Chromosomal localization of PemIK toxin-antitoxin system results in the loss of toxicity - Characterization of pemIKSa1-Sp from Staphylococcus pseudintermedius.

Toxin-antitoxin (TA) systems are ubiquitous in bacteria and on numerous occasions have been postulated to play a role in virulence of pathogens. Some Staphylococcus aureus strains carry a plasmid, which encodes the highly toxic PemIKSa TA system involved in maintenance of the plasmid but also implicated in modulation of gene expression. Here we showed that pemIKSa1-Sp TA system, homologous to the plasmid-encoded PemIKSa, is present in virtually each chromosome of S. pseudintermedius strain, however exhibits sequence heterogeneity. This results in two length variants of the PemKSa1-Sp toxin. The shorter (96 aa), C-terminally truncated toxin is enzymatically inactive, whereas the full length (112 aa) variant is an RNase, though nontoxic to the host cells. The lack of toxicity of the active PemKSa-Sp2 toxin is explained by increased substrate specificity. The pemISa1-Sp antitoxin gene seems pseudogenized, however, the whole pemIKSa1-Sp system is transcriptionally active. When production of N-terminally truncated antitoxins using alternative start codons is assumed, there are five possible length variants. Here we showed that even substantially truncated antitoxins are able to interact with PemKSa-Sp2 toxin and inhibit its RNase activity. Moreover, the antitoxins can rescue bacterial cells from toxic effects of overexpression of plasmid-encoded PemKSa toxin. Collectively, our data indicates that, contrary to the toxic plasmid-encoded PemIKSa TA system, location of pemIKSa1-Sp in the chromosome of S. pseudintermedius results in the loss of its toxicity. Interestingly, the retained RNase activity of PemKSa1-Sp2 toxin and functionality of the putative, N-terminally truncated antitoxins suggest the existence of evolutionary pressure for alleviation/mitigation of the toxin's toxicity and retention of the inhibitory activity of the antitoxin, respectively.

Prevalence of Antibiotic and Heavy Metal Resistance Determinants and Virulence-Related Genetic Elements in Plasmids of Staphylococcus aureus.

The use of antibiotics on a mass scale, particularly in farming, and their release into the environment has led to a rapid emergence of resistant bacteria. Once emerged, resistance determinants are spread by horizontal gene transfer among strains of the same as well as disparate bacterial species. Their accumulation in free-living as well as livestock and community-associated strains results in the widespread multiple-drug resistance among clinically relevant species posing an increasingly pressing problem in healthcare. One of these clinically relevant species is Staphylococcus aureus, a common cause of hospital and community outbreaks. Among the rich diversity of mobile genetic elements regularly occurring in S. aureus such as phages, pathogenicity islands, and staphylococcal cassette chromosomes, plasmids are the major mean for dissemination of resistance determinants and virulence factors. Unfortunately, a vast number of whole-genome sequencing projects does not aim for complete sequence determination, which results in a disproportionately low number of known complete plasmid sequences. To address this problem we determined complete plasmid sequences derived from 18 poultry S. aureus strains and analyzed the prevalence of antibiotic and heavy metal resistance determinants, genes of virulence factors, as well as genetic elements relevant for their maintenance. Some of the plasmids have been reported before and are being found in clinical isolates of strains typical for humans or human ones of livestock origin. This shows that livestock-associated staphylococci are a significant reservoir of resistance determinants and virulence factors. Nevertheless, nearly half of the plasmids were unknown to date. In this group we found a potentially mobilizable plasmid pPA3 being a unique example of accumulation of resistance determinants and virulence factors likely stabilized by a presence of a toxin-antitoxin system.

Do CTRC mutations affect the development of alcoholic chronic pancreatitis and its course among Poles: Preliminary study.

