Bacterial proteases participate in the proteolytic elimination of misfolded or aggregated proteins, carried out by members of the AAA+ protein superfamily such as Hsp100/Clp, Lon, and FtsH. It is estimated that the Clp and Lon families perform around 80% of cellular proteolysis in bacteria. These functions are regulated, in part, through the spatial and/or temporal use of adapter proteins, which participate in the recognition and delivery of specific substrate proteins to proteases. The proteolysis plays an important role in maintaining and controlling the quality of the proteins, avoiding the accumulation and aggregation of unfolded or truncated proteins. However, this is not their only function, since they play an important role in the formation of virulent phenotypes and in the response to different types of stress faced when entering the host or that occur in the environment. This review summarizes the structural and functional aspects of the Clp proteases and their role in Gram-positive microorganisms.

Clostridioides difficile is a Gram-positive pathogen known for its toxin production and spore formation. It is primarily responsible for most cases of antibiotic-associated diarrhea. Bacterial persisters are a small subset of the population that exhibits transient tolerance to bactericidal substances, and they are of significant medical concern due to their association with the emergence of antibiotic resistance and difficult-to-treat chronic or recurrent infections. Vancomycin, the predominant antibiotic utilized in the management of C. difficile infection, is extensively applied in the realm of clinical practice. Previous studies have demonstrated a persister-like phenotype with treatments involving this antibiotic. However, the mechanism in C. difficile remains largely unknown, primarily due to the challenge of isolating this small population at any given time. To better characterize C. difficile persister cells, we present a study that enables the enrichment and characterization of persister cells from bacterial cultures in both the exponential and stationary phases. Moreover, we could differentiate between triggered (induced using antibiotics such as vancomycin) and spontaneous (stochastic) persister cells. Additionally, we observed the involvement of toxin-antitoxin systems and Clp proteases in persister cell formation.

Clostridioides difficile is a Gram-positive bacterium recognized for its ability to produce toxins and form spores. It is mainly accountable for the majority of instances of antibiotic-related diarrhea. Background. Bacterial persister represent a minor fraction of the population that shows temporary tolerance to bactericidal agents, and they pose considerable medical issues because of their link to the rise of antibiotic resistance and challenging chronic or recurrent infections. Our previous research has shown a persister-like phenotype associated with treatments that include pefloxacin. Nonetheless, the mechanism is still mostly unclear, mainly because of the difficulty in isolating this small group of cells. Objectives. To enhance the understanding of C. difficile persister cells, we made an enrichment and characterization of these cells from bacterial cultures during the exponential phase under pefloxacin treatment and lysis treatment. Results. We demonstrate the appearance of cells with lower metabolism and DNA damage. Furthermore, we noted the participation of toxin–antitoxin systems and Clp proteases in the generation of persister cells. Conclusions. This work demonstrates the formation of C. difficile persister cells triggered by a lethal concentration of pefloxacin.

Clostridioides difficile is a Gram-positive pathogen known for its toxin production and spore formation. It is primarily responsible for most cases of antibiotic-associated diarrhea. Bacterial persisters are a small subset of the population that exhibits transient tolerance to bactericidal substances, and they are of significant medical concern due to their association with the emergence of antibiotic resistance and difficult-to-treat chronic or recurrent infections. Vancomycin, the predominant antibiotic utilized in the management of C. difficile infection, is extensively applied in the realm of clinical practice. Previous studies have demonstrated a persister-like phenotype with treatments involving this antibiotic. However, the mechanism in C. difficile remains largely unknown, primarily due to the challenge of isolating this small population at any given time. To better characterize C. difficile persister cells, we present a study that enables the enrichment and characterization of persister cells from bacterial cultures in both exponential and stationary phases. Moreover, we could differentiate between triggered (induced by antibiotics, such as vancomycin) and spontaneous (stochastic) persister cells. Additionally, we observed the involvement of toxin-antitoxin systems and Clp proteases in persister cell formation.

Biofilm formation by bacterial pathogens represents a major challenge in aquaculture, since biofilms promote persistence and increase tolerance to antimicrobial treatments. Yersinia ruckeri, the etiological agent of enteric redmouth disease in salmonids, contributes to recurrent infections in aquaculture. However, the molecular mechanisms governing biofilm development in this pathogen remain poorly understood. In this study, we investigated the role of the global transcriptional regulator Fur in the biofilm formation of Y. ruckeri. To this aim, a fur deletion mutant (Δfur) was phenotypically, structurally, and transcriptionally characterized under biofilm-forming conditions. Fur deletion resulted in impaired motility, reduced flagellar synthesis, and a marked decrease in mature biofilm formation. These changes were accompanied by alterations in three-dimensional architecture, cellular organization, and extracellular matrix production, as revealed by scanning electron and confocal microscopy analyses. Furthermore, transcriptomic profiling via RNA-seq demonstrated that the absence of Fur leads to extensive transcriptional reprogramming. Genes associated with the biosynthesis of flagella and oxidative stress response were significantly down-regulated, whereas genes associated with SOS responses, non-ribosomal peptide biosynthesis, and those related to iron acquisition and siderophore systems exhibited significant up-regulation in the absence of Fur. Interestingly, the strong up-regulation of P2-type prophage genes was also observed in the Δfur strain. Collectively, our findings identify Fur as an important contributor to the normal progression toward mature and spatially organized Y. ruckeri biofilms. Likewise, the Fur-dependent pathways and factors identified here provide candidates for future mechanistic studies and may ultimately help identify strategies to interfere with Y. ruckeri persistence in aquaculture environments.