MYO1F regulates antifungal immunity by regulating acetylation of microtubules.

Opportunistic fungal infections have become one of the leading causes of death among immunocompromised patients, resulting in an estimated 1.5 million deaths each year worldwide. The molecular mechanisms that promote host defense against fungal infections remain elusive. Here, we find that Myosin IF (MYO1F), an unconventional myosin, promotes the expression of genes that are critical for antifungal innate immune signaling and proinflammatory responses. Mechanistically, MYO1F is required for dectin-induced α-tubulin acetylation, acting as an adaptor that recruits both the adaptor AP2A1 and α-tubulin N-acetyltransferase 1 to α-tubulin; in turn, these events control the membrane-to-cytoplasm trafficking of spleen tyrosine kinase and caspase recruitment domain-containing protein 9 Myo1f-deficient mice are more susceptible than their wild-type counterparts to the lethal sequelae of systemic infection with Candida albicans Notably, administration of Sirt2 deacetylase inhibitors, namely AGK2, AK-1, or AK-7, significantly increases the dectin-induced expression of proinflammatory genes in mouse bone marrow-derived macrophages and microglia, thereby protecting mice from both systemic and central nervous system C. albicans infections. AGK2 also promotes proinflammatory gene expression in human peripheral blood mononuclear cells after Dectin stimulation. Taken together, our findings describe a key role for MYO1F in promoting antifungal immunity by regulating the acetylation of α-tubulin and microtubules, and our findings suggest that Sirt2 deacetylase inhibitors may be developed as potential drugs for the treatment of fungal infections.

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes.

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as "phagosome resolution." Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.

The Salmonella effector SseJ disrupts microtubule dynamics when ectopically expressed in normal rat kidney cells.

Salmonella effector protein SseJ is secreted by Salmonella into the host cell cytoplasm where it can then modify host cell processes. Whilst host cell small GTPase RhoA has previously been shown to activate the acyl-transferase activity of SseJ we show here an un-described effect of SseJ protein production upon microtubule dynamism. SseJ prevents microtubule collapse and this is independent of SseJ's acyl-transferase activity. We speculate that the effects of SseJ on microtubules would be mediated via its known interactions with the small GTPases of the Rho family.

Tricellular Tight Junction Protein Tricellulin Is Targeted by the Enteropathogenic Escherichia coli Effector EspG1, Leading to Epithelial Barrier Disruption.

Enteropathogenic Escherichia coli (EPEC)-induced diarrhea is often associated with disruption of intestinal epithelial tight junctions. Although studies have shown alterations in the expression and localization of bicellular tight junction proteins during EPEC infections, little is known about whether tricellular tight junction proteins (tTJs) are affected. Using Caco-2 cell monolayers, we investigated if EPEC is capable of targeting the tTJ protein tricellulin. Our results demonstrated that at 4 h postinfection, EPEC induced a significant reduction in tricellulin levels, accompanied by a significant loss of transepithelial resistance (TEER) and a corresponding increase in paracellular permeability. Conversely, cells overexpressing tricellulin were highly resistant to EPEC-induced barrier disruption. Confocal microscopy revealed the distribution of tricellulin into the plasma membrane of infected epithelial cells and confirmed the localization of EPEC aggregates in close proximity to tTJs. Moreover, infections with EPEC strains lacking genes encoding specific type III secreted effector proteins demonstrated a crucial role for the effector EspG1 in modulating tricellulin expression. Complementation studies suggest that the EspG-induced depletion of tricellulin is microtubule dependent. Overall, our results show that EPEC-induced epithelial barrier dysfunction is mediated in part by EspG1-induced microtubule-dependent depletion of tricellulin.

Two Small Molecules Block Oral Epithelial Cell Invasion by Porphyromons gingivalis.

