ß-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

Interspecies Isobaric Labeling-Based Quantitative Proteomics Reveals Protein Changes in the Ovary of Aedes aegypti Coinfected With ZIKV and Wolbachia.

Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by Ae. aegypti. This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and other clinical aspects such as acute febrile illness and neurologic complications, for example, Guillain-Barré syndrome. One of the most promising strategies to mitigate arbovirus transmission involves releasing Ae. aegypti mosquitoes carrying the maternally inherited endosymbiont bacteria Wolbachia pipientis. The presence of Wolbachia is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits such as fecundity and fertility. However, the mechanisms by which Wolbachia influences metabolic pathways leading to differences in egg production remains poorly known. To investigate the impact of coinfections on the reproductive tract of the mosquito, we applied an isobaric labeling-based quantitative proteomic strategy to investigate the influence of Wolbachia wMel and ZIKV infection in Ae. aegypti ovaries. To the best of our knowledge, this is the most complete proteome of Ae. aegypti ovaries reported so far, with a total of 3913 proteins identified, were also able to quantify 1044 Wolbachia proteins in complex sample tissue of Ae. aegypti ovary. Furthermore, from a total of 480 mosquito proteins modulated in our study, we discuss proteins and pathways altered in Ae. aegypti during ZIKV infections, Wolbachia infections, coinfection Wolbachia/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of Ae. aegypti. The modified aspects mainly were related to the immune priming enhancement by Wolbachia presence and the modulation of the Juvenile Hormone pathway caused by both microorganism's infection.

Recognition of Cell Wall Mannosylated Components as a Conserved Feature for Fungal Entrance, Adaptation and Survival Within Trophozoites of Acanthamoeba castellanii and Murine Macrophages.

Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis.

BACKGROUND: Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. METHODS: AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. RESULTS: AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. CONCLUSIONS: The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.

Comprehensive Quantitative Proteome Analysis of Aedes aegypti Identifies Proteins and Pathways Involved in Wolbachia pipientis and Zika Virus Interference Phenomenon.

Zika virus (ZIKV) is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have had evidence of autochthonous mosquito-borne transmission of ZIKV, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector, Aedes aegypti, to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont Wolbachia pipientis has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which Wolbachia orchestrate resistance to ZIKV infection remain to be elucidated. In this study, we apply isobaric labeling quantitative mass spectrometry (MS)-based proteomics to quantify proteins and identify pathways altered during ZIKV infection; Wolbachia infection; co-infection with Wolbachia/ZIKV in the A. aegypti heads and salivary glands. We show that Wolbachia regulates proteins involved in reactive oxygen species production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of Wolbachia in mosquitoes was determined by MS and corroborates the idea that Wolbachia helps to block ZIKV infections in A. aegypti. The present study offers a rich resource of data that may help to elucidate mechanisms by which Wolbachia orchestrate resistance to ZIKV infection in A. aegypti, and represents a step further on the development of new targeted methods to detect and quantify ZIKV and Wolbachia directly in complex tissues.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.(AU)