An improved expression and purification protocol enables the structural characterization of Mnt1, an antifungal target from Candida albicans.

BACKGROUND: Candida albicans is one of the most prevalent fungi causing infections in the world. Mnt1 is a mannosyltransferase that participates in both the cell wall biogenesis and biofilm growth of C. albicans. While the cell wall performs crucial functions in pathogenesis, biofilm growth is correlated with sequestration of drugs by the extracellular matrix. Therefore, antifungals targeting CaMnt1 can compromise fungal development and potentially also render Candida susceptible to drug therapy. Despite its importance, CaMnt1 has not yet been purified to high standards and its biophysical properties are lacking. RESULTS: We describe a new protocol to obtain high yield of recombinant CaMnt1 in Komagataella phaffii using methanol induction. The purified protein's identity was confirmed by MALDI-TOF/TOF mass spectroscopy. The Far-UV circular dichroism (CD) spectra demonstrate that the secondary structure of CaMnt1 is compatible with a protein formed by α-helices and ß-sheets at pH 7.0. The fluorescence spectroscopy results show that the tertiary structure of CaMnt1 is pH-dependent, with a greater intensity of fluorescence emission at pH 7.0. Using our molecular modeling protocol, we depict for the first time the ternary complex of CaMnt1 bound to its two substrates, which has enabled the identification of residues involved in substrate specificity and catalytic reaction. Our results corroborate the hypothesis that Tyr209 stabilizes the formation of an oxocarbenium ion-like intermediate during nucleophilic attack of the acceptor sugar, opposing the double displacement mechanism proposed by other reports. CONCLUSIONS: The methodology presented here can substantially improve the yield of recombinant CaMnt1 expressed in flask-grown yeasts. In addition, the structural characterization of the fungal mannosyltransferase presents novelties that can be exploited for new antifungal drug's development.

An ELISA-based method for Galleria mellonella apolipophorin-III quantification.

Mammalian models, such as murine, are used widely in pathophysiological studies because they have a high degree of similarity in body temperature, metabolism, and immune response with humans. However, non-vertebrate animal models have emerged as alternative models to study the host-pathogen interaction with minimal ethical concerns. Galleria mellonella is an alternative model that has proved useful in studying the interaction of the host with either bacteria or fungi, performing drug testing, and assessing the immunological response to different microorganisms. The G. mellonella immune response includes cellular and humoral components with structural and functional similarities to the immune effectors found in higher vertebrates, such as humans. An important humoral effector stimulated during infections is apolipophorin III (apoLp-III), an opsonin characterized by its lipid and carbohydrate-binding properties that participate in lipid transport, as well as immunomodulatory activity. Despite some parameters, such as the measurement of phenoloxidase activity, melanin production, hemocytes counting, and expression of antimicrobial peptides genes are already used to assess the G. mellonella immune response to pathogens with different virulence degrees, the apoLp-III quantification remains to be a parameter to assess the immune response in this invertebrate. Here, we propose an immunological tool based on an enzyme-linked immunosorbent assay that allows apoLp-III quantification in the hemolymph of larvae challenged with pathogenic agents. We tested the system with hemolymph coming from larvae infected with Escherichia coli, Candida albicans, Sporothrix schenckii, Sporothrix globosa, and Sporothrix brasiliensis. The results revealed significantly higher concentrations of apoLp-III when each microbial species was inoculated, in comparison with untouched larvae, or inoculated with phosphate-buffered saline. We also demonstrated that the apoLp-III levels correlated with the strains' virulence, which was already reported. To our knowledge, this is one of the first attempts to quantify apoLp-III, using a quick and easy-to-use serological technique.

Case report: Functional characterization of a de novo c.145G>A p.Val49Met pathogenic variant in a case of PIGA-CDG with megacolon.