Porphyromonas gingivalis is a keystone pathogen of periodontitis. One of its bacterial characteristics is the ability to invade various host cells, including nonphagocytic epithelial cells and fibroblasts, which is known to facilitate P. gingivalis adaptation and survival in the gingival environment. In this study, we investigated two small compounds, Alop1 and dynasore, for their role in inhibition of P. gingivalis invasion. Using confocal microscopy, we showed that these two compounds significantly reduced invasion of P. gingivalis and its outer membrane vesicles into human oral keratinocytes in a dose-dependent manner. The inhibitory effects of dynasore, a dynamin inhibitor, on the bacterial entry is consistent with the notion that P. gingivalis invasion is mediated by a clathrin-mediated endocytic machinery. We also observed that microtubule arrangement, but not actin, was altered in the host cells treated with Alop1 or dynasore, suggesting an involvement of microtubule in this inhibitory activity. This work provides an opportunity to develop compounds against P. gingivalis infection.

A Novel Fluorescent Labeling Method Enables Monitoring of Spatio-Temporal Dynamics of Developing Microsporidia.

The microsporidium, Anncaliia algerae (Brachiola algerae), is a eukaryotic obligate intracellular parasite first isolated from mosquitoes and is an important opportunistic human pathogen that can cause morbidity and mortality among immune-compromised individuals including patients with AIDS and those undergoing chemotherapy. There is little known about the Microsporidia-host cell interface in living host cells, due to current approaches being limited by the lack of fluorescent reporters for detecting the parasite lifecycle. Here, we have developed and applied novel vital fluorescent parasite labeling methodologies in conjunction with fluorescent protein-tagged reporters to track simultaneously the dynamics of both parasite and host cell specific components, including the secretory and endocytic trafficking pathways, during the entire infection time period. We have found dramatic changes in the dynamics of host secretory trafficking organelles during the course of infection. The Golgi compartment is gradually disassembled and regenerated into mini-Golgi structures in parallel with cellular microtubule depolymerization. Importantly, we find that Microsporidia progeny are associated with these de novo formed mini-Golgi structures. These host structures appear to create a membrane bound niche environment for parasite development. Our studies presented here provide novel imaging tools and methodologies that will facilitate in understanding the biology of microsporidial parasites in the living host.

Visualization of VirE2 protein translocation by the Agrobacterium type IV secretion system into host cells.

Type IV secretion systems (T4SS) can mediate the translocation of bacterial virulence proteins into host cells. The plant pathogen Agrobacterium tumefaciens uses a T4SS to deliver a VirD2-single stranded DNA complex as well as the virulence proteins VirD5, VirE2, VirE3, and VirF into host cells so that these become genetically transformed. Besides plant cells, yeast and fungi can efficiently be transformed by Agrobacterium. Translocation of virulence proteins by the T4SS has so far only been shown indirectly by genetic approaches. Here we report the direct visualization of VirE2 protein translocation by using bimolecular fluorescence complementation (BiFC) and Split GFP visualization strategies. To this end, we cocultivated Agrobacterium strains expressing VirE2 tagged with one part of a fluorescent protein with host cells expressing the complementary part, either fused to VirE2 (for BiFC) or not (Split GFP). Fluorescent filaments became visible in recipient cells 20-25 h after the start of the cocultivation indicative of VirE2 protein translocation. Evidence was obtained that filament formation was due to the association of VirE2 with the microtubuli.

Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking.

BACKGROUND: The developmental cycle of the obligate intracellular pathogen Chlamydia is dependant on the formation of a unique intracellular niche termed the chlamydial inclusion. The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins. A unique property of the inclusion is its propensity for homotypic fusion. The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion. The chlamydial protein IncA is required for fusion, however the host process involved are uncharacterized. RESULTS: Here, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion. We established that factors involved in trafficking were required for efficient fusion as both disruption of the microtubule network and inhibition of microtubule trafficking reduced the efficiency of fusion. Additionally, fusion occurred at multiple sites in the cell and was delayed when the microtubule minus ends were either no longer anchored at a single MTOC or when a cell possessed multiple MTOCs. CONCLUSIONS: The data presented demonstrates that efficient homotypic fusion requires the inclusions to be in close proximity and that this proximity is dependent on chlamydial microtubule trafficking to the minus ends of microtubules.

