Role of PumB antitoxin as a transcriptional regulator of the PumAB type-II toxin-antitoxin system and its endoribonuclease activity on the PumA (toxin) transcript.

The PumAB type-II toxin-antitoxin (TA) system is encoded by pumAB genes that are organized into an operon. This system is encoded by the pUM505 plasmid, isolated from a Pseudomonas aeruginosa clinical strain. The pumA gene encodes a putative RelE toxin protein (toxic component), whereas the pumB gene encodes a putative HTH antitoxin protein. The expression of the PumAB system in Escherichia coli confers plasmid stability. In addition, PumA toxin overexpression in P. aeruginosa possesses the capability to increase bacterial virulence, an effect that is neutralized by the PumB antitoxin. The aim of this study was to establish the mechanism of regulation of the PumAB toxin-antitoxin system from pUM505. By an in silico analysis of the putative regulatory elements, we identified two putative internal promoters, PpumB and PpumB-AlgU (in addition to the already reported PpumAB), located upstream of pumB. By RT-qPCR assays, we determined that the pumAB genes are transcribed differentially, in that the mRNA of pumB is more abundant than the pumA transcript. We also observed that pumB could be expressed individually and that its mRNA levels decreased under oxidative stress, during individual expression as well as co-expression of pumAB. However, under stressful conditions, the pumA mRNA levels were not affected. This suggests the negative regulation of pumB by stressful conditions. The PumB purified protein was found to bind to a DNA region located between the PpumAB and the pumA coding region, and PumA participates in PumB binding, suggesting that a PumA-PumB complex co-regulates the transcription of the pumAB operon. Interestingly, the pumA mRNA levels decreased after incubation in vitro with PumB protein. This effect was repressed by ribonuclease inhibitors, suggesting that PumB could function as an RNAse toward the mRNA of the toxin. Taken together, we conclude that the PumAB TA system possesses multiple mechanisms to regulate its expression, as well as that the PumB antitoxin generates a decrease in the mRNA toxin levels, suggesting an RNase function. Our analysis provides new insights into the understanding of the control of TA systems from mobile plasmid-encoded genes from a human pathogen.

Rhamnolipid-Based Liposomes as Promising Nano-Carriers for Enhancing the Antibacterial Activity of Peptides Derived from Bacterial Toxin-Antitoxin Systems.

BACKGROUND: Antimicrobial resistance poses substantial risks to human health. Thus, there is an urgent need for novel antimicrobial agents, including alternative compounds, such as peptides derived from bacterial toxin-antitoxin (TA) systems. ParELC3 is a synthetic peptide derived from the ParE toxin reported to be a good inhibitor of bacterial topoisomerases and is therefore a potential antibacterial agent. However, ParELC3 is inactive against bacteria due to its inability to cross the bacterial membranes. To circumvent this limitation we prepared and used rhamnolipid-based liposomes to carry and facilitate the passage of ParELC3 through the bacterial membrane to reach its intracellular target - the topoisomerases. METHODS AND RESULTS: Small unilamellar liposome vesicles were prepared by sonication from three formulations that included 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol. ParELC3 was loaded with high efficiency into the liposomes. Characterization by DLS and TEM revealed the appropriate size, zeta potential, polydispersity index, and morphology. In vitro microbiological experiments showed that ParELC3 loaded-liposomes are more efficient (29 to 11 µmol·L-1) compared to the free peptide (>100 µmol·L-1) at inhibiting the growth of standard E. coli and S. aureus strains. RL liposomes showed high hemolytic activity but when prepared with POPC and Chol this activity had a significant reduction. Independently of the formulation, the vesicles had no detectable cytotoxicity to HepG2 cells, even at the highest concentrations tested (1.3 mmol·L-1 and 50 µmol·L-1 for rhamnolipid and ParELC3, respectively). CONCLUSION: The present findings suggest the potential use of rhamnolipid-based liposomes as nanocarrier systems to enhance the bioactivity of peptides.

Análise do sistema toxina-antitoxina HipA/B na indução da persistência bacteriana de cepas de Escherichia coli extraintestinal / The importance of HipA/B Toxin-Antitoxin system in extraintestinal Escherichia Coli persistence

