Listeria monocytogenes Co-Opts the Host Exocyst Complex To Promote Internalin A-Mediated Entry.

The bacterial pathogen Listeria monocytogenes induces its internalization (entry) into intestinal epithelial cells through interaction of its surface protein, internalin A (InlA), with the human cell-cell adhesion molecule, E-cadherin. While InlA-mediated entry requires bacterial stimulation of actin polymerization, it remains unknown whether additional host processes are manipulated to promote internalization. Here, we show that interaction of InlA with E-cadherin induces the host membrane-trafficking process of polarized exocytosis, which augments uptake of Listeria. Imaging studies revealed that exocytosis is stimulated at sites of InlA-dependent internalization. Experiments inhibiting human N-ethylmaleimide-sensitive factor (NSF) demonstrated that exocytosis is needed for efficient InlA-mediated entry. Polarized exocytosis is mediated by the exocyst complex, which comprises eight proteins, including Sec6, Exo70, and Exo84. We found that Exo70 was recruited to sites of InlA-mediated entry. In addition, depletion of Exo70, Exo84, or Sec6 by RNA interference impaired entry without affecting surface levels of E-cadherin. Similar to binding of InlA to E-cadherin, homophilic interaction of E-cadherin molecules mobilized the exocyst and stimulated exocytosis. Collectively, these results demonstrate that ligation of E-cadherin induces exocytosis that promotes Listeria entry, and they raise the possibility that the exocyst might also control the normal function of E-cadherin in cell-cell adhesion.

Shigella flexneri subverts host polarized exocytosis to enhance cell-to-cell spread.

Shigella flexneri is a gram-negative bacterial pathogen that causes dysentery. Critical for disease is the ability of Shigella to use an actin-based motility (ABM) process to spread between cells of the colonic epithelium. ABM transports bacteria to the periphery of host cells, allowing the formation of plasma membrane protrusions that mediate spread to adjacent cells. Here we demonstrate that efficient protrusion formation and cell-to-cell spread of Shigella involves bacterial stimulation of host polarized exocytosis. Using an exocytic probe, we found that exocytosis is locally upregulated in bacterial protrusions in a manner that depends on the Shigella type III secretion system. Experiments involving RNA interference (RNAi) indicate that efficient bacterial protrusion formation and spread require the exocyst, a mammalian multi-protein complex known to mediate polarized exocytosis. In addition, the exocyst component Exo70 and the exocyst regulator RalA were recruited to Shigella protrusions, suggesting that bacteria manipulate exocyst function. Importantly, RNAi-mediated depletion of exocyst proteins or RalA reduced the frequency of protrusion formation and also the lengths of protrusions, demonstrating that the exocyst controls both the initiation and elongation of protrusions. Collectively, our results reveal that Shigella co-opts the exocyst complex to disseminate efficiently in host cell monolayers.

Role of internalin proteins in the pathogenesis of Listeria monocytogenes.

Listeria monocytogenes is a food-borne bacterium that causes gastroenteritis, meningitis, or abortion. L. monocytogenes induces its internalization (entry) into human cells and either spreads laterally in tissues or transcytoses to traverse anatomical barriers. In this review, we discuss mechanisms by which five structurally related proteins of the "internalin" family of L. monocytogenes (InlA, InlB, InlC, InlF, and InlP) interact with distinct host receptors to promote infection of human cells and/or crossing of the intestinal, blood-brain, or placental barriers. We focus on recent results demonstrating that the internalin proteins InlA, InlB, and InlC exploit exocytic pathways to stimulate transcytosis, entry, or cell-to-cell spread, respectively. We also discuss evidence that InlA-mediated transcytosis contributes to traversal of the intestinal barrier, whereas InlF promotes entry into endothelial cells to breach the blood-brain barrier. InlB also facilitates the crossing of the blood-brain barrier, but does so by extending the longevity of infected monocytes that may subsequently act as a "Trojan horse" to transfer bacteria to the brain. InlA, InlB, and InlP each contribute to fetoplacental infection by targeting syncytiotrophoblast or cytotrophoblast layers of the placenta. This work highlights the diverse functions of internalins and the complex mechanisms by which these structurally related proteins contribute to disease.

The Host GTPase Arf1 and Its Effectors AP1 and PICK1 Stimulate Actin Polymerization and Exocytosis To Promote Entry of Listeria monocytogenes.