Centrosomal targeting of Syk kinase is controlled by its catalytic activity and depends on microtubules and the dynein motor.

The nonreceptor Syk kinase is detected in epithelial cells, where it acts as a tumor suppressor, in addition to its well-established role in immunoreceptor-based signal transduction in hematopoietic cells. Thus, several carcinomas and melanomas have subnormal concentrations of Syk. Although Syk is mainly localized at the plasma membrane, it is also present in centrosomes, where it is involved in the control of cell division. The mechanisms responsible for its centrosomal localization and action are unknown. We used wild-type and mutant fluorescent Syk fusion proteins in live-cell imaging (fluorescence recovery after photobleaching, total internal reflection fluorescence, and photoactivation) combined with mathematical modeling to demonstrate that Syk is actively transported to the centrosomes via the microtubules and that this transport depends on the dynein/dynactin molecular motor. Syk can only target the centrosomes if its kinase activity is intact and it is catalytically active at the centrosomes. We showed that the autophosphorylated Y130 Syk residue helps to uncouple Syk from the plasma membrane and to promote its translocation to the centrosome, suggesting that the subcellular location of Syk depends on its autophosphorylation on specific tyrosine residues. We have thus established the details of how Syk is trafficked intracellularly and found evidence that its targeting to the centrosomes is controlled by autophosphorylation.

Fierce competition between Toxoplasma and Chlamydia for host cell structures in dually infected cells.

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

Wolbachia bacteria reside in host Golgi-related vesicles whose position is regulated by polarity proteins.

Wolbachia pipientis are intracellular symbiotic bacteria extremely common in various organisms including Drosophila melanogaster, and are known for their ability to induce changes in host reproduction. These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We report here that Wolbachia are restricted only to a group of Golgi-related vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes suggesting that cell/tissue polarity genes are required for apical localization of these Golgi-related vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, appeared to be present in these Golgi-related vesicles. Thus, establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site.

EseG, an effector of the type III secretion system of Edwardsiella tarda, triggers microtubule destabilization.

Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. A type III secretion system (T3SS) was recently shown to contribute to pathogenesis, since deletions of various T3SS genes increased the 50% lethal dose (LD(50)) by about 1 log unit in the blue gourami infection model. In this study, we report EseG as the first identified effector protein of T3SS. EseG shares partial homology with two Salmonella T3SS effectors (SseG and SseF) over a conserved domain (amino acid residues 142 to 192). The secretion of EseG is dependent on a functional T3SS and, in particular, requires the chaperone EscB. Experiments using TEM-1 ß-lactamase as a fluorescence-based reporter showed that EseG was translocated into HeLa cells at 35°C. Fractionation of infected HeLa cells demonstrated that EseG was localized to the host membrane fraction after translocation. EseG is able to disassemble microtubule structures when overexpressed in mammalian cells. This phenotype may require a conserved motif of EseG (EseG(142-192)), since truncated versions of EseG devoid of this motif lose their ability to cause microtubule destabilization. By demonstrating the function of EseG, our study contributes to the understanding of E. tarda pathogenesis. Moreover, the approach established in this study to identify type III effectors can be used to identify and characterize more type III and possible type VI effectors in Edwardsiella.

Early intracellular trafficking of Waddlia chondrophila in human macrophages.

