Abstract  Research has demonstrated that metallic nanoparticles produce toxicity in aquatic organisms that is due largely to effects of particulates as opposed to release of dissolved ions. The present research examined the interplay of nanoparticle composition and dissolution on response of the zebrafish gill following exposure to toxic (nanocopper or nanosilver) or nontoxic (nano-TiO2) nanometals. Female zebrafish were exposed to 48-h no observable effects concentration of nanocopper and nanosilver or to soluble Cu and Ag that matched the concentration of dissolved metals released during nanoparticle exposure. Both nanocopper and nanosilver exposures increased metal content associated with gill tissue, though silver concentrations were much higher following nanosilver exposures suggesting that intact silver nanoparticles are associated with the gill. Morphological and transcriptional responses of the gills differed among various nanomaterials and between nanoparticulate and soluble species. Nanocopper increased mean gill filament width by three to fourfold between 24 and 48 h, whereas nanosilver did not alter gill filament width at either time point. Global gene expression analysis demonstrates that the exposure to each nanometal or soluble metal produces a distinct gene expression profile at both 24 and 48 h, suggesting that each exposure is producing biological response by a different mechanism. The differences in responses among the exposures indicates that each particle is having a distinct biological effect that does not appear to be driven solely by release of soluble metal ions into the water column. Based on these results, care should be taken when inferring toxicity of nanomaterials from data on a different material.

Abstract  Nanosilver has well-known antibacterial properties, and is widely used in daily life as various medical and general products. In comparison with silver ion, there is serious lacking of information concerning the biological effects of nanoAg. In this study, we observed the cytotoxic effect of nanoAg in HeLa cells. The nanoAg-induced cytotoxicity was lower than that of AgNO3, used as a silver ion source. Apoptosis evaluated by flowcytometric analysis was associated with this cell death. Further, the expressions of ho-1 and mt-2A, well-known oxidative stress-related genes, were up-regulated by nanoAg treatment. Our results showed that nanoAg possesses the potential for cytotoxicity, therefore, in the case of exposure at high concentrations, we should consider to protect from nanoAg-induced toxicity.

Abstract  We examined the influence of speciation on the kinetics of silver uptake and depuration in the gills of two freshwater fish, the rainbow trout (Oncorhynchus mykiss) which has high branchial Na+ and Cl− uptake rates and is relatively sensitive to silver, and the European eel (Anguilla anguilla, yellow stage) which has low ion uptake rates and is relatively resistant to silver. Fish previously acclimated to the appropriate chloride level were exposed to 110mAgNO3 (1.3 μg l−1, sublethal) for 24 h in synthetic softwater with either low (10 μM) or high (1200 μM) chloride concentration, and then followed over a subsequent 67-day post-exposure period in silver-free water of the same chloride content. The exposures were therefore mainly to the free ion, Ag+ in the low chloride water versus mainly to the neutral aqueous complex, AgClaq in the high chloride water. In trout, but not in eel, water chloride is known to protect against physiological disturbances and toxicity caused by Ag+. In both fish species, at both chloride levels, silver uptake exhibited complex kinetics. Gill silver loading occurred slowly until 6 h, then rose greatly to a peak at 12 h, followed by significant net depuration thereafter during continued exposure. By 24 h, net gill loading was three- to fivefold greater from AgClaq than from Ag+ exposure in both species, and threefold greater in trout than in eel under both conditions, with trout holding a lower fraction of the whole body burden in their gills. During the post-exposure period, depuration of silver from the gills occurred rapidly in trout, but very slowly in eel, such that gill silver burdens were greater in eel throughout the 67-day period on both an absolute and relative basis (e.g. 35% of whole body burden in eel versus <3% in trout at day 8). The kinetics of depuration were described by two phase exponential models, with break points between the fast and slow phases at 1 and 15 days for trout and eel, respectively. We conclude that speciation affects not only uptake rates but also the kinetics of depuration. When silver is loaded from AgClaq it is clearly more labile than from Ag+ exposures, with 1.6–1.8-fold greater loss rates during the fast phases in both species. Differences in branchial silver uptake between eel and trout correlate well with differences in acute toxicity, but are not as large as differences in ion uptake rates. The complex time-dependent patterns of gill loading, and the higher loading from AgClaq than from Ag+, mean that gill total silver burden is not an appropriate endpoint for biotic ligand modelling.