Os sistemas toxina-antitoxinas (TA) compreendem um conjunto de genes que são amplamente difundidos em procariotos. No cromossomo, os sistemas podem estar envolvidos na indução de morte celular em resposta a condições estressantes, indução de persistência, formação de biofilme, colonização de novos nichos, manutenção da mobilidade bacteriana e virulência de bactérias patogênicas. Em E. coli K12, 36 sistemas TA foram descritos, dos quais o do tipo II é o mais abundante e estudado. Dentre as oito toxinas pesquisadas nesse trabalho, o gene da toxina HipA está presente em 76 das 100 cepas de ExPEC estudadas. Apesar da abundância de hipA em ExPEC e em diversos genomas bacterianos, a participação dos sistemas hipA/B na indução da persistência ainda não é clara. Portanto, o sistema hipA/B de duas cepas ExPEC isoladas de infecção sanguínea foi deletado, e estas foram avaliadas quando a indução da persistência bacteriana na presença de antibióticos, formação de biofilme, resistência ao soro e sobrevivência em macrófagos. O sistema TA hipA/B não influenciou no fenótipo de resistência ao soro humano e na sobrevivência intracelular em macrófagos, no entanto, participou da indução da persistência por ciprofloxacino em um isolado (EC182); e da formação de biofilme em superfície de vidro do isolado (EC273)

Toxin-antitoxin (TA) systems comprise a set of genes that are widespread in prokaryotes. On the chromosome, the systems may be involved in the induction of cell death in response to stressful conditions, persistence induction, biofilm formation, colonization of new niches, maintenance of bacterial mobility and virulence. In E. coli K12, 36 TA systems have been described, of which type II is the most abundant. Among the eight toxins searched in this work, hipA is present in 76 bacteria of the 100 ExPEC strains studied. Despite the abundance of hipA in ExPEC and in several bacterial genomes, the participation of hipA/B modules in the persistence is still unclear. Therefore, hipA/B system of two ExPEC strains isolated from blood infection was deleted and consequently evaluated in bacterial persistence induced by antibiotics, serum resistance and macrophage survival. Despite the fact that, the TA hipA/B system did not influence the phenotype of resistance to human serum and intracellular survival in macrophages. Herein, we described that hipA/B was important for persistence induction in one isolate (EC182); and may participate in the biofilm formation on the glass surface in the other studied strain (EC273)

Evaluation of functionality of type II toxin-antitoxin systems of Clostridioides difficile R20291.

Clostridioides difficile is a nosocomial, Gram-positive, strictly anaerobic, spore-forming pathogen capable of colonizing and proliferating in the human intestine. In bacteria, it has been shown that the Toxin-Antitoxin systems mediate the cellular response to external stress by initiating processes such as biofilm formation and programmed cell death. This work aims to evaluate the functionality of four type II TA modules of Clostridioides difficile R20291. We performed bioinformatic analysis to search for putative TA systems using the TADB platform. Then we performed a heterologous expression assay to evaluate the functionality of these systems. Our results showed that the MazEF and RelBE systems were functional, suggesting that their corresponding toxins possess an endoribonuclease activity. In conclusion, MazEF and RelBE systems of C. difficile R20291 are functional in a heterologous expression system.

Virulence Conferred by PumA Toxin from the Plasmid-Encoded PumAB Toxin-Antitoxin System is Regulated by Quorum System.

Toxin-antitoxin (TA) systems are small genetic elements composed of a toxin gene and its cognate antitoxin that are important for plasmid stabilization (plasmid-encoded) and bacterial virulence (chromosome-encoded). These systems are also related to biofilm and persister cell formations. Pseudomonas aeruginosa is an antibiotic-resistant human pathogen that produces virulence factors modulated by quorum sensing (QS) and can form biofilms. The type II PumAB TA system of pUM505, isolated from a clinical strain of P. aeruginosa, confers plasmid stability. Additionally, the PumA toxin increases P. aeruginosa virulence and is neutralized by the PumB antitoxin. In this study, we determined whether virulence conferred by PumA toxin is regulated by QS. The pumA gene was transferred to P. aeruginosa lasI/rhlI, a mutant strain in the LasI and RhlI QS systems, to analyze the effect on virulence of the transformants. pumA transfer did not increase bacterial virulence in lettuce and Caenorhabditis elegans, suggesting that the virulence conferred by PumA requires QS modulation. pumA mRNA levels drastically decreased in the P. aeruginosa lasI/rhlI (pUC_pumA) strain, suggesting positive regulation of pumA gene expression by QS. Supplementation of the growth medium of P. aeruginosa lasI/rhlI (pUC_pumA) with C4-AHL and 3-oxo-C12-AHL autoinducers increased pumA mRNA levels and restored bacterial virulence, suggesting that both autoinducers complemented the mutations and positively regulated the toxic effects of PumA. This strengthened the hypothesis that QS regulates bacterial virulence conferred by the PumA toxin. Thus, this report establishes an important function of QS in the virulence conferred by plasmid-encoded TA systems in bacterial pathogens.

Large-Scale Comparison of Toxin and Antitoxins in Listeria monocytogenes.