Waddlia chondrophila is an obligate intracellular bacterium considered as a potential agent of abortion in both humans and bovines. This member of the order Chlamydiales multiplies rapidly within human macrophages and induces lysis of the infected cells. To understand how this Chlamydia-like micro-organism invades and proliferates within host cells, we investigated its trafficking within monocyte-derived human macrophages. Vacuoles containing W. chondrophila acquired the early endosomal marker EEA1 during the first 30 min following uptake. However, the live W. chondrophila-containing vacuoles never co-localized with late endosome and lysosome markers. Instead of interacting with the endosomal pathway, W. chondrophila immediately co-localized with mitochondria and, shortly after, with endoplasmic reticulum- (ER-) resident proteins such as calnexin and protein disulfide isomerase. The acquisition of mitochondria and ER markers corresponds to the beginning of bacterial replication. It is noteworthy that mitochondrion recruitment to W. chondrophila inclusions is prevented only by simultaneous treatment with the microtubule and actin cytoskeleton-disrupting agents nocodazole and cytochalasin D. In addition, brefeldin A inhibits the replication of W. chondrophila, supporting a role for COPI-dependent trafficking in the biogenesis of the bacterial replicating vacuole. W. chondrophila probably survives within human macrophages by evading the endocytic pathway and by associating with mitochondria and the ER. The intracellular trafficking of W. chondrophila in human macrophages represents a novel route that differs strongly from that used by other members of the order Chlamydiales.

Symmetric and asymmetric mitotic segregation patterns influence Wolbachia distribution in host somatic tissue.

Wolbachia are maternally inherited bacterial endosymbionts that occupy many but not all tissues of adult insects. During the initial mitotic divisions in Drosophila embryogenesis, Wolbachia exhibit a symmetric pattern of segregation. Wolbachia undergo microtubule-dependent and cell-cycle-regulated movement between centrosomes. Symmetric segregation occurs during late anaphase when Wolbachia cluster around duplicated and separating centrosomes. This centrosome association is microtubule-dependent and promotes an even Wolbachia distribution throughout the host embryo. By contrast, during the later embryonic and larval neuroblast divisions, Wolbachia segregate asymmetrically with the apical self-renewing neuroblast. During these polarized asymmetric neuroblast divisions, Wolbachia colocalize with the apical centrosome and apically localized Par complex. This localization depends on microtubules, but not the cortical actin-based cytoskeleton. We also found that Wolbachia concentrate in specific regions of the adult brain, which might be a direct consequence of the asymmetric Wolbachia segregation in the earlier neuroblast divisions. Finally, we demonstrate that the fidelity of asymmetric segregation to the self-renewing neuroblast is lower in the virulent Popcorn strain of Wolbachia.

Outer membrane protein A expression in Enterobacter sakazakii is required to induce microtubule condensation in human brain microvascular endothelial cells for invasion.

Enterobacter sakazakii (ES) causes neonatal meningitis and necrotizing enterocolitis with case-fatality rates among infected infants ranging from 40 to 80%. Very little is known about the mechanisms by which these organisms cause disease. Here, we demonstrate that ES invades human brain microvascular endothelial cells (HBMEC) with higher frequency when compared with epithelial cells and endothelial cells from different origins. The entry of ES into HBMEC requires the expression of outer membrane protein A (OmpA), as the OmpA-deletion mutant was sevenfold less invasive than the wild type ES and the bacterium does not multiply inside HBMEC. Anti-OmpA antibodies generated against the OmpA of Escherichia coli K1, which also recognize the OmpA of ES, did not prevent the invasion of ES in HBMEC. ES invasion depends on microtubule condensation in HBMEC and is independent of actin filament reorganization. Both PI3-kinase and PKC-alpha were activated during ES entry into HBMEC between 15 min and 30 min of infection. Concomitantly, overexpression of dominant negative forms of PI3-kinase and PKC-alpha significantly inhibited the invasion of ES into HBMEC. In summary, ES invasion of HBMEC is dependent on the expression of OmpA similar to that of E. coli K1; however, the epitopes involved in the interaction with HBMEC appears to be different.

Microtubules in basalt glass from Hawaii Scientific Driling Project #2 phase 1 core and Hilina slope, Hawaii: evidence of the occurrence and behavior of endolithic microorganisms.

