Detection of Nucleic Acids of the Fish Pathogen Yersinia ruckeri from Planktonic and Biofilm Samples with a CRISPR/Cas13a-Based Assay.

Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance.

The Small RNA RyhB Homologs from Salmonella Typhimurium Restrain the Intracellular Growth and Modulate the SPI-1 Gene Expression within RAW264.7 Macrophages.

Growing evidence indicates that small noncoding RNAs (sRNAs) play important regulatory roles during bacterial infection. In Salmonella Typhimurium, several sRNAs are strongly up-regulated within macrophages, but little is known about their role during the infection process. Among these sRNAs, the well-characterized paralogs RyhB-1 and RyhB-2 are two regulators of gene expression mainly related with the response to iron availability. To investigate the role of the sRNAs RyhB-1 and RyhB-2 from S. Typhimurium in the infection of RAW264.7 macrophages, we analyzed several phenotypic traits from intracellular mutant strains lacking one and both sRNAs. Deletion of RyhB-1 and/or RyhB-2 resulted in increased intracellular survival and faster replication within macrophages. The bacterial metabolic status inside macrophages was also analyzed, revealing that all the mutant strains exhibited higher intracellular levels of ATP and lower NAD+/NADH ratios than the wild type. Expression analyses from bacteria infecting macrophages showed that RyhB-1 and RyhB-2 affect the intra-macrophage expression of bacterial genes associated with the Salmonella pathogenicity island 1 (SPI-1) and the type III secretion system (T3SS). With a two-plasmid system and compensatory mutations, we confirmed that RyhB-1 and RyhB-2 directly interact with the mRNAs of the invasion chaperone SicA and the regulatory protein RtsB. Altogether, these results indicate that the RyhB homologs contribute to the S. Typhimurium virulence modulation inside macrophages by reducing the intracellular growth and down-regulating the SPI-1 gene expression.

Participation of two sRNA RyhB homologs from the fish pathogen Yersinia ruckeri in bacterial physiology.

Small noncoding RNAs (sRNAs) are important regulators of gene expression and physiology in bacteria. RyhB is an iron-responsive sRNA well characterized in Escherichia coli and conserved in other Enterobacteriaceae. In this study, we identified and characterized two RyhB homologs (named RyhB-1 and RyhB-2) in the fish pathogen Yersinia ruckeri. We found that, as in other Enterobacteriaceae, both RyhB-1 and RyhB-2 are induced under iron starvation, repressed by the Fur regulator, and depend on Hfq for stability. Despite these similarities in expression, the mutant strains of Y. ruckeri lacking RyhB-1 (ΔryhB-1) or RyhB-2 (ΔryhB-2) exhibited differential phenotypes. In comparison with the wild type, the ΔryhB-1 strain showed a hypermotile phenotype, reduced biofilm formation, increased replication rate, faster growth, and increased ATP levels in bacterial cultures. By contrast, in salmon cell cultures, the ΔryhB-1 strain exhibited an increased survival. On the other hand, the ΔryhB-2 strain was non-motile and showed augmented biofilm formation as compared to the wild type. The expression of a subset of RyhB conserved targets, selected from different bacterial species, was analyzed by quantitative RT-PCR in wild type, ΔryhB-1, ΔryhB-2, and ΔryhB-1 ΔryhB-2 strains cultured in iron-depleted media. RyhB-1 negatively affected the expression of most analyzed genes (sodB, acnA, sdhC, bfr, fliF, among others), whose functions are related to metabolism and motility, involving iron-containing proteins. Among the genes analyzed, only sdhC and bfr appeared as targets for RyhB-2. Taken together, these results indicate that Y. ruckeri RyhB homologs participate in the modulation of the bacterial physiology with non-redundant roles.

The cis-encoded antisense RNA IsrA from Salmonella Typhimurium represses the expression of STM0294.1n (iasE), an SOS-induced gene coding for an endoribonuclease activity.

Toxin-antitoxin systems are known to be involved in many bacterial functions that can lead to growth arrest and cell death in response to stress. Typically, toxin and antitoxin genes of type I systems are located in opposite strands, where the antitoxin is a small antisense RNA (sRNA). In the present work we show that the sRNA IsrA from Salmonella Typhimurium down-regulates the expression of its overlapping gene STM0294.1n. Multiple sequence alignment and comparative structure analysis indicated that STM0294.1n belongs to the SymE toxin superfamily, and the gene was renamed iasE (IsrA-overlapping gene with similarity to SymE). The iasE expression was induced in response to mitomycin C, an SOS-inducing agent; conversely, IsrA overexpression repressed the iasE expression even in the presence of mitomycin C. Accordingly, the inactivation of IsrA with an anti-IsrA RNA expressed in trans abrogated the repressive effect of IsrA on the iasE expression. On the other hand, iasE overexpression, as well as the blockage of the antisense IsrA function, negatively affected bacterial growth, arguing for a toxic effect of the iasE gene product. Besides, a bacterial lysate obtained from the iasE-overexpressing strain exhibited endoribonuclease activity, as determined by a fluorometric assay based on fluorescent reporter RNAs. Together, these results indicate that the IasE/IsrA pair of S. Typhimurium constitutes a functional type I toxin-antitoxin system.

A feed-forward loop between SroC and MgrR small RNAs modulates the expression of eptB and the susceptibility to polymyxin B in Salmonella Typhimurium.

Base-pairing small RNAs (sRNAs) regulate gene expression commonly by direct interaction with cognate mRNAs. Nevertheless, recent studies have expanded this knowledge with the discovery of the RNA 'sponges' which are able to interact and repress the functions of classical base-pairing sRNAs. In this work, we present evidence indicating that the sponge RNA SroC from Salmonella enterica serovar Typhimurium base pairs with the MgrR sRNA, thereby antagonizing its regulatory effects on both gene expression and resistance to the antimicrobial peptide polymyxin B (PMB). By a predictive algorithm, we determined putative SroC-MgrR base-pairing regions flanking the interaction area between MgrR and its target mRNA, eptB, encoding a LPS-modifying enzyme. With a two-plasmid system and compensatory mutations, we confirmed that SroC directly interacts and down-regulates the levels of MgrR, thus relieving the MgrR-mediated repression of eptB mRNA. Since it was previously shown that an Escherichia coli strain carrying an mgrR deletion is more resistant to PMB, we assessed the significance of SroC in the susceptibility of S. Typhimurium to PMB. Whereas the sroC deletion increased the sensitivity to PMB, as compared to the wild-type, the resistance phenotypes between the ΔmgrR and ΔsroCΔmgrR strains were comparable, evidencing that mgrR mutation is epistatic to the sroC mutation. Together, these results indicate that both SroC and MgrR sRNAs compose a coherent feed-forward loop controlling the eptB expression and hence the LPS modification in S. Typhimurium.