BACKGROUND: Genetic mutations are one of the etiological factors that predispose people to develop chronic pancreatitis. OBJECTIVES: The aim of our study was to examine the effect of p.Trp55*, p.Arg254Trp and c.738_761del mutations in the chemotrypsin gene (CTRC) on the development of alcoholic chronic pancreatitis (ACP) in order to answer the questions whether these mutations vary between gender groups, whether they were related to the age when ACP was first diagnosed, and whether they affected the morphological changes in the pancreas and the course of ACP. MATERIAL AND METHODS: The study included 124 patients with ACP, 52 with nonalcoholic pancreatitis and 52 controls. The p.Trp55*, c.738_761del and p.Arg254Trp mutations in the CTRC gene were tested by the polymerase chain reaction (PCR). RESULTS: The c.738_761del and p.Arg254Trp mutations occurred in 3.07% and 1.31% of cases, respectively. None of the examined patients were found to have the p.Trp55* mutation. The frequency of detected mutations did not significantly differ between the study groups. The c.738_761del mutation was detected more frequently in women than in men. No significant differences were found in the age at ACP onset, morphological changes affecting the pancreas, or in the course of ACP between the patients with and without the 2 examined mutations. The c.738_761del mutation was significantly more frequent in the diabetic patients than in the non-diabetics. The patients with this mutation more frequently required surgery than those without the c.738_761del mutation. CONCLUSIONS: No relationship between the c.738_761del and p.Arg254Trp mutations and the development of APC was found. The c.738_761del mutation was more frequent in females than in males. Neither mutation affected the patient's age at ACP onset or its course. In contrast to p.Arg254Trp, the c.738_761del mutation correlated with diabetes development and the need for surgery in the course of ACP.

Joint Genomic and Proteomic Analysis Identifies Meta-Trait Characteristics of Virulent and Non-virulent Staphylococcus aureus Strains.

Staphylococcus aureus is an opportunistic pathogen of humans and warm-blooded animals and presents a growing threat in terms of multi-drug resistance. Despite numerous studies, the basis of staphylococcal virulence and switching between commensal and pathogenic phenotypes is not fully understood. Using genomics, we show here that S. aureus strains exhibiting virulent (VIR) and non-virulent (NVIR) phenotypes in a chicken embryo infection model genetically fall into two separate groups, with the VIR group being much more cohesive than the NVIR group. Significantly, the genes encoding known staphylococcal virulence factors, such as clumping factors, are either found in different allelic variants in the genomes of NVIR strains (compared to VIR strains) or are inactive pseudogenes. Moreover, the pyruvate carboxylase and gamma-aminobutyrate permease genes, which were previously linked with virulence, are pseudogenized in NVIR strain ch22. Further, we use comprehensive proteomics tools to characterize strains that show opposing phenotypes in a chicken embryo virulence model. VIR strain CH21 had an elevated level of diapolycopene oxygenase involved in staphyloxanthin production (protection against free radicals) and expressed a higher level of immunoglobulin-binding protein Sbi on its surface compared to NVIR strain ch22. Furthermore, joint genomic and proteomic approaches linked the elevated production of superoxide dismutase and DNA-binding protein by NVIR strain ch22 with gene duplications.

Identification of novel mazEF/pemIK family toxin-antitoxin loci and their distribution in the Staphylococcus genus.

The versatile roles of toxin-antitoxin (TA) systems in bacterial physiology and pathogenesis have been investigated for more than three decades. Diverse TA loci in Bacteria and Archaea have been identified in genome-wide studies. The advent of massive parallel sequencing has substantially expanded the number of known bacterial genomic sequences over the last 5 years. In staphylococci, this has translated into an impressive increase from a few tens to a several thousands of available genomes, which has allowed us for the re-evalution of prior conclusions. In this study, we analysed the distribution of mazEF/pemIK family TA system operons in available staphylococcal genomes and their prevalence in mobile genetic elements. 10 novel m azEF/pemIK homologues were identified, each with a corresponding toxin that plays a potentially different and undetermined physiological role. A detailed characterisation of these TA systems would be exceptionally useful. Of particular interest are those associated with an SCCmec mobile genetic element (responsible for multidrug resistance transmission) or representing the joint horizontal transfer of TA systems and determinants of vancomycin resistance from enterococci. The involvement of TA systems in maintaining mobile genetic elements and the associations between novel mazEF/pemIK loci and those which carry drug resistance genes highlight their potential medical importance.