A-to-I RNA Editing in Klebsiella pneumoniae Regulates Quorum Sensing and Affects Cell Growth and Virulence.

Millions of adenosine (A) to inosine (I) RNA editing events are reported and well-studied in eukaryotes; however, many features and functions remain unclear in prokaryotes. By combining PacBio Sequel, Illumina whole-genome sequencing, and RNA Sequencing data of two Klebsiella pneumoniae strains with different virulence, a total of 13 RNA editing events are identified. The RNA editing event of badR is focused, which shows a significant difference in editing levels in the two K. pneumoniae strains and is predicted to be a transcription factor. A hard-coded Cys is mutated on DNA to simulate the effect of complete editing of badR. Transcriptome analysis identifies the cellular quorum sensing (QS) pathway as the most dramatic change, demonstrating the dynamic regulation of RNA editing on badR related to coordinated collective behavior. Indeed, a significant difference in autoinducer 2 activity and cell growth is detected when the cells reach the stationary phase. Additionally, the mutant strain shows significantly lower virulence than the WT strain in the Galleria mellonella infection model. Furthermore, RNA editing regulation of badR is highly conserved across K. pneumoniae strains. Overall, this work provides new insights into posttranscriptional regulation in bacteria.

Selection of Reference Genes for Normalization of Gene Expression in Thermobia domestica (Insecta: Zygentoma: Lepismatidae).

Zygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (qPCR) studies from Thermobia domestica, a representative species of Zygentoma, including Actin 5C (Actin5C), Elongation factor-1 alpha (EF1A), Ribosome protein S26 (RPS26), Ribosome protein L32 (RPL32), and Superoxide dismutase 2 (SOD2), at different developmental stages, in various body parts, and under dsRNA microinjection and starvation stresses, using four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) and a comparative algorithm (RefFinder). Specific suitable reference genes were recommended across specific experimental conditions, and the combination of RPS26 and RPL32 was appropriate for all tested samples. Employing our selected reference gene combination, we investigated the gene expression pattern of Myoglianin (Myo), a crucial gene-regulating insect metamorphosis, in ametabolous T. domestica, and demonstrated the efficiency of RNA interference (RNAi) in firebrat nymphs. This study provides a basis for reliable quantitative studies of genes and greatly benefits evolutionary and functional genomics studies in Zygentoma.

Clinical and Microbiological Characterization of Invasive Pulmonary Aspergillosis Caused by Aspergillus lentulus in China.

Invasive aspergillosis (IA) due to Aspergillus lentulus is associated with high mortality. In this study, we investigated the clinical and microbiological characteristics of 6 fatal cases of proven or probable IA caused by A. lentulus in China. Underlying immunosuppression, prior antifungal exposure, and intensive care unit (ICU) hospitalization were important risk factors for invasive A. lentulus infection. Phenotypic differences were observed for A. lentulus isolates including slower growth, reduced sporulation, and inability to grow at 48°C, compared with Aspergillus fumigatus complex. ITS sequencing was unable to distinguish A. lentulus from A. fumigatus, but sequencing of the benA, CaM, and rod A loci enabled reliable distinction of these closely related species. Phylogenetic analysis further confirmed that the ITS region had little variation within the Aspergillus section Fumigati while the benA gene offered the highest intraspecific discrimination. Microsatellite typing results revealed that only loci on chromosomes 1, 3, 5, and 6b generated detectable amplicons for identification. All A. lentulus isolates showed in vitro resistance to multiple antifungal drugs including amphotericin B (MIC range 4 to 8 µg/ml), itraconazole (MIC 2 µg/ml), voriconazole (MIC of 4-16 µg/ml), and posaconazole (MIC of 0.5-1 µg/ml). However, MECs for the echinocandin drugs ranged from 0.03-0.25, ≤0.008-0.015, and ≤0.015-0.03 µg/ml for caspofungin, micafungin, and anidulafungin, respectively. A. lentulus is an emerging fungal pathogen in China, causing fatal disease, and clinicians as well as laboratories should be alert to their increasing presence.