Biology subjects

Oliveira Paiva, A. M.

Publications and source records attributed to Oliveira Paiva, A. M..

3 recordsLinked to original sources

bioRxiv2024

The L,D-transpeptidation pathway is inhibited by antibiotics of the β-lactam class in Clostridioides difficile

The resistance of Clostridioides difficile to the {beta}-lactam antibiotics cephalosporins, which target the peptidoglycan (PG) assembly, is a leading contributor to the development of C. difficile infections. C. difficile has an original PG structure with a predominance of 3[->]3 cross-links generated by L,D-transpeptidases (LDTs). C. difficile forms spores and we show that the spore cortex PG contains exclusively 3[->]3 cross-links. PG and spore cortex of C. difficile cells were largely unaffected by the deletion of the three predicted LDTs, revealing the implication of a new family of LDTS. The D,D-carboxypeptidases producing the essential LDT substrate were inactivated by cephalosporins, resulting in the inhibition of the L,D-transpeptidation pathway. In contrast, the participation of penicillin-binding proteins (PBPs) to PG cross-linking increased in the presence of the antibiotics. Our findings highlight that cephalosporin resistance is not primarily mediated by LDTs and illustrate the plasticity of the PG biosynthesis machinery in C. difficile.

microbiology↗

bioRxiv2021

Practical observations on the use of fluorescent reporter systems in C. difficile

ObjectivesFluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherryOpt and phiLOV2.1, and the self-labelling tags SNAPCd and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. MethodsWe performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. ResultsWe show that the intrinsic fluorescence of C. difficile cells increases during growth, independent from sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherryOpt were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. ConclusionsThe present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAPCd appears the most suitable candidate for live-cell imaging in C. difficile to date. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/448366v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@a19b90org.highwire.dtl.DTLVardef@1ec1db9org.highwire.dtl.DTLVardef@939510org.highwire.dtl.DTLVardef@11f623f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2018

The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile

The maintenance and organization of the chromosome plays an important role in the development and survival of bacteria. Bacterial chromatin proteins are architectural proteins that bind DNA, modulate its conformation and by doing so affect a variety of cellular processes. No bacterial chromatin proteins of C. difficile have been characterized to date.\n\nHere, we investigate aspects of the C. difficile HupA protein, a homologue of the histone-like HU proteins of Escherichia coli. HupA is a 10 kDa protein that is present as a homodimer in vitro and self-interacts in vivo. HupA co-localizes with the nucleoid of C. difficile. It binds to the DNA without a preference for the DNA G+C content. Upon DNA binding, HupA induces a conformational change in the substrate DNA in vitro and leads to compaction of the chromosome in vivo.\n\nThe present study is the first to characterize a bacterial chromatin protein in C. difficile and opens the way to study the role of chromosomal organization in DNA metabolism and on other cellular processes in this organism.

microbiology↗