Listeria monocytogenes is a foodborne bacterium that causes gastroenteritis, meningitis, or abortion. Listeria induces its internalization (entry) into some human cells through interaction of the bacterial surface protein InlB with its host receptor, the Met tyrosine kinase. InlB and Met promote entry through stimulation of localized actin polymerization and exocytosis. How actin cytoskeletal changes and exocytosis are controlled during entry is not well understood. Here, we demonstrate important roles for the host GTPase Arf1 and its effectors AP1 and PICK1 in actin polymerization and exocytosis during InlB-dependent uptake. Depletion of Arf1 by RNA interference (RNAi) or inhibition of Arf1 activity using a dominant-negative allele impaired InlB-dependent internalization, indicating an important role for Arf1 in this process. InlB stimulated an increase in the GTP-bound form of Arf1, demonstrating that this bacterial protein activates Arf1. RNAi and immunolocalization studies indicated that Arf1 controls exocytosis and actin polymerization during entry by recruiting the effectors AP1 and PICK1 to the plasma membrane. In turn, AP1 and PICK1 promoted plasma membrane translocation of both Filamin A (FlnA) and Exo70, two host proteins previously found to mediate exocytosis during InlB-dependent internalization (M. Bhalla, H. Van Ngo, G. C. Gyanwali, and K. Ireton, Infect Immun 87:e00689-18, 2018, https://doi.org/10.1128/IAI.00689-18). PICK1 mediated recruitment of Exo70 but not FlnA. Collectively, these results indicate that Arf1, AP1, and PICK1 stimulate exocytosis by redistributing FlnA and Exo70 to the plasma membrane. We propose that Arf1, AP1, and PICK1 are key coordinators of actin polymerization and exocytosis during infection of host cells by Listeria.

Host endoplasmic reticulum COPII proteins control cell-to-cell spread of the bacterial pathogen Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen that uses actin-dependent motility to spread between human cells. Cell-to-cell spread involves the formation by motile bacteria of plasma membrane-derived structures termed 'protrusions'. In cultured enterocytes, the secreted Listeria protein InlC promotes protrusion formation by binding and inhibiting the human scaffolding protein Tuba. Here we demonstrate that protrusions are controlled by human COPII components that direct trafficking from the endoplasmic reticulum. Co-precipitation experiments indicated that the COPII proteins Sec31A and Sec13 interact directly with a Src homology 3 domain in Tuba. This interaction was antagonized by InlC. Depletion of Sec31A or Sec13 restored normal protrusion formation to a Listeria mutant lacking inlC, without affecting spread of wild-type bacteria. Genetic impairment of the COPII component Sar1 or treatment of cells with brefeldin A affected protrusions similarly to Sec31A or Sec13 depletion. These findings indicated that InlC relieves a host-mediated restriction of Listeria spread otherwise imposed by COPII. Inhibition of Sec31A, Sec13 or Sar1 or brefeldin A treatment also perturbed the structure of cell-cell junctions. Collectively, these findings demonstrate an important role for COPII in controlling Listeria spread. We propose that COPII may act by delivering host proteins that generate tension at cell junctions.

Impact of the Listeria monocytogenes protein InlC on infection in mice.

The bacterial pathogen Listeria monocytogenes causes serious food-borne illnesses in pregnant women and the immunocompromised. L. monocytogenes promotes its internalization into host epithelial cells and then uses an F-actin-dependent motility process to spread from infected cells to surrounding healthy cells. In cultured enterocytes, efficient spread of L. monocytogenes requires the secreted bacterial protein InlC. InlC promotes dissemination by physically interacting with and antagonizing the function of the human adaptor protein Tuba. Here we examine the role of InlC and its interaction with host Tuba during infection in mice. The study took advantage of a single-amino-acid substitution (K173A) in InlC that impairs binding to human Tuba but does not affect InlC-mediated inhibition of the NF-κB pathway. Mice were inoculated intravenously with the wild-type L. monocytogenes strain EGD, an isogenic strain deleted for the inlC gene (ΔinlC), or a strain expressing K173A mutant InlC (inlC.K173A). The 50% lethal doses (LD(50)) for the ΔinlC or inlC.K173A mutant strain were approximately 4- or 6-fold greater than that for the wild-type strain, indicating a role for inlC in virulence. Compared to the wild-type strain, the inlC.K173A mutant strain exhibited lower bacterial loads in the liver. Histological analysis of livers indicated that the two inlC mutant strains produced smaller foci of infection than did the wild-type strain. These smaller foci are consistent with a role for InlC in cell-to-cell spread in vivo. Taken together, these results provide evidence that interaction of InlC with host Tuba is important for full virulence.

