No abstract

The interaction of Magnaporthe oryzae, the rice blast fungus, and rice begins when M. oryzae establishes contact with the host plant surface. On perception of appropriate surface signals, M. oryzae forms appressoria and initiates host invasion. Pth11, an important G-protein-coupled receptor necessary for appressorium formation in M. oryzae, contains seven transmembrane regions and a CFEM (common in several fungal extracellular membrane proteins) domain with the characteristic eight cysteine residues. We focused on gaining further insight into the role of the CFEM domain in the putative surface sensing/response function of Pth11. Increased/constitutive expression of CFEM resulted in precocious, albeit defective, appressoria formation in wild-type M. oryzae. The Pth11 mutant, probably with disrupted disulfide bonds in the CFEM, showed delayed appressorium formation and reduced virulence. Furthermore, the accumulation of reactive oxygen species (ROS) was found to be altered in the pth11Δ strain. Strikingly, antioxidant treatment induced appressorium formation in pth11Δ. The Gα subunit MagB and the mitogen-activated protein (MAP) kinase Pmk1 were required for the formation of antioxidant-induced appressoria. We conclude that the CFEM domain of Pth11 is required for proper development of the appressoria, appressoria-like structures and pathogenicity. Highly regulated ROS homeostasis is important for Pth11-mediated appressorium formation in M. oryzae.

Hypervirulent Klebsiella pneumoniae (hvKP) causes Klebsiella‐induced liver abscess. Capsule is important for the pathogenesis of Klebsiella in systemic infection, but its role in gut colonisation is not well understood. By generating ΔwcaJ, Δwza and Δwzy capsule‐null mutants in a prototypical K1 hypervirulent isolate, we show that inactivation of wza (capsule exportase) and wzy (capsule polymerase) confer cell envelope defects in addition to capsule loss, making them susceptible to bile salts and detergent stress. Bile salt resistance is restored when the initial glycosyltransferase wcaJ was inactivated together with wzy, indicating that build‐up of capsule intermediates contribute to cell envelope defects. Mouse gut colonisation competition assays show that the capsule and its regulator RmpA were not required for hvKP to persist in the gut, although initial colonisation was decreased in the mutants. Both ΔrmpA and ΔwcaJ mutants gradually outcompeted the wild type in the gut, whereas Δwza and Δwzy mutants were less fit than wild type. Together, our results advise caution in using the right capsule‐null mutant for determination of capsule's role in bacterial pathogenesis. With the use of ΔwcaJ mutant, we found that although the capsule is important for bacterial survival outside the gut environment, it imposes a fitness cost in the gut.

T he rise of multidrug-resistant (MDR) Enterobacteriaceae prompted the World Health Organization to classify carbapenem-resistant Enterobacteriaceae, of which Klebsiella is the most common genus, on the global priority list of antibiotic-resistant bacteria in 2017 (1). Carbapenem-resistant K. pneumoniae (CRKP, also including K. quasipneumoniae) infections are generally hospital acquired, particularly among elderly and immunocompromised patients (2,3). The major carbapenemases include K. pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase, and carbapenem-hydrolyzing class D β-lactamase (OXA), all of which have spread globally (4-7). The Carbapenemase-Producing Enterobacteriaceae in Singapore (CaPES) study initiated in 2013 revealed that the rate of incident carbapenem-resistant Enterobacteriaceae clinical cultures in government hospitals in Singapore increased during 2011-2013 and plateaued thereafter (8). The number of cases of hypervirulent K. pneumoniae has increased in the past 3 decades in parts of Asia, and likewise, the number of cases of monomicrobial Klebsiella-induced liver abscesses has also increased (9,10). The prevalence of antimicrobial resistance among hypervirulent K. pneumoniae isolates is rare compared with that of standard isolates (11,12); hypervirulent K. pneumoniae and CRKP seem to have their own particular reservoirs and remain mostly segregated from each other. However, hypervirulent K. pneumoniae and CRKP isolates can converge in the same organism, leading to the emergence of superbugs resistant to antimicrobial drugs of even the last line of treatment that are capable of infecting healthy persons. This emergence has already been reported in China, Brazil, and the United Kingdom (13-15). The fatal outbreak that occurred in a hospital in China in 2016 was caused by a carbapenem-resistant hypervirulent K. pneumoniae strain that had acquired a virulence plasmid by a classic sequence type (ST) 11 strain (16). In a study of a collection of >2,200 K. pneumoniae genomes, distinct evolutionary patterns of horizontal gene transfer were observed in MDR isolates versus

Klebsiella pneumoniae-induced liver abscess (KLA) is emerging as a leading cause of pyogenic liver abscess worldwide. In recent years, the emergence of hypervirulent K. pneumoniae (hvKp) has been strongly associated with KLA. Unlike classical K. pneumoniae, which generally infects the immunocompromised population, hvKp can cause serious and invasive infections in young and healthy individuals. hvKp isolates are often associated with the K1/K2 capsular types and possess hypermucoviscous capsules. KLA is believed to be caused by K. pneumoniae colonizing the gastrointestinal tract of the host and translocating across the intestinal barrier via the hepatic portal vein into the liver to cause liver abscess. We optimized the isolation of the liver-resident macrophages called Kupffer cells in mice and examined their importance in controlling bacterial loads during hvKp infection in healthy mice. Our study reveals the high capability of Kupffer cells to kill hvKp in vitro despite the presence of the bacterial hypermucoviscous capsule, in contrast to other macrophages, which were unable to phagocytose the bacteria efficiently. Depletion of Kupffer cells and macrophages with liposome-encapsulated clodronate (liposomal clodronate) in both an intraperitoneal and an oral mouse infection model resulted in increased bacterial loads in the livers, spleens, and lungs and increased mortality of the infected mice. Thus, Kupffer cells and macrophages are critical for the control of hvKp infection.