From anatomy to immunity in the gastrointestinal system.

The gastrointestinal system is classically known for its function in digesting food for nutrient uptake, but it plays a much larger role in the general health of organisms. Understanding the relationships between the gastrointestinal tract and inflammation, the nervous system, diseases caused through disregulation of molecular components as well as its association with beneficial and pathogenic microbes have been the focus of intense research over the many decades. In this Special Issue we delve into histological, molecular, and evolutionary aspects of gastrointestinal system components in healthy and diseased tissues, to give a broad perspective on the different organs that make-up this system.

Ube2N is present and functions within listeria Actin-rich structures and lamellipodia: A localization and pharmacological inhibition study.

The actin cytoskeleton forms much of the structure needed for the intracellular motility of an assortment of microbes as well as entire cells. The co-factor to the ubiquitin conjugating enzyme Ube2N (Ube2V1) has been implicated in both cancer cell metastasis and lysine-63 ubiquitylation of ß actin. As this protein complexes with Ube2N, we sought to investigate whether Ube2N itself was involved in actin-based events occurring during the Listeria monocytogenes infections as well as within motile whole cells. Through examination of L. monocytogenes actin clouds, comet tails and membrane protrusions as well as lamellipodia in migrating cells, we show that Ube2N is recruited to actin-rich structures. When pharmacologically inhibited we demonstrate that Ube2N is crucial for the function of actin-rich structures when associated with the plasma membrane.

Localization of host endocytic and actin-associated proteins during Shigella flexneri intracellular motility and intercellular spreading.

Shigella flexneri (S. flexneri), the causative agent of bacillary dysentery, uses an effector-mediated strategy to hijack host cells and cause disease. To propagate and spread within human tissues, S. flexneri bacteria commandeer the host actin cytoskeleton to generate slender actin-rich comet tails to move intracellularly, and later, plasma membrane actin-based protrusions to move directly between adjacent host cells. To facilitate intercellular bacterial spreading, large micron-sized endocytic-like membrane invaginations form at the periphery of neighboring host cells that come into contact with S. flexneri-containing membrane protrusions. While S. flexneri comet tails and membrane protrusions consist primarily of host actin cytoskeletal proteins, S. flexneri membrane invaginations remain poorly understood with only clathrin and the clathrin adapter epsin-1 localized to the structures. Tangentially, we recently reported that Listeria monocytogenes, another actin-hijacking pathogen, exploits an assortment of caveolar and actin-bundling proteins at their micron-sized membrane invaginations formed during their cell-to-cell movement. Thus, to further characterize the S. flexneri disease process, we set out to catalog the distribution of a variety of actin-associated and caveolar proteins during S. flexneri actin-based motility and cell-to-cell spreading. Here we show that actin-associated proteins found at L. monocytogenes comet tails and membrane protrusions mimic those present at S. flexneri comet tails with the exception of α-actinins 1 and 4, which were shed from S. flexneri membrane protrusions. We also demonstrate that all known host endocytic components found at L. monocytogenes membrane invaginations are also present at those formed during S. flexneri infections.

mDia1 Assembles a Linear F-Actin Coat at Membrane Invaginations To Drive Listeria monocytogenes Cell-to-Cell Spreading.

Direct cell-to-cell spreading of Listeria monocytogenes requires the bacteria to induce actin-based finger-like membrane protrusions in donor host cells that are endocytosed through caveolin-rich membrane invaginations by adjacent receiving cells. An actin shell surrounds these endocytic sites; however, its structure, composition, and functional significance remain elusive. Here, we show that the formin mDia1, but surprisingly not the Arp2/3 complex, is enriched at the membrane invaginations generated by L. monocytogenes during HeLa and Jeg-3 cell infections. Electron microscopy reveals a band of linear actin filaments that run along the longitudinal axis of the invagination membrane. Mechanistically, mDia1 expression is vital for the assembly of this F-actin shell. mDia1 is also required for the recruitment of Filamin A, a caveola-associated F-actin cross-linking protein, and caveolin-1 to the invaginations. Importantly, mixed-cell infection assays show that optimal caveolin-based L. monocytogenes cell-to-cell spreading correlates with the formation of the linear actin filament-containing shell by mDia1. IMPORTANCE Listeria monocytogenes spreads from one cell to another to colonize tissues. This cell-to-cell movement requires the propulsive force of an actin-rich comet tail behind the advancing bacterium, which ultimately distends the host plasma membrane into a slender bacterium-containing membrane protrusion. These membrane protrusions induce a corresponding invagination in the membrane of the adjacent host cell. The host cell that receives the protrusion utilizes caveolin-based endocytosis to internalize the structures, and filamentous actin lines these membrane invaginations. Here, we set out to determine the structure and function of this filamentous actin "shell." We demonstrate that the formin mDia1, but not the Arp2/3 complex, localizes to the invaginations. Morphologically, we show that this actin is organized into linear arrays and not branched dendritic networks. Mechanistically, we show that the actin shell is assembled by mDia1 and that mDia1 is required for efficient cell-to-cell transfer of L. monocytogenes.