Abstract  To fully assess the impact of pollutant releases into the environment, it is necessary to determine both the concentration of chemicals accumulating in biota and the biological effects they give rise to. Owing to time, expertise, and cost constraints, this is, however, rarely achieved. Here, quick, simple to perform, and inexpensive biomarkers and chemical immunoassays were combined in a rapid assessment approach to measure exposure to and effects of organic and metal pollutants on the ribbed mussel (Geukensia demmissa) from New Bedford Harbor, MA. Significant differences in polychlorinated biphenyl (PCB) and polyaromatic hydrocarbon (PAH) tissue residue concentrations were detected among sites using RaPID immunoassay. Selected analyses were verified using GC/MS. No significant differences were observed in metal concentrations (Cu, Cd, Pb, As, Hg, Ni) throughout the area. While causality cannot be attributed, multivariate canonical correlation analysis indicated that PCB and PAH concentrations were strongly associated with the induction of biomarkers of genotoxicity (micronucleus formation), immunotoxicity (spontaneous cytotoxicity), and physiological impairment (heart rate). It is concluded thatthe incorporation of chemical immunoassays with biological monitoring tools into routine management procedures is clearly viable and valuable as a means of identifying toxic impacts of pollutants on biota in situ.

Abstract  Background: Studies in monkeys with intranasally instilled gold ultrafine particles (UFPs; < 100 nm) and in rats with inhaled carbon UFPs suggested that solid UFPs deposited in the nose travel along the olfactory nerve to the olfactory bulb. Methods: To determine if olfactory translocation occurs for other solid metal UFPs and assess potential health effects, we exposed groups of rats to manganese (Mn) oxide UFPs (30 nm; ~ 500 μg/m3) with either both nostrils patent or the right nostril occluded. We analyzed Mn in lung, liver, olfactory bulb, and other brain regions, and we performed gene and protein analyses. Results: After 12 days of exposure with both nostrils patent, Mn concentrations in the olfactory bulb increased 3.5-fold, whereas lung Mn concentrations doubled; there were also increases in striatum, frontal cortex, and cerebellum. Lung lavage analysis showed no indications of lung inflammation, whereas increases in olfactory bulb tumor necrosis factor-α mRNA (~ 8-fold) and protein (~ 30-fold) were found after 11 days of exposure and, to a lesser degree, in other brain regions with increased Mn levels. Macrophage inflammatory protein-2, glial fibrillary acidic protein, and neuronal cell adhesion molecule mRNA were also increased in olfactory bulb. With the right nostril occluded for a 2-day exposure, Mn accumulated only in the left olfactory bulb. Solubilization of the Mn oxide UFPs was < 1.5% per day. Conclusions: We conclude that the olfactory neuronal pathway is efficient for translocating inhaled Mn oxide as solid UFPs to the central nervous system and that this can result in inflammatory changes. We suggest that despite differences between human and rodent olfactory systems, this pathway is relevant in humans.

Abstract  A novel biological method for the synthesis of silver nanoparticles using the fungus Verticillium is reported. Exposure of the fungal biomass to aqueous Ag+ ions resulted in the intracellular reduction of the metal ions and formation of silver nanoparticles of dimensions 25 ± 12 nm. Electron microscopy analysis of thin sections of the fungal cells indicated that the silver particles were formed below the cell wall surface, possibly due to reduction of the metal ions by enzymes present in the cell wall membrane. The metal ions were not toxic to the fungal cells and the cells continued to multiply after biosynthesis of the silver nanoparticles.

Abstract  This study aims at investigating feasibility and challenges associated with conducting a human health risk assessment for nano-silver based on the open literature by following an approach similar to a classical regulatory risk assessment. Gaps in the available data set, both in relation to exposures and hazard, do not allow reaching any definite conclusions that could be used for regulatory decision making. Results show that repeated inhalation in the workplace and possibly consumer inhalation may cause risks. Also (uncontrolled) nano-silver drug intake and burn treatment of large parts of the body with wound dressings may cause risks. Main future work should focus on generating occupational and consumer exposure data, as well as toxicity data on absorption (are particles or only ions absorbed?), information on genetoxicity, and further information on the toxicity following inhalation exposure to sizes and agglomeration states as uncounted in the workplace.

