this control with trxA1B1 during sporulation. These ferredoxins, which are detected in the proteome of the vegetative cells for CD3605.1 [72] and of the spores for CD3605.1 and CD1595.1, are good candidates as main partners of TrxB1.…”

Section: Discussionmentioning

confidence: 99%

The multiplicity of Thioredoxin systems meets the specific needs of Clostridia

Anjou,

Lotoux,

Zhukova

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Oxidative stress is a highly common stress for cells, which targets proteins with oxidation of cysteine residues. The thioredoxin (Trx) system, which is a ubiquitous system for thiol- and protein-repair, is composed of a thioredoxin (TrxA) and a thioredoxin-reductase (TrxB). TrxAs reduce disulfide bonds of oxidized proteins and are then usually recycled by a single pleiotropic NAD(P)H-dependent TrxB (NTR). However, some Clostridia have also ferredoxin-dependent TrxBs. In this work, we first analyzed the composition of Trx systems across Bacteria. Most of bacteria have only one NTR, but organisms in some Phyla including Firmicutes have several TrxBs. In Firmicutes, this multiplicity of TrxBs is observed only in Clostridia. We thus used Clostridioides difficile as a model to investigate the biological relevance of TrxB multiplicity by studying the physiological roles of the Trx systems in this gut pathogen. Three TrxAs and three TrxBs are present in the 630Δerm strain. We showed that two systems were involved in response to infection-related stresses, allowing survival of vegetative cells to exposure to oxygen, inflammation-related molecules and bile salts. A supplementary TrxB copy present in some C. difficile strains also contributes to this stress-response arsenal. One of the conserved stress-response Trx system was also found to be present in the spore via a dual transcriptional control by different sigma factors. This system contributes to spore survival to hypochlorite and ensure proper germination in the presence of oxygen. Finally, we found that the third Trx system was contributing to sporulation. This involvement was likely linked to the recycling of the glycine-reductase, a Stickland pathway enzyme that allows consumption of glycine, a spore co-germinant. Altogether, our results showed that the multiplicity of Trx systems produced under the control of different regulatory signals and networks and the diversity of TrxBs meet specific needs of Clostridia, i.e., adaptation to strong stress exposure, sporulation and Stickland pathways. More broadly, this multiplicity responds to cell compartmentation and differentiation, which can be transposed to other multiple-TrxBs organisms such as Cyanobacteria or eukaryotes.

show abstract

Physiological role and complex regulation of O ₂ -reducing enzymes in the obligate anaerobe Clostridioides difficile

Caulat,

Lotoux,

Martins

et al. 2024

mBio

View full text Add to dashboard Cite

Clostridioides difficile , the major cause of antibiotic-associated diarrhea, is a strict anaerobic, sporulating Firmicutes. However, during its infectious cycle, this anaerobe is exposed to low oxygen (O 2 ) tensions, with a longitudinal decreasing gradient along the gastrointestinal tract and a second lateral gradient with higher O 2 tensions in the vicinity of the cells. A plethora of enzymes involved in oxidative stress detoxication has been identified in C. difficile , including four O 2 -reducing enzymes: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). Here, we investigated the role of the four O 2 -reducing enzymes in the tolerance to increasing physiological O 2 tensions and air. The four enzymes have different, yet overlapping, spectra of activity. revRbr2 is specific to low O 2 tensions (<0.4%), FdpA to low and intermediate O 2 tensions (0.4%–1%), revRbr1 has a wider spectrum of activity (0.1%–4%), and finally FdpF is more specific to tensions > 4% and air. These different O 2 ranges of action partly arise from differences in regulation of expression of the genes encoding those enzymes. Indeed, we showed that revrbr2 is under the dual control of σ A and σ B . We also identified a regulator of the Spx family that plays a role in the induction of fdp and revrbr genes upon O 2 exposure. Finally, fdpF is regulated by Rex, a regulator sensing the NADH/NAD + ratio. Our results demonstrate that the multiplicity of O 2 -reducing enzymes of C. difficile is associated with different roles depending on the environmental conditions, stemming from a complex multi-leveled network of regulation. IMPORTANCE The gastrointestinal tract is a hypoxic environment, with the existence of two gradients of O 2 along the gut, one longitudinal anteroposterior decreasing gradient and one proximodistal increasing from the lumen to the epithelial cells. O 2 is a major source of stress for an obligate anaerobe such as the enteropathogen C. difficile . This bacterium possesses a plethora of enzymes capable of scavenging O 2 and reducing it to H 2 O. In this work, we identified the role of the four O 2 -reducing enzymes in the tolerance to the physiological O 2 tensions faced by C. difficile during its infectious cycle. These four enzymes have different spectra of action and protect the vegetative cells over a large range of O 2 tensions. These differences are associated with a distinct regulation of each gene encoding those enzymes. The complex network of regulation is crucial for C. difficile to adapt to the various O 2 tensions encountered during infection.

show abstract

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

Cited by 7 publications

References 101 publications

Dynamic protein phosphorylation inStreptococcus pyogenesduring growth, stationary phase and starvation

Dynamic protein phosphorylation inStreptococcus pyogenesduring growth, stationary phase and starvation

The multiplicity of Thioredoxin systems meets the specific needs of Clostridia

Physiological role and complex regulation of O ₂ -reducing enzymes in the obligate anaerobe Clostridioides difficile

Contact Info

Product

Resources

About

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

Cited by 7 publications

References 101 publications

Dynamic protein phosphorylation inStreptococcus pyogenesduring growth, stationary phase and starvation

Dynamic protein phosphorylation inStreptococcus pyogenesduring growth, stationary phase and starvation

The multiplicity of Thioredoxin systems meets the specific needs of Clostridia

Physiological role and complex regulation of O 2 -reducing enzymes in the obligate anaerobe Clostridioides difficile

Contact Info

Product

Resources

About

Physiological role and complex regulation of O ₂ -reducing enzymes in the obligate anaerobe Clostridioides difficile