RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence.

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.

A Vibrio cholerae Anti-Phage System Depletes Nicotinamide Adenine Dinucleotide to Restrict Virulent Bacteriophages.

Bacteria and their predatory viruses (bacteriophages or phages) are in a perpetual molecular arms race. This has led to the evolution of numerous phage defensive systems in bacteria that are still being discovered, as well as numerous ways of interference or circumvention on the part of phages. Here, we identify a unique molecular battle between the classical biotype of Vibrio cholerae and virulent phages ICP1, ICP2, and ICP3. We show that classical biotype strains resist almost all isolates of these phages due to a 25-kb genomic island harboring several putative anti-phage systems. We observed that one of these systems, Nezha, encoding SIR2-like and helicase proteins, inhibited the replication of all three phages. Bacterial SIR2-like enzymes degrade the essential metabolic coenzyme nicotinamide adenine dinucleotide (NAD+), thereby preventing replication of the invading phage. In support of this mechanism, we identified one phage isolate, ICP1_2001, which circumvents Nezha by encoding two putative NAD+ regeneration enzymes. By restoring the NAD+ pool, we hypothesize that this system antagonizes Nezha without directly interacting with either protein and should be able to antagonize other anti-phage systems that deplete NAD+.

Identifying essential genes in Schaalia odontolytica using a highly-saturated transposon library.

The unique epibiotic-parasitic relationship between Nanosynbacter lyticus type strain TM7x, a member of the newly identified Candidate Phyla Radiation, now referred to as Patescibacteria, and its basibiont, Schaalia odontolytica strain XH001 (formerly Actinomyces odontolyticus), require more powerful genetic tools for deeper understanding of the genetic underpinnings that mediate their obligate relationship. Previous studies have mainly characterized the genomic landscape of XH001 during or post TM7x infection through comparative genomic or transcriptomic analyses followed by phenotypic analysis. Comprehensive genetic dissection of the pair is currently cumbersome due to the lack of robust genetic tools in TM7x. However, basic genetic tools are available for XH001 and this study expands the current genetic toolset by developing high-throughput transposon insertion sequencing (Tn-seq). Tn-seq was employed to screen for essential genes in XH001 under laboratory conditions. A highly saturated Tn-seq library was generated with nearly 660,000 unique insertion mutations, averaging one insertion every 2-3 nucleotides. 203 genes, 10.5% of the XH001 genome, were identified as putatively essential.

Mechanisms of the evolutionary arms race between Vibrio cholerae and Vibriophage clinical isolates

This review highlights recent findings on the evolutionary arms race between the causative agent of cholera Vibrio cholerae and virulent bacteriophages (phages) ICP1, ICP2, and ICP3 isolated from cholera patient stool samples. We discuss mechanisms of phage resistance such as a unique phage-inhibitory chromosomal island and mutations that affect phage receptor expression. We also discuss the molecular characterization of ICP1 and its unique CRISPR-Cas system, which it uses to combat the phage-inhibitory chromosomal island. The role of phages in the life cycle of V. cholerae has been increasingly recognized and investigated in the past decade. This article will review hypotheses as to how the predator-prey relationship may have an impact on infections within individuals and on the self-limiting nature of cholera epidemics. In addition, we put forth a strategy of using phages as an intervention to reduce household transmission of cholera within a community (AU)

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