Abstract  Some adjustments are needed in the European chemicals legislation REACH to assess and control the risks of nanomaterials. The information on substances to be provided under REACH is not sufficient to determine the specific properties of nanomaterials, nor to assess how these properties affect their behaviour and effects in humans and the environment. RIVM concluded this following research into the suitability of REACH for nanomaterials. RIVM therefore proposes an adapted set of minimum information requirements, to be applied to all nanomaterials to be registered under REACH, independent of their volume of production and import. These requirements allow a risk assessment of nanomaterials. Over the last years the use of nanomaterials has strongly increased. As yet, nanomaterials are defined as substances of which the discrete parts have at least one dimension smaller than one hundred nanometres. Due to their nanosize they have specific properties. Legislation should focus on controlling the potential hazards and risks of these nanomaterials. By conducting a hypothetical registration of nanosilver it was investigated whether REACH is suitable for assessing the safe use of nanomaterials. From this it appeared that no definition of a nanomaterial is present, and that a relevant measure for expressing harmfulness and exposure is as yet not known. In addition, the standard information requirements are insufficient to assess hazard and exposure. They are also insufficient for a proper characterisation of the nanomaterial. Consequently, it cannot be determined to what extent the nanoform of a substance corresponds to the non-nanoform of the same substance. Furthermore, it is unclear whether current risk reduction measures and extrapolation methods in risk assessment, as established for non-nanomaterials, are applicable to nanomaterials.

Abstract  Exposure assessment is crucial for risk assessment for nanomaterials. We propose a framework to aid exposure assessment in consumer products. We determined the location of the nanomaterials and the chemical identify of the 580 products listed in the inventory maintained by the Woodrow Wilson International Center for Scholars, of which 37% used nanoparticles suspended in liquids, whereas <1% contained "free airborne nanoparticles". C(60) is currently only used as suspended nanoparticles in liquids and nanosilver is used more as surface bound nanoparticles than as particles suspended in liquids. Based on the location of the nanostructure we were able to further group the products into categories of: (1) expected, (2) possible, and (3) no expected exposure. Most products fall into the category of expected exposure, but we were not able to complete a quantitative exposure assessment mainly due to the lack of information on the concentration of the nanomaterial in the products--a problem that regulators and industry will have to address if we are to have realistic exposure assessment in the future. To illustrate the workability of our procedure, we applied it to four product scenarios using the best estimates available and/or worst-case assumptions. Using the best estimates available and/or worst-case assumptions we estimated the consumer exposure to be 26, 15, and 44 microg kg(-1) bw year(-1) for a facial lotion, a fluid product, and a spray product containing nanoparticles, respectively. The application of sun lotion containing 2% nanoparticles result in an exposure of 56.7 mg kg(-1) bw d(-1) for a 2-year-old child, if the amounts applied correspond to the European Commission recommendations on use of sunscreen.

Abstract  There is an emerging literature reporting toxic effects of manufactured nanomaterials (NMs) and nanoparticles (NPs) in fish, but the mechanistic basis of both exposure and effect are poorly understood. This paper critically evaluates some of the founding assumptions in fish toxicology, and likely mechanisms of absorption, distribution, metabolism and excretion (ADME) of NPs in fish compared to other chemicals. Then, using a case study approach, the paper compares these assumptions for two different NPs; TiO2 and C60 fullerenes. Adsorption of NPs onto the gill surface will involve similar processes in the gill microenvironment and mucus layer to other substances, but the uptake mechanisms for NPs by epithelial cells are more likely to occur via vesicular processes (e.g., endocytosis) than uptake on membrane transporters or by diffusion through the cell membranes. Target organs may include the gills, gut, liver and sometimes the brain. Information on metabolism and excretion of NPs in fish is limited; but hepatic excretion into the bile seems a more likely mechanism, rather than mainly by renal or branchial excretion. TiO2 and C60 share some common chemical properties that appear to be associated with some similar toxic effects, but there are also differences, that highlight the notion that chemical reactivity can inform toxic effect of NPs in a fundamentally similar way to other chemicals. In this paper we identify many knowledge gaps including the lack of field observations on fish and other wildlife species for exposure and effects of manufactured NMs. Systematic studies of the abiotic factors that influence bioavailability, and investigation of the cell biology that informs on the mechanisms of metabolism and excretion of NMs, will greatly advance our understanding of the potential for adverse effects. There are also opportunities to apply existing tools and techniques to fundamental studies of fish toxicology with NPs, such as perfused organs and fish cell culture systems.

Abstract  Environmental toxicologists, chemists and social scientists have identified three priorities for research into the impact of engineered nanoparticles on the environment.

