The distinctive roles played by the superoxide dismutases of the extremophile Acinetobacter sp. Ver3.

Acinetobacter sp. Ver3 is a polyextremophilic strain characterized by a high tolerance to radiation and pro-oxidants. The Ver3 genome comprises the sodB and sodC genes encoding an iron (AV3SodB) and a copper/zinc superoxide dismutase (AV3SodC), respectively; however, the specific role(s) of these genes has remained elusive. We show that the expression of sodB remained unaltered in different oxidative stress conditions whereas sodC was up-regulated in the presence of blue light. Besides, we studied the changes in the in vitro activity of each SOD enzyme in response to diverse agents and solved the crystal structure of AV3SodB at 1.34 Å, one of the highest resolutions achieved for a SOD. Cell fractionation studies interestingly revealed that AV3SodB is located in the cytosol whereas AV3SodC is also found in the periplasm. Consistently, a bioinformatic analysis of the genomes of 53 Acinetobacter species pointed out the presence of at least one SOD type in each compartment, suggesting that these enzymes are separately required to cope with oxidative stress. Surprisingly, AV3SodC was found in an active state also in outer membrane vesicles, probably exerting a protective role. Overall, our multidisciplinary approach highlights the relevance of SOD enzymes when Acinetobacter spp. are confronted with oxidizing agents.

Acinetobacter defence mechanisms against biological aggressors and their use as alternative therapeutic applications.

Several Acinetobacter strains are important nosocomial pathogens, with Acinetobacter baumannii being the species of greatest worldwide concern due to its multi-drug resistance and the recent appearance of hyper-virulent strains in the clinical setting. Colonisation of this environment is associated with a multitude of bacterial factors, and the molecular features that promote environmental persistence in abiotic surfaces, including intrinsic desiccation resistance, biofilm formation and motility, have been previously addressed. On the contrary, mechanisms enabling Acinetobacter spp. survival when faced against other biological competitors are starting to be characterised. Among them, secretion systems (SS) of different types, such as the T5bSS (Contact-dependent inhibition systems) and the T6SS, confer adaptive advantages against bacterial aggressors. Regarding mechanisms of defence against bacteriophages, such as toxin-antitoxin, restriction-modification, Crispr-Cas and CBASS, among others, have been identified but remain poorly characterised. In view of this, we aimed to summarise the present knowledge on defence mechanisms that enable niche establishment in members of the Acinetobacter genus. Different proposals are also described for the use of some components of these systems as molecular tools to treat Acinetobacter infections.

Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2.

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55-100) positive samples and 104/105 (99.05%; 95% CI: 94.81-99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18-99.88) positive samples and 9/9 (100%; 95% CI: 66.37-100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45-36.90) and dilutions of heat-inactivated virus (range: 2.5-100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

CRISPR-based platform for carbapenemases and emerging viruses detection using Cas12a (Cpf1) effector nuclease.

CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.

Catalases of the polyextremophylic Andean isolate Acinetobacter sp. Ver 3 confer adaptive response to H2 O2 and UV radiation.

The polyextremophilic strain Acinetobacter sp. Ver3 isolated from high-altitude Andean lakes exhibits elevated tolerance to UV-B radiation and to pro-oxidants, a feature that has been correlated to its unusually high catalase activity. The Ver3 genome sequence analysis revealed the presence of two genes coding for monofunctional catalases: AV3 KatE1 and AV3 KatE2, the latter harboring an N-terminal signal peptide. We show herein that AV3 KatE1 displays one of the highest catalytic activities reported so far and is constitutively expressed at relatively high amounts in the cytosol, acting as the main protecting catalase against H2 O2 and UV-B radiation. The second catalase, AV3 KatE2, is a periplasmic enzyme strongly induced by both peroxide and UV, conferring supplementary protection against pro-oxidants. The N-terminal signal present in AV3 KatE2 was required not only for transport to the periplasm via the twin-arginine translocation pathway, but also for proper folding and subsequent catalytic activity. The analysis of catalase distribution among 114 Acinetobacter complete genomes revealed a great variability in the catalase classes, with A. baumannii clinical isolates exhibiting higher numbers of isoenzymes and the most variable profiles.