A subgroup of congenital disorders of glycosylation (CDGs) includes inherited GPI-anchor deficiencies (IGDs) that affect the biosynthesis of glycosylphosphatidylinositol (GPI) anchors, including the first reaction catalyzed by the X-linked PIGA. Here, we show the first PIGA-CDG case reported in Mexico in a male child with a moderate-to-severe phenotype characterized by neurological and gastrointestinal symptoms, including megacolon. Exome sequencing identified the hemizygous variant PIGA c.145G>A (p.Val49Met), confirmed by Sanger sequencing and characterized as de novo. The pathogenicity of this variant was characterized by flow cytometry and complementation assays in PIGA knockout (KO) cells.

Moonlighting proteins in medically relevant fungi.

Moonlighting proteins represent an intriguing area of cell biology, due to their ability to perform two or more unrelated functions in one or many cellular compartments. These proteins have been described in all kingdoms of life and are usually constitutively expressed and conserved proteins with housekeeping functions. Although widely studied in pathogenic bacteria, the information about these proteins in pathogenic fungi is scarce, but there are some reports of their functions in the etiological agents of the main human mycoses, such as Candida spp., Paracoccidioides brasiliensis, Histoplasma capsulatum, Aspergillus fumigatus, Cryptococcus neoformans, and Sporothrix schenckii. In these fungi, most of the described moonlighting proteins are metabolic enzymes, such as enolase and glyceraldehyde-3-phosphate dehydrogenase; chaperones, transcription factors, and redox response proteins, such as peroxiredoxin and catalase, which moonlight at the cell surface and perform virulence-related processes, contributing to immune evasion, adhesions, invasion, and dissemination to host cells and tissues. All moonlighting proteins and their functions described in this review highlight the limited information about this biological aspect in pathogenic fungi, representing this a relevant opportunity area that will contribute to expanding our current knowledge of these organisms' pathogenesis.

Escherichia coli transcription factors of unknown function: sequence features and possible evolutionary relationships.

Organisms need mechanisms to perceive the environment and respond accordingly to environmental changes or the presence of hazards. Transcription factors (TFs) are required for cells to respond to the environment by controlling the expression of genes needed. Escherichia coli has been the model bacterium for many decades, and still, there are features embedded in its genome that remain unstudied. To date, 58 TFs remain poorly characterized, although their binding sites have been experimentally determined. This study showed that these TFs have sequence variation at the third codon position G+C content but maintain the same Codon Adaptation Index (CAI) trend as annotated functional transcription factors. Most of these transcription factors are in areas of the genome where abundant repetitive and mobile elements are present. Sequence divergence points to groups with distinctive sequence signatures but maintaining the same type of DNA binding domain. Finally, the analysis of the promoter sequences of the 58 TFs showed A+T rich regions that agree with the features of horizontally transferred genes. The findings reported here pave the way for future research of these TFs that may uncover their role as spare factors in case of lose-of-function mutations in core TFs and trace back their evolutionary history.

Overexpression of the celA1 gene in BCG modifies surface pellicle, glucosamine content in biofilms, and affects in vivo replication.

Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.

Coinfection of domestic felines by distinct Sporothrix brasiliensis in the Brazilian sporotrichosis hyperendemic area.