Toxin-antitoxin systems (TASs) are widely distributed in prokaryotes and encode pairs of genes involved in many bacterial biological processes and mechanisms, including pathogenesis. The TASs have not been extensively studied in Listeria monocytogenes (Lm), a pathogenic bacterium of the Firmicutes phylum causing infections in animals and humans. Using our recently published TASmania database, we focused on the known and new putative TASs in 352 Listeria monocytogenes genomes and identified the putative core gene TASs (cgTASs) with the Pasteur BIGSdb-Lm database and, by complementarity, the putative accessory gene TAS (acTASs). We combined the cgTASs with those of an additional 227 L. monocytogenes isolates from our previous studies containing metadata information. We discovered that the differences in 14 cgTAS alleles are sufficient to separate the four main lineages of Listeria monocytogenes. Analyzing these differences in more details, we uncovered potentially co-evolving residues in some pairs of proteins in cgTASs, probably essential for protein-protein interactions within the TAS complex.

Toxin-antitoxin systems shows variability among Mycobacterium tuberculosis lineages.

The toxin-antitoxin (TA) systems are operons involved in the formation of persistent cells and in stress situations in microorganism. TA systems are widely distributed in Mycobacterium tuberculosis (MTB). The objective of this study was to determine the distribution and variability of protein sequences of TA systems in seven MTB lineages. Protein prediction on 73 genomes of different lineage was made using Prodigal, and profile hidden Markov models (PHMMs) of 225 reference TA proteins were constructed with HMMER. An homology search was made comparing the predicted proteins to PHMMs. Consecutive proteins that matched PHMMs (forming an operon) were selected. Thereafter, clustering of orthologous genes was made for further mutation scanning through multiple alignments. All proteins found belong to TA types II and IV, and 45 proteins were found completely conserved. Six uncharacterized antitoxins homologous to VapB11, VapB5, VapB45, VapB13, ParD1 and RelB were found. Multiple alignments revealed differences among lineages with specific mutations suitable for genotyping. Significant changes in amino acid sequences caused by frameshift mutations were found in 46 TA proteins.

Plasmid pUM505 encodes a Toxin-Antitoxin system conferring plasmid stability and increased Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin-Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET-pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans, indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence.

Allodemic distribution of plasmids co-harbouring blaCTX-M-15/aac(6')-Ib-cr/qnrB in Klebsiella pneumoniae is the main source of extended-spectrum ß-lactamases in Uruguay's paediatric hospital.

OBJECTIVES: This study aimed to describe the characteristics of clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing enterobacteria (EPE) in Uruguay's paediatric hospital. METHODS: ESBLs, qnr alleles and aac(6')-Ib-cr were sought and characterised in EPE isolated between March 2010 and March 2012. Transfer of resistance determinants was assessed by conjugation. Incompatibility (Inc) groups, plasmid toxin-antitoxin systems (TAS) and plasmid size were determined in transconjugants. Clonality was analysed by pulsed-field gel electrophoresis. Multilocus sequence typing was done for ESBL-producing Klebsiella pneumoniae. RESULTS: A total of 77 EPE isolates were characterised, comprising 43% K. pneumoniae, 19.5% Serratia marcescens, 19.5% Escherichia coli, 17% Enterobacter cloacae and 1% Klebsiella oxytoca. ESBLs belonged mainly to the blaCTX-M family (69.6%) [blaCTX-M-15 (45%) and blaCTX-M-2 (31%)]. The aac(6')-Ib-cr/qnrB duplex was the most frequently detected plasmid-mediated quinolone resistance mechanism; this association was detected in K. pneumoniae harbouring blaCTX-M-15. Transconjugants were obtained for 71% of the EPE. Amongst transconjugants, certain combinations were found between ESBLs and Inc group, e.g. IncA/C-blaCTX-M-2, IncHI1/HI2-blaCTX-M-9 and IncHI1/HI2-blaSHV-12. In addition, the combination ccdAB-blaCTX-M-15 was also found. K. pneumoniae isolates harbouring blaCTX-M-15/aac(6')-Ib-cr/qnrB showed allodemic behaviour, with a predominance of ST14, ST45 and ST48. CONCLUSIONS: In this study, epidemiological changes in ESBL distribution could be explained by the spread of K. pneumoniae harbouring blaCTX-M-15/aac(6')-Ib-cr/qnrB, encoded mainly on conjugative plasmids featuring ccdAB TAS. Since reports of TAS in K. pneumoniae plasmids are scarce, new strategies are needed to combat intrinsic selection pressure exerted by the association, in conjugative plasmids, of resistance mechanisms with TAS.