The X-linked intellectual disability protein TSPAN7 regulates excitatory synapse development and AMPAR trafficking.

Mutations in TSPAN7--a member of the tetraspanin protein superfamily--are implicated in some forms of X-linked intellectual disability. Here we show that TSPAN7 overexpression promotes the formation of filopodia and dendritic spines in cultured hippocampal neurons from embryonic rats, whereas TSPAN7 silencing reduces head size and stability of spines and AMPA receptor currents. Via its C terminus, TSPAN7 interacts with the PDZ domain of protein interacting with C kinase 1 (PICK1), to regulate PICK1 and GluR2/3 association and AMPA receptor trafficking. These findings indicate that, in hippocampal neurons, TSPAN7 regulates AMPA receptor trafficking by limiting PICK1 accessibility to AMPA receptors and suggest an additional mechanism for the functional maturation of glutamatergic synapses, whose impairment is implicated in intellectual disability.

Identification of components of the host type IA phosphoinositide 3-kinase pathway that promote internalization of Listeria monocytogenes.

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria promotes its internalization into some human cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. The interaction of InlB with the Met receptor stimulates host signaling pathways that promote cell surface changes driving bacterial uptake. One human signaling protein that plays a critical role in Listeria entry is type IA phosphoinositide 3-kinase (PI 3-kinase). The molecular mechanism by which PI 3-kinase promotes bacterial internalization is not understood. Here we perform an RNA interference (RNAi)-based screen to identify components of the type IA PI 3-kinase pathway that control the entry of Listeria into the human cell line HeLa. The 64 genes targeted encode known upstream regulators or downstream effectors of type IA PI 3-kinase. The results of this screen indicate that at least 9 members of the PI 3-kinase pathway play important roles in Listeria uptake. These 9 human proteins include a Rab5 GTPase, several regulators of Arf or Rac1 GTPases, and the serine/threonine kinases phosphoinositide-dependent kinase 1 (PDK1), mammalian target of rapamycin (mTor), and protein kinase C-ζ. These findings represent a key first step toward understanding the mechanism by which type IA PI 3-kinase controls bacterial internalization.

A circadian clock in hippocampus is regulated by interaction between oligophrenin-1 and Rev-erbα.

Oligophrenin-1 regulates dendritic spine morphology in the brain. Mutations in the oligophrenin-1 gene (OPHN1) cause intellectual disability. We discovered a previously unknown partner of oligophrenin-1, Rev-erbα, a nuclear receptor that represses the transcription of circadian oscillators. We found that oligophrenin-1 interacts with Rev-erbα in the mouse brain, causing it to locate to dendrites, reducing its repressor activity and protecting it from degradation. Our results indicate the presence of a circadian oscillator in the hippocampus, involving the clock gene Bmal1 (also known as Arntl), that is modulated by Rev-erbα and requires oligophrenin-1 for normal oscillation. We also found that synaptic activity induced Rev-erbα localization to dendrites and spines, a process that is mediated by AMPA receptor activation and requires oligophrenin-1. Our data reveal new interactions between synaptic activity and circadian oscillators, and delineate a new means of communication between nucleus and synapse that may provide insight into normal plasticity and the etiology of intellectual disability.

Critical role for the host GTPase-activating protein ARAP2 in InlB-mediated entry of Listeria monocytogenes.

The bacterial pathogen Listeria monocytogenes causes food-borne illnesses culminating in gastroenteritis, meningitis, or abortion. Listeria induces its internalization into some mammalian cells through binding of the bacterial surface protein InlB to the host receptor tyrosine kinase Met. Interaction of InlB with the Met receptor elicits host downstream signaling pathways that promote F-actin cytoskeletal changes responsible for pathogen engulfment. Here we show that the mammalian signaling protein ARAP2 plays a critical role in cytoskeletal remodeling and internalization of Listeria. Depletion of ARAP2 through RNA interference (RNAi) caused a marked inhibition of InlB-mediated F-actin rearrangements and bacterial entry. ARAP2 contains multiple functional domains, including a GTPase-activating protein (GAP) domain that antagonizes the GTPase Arf6 and a domain capable of binding the GTPase RhoA. Genetic data indicated roles for both the Arf GAP and RhoA binding domains in Listeria entry. Experiments involving Arf6 RNAi or a constitutively activated allele of Arf6 demonstrated that one of the ways in which ARAP2 promotes bacterial uptake is by restraining the activity of Arf6. Conversely, Rho activity was dispensable for Listeria internalization, suggesting that the RhoA binding domain in ARAP2 acts by engaging a host ligand other than Rho proteins. Collectively, our findings indicate that ARAP2 promotes InlB-mediated entry of Listeria, in part, by antagonizing the host GTPase Arf6.