Abstract  It is inevitable that, during their use, engineered nanoparticles will be released into soils and waters. There is therefore increasing concern over the potential impacts of engineered nanoparticles in the environment on aquatic and terrestrial organisms and on human health. Once released into the environment, engineered nanoparticles will aggregate to some degree; they might also associate with suspended solids, sediment, be accumulated by organisms and enter drinking water sources and food materials. These fate processes are dependent on the characteristics of the particle and the characteristics of the environmental system. A range of ecotoxicological effects have also been reported, including effects on microbes, plants, invertebrates and fish. Although available data indicate that current risks of engineered nanoparticles in the environment to environmental and human health are probably low, our knowledge of the potential impacts of engineered nanoparticles in the environment on human health is still limited. There is therefore a need for continued work to develop an understanding of the exposure levels for engineered nanoparticles in environmental systems and to begin to explore the implications of these levels in terms of the ecosystem and human health. This will require research in a range of areas, including detection and characterization, environmental fate and transport, ecotoxicology and toxicology.

Abstract  Ultrafine particles (UFP, particles <100 nm) are ubiquitous in ambient urban and indoor air from multiple sources and may contribute to adverse respiratory and cardiovascular effects of particulate matter (PM). Depending on their particle size, inhaled UFP are efficiently deposited in nasal, tracheobronchial, and alveolar regions due to diffusion. Our previous rat studies have shown that UFP can translocate to interstitial sites in the respiratory tract as well as to extrapulmonary organs such as liver within 4 to 24h postexposure. There were also indications that the olfactory bulb of the brain was targeted. Our objective in this follow-up study, therefore, was to determine whether translocation of inhaled ultrafine solid particles to regions of the brain takes place, hypothesizing that UFP depositing on the olfactory mucosa of the nasal region will translocate along the olfactory nerve into the olfactory bulb. This should result in significant increases in that region on the days following the exposure as opposed to other areas of the central nervous system (CNS). We generated ultrafine elemental 13C particles (CMD = 36 nm; GSD = 1.66) from [13C] graphite rods by electric spark discharge in an argon atmosphere at a concentration of 160 microg/m3. Rats were exposed for 6 h, and lungs, cerebrum, cerebellum and olfactory bulbs were removed 1, 3, 5, and 7 days after exposure. 13C concentrations were determined by isotope ratio mass spectroscopy and compared to background 13C levels of sham-exposed controls (day 0). The background corrected pulmonary 13C added as ultrafine 13C particles on day 1 postexposure was 1.34 microg/lung. Lung 13C concentration decreased from 1.39 microg/g (day 1) to 0.59 microg/g by 7 days postexposure. There was a significant and persistent increase in added 13C in the olfactory bulb of 0.35 microg/g on day 1, which increased to 0.43 microg/g by day 7. Day 1 13C concentrations of cerebrum and cerebellum were also significantly increased but the increase was inconsistent, significant only on one additional day of the postexposure period, possibly reflecting translocation across the blood-brain barrier in certain brain regions. The increases in olfactory bulbs are consistent with earlier studies in nonhuman primates and rodents that demonstrated that intranasally instilled solid UFP translocate along axons of the olfactory nerve into the CNS. We conclude from our study that the CNS can be targeted by airborne solid ultrafine particles and that the most likely mechanism is from deposits on the olfactory mucosa of the nasopharyngeal region of the respiratory tract and subsequent translocation via the olfactory nerve. Depending on particle size, >50% of inhaled UFP can be depositing in the nasopharyngeal region during nasal breathing. Preliminary estimates from the present results show that ~20% of the UFP deposited on the olfactory mucosa of the rat can be translocated to the olfactory bulb. Such neuronal translocation constitutes an additional not generally recognized clearance pathway for inhaled solid UFP, whose significance for humans, however, still needs to be established. It could provide a portal of entry into the CNS for solid UFP, circumventing the tight blood-brain barrier. Whether this translocation of inhaled UFP can cause CNS effects needs to be determined in future studies.

Abstract  The rapid development and potential release of engineered nanoparticles (ENPs) have raised considerable concerns due to the unique properties of nanomaterials. An important aspect of the risk assessment of ENPs is to understand the interactions of ENPs with plants, an essential base component of all ecosystems. The impact of ENPs on plant varies, depending on the composition, concentration, size and other important physical chemical properties of ENPs and plant species. Both enhancive and inhibitive effects of ENPs on plant growth at different developmental stages have been documented. ENPs could be potentially taken up by plant roots and transported to shoots through vascular systems depending upon the composition, shape, size of ENPs and plant anatomy. Despite the insights gained through many previous studies, many questions remain concerning the fate and behavior of ENPs in plant systems such as the role of surface area or surface activity of ENPs on phytotoxicity, the potential route of entrance to plant vascular tissues and the role of plant cell walls in internalization of ENPs. This article reviewed the current knowledge on the phytotoxicity and interactions of ENPs with plants at seedling and cellular levels and discussed the information gap and some immediate research needs to further our knowledge on this topic.

Abstract  The cardiovascular system is currently considered a target for particulate matter, especially for ultrafine particles. In addition to autonomic or cytokine mediated effects, the direct interaction of inhaled materials with the target tissue must be examined to understand the underlying mechanisms. In the first approach, pulmonary and systemic distribution of inhaled ultrafine elemental silver (EAg) particles was investigated on the basis of morphology and inductively coupled plasma mass spectrometry (ICP-MS) analysis. Rats were exposed for 6 hr at a concentration of 133 Ág EAg m3 (3 * 106 cm3, 15 nm modal diameter) and were sacrificed on days 0, 1, 4, and 7. ICP-MS analysis showed that 1.7 Ág Ag was found in the lungs immediately after the end of exposure. Amounts of Ag in the lungs decreased rapidly with time, and by day 7 only 4% of the initial burden remained. In the blood, significant amounts of Ag were detected on day 0 and thereafter decreased rapidly. In the liver, kidney, spleen, brain, and heart, low concentrations of Ag were observed. Nasal cavities, especially the posterior portion, and lung-associated lymph nodes showed relatively high concentrations of Ag. For comparison, rats received by intratracheal instillation either 150 ÁL aqueous solution of 7 Ág silver nitrate (AgNO3) (4.4 Ág Ag) or 150 ÁL aqueous suspension of 50 Ág agglomerated ultrafine EAg particles. A portion of the agglomerates remained undissolved in the alveolar macrophages and in the septum for at least 7 days. In contrast, rapid clearance of instilled water-soluble AgNO3 from the lung was observed. These findings show that although instilled agglomerates of ultrafine EAg particles were retained in the lung, Ag was rapidly cleared from the lung after inhalation of ultrafine EAg particles, as well as after instillation of AgNO3, and entered systemic pathways.

Abstract  National Institute of Environmental Health Sciences. INTRODUCTION: Efficient elimination of inhaled soluble and insoluble particulate compounds deposited in the respiratory tract is necessary to keep its mucosal surfaces clean and functionally intact. In addition, the characteristic clearance processes and kinetics prevailing in different regions of the respiratory tract determine the retained dose of an inhaled substance in structures of the respiratory system. Thus, whether deposition and subsequent retention of inhaled particles is considered in clinical situations involving diagnostic or therapeutic aerosols or in toxicology with occupational or environmental pollutant aerosols, knowledge of the clearance of these substances is crucial in understanding their respiratory tract dosimetry. Clearance mechanisms are not only different for different regions of the respiratory tract, but also depend on physico-chemical characteristics of the deposited particulate material. Several recent review articles provide detailed descriptions and discussions of different processes governing the removal of inhaled solid and solute particles after deposition in the respiratory tract (Lauweryns and Baert, 1977; Morrow, 1977; Camner, 1980; Jones et al., 1982; Effros and Mason, 1983; Jones, 1984; Pavia, 1984; Morrow and Yu, 1985; Schlesinger, 1985; Brain, 1985, 1986; Cuddihy and Yeh, 1988). Because of the multitude and complexity of the diverse respiratory tract clearance processes, the present article will selectively review aspects of soluble and insoluble particle removal from tracheobronchial and deep lung regions. In addition, the article emphasizes clinical and toxicological implications of these removal processes and considers some unresolved issues related to lung clearance.

