Integration of linear and dendritic actin nucleation in Nck-induced actin comets

The Nck adaptor protein recruits cytosolic effectors such as N-WASP that induce localized actin polymerization. Experimental aggregation of Nck SH3 domains at the membrane induces actin comet tails-dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. Here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of Nck-induced actin comets. Inhibition of linear, form-in-based nucleation with the small-molecule inhibitor SMIFH2 or overexpression of the formin FH1 domain resulted in formation of predominantly circular-shaped actin structures with low mobility (actin blobs). These results indicate that formin-based linear actin polymerization is critical for the formation and maintenance of Nck-dependent actin comet tails. Consistent with this, aggregation of an exclusively branched nucleation-promoting factor (the VCA domain of N-WASP), with density and turnover similar to those of N-WASP in Nck comets, did not reconstitute dynamic, elongated actin comets. Furthermore, enhancement of branched Arp2/3-mediated nucleation by N-WASP overexpression caused loss of the typical actin comet tail shape induced by Nck aggregation. Thus the ratio of linear to dendritic nucleation activity may serve to distinguish the properties of actin structures induced by various viral and bacterial pathogens.Fil: Surtayeva, Sofya. University of Connecticut School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Velle, Katrina B.. University of Connecticut; Estados UnidosFil: Campellone, Kenneth G.. University of Connecticut; Estados UnidosFil: Talman, Arthur. Yale School of Medicine; Estados UnidosFil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Agaisse, Hervé. Yale School of Medicine; Estados UnidosFil: Wu, Yi I.. University of Connecticut School of Medicine; Estados UnidosFil: Loew, Leslie M.. University of Connecticut School of Medicine; Estados UnidosFil: Mayer, Bruce J.. University of Connecticut School of Medicine; Estados Unido

Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri

Background The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely. Results Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system. Conclusions In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.publishedVersio

Extracellular motility and cell-to-cell transmission of enterohemorrhagic E. coli is driven by EspFU-mediated actin assembly.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are closely-related pathogens that attach tightly to intestinal epithelial cells, efface microvilli, and promote cytoskeletal rearrangements into protrusions called actin pedestals. To trigger pedestal formation, EPEC employs the tyrosine phosphorylated transmembrane receptor Tir, while EHEC relies on the multivalent scaffolding protein EspFU. The ability to generate these structures correlates with bacterial colonization in several animal models, but the precise function of pedestals in infection remains unclear. To address this uncertainty, we characterized the colonization properties of EPEC and EHEC during infection of polarized epithelial cells. We found that EPEC and EHEC both formed distinct bacterial communities, or "macrocolonies," that encompassed multiple host cells. Tir and EspFU, as well as the host Arp2/3 complex, were all critical for the expansion of macrocolonies over time. Unexpectedly, EspFU accelerated the formation of larger macrocolonies compared to EPEC Tir, as EspFU-mediated actin assembly drove faster bacterial motility to cell junctions, where bacteria formed a secondary pedestal on a neighboring cell and divided, allowing one of the daughters to disengage and infect the second cell. Collectively, these data reveal that EspFU enhances epithelial colonization by increasing actin-based motility and promoting an efficient method of cell-to-cell transmission

Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance.

Enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC) are closely related extracellular pathogens that reorganize host cell actin into "pedestals" beneath the tightly adherent bacteria. This pedestal-forming activity is both a critical step in pathogenesis, and it makes EPEC and EHEC useful models for studying the actin rearrangements that underlie membrane protrusions. To generate pedestals, EPEC relies on the tyrosine phosphorylated bacterial effector protein Tir to bind host adaptor proteins that recruit N-WASP, a nucleation-promoting factor that activates the Arp2/3 complex to drive actin polymerization. In contrast, EHEC depends on the effector EspFU to multimerize N-WASP and promote Arp2/3 activation. Although these core pathways of pedestal assembly are well-characterized, the contributions of additional actin nucleation factors are unknown. We investigated potential cooperation between the Arp2/3 complex and other classes of nucleators using chemical inhibitors, siRNAs, and knockout cell lines. We found that inhibition of formins impairs actin pedestal assembly, motility, and cellular colonization for bacteria using the EPEC, but not the EHEC, pathway of actin polymerization. We also identified mDia1 as the formin contributing to EPEC pedestal assembly, as its expression level positively correlates with the efficiency of pedestal formation, and it localizes to the base of pedestals both during their initiation and once they have reached steady state. Collectively, our data suggest that mDia1 enhances EPEC pedestal biogenesis and maintenance by generating seed filaments to be used by the N-WASP-Arp2/3-dependent actin nucleation machinery and by sustaining Src-mediated phosphorylation of Tir

EHEC Tir and EspF_U promote more efficient colonization of polarized epithelial cells than EPEC Tir.