Direct microfabrication of topographical and chemical cues for the guided growth of neural cell networks on polyamidoamine hydrogels.

Cell patterning is an important tool for organizing cells in surfaces and to reproduce in a simple way the tissue hierarchy and complexity of pluri-cellular life. The control of cell growth, proliferation and differentiation on solid surfaces is consequently important for prosthetics, biosensors, cell-based arrays, stem cell therapy and cell-based drug discovery concepts. We present a new electron beam lithography method for the direct and simultaneous fabrication of sub-micron topographical and chemical patterns, on a biocompatible and biodegradable PAA hydrogel. The localized e-beam modification of a hydrogel surface makes the pattern able to adsorb proteins in contrast with the anti-fouling surface. By also exploiting the selective attachment, growth and differentiation of PC12 cells, we fabricated a neural network of single cells connected by neuritis extending along microchannels. E-beam microlithography on PAA hydrogels opens up the opportunity of producing multifunctional microdevices incorporating complex topographies, allowing precise control of the growth and organization of individual cells.

A postsynaptic signaling pathway that may account for the cognitive defect due to IL1RAPL1 mutation.

BACKGROUND: Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) gene mutations are associated with cognitive impairment ranging from nonsyndromic X-linked mental retardation to autism. IL1RAPL1 belongs to a novel family of Toll/IL-1 receptors, whose expression in the brain is upregulated by neuronal activity. Currently, very little is known about the function of this protein. We previously showed that IL1RAPL1 interacts with the neuronal calcium sensor NCS-1 and that it regulates voltage-gated calcium channel activity in PC12 cells. RESULTS: Here we show that IL1RAPL1 is present in dendritic spine where it interacts with PSD-95, a major component of excitatory postsynaptic compartment. Using gain- and loss-of-function experiments in neurons, we demonstrated that IL1RAPL1 regulates the synaptic localization of PSD-95 by controlling c-Jun terminal kinase (JNK) activity and PSD-95 phosphorylation. Mice carrying a null mutation of the mouse Il1rapl1 gene show a reduction of both dendritic spine density and excitatory synapses in the CA1 region of the hippocampus. These structural abnormalities are associated with specific deficits in hippocampal long-term synaptic plasticity. CONCLUSION: The interaction of IL1RAPL1 with PSD-95 discloses a novel pathophysiological mechanism of cognitive impairment associated with alterations of the JNK pathway leading to a mislocalization of PSD-95 and abnormal synaptic organization and function.

Biomimetic poly(amidoamine) hydrogels as synthetic materials for cell culture.

BACKGROUND: Poly(amidoamine)s (PAAs) are synthetic polymers endowed with many biologically interesting properties, being highly biocompatible, non toxic and biodegradable. Hydrogels based on PAAs can be easily modified during the synthesis by the introduction of functional co-monomers. Aim of this work is the development and testing of novel amphoteric nanosized poly(amidoamine) hydrogel film incorporating 4-aminobutylguanidine (agmatine) moieties to create RGD-mimicking repeating units for promoting cell adhesion. RESULTS: A systematic comparative study of the response of an epithelial cell line was performed on hydrogels with agmatine and on non-functionalized amphoteric poly(amidoamine) hydrogels and tissue culture plastic substrates. The cell adhesion on the agmatine containing substrates was comparable to that on plastic substrates and significantly enhanced with respect to the non-functionalized controls. Interestingly, spreading and proliferation on the functionalized supports are slower than on plastic exhibiting the possibility of an easier control of the cell growth kinetics. In order to favor the handling of the samples, a procedure for the production of bi-layered constructs was also developed by means the deposition via spin coating of a thin layer of hydrogel on a pre-treated cover slip. CONCLUSION: The obtained results reveal that PAAs hydrogels can be profitably functionalized and, in general, undergo physical and chemical modifications to meet specific requirements. In particular the incorporation of agmatine warrants good potential in the field of cell culturing and the development of supported functionalized hydrogels on cover glass are very promising substrates for applications in cell screening devices.