Thiago P. Silva

Visualizing aquatic bacteria by light and transmission electron microscopy

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.

Natural Schistosoma mansoni Infection in the Wild Reservoir Nectomys squamipes Leads to Excessive Lipid Droplet Accumulation in Hepatocytes in the Absence of Liver Functional Impairment

Schistosomiasis is a neglected tropical disease of a significant public health impact. The water rat Nectomys squamipes is one of the most important non-human hosts in the schistosomiasis mansoni transmission in Brazil, being considered a wild reservoir. Cellular mechanisms that contribute to the physiological adaptation of this rodent to the Schistosoma mansoni parasite are poorly understood. Here we identified, for the first time, that a hepatic steatosis, a condition characterized by excessive lipid accumulation with formation of lipid droplets (LDs) within hepatocytes, occurs in response to the natural S. mansoni infection of N. squamipes, captured in an endemic region. Significant increases of LD area in the hepatic tissue and LD numbers/hepatocyte, detected by quantitative histopathological and ultrastructural analyses, were paralleled by increased serum profile (total cholesterol and triglycerides) in infected compared to uninfected animals. Raman spectroscopy showed high content of polyunsaturated fatty acids (PUFAs) in the liver of both groups. MALDI-TOFF mass spectroscopy revealed an amplified pool of omega-6 PUFA arachidonic acid in the liver of infected animals. Assessment of liver functional activity by the levels of hepatic transaminases (ALT and AST) did not detect any alteration during the natural infection. In summary, this work demonstrates that the natural infection of the wild reservoir N. squamipes with S. mansoni elicits hepatic steatosis in the absence of liver functional harm and that accumulation of lipids, markedly PUFAs, coexists with low occurrence of inflammatory granulomatous processes, suggesting that lipid stores may be acting as a protective mechanism for dealing with the infection.

Extracellular Microvesicle Production by Human Eosinophils Activated by “Inflammatory” Stimuli

A key function of human eosinophils is to secrete cytokines, chemokines and cationic proteins, trafficking, and releasing these mediators for roles in inflammation and other immune responses. Eosinophil activation leads to secretion of pre-synthesized granule-stored mediators through different mechanisms, but the ability of eosinophils to secrete extracellular vesicles (EVs), very small vesicles with preserved membrane topology, is still poorly understood. In the present work, we sought to identify and characterize EVs released from human eosinophils during different conditions: after a culturing period or after isolation and stimulation with inflammatory stimuli, which are known to induce eosinophil activation and secretion: CCL11 (eotaxin-1) and tumor necrosis factor alpha (TNF-α). EV production was investigated by nanoscale flow cytometry, conventional transmission electron microscopy (TEM) and pre-embedding immunonanogold EM. The tetraspanins CD63 and CD9 were used as EV biomarkers for both flow cytometry and ultrastructural immunolabeling. Nanoscale flow cytometry showed that human eosinophils produce EVs in culture and that a population of EVs expressed detectable CD9, while CD63 was not consistently detected. When eosinophils were stimulated immediately after isolation and analyzed by TEM, EVs were clearly identified as microvesicles (MVs) outwardly budding off the plasma membrane. Both CCL11 and TNF-α induced significant increases of MVs compared to unstimulated cells. TNF-α induced amplified release of MVs more than CCL11. Eosinophil MV diameters varied from 20 to 1000 nm. Immunonanogold EM revealed clear immunolabeling for CD63 and CD9 on eosinophil MVs, although not all MVs were labeled. Altogether, we identified, for the first time, that human eosinophils secrete MVs and that this production increases in response to inflammatory stimuli. This is important to understand the complex secretory activities of eosinophils underlying immune responses. The contribution of the eosinophil-derived MVs to the regulation of immune responses awaits further investigation.

Potential effects of UV radiation on photosynthetic structures of the bloom-forming cyanobacterium Cylindrospermopsis raciborskii CYRF-01

Cyanobacteria are aquatic photosynthetic microorganisms. While of enormous ecological importance, they have also been linked to human and animal illnesses around the world as a consequence of toxin production by some species. Cylindrospermopsis raciborskii, a filamentous nitrogen-fixing cyanobacterium, has attracted considerable attention due to its potential toxicity and ecophysiological adaptability. We investigated whether C. raciborskii could be affected by ultraviolet (UV) radiation. Non-axenic cultures of C. raciborskii were exposed to three UV treatments (UVA, UVB, or UVA + UVB) over a 6 h period, during which cell concentration, viability and ultrastructure were analyzed. UVA and UVA + UVB treatments showed significant negative effects on cell concentration (decreases of 56.4 and 64.3%, respectively). This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe. Over 90% of UVA + UVB- and UVA-treated cells died. UVB did not alter cell concentration, but reduced cell viability in almost 50% of organisms. Transmission electron microscopy (TEM) revealed a drastic loss of thylakoids, membranes in which cyanobacteria photosystems are localized, after all treatments. Moreover, other photosynthetic- and metabolic-related structures, such as accessory pigments and polyphosphate granules, were damaged. Quantitative TEM analyses revealed a 95.8% reduction in cell area occupied by thylakoids after UVA treatment, and reduction of 77.6 and 81.3% after UVB and UVA + UVB treatments, respectively. Results demonstrated clear alterations in viability and photosynthetic structures of C. raciborskii induced by various UV radiation fractions. This study facilitates our understanding of the subcellular organization of this cyanobacterium species, identifies specific intracellular targets of UVA and UVB radiation and reinforces the importance of UV radiation as an environmental stressor.

Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation

Secretion of membrane vesicles is an important biological process of both eukaryotic and prokaryotic cells. This process has been characterized in pathogenic bacteria, but is less clear in non-pathogenic bacteria from aquatic ecosystems. Here, we investigated, for the first time, the process of formation of outer membranes vesicles (OMVs), nanoscale vesicles extruded from the outer membrane (OM) of gram-negative bacteria, in cultures of freshwater bacteria after exposure or not to ultraviolet radiation (UVR) as an environmental stressor. Non-axenic cultures of freshwater bacteria isolated from a Brazilian aquatic ecosystem (Funil reservoir) were exposed or not to UVR (UVA+UVB) over a 3h period, during which cell density, viability and ultrastructure were analyzed. First, we showed that UVR induce bacterial death. UVR triggered significant negative effect on cell density after 3h of UVR treatment. This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe that enables the distinction of live/dead bacteria. Transmission electron microscopy (TEM) revealed changes indicative of cell death after 3h of UVR exposure, with significant increase of damaged cells compared to the control group. Second, we demonstrated that gram-negative bacteria release OMVs during normal growth and after UVR exposure. OMVs were clearly identified as round, membrane-bound vesicles budding off from the bacterial OM as isolated or clustered vesicles or free in the extracellular medium. Remarkably, quantitative TEM analyses showed that bacteria respond to UVR with increased formation of OMVs. Moreover, while OMVs numbers per intact or damaged cell did not differ in the untreated group, UVR led to a higher vesiculation by bacteria in process of death. This means that degenerating bacteria release OMVs before lysis and that this secretion might be an adaptive/protective response to rapid changes in environmental conditions such as UV radiation.

Histological assessment of granulomas in natural and experimental Schistosoma mansoni infections using whole slide imaging

The pathology of schistosomiasis mansoni, a neglected tropical disease of great clinical and socioeconomic importance, results from the parasite eggs that become trapped in host tissues, particularly in the liver and intestines. Continuous antigenic stimulation from these eggs leads to recruitment of inflammatory cells to the sites of infection with formation of periovular granulomas. These complex structures have variable size and composition and are the most striking histopathological feature of schistosomiasis mansoni. However, evaluation of granulomas by conventional microscopy methods is time-consuming and limited, especially in large-scale studies. Here, we used high resolution Whole Slide Imaging (WSI), which allows fast scanning of entire histological slides, and multiple morphometric evaluations, to assess the granulomatous response elicited in target organs (liver, small and large intestines) of two models of schistosomiasis mansoni. One of the advantages of WSI, also termed virtual microscopy, is that it generates images that simultaneously offer high resolution and a wide field of observation. By using a model of natural (Nectomys squamipes, a wild reservoir captured from endemic areas in Brazil) and experimental (Swiss mouse) infection with Schistosoma mansoni, we provided the first detailed WSI characterization of granulomas and other pathological aspects. WSI and quantitative analyses enabled a fast and reliable assessment of the number, evolutional types, frequency and areas of granulomas and inflammatory infiltrates and revealed that target organs are differentially impacted by inflammatory responses in the natural and experimental infections. Remarkably, high-resolution analysis of individual eosinophils, key cells elicited by this helminthic infection, showed a great difference in eosinophil numbers between the two infections. Moreover, features such as the intestinal egg path and confluent granulomas were uncovered. Thus, WSI may be a suitable tool for detailed and precise histological analysis of granulomas and other pathological aspects for clinical and research studies of schistosomiasis.

Cytotoxicity and bacterial membrane destabilization induced by Annona squamosa L. extracts

This study aimed to further investigate the cytotoxicity against tumor cell lines and several bacterial strains of Annona squamosa and its mode of action. Methanol extracts of A. squamosa leaves (ASL) and seeds (ASS) were used. ASL showed significant antibacterial activity against S. aureus, K. pneumoniae and E. faecalis with MIC values of 78, 78 and 39 µg/mL respectively. Moreover, ASL exhibited significant biofilm disruption, rapid time dependent kinetics of bacterial killing, increased membrane permeability and significantly reduced the cell numbers and viability. Regarding the cytotoxicity against tumor cell lines, ASS was more active against Jurkat and MCF-7 cells, with CI50 1.1 and 2.1 µg/mL, respectively. ASL showed promising activity against Jurkat and HL60, with CI50 4.2 and 6.4 µg/mL, respectively. Both extracts showed lower activity against VERO cells and reduced the clonogenic survival at higher concentrations (IC90) to MCF-7 and HCT-116 lineages. The alkaloids anonaine, asimilobine, corypalmine, liriodenine nornuciferine and reticuline were identified in extracts by UPLC-ESI-MS/MS analysis. This study reinforced that A. squamosa presents a remarkable phytomedicinal potential and revealed that its antimicrobial mechanism of action is related to bacterial membrane destabilization.