<p>(A) Polarized Caco-2 monolayers were infected with KC12 and EPEC strains for 6 h, fixed, and stained to visualize bacteria, F-actin, and DNA. Scale circles, 100, 500, 1000 μm². (B) Macrocolony sizes >100 μm² from experiments described in (A) were measured and binned into size groups. Each bar represents the mean number of macrocolonies (+/- SE) from 3 experiments, spanning 225–275 fields of view (FOV) and 4658–8434 colonies. All p-value significance is in reference to the 100–250 μm² bins. *p<0.05, **p<0.01 (ANOVA, Tukey post-hoc tests). (C) Data collected in (B) were reorganized to compare the strains within each category. Bars represent the mean (+/- SD) for 3 experiments, of the % of colonies falling into each bin. Asterisks are in reference to KC12+EspF_U. *p<0.05, **p<0.01 (ANOVA, Tukey post-hoc tests). (D) Macrocolony sizes measured from part (B) were averaged. Each bar represents the mean (+/- SD) from 3 experiments. All p-values are in reference to KC12+EspF_U. (E) Experiments were performed as in (A), but for 7 h. Each bar represents the mean (+/- SE) of the % of monolayer area infected for 58–60 FOVs. (F) The number of infected cells per macrocolony was calculated. Each bar represents the mean (+/- SE) calculated from 238–497 macrocolonies, taken from 17–19 representative fields of view from the images quantified in (E). *p<0.05, **p<0.01 (ANOVA, Tukey post-hoc tests).</p

EspF_U can enhance macrocolony size using either the EHEC or EPEC version of Tir.

<p>(A) Polarized Caco-2 monolayers were infected for 6 h with the indicated KC12 and KC12Δ<i>tir</i> strains, fixed, and stained to visualize bacteria, DNA, and F-actin. (B) Experiments described in (A) were quantified. Each bar represents the mean macrocolony size (+/- SE) calculated from 6 coverslips (2025–3179 colonies). (C) The experiments in (B) were also used to quantify the % of monolayer area infected. Each bar represents the mean (+/- SE) from 59–60 FOVs. (D-E) Polarized Caco-2 monolayers were infected with EHEC strains for 8 h, fixed, and stained as in (A). Bars represent the mean macrocolony size (+/- SE) calculated from 315–617 macrocolonies. (F-G) Polarized Caco-2 monolayers were infected with WT EPEC strains with or without EspF_U. Each bar represents the mean macrocolony size (+/- SE) calculated from 1163–2722 macrocolonies. KC12+EspF_U is shown in purple. For all panels, scale circles, 100, 500, 1000 μm². ** p <0.01, *** p <0.001 (ANOVA, Tukey post-hoc tests). To allow for a sufficient number of Δ<i>tir</i> colonies that could be analyzed, colonies larger than 50 μm² were included in quantification, unlike <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006501#ppat.1006501.g004" target="_blank">Fig 4</a> where 100 μm² was the lower limit.</p

EspF_U-dependent actin pedestals allow for an efficient pathway of cell-to-cell transmission.

<p>(A) JEG-3 and HeLa cells infected for 6 h and 5 h, respectively, were stained to show bacteria (blue), F-actin (red), and HA-Tir (green). Individual events were ordered into the following sequence based on live imaging in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006501#ppat.1006501.g007" target="_blank">Fig 7</a>: bacteria (i) use pedestals to protrude and contact an uninfected neighboring cell, (ii) translocate effectors including Tir (arrowheads) into the second cell, (iii) polymerize a second pedestal (arrows), and (iv) use the secondary pedestal to dock bacteria at junctions as the bacteria divide. Scale bars, 3 μm. (B) The proposed model is based on data from experiments in Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006501#ppat.1006501.g007" target="_blank">7</a> and 8, and is shown to incorporate every step of the infectious life cycle. Green circles represent translocated effectors, red lines indicate F-actin in pedestals, and green ovals represent Tir.</p

The actin pedestals assembled by KC12+EspF_U and EPEC Y474* promote motility and exploration of the host cell surface.

<p>(A) NIH3T3 cells stably expressing mCherry-actin were infected with EPEC expressing GFP for 3 h and imaged live for 45 min. Scale bar, 10 μm. (B) mCherry-actin expressing cells were infected with the indicated strains for 3 h and imaged live for 18–20 min. 15–20 bacteria per host cell for each strain were tracked, and data from representative cells were plotted such that points starting at t = 0 were centered at the origin. (C) Bacterial motility rates were quantified from cells infected as in (B). Each bar represents the mean speed (+/- SE) of bacteria on 6–20 host cells (95–293 total bacteria). ** p<0.01, *p<0.05 (ANOVA, Tukey post-hoc tests). (D) The fraction of pedestals that were considered moving was quantified, using the average speed of the pedestal deficient counterpart strain as a minimum cutoff to define movement. Each bar represents the mean (+/- SE) from 227–320 pedestals. p = 0.3 (Fisher’s exact test). (E) Directional persistence of pedestals was calculated by dividing the maximum displacement by the total path length for pedestals considered to be moving. Each bar represents the mean (+/- SE) for 205–297 pedestals on 19–20 cells. p = 0.1 (unpaired <i>t</i> test) (F) Cells were infected with EHEC strains with or without EspF_U and imaged live. Each bar represents the mean speed (+/- SE) of bacteria on 6 cells (60 bacteria). *p<0.05 (ANOVA, Tukey post-hoc test).</p