Klebsiella pneumoniae raises significant concerns to the health care industry as these microbes are the source of widespread contamination of medical equipment, cause pneumonia as well as other multiorgan metastatic infections and have gained multidrug resistance. Despite soaring mortality rates, the host cell alterations occurring during these infections remain poorly understood. Here, we show that during in vitro and in vivo K. pneumoniae infections of lung epithelia, microtubules are severed and then eliminated. This destruction does not require direct association of K. pneumoniae with the host cells, as microtubules are disassembled in cells that are distant from the infecting bacteria. This microtubule dismantling is dependent on the K. pneumoniae (Kp) gene ytfL as non‐pathogenic Escherichia coli expressing Kp ytfL disassemble microtubules in the absence of K. pneumoniae itself. Our data points to the host katanin catalytic subunit A like 1 protein (KATNAL1) and the katanin regulatory subunit B1 protein (KATNB1) as the gatekeepers to the microtubule severing event as both proteins localise specifically to microtubule cut sites. Infected cells that had either of these proteins knocked out maintained intact microtubules. Taken together, we have identified a novel mechanism that a bacterial pathogen has exploited to cause microtubule destruction within the host epithelia.

Klebsiella pneumoniae has become a growing concern within hospitals due to multidrug resistant strains and increasing mortality rates. Recently, we showed that at the subcellular level, K. pneumoniae compromises the integrity of the epithelia by disassembling the microtubule networks of cells through the actions of katanin microtubule severing proteins. In this study, we report on the observation that mitotic cells are targeted by K. pneumoniae and that during infections, the katanin proteins are excluded from the microtubule organizing centers of dividing cells, resulting in the alteration of the microtubule cytoskeleton. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1859–1864, 2020. © 2019 American Association for Anatomy

Hospitals have raised a growing concern for the rampant spread of the bacterial pathogen Klebsiella pneumoniae, which can cause a wide array of symptoms such as pneumonia, urinary tract infections, septicemia, and pyogenic liver abscesses. Moreover, the misuse of antibiotics has contributed to the surge of multidrug‐resistant hypervirulent strains. As a result, therapeutics for K. pneumoniae infections are becoming ineffective leading to lower survival rates and higher mortality rates in highly infected individuals. In our laboratory, we have shown that K. pneumoniae activates katanin microtubule severing enzymes to induce microtubule disassembly in lung epithelial cells. In contrast, microtubule associated proteins (MAPs) in host cells regulate the stability of microtubules and specific MAPs such as Tau (MAPTau) can protect microtubules from katanin‐induced severing. Since Tau plays a role in the stability of microtubules, we hypothesized that Tau protein levels may prevent microtubule disassembly during K. pneumoniae infections. To test this hypothesis, we initially determined if K. pneumoniae targets Tau in infected A549 lung epithelial cells. We lysed uninfected and infected cells and we found that a higher molecular weight Tau species was present in the infected cell lysates. Hyperphosphorylation of Tau typically regulates its binding to microtubules, and thus, we then determined if overexpressing Tau constructs that have different phosphorylation abilities could protect microtubules from K. pneumoniae‐induced katanin‐mediated severing. To do this, we obtained three green fluorescent protein (EGFP)‐tagged tau constructs: wildtype Tau (wt Tau), hyperphosphorylated Tau (E14 Tau), and Tau that cannot be phosphorylated (AP Tau). We then expressed these plasmids in A549 cells and infected the cells with K. pneumoniae. If Tau protects microtubules from katanin‐severing, E14 Tau should have no effect on K. pneumoniae‐induced severing while wild‐type Tau and AP Tau should prevent microtubule severing. During these infections no rescue of the microtubule phenotype was observed thus, neither wild‐type Tau nor AP Tau protected microtubules from K. pneumoniae‐triggered microtubule disassembly. Our findings show that Tau overexpression plays no discernible role in protecting microtubules during Klebsiella pneumoniae infections.Support or Funding InformationThis study was funded by SFU institutional funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microtubules play an important role in maintaining cell shape, protein and organellar transport, and cell division. The integrity of the microtubule network is crucial for the viability of mammalian cells. Consequently, the cell has evolved many regulatory mechanisms to ensure the proper regulation of microtubules, especially during the cell cycle. The katanin family of microtubule severing proteins maintain microtubule lengths during interphase. When mitosis occurs, these katanins are recruited to the microtubule organizing centers (MTOCs) to facilitate DNA separation. Previously, we have shown that the microtubules networks in lung epithelia were disassembled by the bacterial pathogen Klebsiella pneumoniae. Because the microtubules were targeted by disease‐causing proteins originating from K. pneumoniae, we hypothesized that K. pneumoniae has devised strategies to manipulate the katanin microtubule severing enzymes to ultimately cause cell cycle arrest. To test this hypothesis, we infected A549 lung cells with K. pneumoniae and immunolocalized the katanin proteins ‐ katanin catalytic subunit A1 protein (KATNA1), katanin catalytic subunit like protein A1 like protein (KATNAL1), katanin regulatory subunit B1 (KATNB1), and katanin regulatory subunit B1 like protein (KATNBL1). In uninfected cells, these proteins immunolocalized to the MTOCs and the cleavage furrow during cell division, but in infected dividing cells, these proteins were absent. This suggested that mitosis was halted. Thus, we examined if these infected cells were still viable and able to proliferate. Using a live/dead staining kit, we found that at this point of the infections, the cells were non‐viable and cell death has occurred. Given that host cells could be killed by the microbes and that microtubules were disassembled during the infections, we investigated whether microtubules could be destroyed in vincristine‐ and vinblastine‐resistant H69AR lung cancer epithelial cells. Microtubule disassembly occurred in these H69AR cells and taken together, we have identified a potent mechanism for destroying the microtubule cytoskeleton and ultimately killing lung cancer cells that are resistant to conventional therapeutics.Support or Funding InformationThis study was funded by SFU institutional funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Klebsiella pneumoniae is an enteric bacterium known to cause pneumonia, urinary tract infections and pyogenic liver abscesses. Of these infections, patients who develop pneumonia have poor survival rates and alarming mortality rates of up to 44% in highly infected individuals. Many studies have focused on identifying bacterial components crucial to the disease process, but the underlying sub‐cellular mechanisms for disease progression have remained elusive. To begin to study these mechanisms, we assessed the cytoskeletal integrity of infected A549 lung epithelial cells and lung epithelia from infected C57Bl/6J mice. We found that the microtubule networks of the entire epithelia were either severed or fully disassembled even when there were no bacteria directly attached on the host cells. Since bacterial attachment was not necessary to cause microtubule disassembly, we hypothesized that a novel K. pneumoniae protein activates signalling cascades that target microtubule‐severing activity within these lung cells. To test this hypothesis, we first infected A549 cells with K. pneumoniae mutants deficient for known bacterially generated pathogenic proteins and found that none of those proteins triggered microtubule severing. Then, we created a genomic library of the entire K. pneumoniae genome. Out of the ~3000 known K. pneumoniae genes, we found that K. pneumoniae ytfL (KP ytfL) consistently triggered microtubule severing in infected A549 cells. Bioinformatic analysis showed that the C‐terminal domain of KP YtfL has a catalytic region that could activate host signalling cascades. We then sought to identify the host microtubule‐severing enzyme responsible for the epithelial cell phenotype. Through immunolocalization studies, we discovered that the katanin catalytic subunit A1 like 1 protein (KATNAL1) and the katanin regulatory subunit B1 protein (KATNB1) were at the precise sites of microtubule severing. To determine if KATNAL1 and KATNB1 were responsible for K. pneumoniae‐induced severing, we deleted the gene for either KATNAL1 or KATNB1 in A549 cells using CRISPR. We then infected the cells and when all the infected wild type cells had disassembled microtubules, KATNAL1 and KATNB1‐deficient cells retained intact microtubules. Thus, KATNAL1 and KATNB1 contribute to the microtubule severing activity during K. pneumoniae infections. Taken together, our data indicates that KP YtfL is a novel protein effector that triggers the activation of KATNAL1 and KATNB1 to sever microtubules in infected lung epithelial cells. Through our study, we identified a novel bacterial mechanism to destabilize the microtubule networks of an entire epithelium through the activation of host cell microtubule severing enzymes.Support or Funding InformationThis study was funded by NSERC, Taiwan NHRI and SFU institutional funds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.