TEM as an Important Tool to Study Aquatic Microorganisms and their Relationships with Ecological Processes

Microorganisms are critically important for ecological processes in aquatic environments. Bacteria and viruses are key components of the microbial loop and are central for biogeo‐ chemical cycles in aquatic ecosystems. Our group has been using transmission electron microscopy (TEM) to study aquatic microorganisms in both natural tropical ecosystems and cultures. In this review, we highlight structural aspects of freshwater bacteria, based on TEM findings that have provided insights into the functional capabilities of these cells in aquatic tropical ecosystems. First, we focus on TEM applied to the study of the ultra‐ structural diversity and morphological alterations of bacteria in response to environmen‐ tal stress. Second, we address the relationship between viruses and bacteria in freshwater ecosystems. Third, we demonstrate by TEM that outer membrane vesicles (OMVs), struc‐ tures associated with cell secretion and cell communication, are released by aquatic bacte‐ ria into natural ecosystems and cultures. Thus, TEM has proven to be a powerful technique to study aquatic microorganisms, contributing to the understanding of ecologi‐ cal processes, including regulation of bacterial populations, during different environmen‐ tal conditions.

The Cyanobacterium Cylindrospermopsis raciborskii (CYRF-01) Responds to Environmental Stresses with Increased Vesiculation Detected at Single-Cell Resolution

Secretion of membrane-limited vesicles, collectively termed extracellular vesicles (EVs), is an important biological process of both eukaryotic and prokaryotic cells. This process has been observed in bacteria, but remains to be better characterized at high resolution in cyanobacteria. In the present work, we address the release of EVs by Cylindrospermopsis raciborskii (CYRF-01), a filamentous bloom-forming cyanobacterium, exposed to environmental stressors. First, non-axenic cultures of C. raciborskii (CYRF-01) were exposed to ultraviolet radiation (UVA + UVB) over a 6 h period, which is known to induce structural damage to this species. Second, C. raciborskii was co-cultured in interaction with another cyanobacterium species, Microcystis aeruginosa (MIRF-01), over a 24 h period. After the incubation times, cell density and viability were analyzed, and samples were processed for transmission electron microscopy (TEM). Our ultrastructural analyses revealed that C. raciborskii constitutively releases EVs from the outer membrane during its normal growth and amplifies such ability in response to environmental stressors. Both situations induced significant formation of outer membrane vesicles (OMVs) by C. raciborskii compared to control cells. Quantitative TEM revealed an increase of 48% (UV) and 60% (interaction) in the OMV numbers compared to control groups. Considering all groups, the OMVs ranged in size from 20 to 300 nm in diameter, with most OMVs showing diameters between 20 and 140 nm. Additionally, we detected that OMV formation is accompanied by phosphatidylserine exposure, a molecular event also observed in EV-secreting eukaryotic cells. Altogether, we identified for the first time that C. raciborskii has the competence to secrete OMVs and that under different stress situations the genesis of these vesicles is increased. The amplified ability of cyanobacteria to release OMVs may be associated with adaptive responses to changes in environmental conditions and interspecies cell communication.

Antibacterial and Antibiofilm Activities of Psychorubrin, a Pyranonaphthoquinone Isolated From Mitracarpus frigidus (Rubiaceae)

Psychorubrin, a natural pyranonaphthoquinone found in different plants, has become an interesting compound in the search for new antimicrobial therapeutic agents. Here, we investigated the potential antagonistic activity of psychorubrin against planktonic and biofilm bacteria. First, psychorubrin was tested against several Gram-positive and Gram-negative bacteria strains by a broth microdilution susceptibility method. Second, bacterial killing assay, bacterial abundance, and membrane viability were evaluated. The nucleotide leakage assay was used to verify membrane destabilization while antibiofilm activities were analyzed by the effect on established biofilm, static biofilm formation, isolation of biofilm matrix assay and scanning electron microscopy. In parallel, the combinatorial effect of psychorubrin and chloramphenicol was evaluated by the checkerboard method. Psychorubrin was active against Gram-positive bacteria, showing rapid time-dependent kinetics of bacterial killing, amplified nucleotide leakage, and greater activity against the methicillin-resistant species (MRSA) Staphylococcus aureus 33591 and 33592 and Staphylococcus pyogenes 10096. Psychorubrin also interfered with the composition of the biofilm matrix by reducing the total content of carbohydrates and proteins. A synergic effect between psychorubrin and chloramphenicol was observed for S. aureus 33592 and S. pyogenes 10096 while an additive effect was detected for S. aureus 33591. Our findings demonstrate, for the first time, an antagonistic activity of psychorubrin against bacteria not only in their planktonic forms but also in biofilms, and identify bacterial membranes as primary targets for this compound. Based on these observations, psychorubrin has a good potential for the design of novel antimicrobial agents.