Distribution of PDLIM1 at actin-rich structures generated by invasive and adherent bacterial pathogens.

The enteric bacterial pathogens Listeria monocytogenes (Listeria) and enteropathogenic Escherichia coli (EPEC) remodel the eukaryotic actin cytoskeleton during their disease processes. Listeria generate slender actin-rich comet/rocket tails to move intracellularly, and later, finger-like membrane protrusions to spread amongst host cells. EPEC remain extracellular, but generate similar actin-rich membranous protrusions (termed pedestals) to move atop the host epithelia. These structures are crucial for disease as diarrheal (and systemic) infections are significantly abrogated during infections with mutant strains that are unable to generate the structures. The current repertoire of host components enriched within these structures is vast and diverse. In this protein catalog, we and others have found that host actin crosslinkers, such as palladin and α-actinin-1, are routinely exploited. To expand on this list, we set out to investigate the distribution of PDLIM1, a scaffolding protein and binding partner of palladin and α-actinin-1, during bacterial infections. We show that PDLIM1 localizes to the site of initial Listeria entry into cells. Following this, PDLIM1 localizes to actin filament clouds surrounding immotile bacteria, and then colocalizes with actin once the comet/rocket tails are generated. Unlike palladin or α-actinin-1, PDLIM1 is maintained within the actin-rich core of membrane protrusions. Conversely, α-actinin-1, but not PDLIM1 (or palladin), is enriched at the membrane invagination that internalizes the Listeria-containing membrane protrusion. We also show that PDLIM1 is a component of the EPEC pedestal core and that its recruitment is dependent on the bacterial effector Tir. Our findings highlight PDLIM1 as another protein present within pathogen-induced actin-rich structures.

Localization of alpha-actinin-4 during infections by actin remodeling bacteria.

Bacterial pathogens cause disease by subverting the structure and function of their target host cells. Several foodborne agents such as Listeria monocytogenes (L. monocytogenes), Shigella flexneri (S. flexneri), Salmonella enterica serovar Typhimurium (S. Typhimurium) and enteropathogenic Escherichia coli (EPEC) manipulate the host actin cytoskeleton to cause diarrheal (and systemic) infections. During infections, these invasive and adherent pathogens hijack the actin filaments of their host cells and rearrange them into discrete actin-rich structures that promote bacterial adhesion (via pedestals), invasion (via membrane ruffles and endocytic cups), intracellular motility (via comet/rocket tails) and/or intercellular dissemination (via membrane protrusions and invaginations). We have previously shown that actin-rich structures generated by L. monocytogenes contain the host actin cross-linker α-actinin-4. Here we set out to examine α-actinin-4 during other key steps of the L. monocytogenes infectious cycle as well as characterize the subcellular distribution of α-actinin-4 during infections with other model actin-hijacking bacterial pathogens (S. flexneri, S. Typhimurium and EPEC). Although α-actinin-4 is absent at sites of initial L. monocytogenes invasion, we show that it is a new component of the membrane invaginations formed during secondary infections of neighboring host cells. Importantly, we reveal that α-actinin-4 also localizes to the major actin-rich structures generated during cell culture infections with S. flexneri (comet/rocket tails and membrane protrusions), S. Typhimurium (membrane ruffles) and EPEC (pedestals). Taken together, these findings suggest that α-actinin-4 is a host factor that is exploited by an assortment of actin-hijacking bacterial pathogens.

Listeria monocytogenes Exploits Host Caveolin for Cell-to-Cell Spreading.

Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens.IMPORTANCEListeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1-based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1-based internalization event can exceed the theoretical size limit for this endocytic pathway.

Klebsiella pneumoniae Redistributes Katanin Severing Proteins and Alters Astral Microtubules during Mitosis.

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.

Edwardsiella induces microtubule-severing in host epithelial cells.

