Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a concerning global pathogen. hvKp is more virulent than classical K. pneumoniae (cKp) and capable of causing community‐acquired infections, often in healthy individuals. hvKp is carried in the gastrointestinal tract, which contributes to its spread in the community and healthcare settings. First recognized in Asia, hvKp arose as a leading cause of pyogenic liver abscesses. In the decades since, hvKp has spread globally and causes a variety of infections. In addition to liver abscesses, hvKp is distinct from cKp in its ability to metastasize to distant sites, including most commonly the eye, lung and central nervous system (CNS). hvKp has also been implicated in primary extrahepatic infections including bacteremia, pneumonia and soft tissue infections. The genetic determinants of hypervirulence are often found on large virulence plasmids as well as chromosomal mobile genetic elements which can be used as biomarkers to distinguish hvKp from cKp clinical isolates. These distinct virulence determinants of hvKp include up to four siderophore systems for iron acquisition, increased capsule production, K1 and K2 capsule types, and the colibactin toxin. Additionally, hvKp strains demonstrate hypermucoviscosity, a phenotypic description of hvKp in laboratory conditions that has become a distinguishing feature of many hypervirulent isolates. Alarmingly, multidrug‐resistant hypervirulent strains have emerged, creating a new challenge in combating this already dangerous pathogen.

The CRISPR/Cas system known to aid bacterial defences by targeting invading DNA can also act to evade eukaryotic defences through a different class of small RNAs downregulating an endogenous immunogenic bacterial lipoprotein. Bacteria handle unwelcome exogenous material from plasmids and phages using a series of small RNAs to degrade incoming DNA. It has been suggested that the system involved, known as CRISPR/CAS, has other functions too. Here David Weiss and colleagues show that the pathogenic bacterium Francisella novicida, which is able to invade the cells of its eukaryote hosts, uses its CRISPR/CAS system to suppress production of one of its own lipoproteins, which would otherwise be recognized as foreign by the host's innate immune system. Other pathogens, including Neisseria spp. and Campylobacter spp., also have active CRISPR/CAS systems that might serve similar anti-immunity functions, raising the possibility of a previously untapped source of targets for antibacterial interventions. CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids1,2,3,4. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation5,6,7,8. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes9,10. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens11,12, CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.

Significance: The clustered, regularly interspaced, short palindromic repeats associated endonuclease, Cas9, has quickly become a revolutionary tool in genome engineering. Utilizing small guiding RNAs, Cas9 can be targeted to specific DNA sequences of interest, where it catalyzes DNA cleavage. We now demonstrate that Cas9 from the Gram-negative bacterium Francisella novicida (FnCas9) can be reprogrammed to target a specific RNA substrate, the genome of the +ssRNA virus, hepatitis C virus, in eukaryotic cells. Further, this targeting results in inhibition of viral protein production. Overall, programmable Cas9-mediated viral RNA targeting likely represents one of myriad potential applications of FnCas9 in RNA targeting in eukaryotic cells.

Acinetobacter baumannii is an opportunistic pathogen that is a cause of clinically significant nosocomial infections. Increasingly, clinical isolates of A. baumannii are extensively resistant to numerous antibiotics, and the use of polymyxin antibiotics against these infections is often the final treatment option. Historically, the polymyxins have been thought to kill bacteria through membrane lysis. Here, we present an alternative mechanism based on data demonstrating that polymyxins induce rapid cell death through hydroxyl radical production. Supporting this notion, we found that inhibition of radical production delays the ability of polymyxins to kill A. baumannii . Notably, we demonstrate that this mechanism of killing occurs in multidrug-resistant clinical isolates of A. baumannii and that this response is not induced in a polymyxin-resistant isolate. This study is the first to demonstrate that polymyxins induce rapid killing of A. baumannii and other Gram-negatives through hydroxyl radical production. This significantly augments our understanding of the mechanism of polymyxin action, which is critical knowledge toward the development of adjunctive therapies, particularly given the increasing necessity for treatment with these antibiotics in the clinical setting.