Elongate, fine tubes, approximately 1 microm wide and up to 200 microm long, extend from fractured surfaces, vesicle walls, and internal fractures into fragments of basalt glass in samples from the Hawaii Scientific Drilling Project #2 phase 1 (HSDP #2(1)) core and the Hilina slope, Hawaii. Several features indicate that these tubes are microbial endolithic microborings: the tubes resemble many described microborings from oceanic basalt glass, their formation is postdepositional but restricted to certain but different ranges of time in the two sets of samples, and they are not uniformly distributed throughout glass fragments. Microtubules record several characteristic behaviors including boring into glass, mining, seeking olivine, and avoiding plagioclase. They also are highly associated with a particular form of glass-replacing smectite. Evidence of behavior should join morphological and geochemical criteria in indicating microbial alteration of basalt glass. In some samples, steeply conical tubes, approximately 10-20 microm in diameter tapering to 1 microm and commonly filled with smectite, appear to be modifications or elaborations of the microtubules. These also curve toward olivine and are associated with replacement smectite. In HSDP #2(1) samples, microtubules initiated at margins of shards before palagonite replaced those margins and are preserved during palagonitization. In fact, microtubules appear to have provided routes that enhanced the efficiency of water's reaching of unaltered glass. In Hilina Slope samples, the microtubules appear to postdate palagonitization because they initiate at the boundary between palagonite and unaltered sideromelane. Preservation of microtubules during palagonitization in samples together with recognition of other associated characteristics representing behavior suggests that such features may be recognizable in more heavily altered ancient rocks.

A cellular basis for Wolbachia recruitment to the host germline.

Wolbachia are among the most widespread intracellular bacteria, carried by thousands of metazoan species. The success of Wolbachia is due to efficient vertical transmission by the host maternal germline. Some Wolbachia strains concentrate at the posterior of host oocytes, which promotes Wolbachia incorporation into posterior germ cells during embryogenesis. The molecular basis for this localization strategy is unknown. Here we report that the wMel Wolbachia strain relies upon a two-step mechanism for its posterior localization in oogenesis. The microtubule motor protein kinesin-1 transports wMel toward the oocyte posterior, then pole plasm mediates wMel anchorage to the posterior cortex. Trans-infection tests demonstrate that factors intrinsic to Wolbachia are responsible for directing posterior Wolbachia localization in oogenesis. These findings indicate that Wolbachia can direct the cellular machinery of host oocytes to promote germline-based bacterial transmission. This study also suggests parallels between Wolbachia localization mechanisms and those used by other intracellular pathogens.

Intracellular Salmonella enterica redirect exocytic transport processes in a Salmonella pathogenicity island 2-dependent manner.

During intracellular life, Salmonella enterica proliferate within a specialized membrane compartment, the Salmonella-containing vacuole (SCV), and interfere with the microtubule cytoskeleton and cellular transport. To characterize the interaction of intracellular Salmonella with host cell transport processes, we utilized various model systems to follow microtubule-dependent transport. The vesicular stomatitis virus glycoprotein (VSVG) is a commonly used marker to follow protein transport from the Golgi to the plasma membrane. Using a VSVG-GFP fusion protein, we observed that virulent intracellular Salmonella alter exocytotic transport and recruit exocytotic transport vesicles to the SCV. This virulence function was dependent on the function of the type III secretion system encoded by Salmonella Pathogenicity Island 2 (SPI2) and more specifically on a subset of SPI2 effector proteins. Furthermore, the Golgi to plasma membrane traffic of the shingolipid C(5)-ceramide was redirected to the SCV by virulent Salmonella. We propose that Salmonella modulates the biogenesis of the SCV by deviating this compartment from the default endocytic pathway to an organelle that interacts with the exocytic pathway. This observation might reveal a novel element of the intracellular survival and replication strategy of Salmonella.

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum.

Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria.