21. Chua, MD; Liou, CH; Bogdan, AC; Law, HT; Yeh, KM; Lin, JC; Siu, LK; Guttman, JA. (2019) Klebsiella pneumoniae disassembles host microtubules in lung epithelial cells.Cell Microbiol. 21 Klebsiella pneumoniae disassembles host microtubules in lung epithelial cells
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. DOI PubMed
20. Dhanda, AS; Lulic, KT; Vogl, AW; Mc Gee, MM; Chiu, RH; Guttman, JA. (2019) Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading.J. Infect. Dis. 219 Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading
cell-to-cell spread; cyclophilin A; Listeria; Listeria monocytogenes
Background. Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighboring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. Methods. In this study, we demonstrate that the prolyl cis-trans isomerase (PPIase) cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. Results. Cyclophilin A localizes within the F-actin of these structures and is crucial for their proper formation, as cells depleted of CypA have extended actin-rich structures that are misshaped and are collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Conclusions. Our results demonstrate a crucial role for CypA during Listeria infections. DOI PubMed
19. Dhanda, AS; Vogl, AW; Albraiki, SE; Otey, CA; Beck, MR; Guttman, JA. (2018) Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections.mBio 9 Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections
actin nucleation; actin polymerization; Listeria monocytogenes
Palladin is an important component of motile actin-rich structures and nucleates branched actin filament arrays in vitro. Here we examine the role of palladin during Listeria monocytogenes infections in order to tease out novel functions of palladin. We show that palladin is co-opted by L. monocytogenes during its cellular entry and intracellular motility. Depletion of palladin resulted in shorter and misshapen comet tails, and when actin-or VASP-binding mutants of palladin were overexpressed in cells, comet tails disintegrated or became thinner. Comet tail thinning resulted in parallel actin bundles within the structures. To determine whether palladin could compensate for the Arp2/3 complex, we overexpressed palladin in cells treated with the Arp2/3 inhibitor CK-666. In treated cells, bacterial motility could be initiated and maintained when levels of palladin were increased. To confirm these findings, we utilized a cell line depleted of multiple Arp2/3 complex subunits. Within these cells, L. monocytogenes failed to generate comet tails. When palladin was overexpressed in this Arp2/3 functionally null cell line, the ability of L. monocytogenes to generate comet tails was restored. Using purified protein components, we demonstrate that L. monocytogenes actin clouds and comet tails can be generated ( in a cell-free system) by palladin in the absence of the Arp2/3 complex. Collectively, our results demonstrate that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility. IMPORTANCE Structures containing branched actin filaments require the Arp2/3 complex. One of the most commonly used systems to study intracellular movement generated by Arp2/3-based actin motility exploits actin-rich comet tails made by Listeria. Using these infections together with live imaging and cell-free protein reconstitution experiments, we show that another protein, palladin, can be used in place of Arp2/3 to form actin-rich structures. Additionally, we show that palladin is needed for the structural integrity of comet tails as its depletion or mutation of critical regions causes dramatic changes to comet tail organization. These findings are the first to identify a protein that can functionally replace the Arp2/3 complex and have implications for all actin-based structures thought to exclusively use that complex. DOI PubMed
10. Law, HT; Bonazzi, M; Jackson, J; Cossart, P; Guttman, JA. (2012) Nexilin is a dynamic component of Listeria monocytogenes and enteropathogenic Escherichia coli actin-rich structures.Cellular Microbiology 14: 1097-1108 Nexilin is a dynamic component of Listeria monocytogenes and enteropathogenic Escherichia coli actin-rich structures
The bacterial pathogens Listeria monocytogenes and enteropathogenic Escherichia coli (EPEC) generate motile actin-rich structures (comet tails and pedestals) as part of their infectious processes. Nexilin, an actin-associated protein and a component of focal adhesions, has been suggested to be involved in actin-based motility. To determine whether nexilin is commandeered during L. monocytogenes and EPEC infections, we infected cultured cells and found that nexilin is crucial for L. monocytogenes invasion as levels of internalized bacteria were significantly decreased in nexilin-targeted siRNA-treated cells. In addition, nexilin is a component of the machinery that drives the formation of L. monocytogenes comet tails and EPEC pedestals. Nexilin colocalizes with stationary bacteria and accumulates at the distal portion of comet tails and pedestals of motile bacteria. We also show that nexilin is crucial for efficient comet tail formation as cells pre-treated with nexilin siRNA generate malformed comet tails, whereas nexilin is dispensable during EPEC pedestal generation. These findings demonstrate that nexilin is required for efficient infection with invasive and adherent bacteria and is key to the actin-rich structures these microbes generate. DOI
9. Lin, AE; Benmerah, A; Guttman, JA. (2011) Eps15 and Epsin1 Are Crucial for Enteropathogenic Escherichia coli Pedestal Formation Despite the Absence of Adaptor Protein 2.Journal of Infectious Diseases 204: 695-703 Eps15 and Epsin1 Are Crucial for Enteropathogenic Escherichia coli Pedestal Formation Despite the Absence of Adaptor Protein 2
Enteropathogenic Escherichia coli (EPEC) are primarily extracellular pathogens that generate actin-rich structures known as pedestals during their pathogenesis. Surprising evidence has demonstrated that despite maintaining an extracellular location, EPEC require the endocytic protein, clathrin, for pedestal formation. To evaluate the strategies EPEC use to usurp endocytic machinery, we investigated the roles of a number of clathrin-coated pits components, adaptor protein 2 (AP-2), Eps15 and epsin1, during EPEC infections. We demonstrated that in conjunction with clathrin, pedestal formation also required the recruitment of Eps15 and epsin1 but not AP-2. Because AP-2 orchestrates the recruitment of clathrin, Eps15, and epsin1, as well as other adaptors, during assembly of clathrin-coated pits at the plasma membrane, our findings reveal a novel internalization subversion strategy employed by EPEC. These results further emphasize the recent paradigm that endocytic proteins are important for EPEC-mediated disease. DOI
8.Guttman, JA; Lin, AEJ; Li, YL; Bechberger, J; Naus, CC; Vogl, AW; Finlay, BB. (2010) Gap junction hemichannels contribute to the generation of diarrhoea during infectious enteric disease.Gut 59: 218-226 Gap junction hemichannels contribute to the generation of diarrhoea during infectious enteric disease
Objective The attaching and effacing (A/E) pathogens enterohaemorrhagic Escherichia coli, enteropathogenic E coli and Citrobacter rodentium colonise intestinal tracts, attach to enterocytes, collapse infected cell microvilli and alter numerous host cell processes during infection. Enterocyte alterations result in numerous small molecules being released from host cells that likely contribute to diarrhoeal phenotypes observed during these infections. One possible route for small molecules to be released from intestinal cells may be through functional gap junction hemichannels. Here we examine the involvement of these hemichannels during the diarrhoeal disease caused by A/E pathogens in vivo. Design Mice were infected with the diarrhoea-causing murine A/E pathogen C rodentium for 7 days. Connexin43 (Cx43) protein levels and immunolocalisation in the colon were initially used to determine alterations during A/E bacterial infections in vivo. Connexin mimetic peptides and connexin permeable tracer molecules were used to gage the presence and function of unpaired connexin hemichannels. The role of Cx43 in diarrhoea generation was assessed by comparing infections of wild-type mice to Cx43 mutant mice and determining the water abundance in the colonic luminal material. Results We demonstrate that Cx43 protein levels are increased in colonocytes during in vivo A/E bacterial infections, resulting in functionally open connexon hemichannels in apical membranes of infected cells. Moreover, infected Cx43 +/- mice do not suffer from diarrhoeal disease. Conclusions This study provides the first evidence that functional connexon hemichannels can occur in the intestine and are a novel molecular mechanism of water release during infectious diarrhoea. DOI
7. Shames, SR; Deng, WY; Guttman, JA; de Hoog, CL; Li, YL; Hardwidge, PR; Sham, HP; Vallance, BA; Foster, LJ; Finlay, BB. (2010) The pathogenic E-coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling.Cellular Microbiology 12: 1322-1339 The pathogenic E-coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling
Cell Biology; Microbiology
Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are diarrhoeal pathogens that cause the formation of attaching and effacing (A/E) lesions on infected host cells. These pathogens encode a type III secretion system (T3SS) used to inject effector proteins directly into host cells, an essential requirement for virulence. In this study, we identified a function for the type III secreted effector EspZ. Infection with EPEC Delta espZ caused increased cytotoxicity in HeLa and MDCK cells compared with wild-type EPEC, and expressing espZ in cells abrogated this effect. Using yeast two-hybrid, proteomics, immunofluorescence and co-immunoprecipitation, it was demonstrated that EspZ interacts with the host protein CD98, which contributes to protection against EPEC-mediated cytotoxicity. EspZ enhanced phosphorylation of focal adhesion kinase (FAK) and AKT during infection with EPEC, but CD98 only appeared to facilitate FAK phosphorylation. This study provides evidence that EspZ and CD98 promote host cell survival mechanisms involving FAK during A/E pathogen infection. DOI
6. Menendez, A; Arena, ET; Guttman, JA; Thorson, L; Vallance, BA; Vogl, W; Finlay, BB. (2009) Salmonella Infection of Gallbladder Epithelial Cells Drives Local Inflammation and Injury in a Model of Acute Typhoid Fever.Journal of Infectious Diseases 200: 1703-1713 Salmonella Infection of Gallbladder Epithelial Cells Drives Local Inflammation and Injury in a Model of Acute Typhoid Fever
ACUTE ACALCULOUS CHOLECYSTITIS; IN-VIVO; PEYERS-PATCHES; BILIARY-TRACT; TYPHIMURIUM; BILE; MACROPHAGES; RESISTANCE; IDENTIFICATION; PERFORATION
The gallbladder is often colonized by Salmonella during typhoid fever, yet little is known about bacterial pathogenesis in this organ. With use of a mouse model of acute typhoid fever, we demonstrate that Salmonella infect gallbladder epithelial cells in vivo. Bacteria in the gallbladder showed a unique behavior as they replicated within gallbladder epithelial cells and remained confined to those cells without translocating to the mucosa. Infected gallbladders showed histopathological damage characterized by destruction of the epithelium and massive infiltration of neutrophils, accompanied by a local increase of proinflammatory cytokines. Damage was determined by the ability of Salmonella to invade gallbladder epithelial cells and was independent of high numbers of replication-competent, although invasion-deficient, bacteria in the lumen. Our results establish gallbladder epithelial cells as a novel niche for in vivo replication of Salmonella and reveal the involvement of these cells in the pathogenesis of Salmonella in the gallbladder during the course of acute typhoid fever. DOI
5. Valdez, Y; Grassl, GA; Guttman, JA; Coburn, B; Gros, P; Vallance, BA; Finlay, BB. (2009) Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice.Cellular Microbiology 11: 351-362 Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice
ENTERICA SEROTYPE TYPHIMURIUM; SLC11A1 FORMERLY NRAMP1; NATURAL-RESISTANCE; SEROVAR TYPHIMURIUM; INTRACELLULAR PARASITES; DENDRITIC CELLS; BCG/ITY/LSH LOCUS; CANDIDA-ALBICANS; GENETIC-CONTROL; C57BL/6J MICE