Edwardsiella bacteria cause economic losses to a variety of commercially important fish globally. Human infections are rare and result in a gastroenteritis-like illness. Because these bacteria are evolutionarily related to other Enterobacteriaceae and the host cytoskeleton is a common target of enterics, we hypothesized that Edwardsiella may cause similar phenotypes. Here we use HeLa and Caco-2 infection models to show that microtubules are severed during the late infections. This microtubule alteration phenotype was not dependant on the type III or type VI secretion system (T3SS and T6SS) of the bacteria as ΔT3SS and ΔT6SS mutants of E. piscicida EIB202 and E. tarda ATCC15947 that lacks both also caused microtubule disassembly. Immunolocalization experiments showed the host katanin catalytic subunits A1 and A like 1 proteins at regions of microtubule severing, suggesting their involvement in the microtubule disassembly events. To identify bacterial components involved in this phenotype, we screened a 2,758 transposon library of E. piscicida EIB202 and found that 4 single mutations in the atpFHAGDC operon disrupted microtubule disassembly in HeLa cells. We then constructed three atp deletion mutants; they all could not disassemble host microtubules. This work provides the first clear evidence of host cytoskeletal alterations during Edwardsiella infections.

Distribution of CD147 During Enteropathogenic Escherichia coli and Salmonella enterica Serovar Typhimurium Infections.

Enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are highly infectious gastrointestinal human pathogens. These microbes inject bacterial-derived effector proteins directly into the host cell cytosol as part of their disease processes. A common host subcellular target of these pathogens is the actin cytoskeleton, which is commandeered by the bacteria and is used during their attachment onto (EPEC) or invasion into (S. Typhimurium) the host cells. We previously demonstrated that the host enzyme cyclophilin A (CypA) is recruited to the actin-rich regions of EPEC pedestals and S. Typhimurium membrane ruffles. To further expand the growing catalogue of host proteins usurped by actin-hijacking bacteria, we examined the host plasma membrane protein and cognate receptor of CypA, CD147, during EPEC and S. Typhimurium infections. Here, we show that CD147 is enriched at the basolateral regions of pedestals but, unlike CypA, it is absent from their actin-rich core. We show that the CD147 recruitment to these areas requires EPEC pedestal formation and not solely bacteria-host cell contact. Additionally, we demonstrate that the depletion of CD147 by siRNA does not alter the formation of pedestals. Finally, we show that CD147 is also a component of actin-rich membrane ruffles generated during S. Typhimurium invasion of host cells. Collectively, our findings establish CD147 as another host component present at dynamic actin-rich structures formed during bacterial infections. Anat Rec, 302:2224-2232, 2019. © 2019 American Association for Anatomy.

Listeria monocytogenes hijacks CD147 to ensure proper membrane protrusion formation and efficient bacterial dissemination.

Efficient cell-to-cell transfer of Listeria monocytogenes (L. monocytogenes) requires the proper formation of actin-rich membrane protrusions. To date, only the host proteins ezrin, the binding partner of ezrin, CD44, as well as cyclophilin A (CypA) have been identified as crucial components for L. monocytogenes membrane protrusion stabilization and, thus, efficient cell-to-cell movement of the microbes. Here, we examine the classical binding partner of CypA, CD147, and find that this membrane protein is also hijacked by the bacteria for their cellular dissemination. CD147 is enriched at the plasma membrane surrounding the membrane protrusions as well as the resulting invaginations generated in neighboring cells. In cells depleted of CD147, these actin-rich structures appear similar to those generated in CypA depleted cells as they are significantly shorter and more contorted as compared to their straighter counterparts formed in wild-type control cells. The presence of malformed membrane protrusions hampers the ability of L. monocytogenes to efficiently disseminate from CD147-depleted cells. Our findings uncover another important host protein needed for L. monocytogenes membrane protrusion formation and efficient microbial dissemination.

Listeria Membrane Protrusion Collapse: Requirement of Cyclophilin A for Listeria Cell-to-Cell Spreading.

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.

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.

Cyclophilin A Controls Salmonella Internalization Levels and is Present at E. coli Actin-Rich Pedestals.

Salmonella enterica serovar Typhimurium (S. Typhimurium), enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) commandeer the actin cytoskeleton of their host cells as a crucial step in their infectious processes. These pathogens depend on the injection of their own effectors directly into target host cells in order to usurp cellular signaling pathways that lead to morphological actin rearrangements in those cells. Here we show that the PPIase Cyclophilin A (CypA) is a novel component of S. Typhimurium-induced membrane ruffles and functions to restrict bacterial invasion levels, as in cells depleted of CypA, bacterial loads increase. We also demonstrate that CypA requires the EPEC effector Tir as well as pedestal formation for its recruitment to bacterial attachment sites and that its presence at pedestals also holds during EHEC infections. Finally, we demonstrate that CypA is found at lamellipodia; actin-rich structures at the leading edge of motile cells. Our findings further establish CypA as a component of dynamic actin-rich structures formed during bacterial infections and within cells in general. Anat Rec, 301:2086-2094, 2018. © 2018 Wiley Periodicals, Inc.