Abstract  Two control and eight field-contaminated, metal-polluted soils were inoculated with Eisenia fetida (Savigny, 1826). Three, 7, 14, 21, 28 and 42 days after inoculation, earthworm survival, body weight, cocoon production and hatching rate were measured. Seventeen metals were analysed in E.fetida tissue, bulk soil and soil solution. Soil organic carbon content, texture, pH and cation exchange capacity were also measured. Cocoon production and hatching rate were more sensitive to adverse conditions than survival or weight change. Soil properties other than metal concentration impacted toxicity. The most toxic soils were organic-poor (1-10 g C kg(-1)), sandy soils (c. 74% sand), with intermediate metal concentrations (e.g. 7150-13, 100 mg Ph kg(-1), 2970-53,400 mg Zn kg(-1)). Significant relationships between soil properties and the life cycle parameters were determined. The best coefficients of correlation were generally found for texture, pH, Ag, Cd, Mg, Pb, Tl, and Zn both singularly and in multivariate regressions. Studies that use metal-amended artificial soils are not useful to predict toxicity of field multi-contaminated soils. (c) 2007 Elsevier Ltd. All rights reserved.

Abstract  The Models Knowledge Base is an inventory of the computational models that are developed, used or supported by EPA's offices. For each model, the Models Knowledge Base provides information on: its development; its conceptual basis, scientific detail and evaluation; technical requirements and how to use it; information on the its inputs and outputs; and directions for downloading it and links to further information.

Abstract  Nanotechnology is expected to open new avenues to fight and prevent disease using atomic scale tailoring of materials. Among the most promising nanomaterials with antibacterial properties are metallic nanoparticles, which exhibit increased chemical activity due to their large surface to volume ratios and crystallographic surface structure. The study of bactericidal nanomaterials is particularly timely considering the recent increase of new resistant strains of bacteria to the most potent antibiotics. This has promoted research in the well known activity of silver ions and silver-based compounds, including silver nanoparticles. The present work studies the effect of silver nanoparticles in the range of 1-100 nm on Gram-negative bacteria using high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM). Our results indicate that the bactericidal properties of the nanoparticles are size dependent, since the only nanoparticles that present a direct interaction with the bacteria preferentially have a diameter of [?]1-10 nm.

Abstract  In this work we investigated the antibacterial properties of differently shaped silver nanoparticles against the gram-negative bacterium Escherichia coli, both in liquid systems and on agar plates. Energy-filtering transmission electron microscopy images revealed considerable changes in the cell membranes upon treatment, resulting in cell death. Truncated triangular silver nanoplates with a {111} lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles and with Ag+ (in the form of AgNO3). It is proposed that nanoscale size and the presence of a {111} plane combine to promote this biocidal property. To our knowledge, this is the first comparative study on the bactericidal properties of silver nanoparticles of different shapes, and our results demonstrate that silver nanoparticles undergo a shape-dependent interaction with the gram-negative organism E. coli.

Abstract  The antimicrobial activity of silver nanoparticles against E. coli was investigated as a model for Gram-negative bacteria. Bacteriological tests were performed in Luria?Bertani (LB) medium on solid agar plates and in liquid systems supplemented with different concentrations of nanosized silver particles. These particles were shown to be an effective bactericide. Scanning and transmission electron microscopy (SEM and TEM) were used to study the biocidal action of this nanoscale material. The results confirmed that the treated E. coli cells were damaged, showing formation of ?pits? in the cell wall of the bacteria, while the silver nanoparticles were found to accumulate in the bacterial membrane. A membrane with such a morphology exhibits a significant increase in permeability, resulting in death of the cell. These nontoxic nanomaterials, which can be prepared in a simple and cost-effective manner, may be suitable for the formulation of new types of bactericidal materials.

Abstract  The cell envelope of Gram-negative bacteria is composed of two membranes, which are separated by the peptidoglycan-containing periplasm. Whereas the envelope forms an essential barrier against harmful substances, it is nevertheless a compartment of intense traffic for large proteins such as enzymes and toxins. Numerous studies dealing with the molecular mechanism of protein secretion have revealed that Gram-negative bacteria evolved different strategies to achieve this process. Among them, the type II secretion mechanism is part of a two-step process. Exoproteins following this pathway are synthesized as signal peptide-containing precursors. After cleavage of the signal peptide, the mature exoproteins are released into the periplasm, where they fold. The type II machinery, also known as the secreton, is responsible for the translocation of the periplasmic intermediates across the OM. The type II system is broadly conserved in Gram-negative bacteria and involves a set of 12–16 different proteins named GspC-M, GspAB, GspN, GspO, and GspS. The type II secretion system is highly reminiscent of the type IV piliation assembly system. Based on findings about the subcellular localisation of the Gsp components, protein–protein interactions between Gsps and their multimerisation status, structural data and electron microscopy observation, it could be proposed a working model that strikingly runs both systems in parallel.