A recently developed model for enterocolitis in mice involves pre-treatment with the antibiotic streptomycin prior to infection with Salmonella enterica serovar Typhimurium (S. Typhimurium). The contribution of Nramp1/Slc11a1 protein, a critical host defence mechanism against S. Typhimurium, to the development of inflammation in this model has not been studied. Here, we analysed the impact of Nramp1 expression on the early development of colitis using isogenic Nramp1(+/+) and Nramp1(-/-) mice. We hypothesized that Nramp1 acts by rapidly inducing an inflammatory response in the gut mucosa creating an antibacterial environment and limiting spread of S. Typhimurium to systemic sites. We observed that Nramp1(+/+) mice showed lower numbers of S. Typhimurium in the caecum compared with Nramp1(-/-) mice at all times analysed. Acute inflammation was much more pronounced in Nramp1(+/+) mice 1 day after infection. The effect of Nramp1 on development of colitis was characterized by higher secretion of the pro-inflammatory cytokines IFN-gamma, TNF-alpha and MIP-1 alpha and a massive infiltration of neutrophils and macrophages, compared with Nramp1(-/-) animals. These data show that an early and rapid inflammatory response results in protection against pathological effects of S. Typhimurium infection in Nramp1(+/+) mice. DOI
3. Valdez, Y; Diehl, GE; Vallance, BA; Grassl, GA; Guttman, JA; Brown, NF; Rosenberger, CM; Littman, DR; Gros, P; Finlay, BB. (2008) Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection.Cellular Microbiology 10: 1646-1661 Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection
Host resistance against Salmonella enterica serovar Typhimurium (S. Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S. Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c(+)CD103(-)) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c(+)CD103(-) DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-alpha) than Nramp1-/-, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium. DOI
2. Bhavsar A, Guttman, JA., Finlay, BB. (2007) Manipulation of host-cell pathways by bacterial pathogens.Nature 449:827-834. Manipulation of host-cell pathways by bacterial pathogens.
Bacterial pathogens operate by attacking crucial intracellular pathways in their hosts. These pathogens
usually target more than one intracellular pathway and often interact at several points in each of these
pathways to commandeer them fully. Although different bacterial pathogens tend to exploit similar pathway
components in the host, the way in which they ‘hijack’ host cells usually differs. Knowledge of how pathogens
target distinct cytoskeletal components and immune-cell signalling pathways is rapidly advancing, together
with the understanding of bacterial virulence at a molecular level. Studying how these bacterial pathogens
subvert host-cell pathways is central to understanding infectious disease. PDF DOI
1. Leavitt, BR; Guttman, JA; Hodgson, JG; Kimel, GH; Singaraja, R; Vogl, AW; Hayden, MR. (2001) Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo.American Journal of Human Genetics 68: 313-324 Wild-type huntingtin reduces the cellular toxicity of mutant huntingtin in vivo
We have developed yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 or YAC72) human huntingtin (htt), in a developmental- and tissue-specific manner, that is identical to endogenous htt. YAC72 mice develop selective degeneration of medium spiny projection neurons in the lateral striatum, similar to what is observed in Huntington disease. Mutant human htt expressed by YAC transgenes can compensate for the absence of endogenous htt and can rescue the embryonic lethality that characterizes mice homozygous for targeted disruption of the endogenous Hdh gene (-/-). YAC72 mice lacking endogenous htt (YAC72 -/-) manifest a novel phenotype characterized by infertility, testicular atrophy, aspermia, and massive apoptotic cell death in the testes. The testicular cell death in YAC72 -/- mice can be markedly reduced by increasing endogenous htt levels. YAC72 mice with equivalent levels of both wild-type and mutant htt (YAC72 +/+) breed normally and have no evidence of increased testicular cell death. Similar findings are seen in YAC46 -/- mice compared with YAC46 +/+ mice, in which wild-type htt can completely counteract the proapoptotic effects of mutant htt. YAC18 -/- mice display no evidence of increased cellular apoptosis, even in the complete absence of endogenous htt, demonstrating that the massive cellular apoptosis observed in YAC46 -/- mice and YAC72 -/- mice is polyglutamine-mediated toxicity from the mutant transgene. These data provide the first direct in vivo evidence of a role for wild-type htt in decreasing the cellular toxicity of mutant htt. DOI