An Introduction to Actin and Actin-Rich Structures.

The actin cytoskeleton has long been recognized as a crucial sub-cellular filament system that is responsible for governing fundamental events ranging from cell division and muscle contraction to whole cell motility and the maintenance of tissue integrity. Consequently, it is not surprising that this network is the focus of over 100,000 different manuscripts. Alterations in the actin cytoskeleton lead to an assortment of diseases and serve as a target for a variety of pathogens. Here we have brought together a collection of primary research articles and reviews that underscore the broad influence this filament system has on organisms. Anat Rec, 301:1986-1990, 2018. © 2018 Wiley Periodicals, Inc.

Hsc70 is a Component of Bacterially Generated Actin-Rich Structures: An Immunolocalization Study.

Enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium, and Listeria monocytogenes usurp the actin cytoskeleton for their attachment, internalization and transport within and amongst infected cells. To try to gain a greater understanding of the molecular components utilized by these microbes during their infections we previously concentrated actin-rich structures generated during EPEC infections (called pedestals) and identified the heat shock cognate 70 protein (Hsc70) as a potential candidate. This multifunctional protein classically acts as a chaperone for the proper folding of a variety of proteins and is involved in uncoating clathrin from coated pits. Here we demonstrated that Hsc70 is recruited to actin structures generated during EPEC, Listeria and Salmonella infections, but not to the same location as clathrin. Anat Rec, 301:2095-2102, 2018. © 2018 Wiley Periodicals, Inc.

Calponins Are Recruited to Actin-Rich Structures Generated by Pathogenic Escherichia coli, Listeria, and Salmonella.

The ingestion of enteropathogenic Escherichia coli (EPEC), Listeria monocytogenes, or Salmonella enterica serovar Typhimurium leads to their colonization of the intestinal lumen, which ultimately causes an array of ailments ranging from diarrhea to bacteremia. Once in the intestines, these microbes generate various actin-rich structures to attach, invade, or move within the host intestinal epithelial cells. Although an assortment of actin-associated proteins has been identified to varying degrees at these structures, the localization of many actin stabilizing proteins have yet to be analyzed. Here, we examined the recruitment of the actin-associated proteins, calponin 1 and 2 at EPEC pedestals, L. monocytogenes actin clouds, comet tails and listeriopods, and S. Typhimurium membrane ruffles. In other systems, calponins are known to bind to and stabilize actin filaments. In EPEC pedestals, calponin 1 was recruited uniformly throughout the structures while calponin 2 was enriched at the apical tip. During L. monocytogenes infections, calponin 1 was found through all the actin-rich structures generated by the bacteria, while calponin 2 was only present within actin-rich structures formed by L. monocytogenes near the host cell membrane. Finally, both calponins were found within S. Typhimurium-generated membrane ruffles. Taken together, we have shown that although calponin 1 is recruited to actin-rich structures formed by the three bacteria, calponin 2 is specifically recruited to only membrane-bound actin-rich structures formed by the bacteria. Thus, our findings suggest that calponin 2 is a novel marker for membrane-bound actin structures formed by pathogenic bacteria. Anat Rec, 301:2103-2111, 2018. © 2018 Wiley Periodicals, Inc.

SM22 is required for the maintenance of actin-rich structures generated during bacterial infections.

The host actin cytoskeleton is utilized by an assortment of pathogenic bacteria to colonize and cause disease in their hosts. Two prominently studied actin-hijacking bacteria are enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes. EPEC form actin-rich pedestals atop its host cells to move across the intestinal epithelia, while Listeria monocytogenes generate branched actin networks arranged as actin clouds around the bacteria and as comet tails for propulsion within and amongst their host cells. Previous mass spectrometry analysis revealed that a member of the calponin family of actin-bundling proteins, transgelin/SM22 was enriched in EPEC pedestals. To validate that finding and examine the role of SM22 during infections, we initially immunolocalized SM22 in EPEC and L. monocytogenes infected cells, used siRNA to deplete SM22 and EGFP-SM22 to overexpress SM22, then quantified the alterations to the bacterially generated actin structures. SM22 concentrated at all bacterially-generated actin structures. Depletion of SM22 resulted in fewer pedestals and comet tails and caused comet tails to shorten. The decrease in comet tail abundance caused a proportional increase in actin clouds whereas overexpression of SM22 reversed the actin cloud to comet tail proportions and increased comet tail length, while not influencing EPEC pedestal abundance. Thus, we demonstrate that SM22 plays a role in regulating the transitions and morphological appearance of bacterially generated actin-rich structures during infections.

Palladin Compensates for the Arp2/3 Complex and Supports Actin Structures during Listeria Infections.

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.