Microbial interactions may impact patient's diagnosis, prognosis and treatment. Sporotrichosis is a hyperendemic neglected zoonosis in Brazil, caused by Sporothrix brasiliensis. Four pairs of clinical isolates of Sporothrix were recovered from four diseased cats (CIM01-CIM04, two isolates per animal) raising the possibility of coinfection in a sporotrichosis hyperendemic area, Brazil. Each isolate of the pair had distinct pigmentation in mycological culture, and was designated as "Light" or "Dark", for low and high pigmentation, respectively. Dark isolates reacted strongly with monoclonal antibodies to melanin (p ≤ 0.05) by both ELISA and FACS quantitation, and displayed a ring pattern with some regions exhibiting higher punctuated labeling at cell wall by immunofluorescence. In turn, Light isolates reacted less intensely, with few and discrete punctuated labeling at the cell wall. PCR identified all isolates as S. brasiliensis, MAT1-2 idiomorph. Sequencing of ß-tubulin and calmodulin genes followed by phylogenetic analysis placed all eight isolates within the same cluster as others from the Brazilian hyperendemic area. The ability of these strains to stimulate cytokine production by human PBMCs (Peripheral blood mononuclear cells) was also analyzed. CIM01 and CIM03 Light and Dark isolates showed similar cytokine profiles to the control strain, while CIM02 and CIM04 behaved differently (p < 0.001), suggesting that differences in the surface of the isolates can influence host-fungus interaction. MICs for amphotericin B, terbinafine, caspofungin, micafungin, itraconazole, fluconazole, and voriconazole were obtained (CLSI M38-A2/M27-A3). Pairwise comparisons showed distinct MICs between Sporothrix Light and Dark isolates, higher than at least two-fold dilutions, to at least one of the antifungals tested. Isolates from the same pair displayed discrepancies in relation to fungistatic or fungicidal drug activity, notably after itraconazole exposure. Since S. brasiliensis Light and Dark isolates show disparate phenotypic parameters it is quite possible that coinfection represents a common occurrence in the hyperendemic area, with potential clinical implications on feline sporotrichosis dynamics. Alternatively, future studies will address if this specie may have, as reported for other fungi, broad phenotypic plasticity.

Role of protein phosphomannosylation in the Candida tropicalis-macrophage interaction.

Candida tropicalis is an opportunistic fungal pathogen responsible for mucosal and systemic infections. The cell wall is the initial contact point between a fungal cell and the host immune system, and mannoproteins are important components that play key roles when interacting with host cells. In Candida albicans, mannans are modified by mannosyl-phosphate moieties, named phosphomannans, which can work as molecular scaffolds to synthesize ß1,2-mannooligosaccharides, and MNN4 is a positive regulator of the phosphomannosylation pathway. Here, we showed that C. tropicalis also displays phosphomannans on the cell surface, but the amount of this cell wall component varies depending on the fungal strain. We also identified a functional ortholog of CaMNN4 in C. tropicalis. Disruption of this gene caused depletion of phosphomannan content. The C. tropicalis mnn4Δ did not show defects in the ability to stimulate cytokine production by human mononuclear cells but displayed virulence attenuation in an insect model of candidiasis. When the mnn4Δ-macrophage interaction was analyzed, results showed that presence of cell wall phosphomannan was critical for C. tropicalis phagocytosis. Finally, our results strongly suggest a differential role for phosphomannans during phagocytosis of C. albicans and C. tropicalis.

Tenebrio molitor (Coleoptera: Tenebrionidae) as an alternative host to study fungal infections.

Models of host­pathogen interactions are crucial for the analysis of microbial pathogenesis. In this context, invertebrate hosts, including Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode) and Galleria mellonella (moth), have been used to study the pathogenesis of fungi and bacteria. Each of these organisms offers distinct benefits in elucidating host­pathogen interactions. In this study,we present a newinvertebrate infection model to study fungal infections: the Tenebrio molitor (beetle) larvae. Here we performed T. molitor larvae infection with one of two important fungal human pathogens, Candida albicans or Cryptococcus neoformans, and analyzed survival curves and larva infected tissues.We showed that increasing concentrations of inoculum of both fungi resulted in increased mortality rates, demonstrating the efficiency of the method to evaluate the virulence of pathogenic yeasts. Additionally, following 12 h post-infection, C. albicans formsmycelia, spreading its hyphae through the larva tissue,whilst GMS stain enabled the visualization of C. neoformans yeast and theirmelanin capsule. These larvae are easier to cultivate in the laboratory than G. mellonella larvae, and offer the same benefits. Therefore, this insect model could be a useful alternative tool to screen clinical pathogenic yeast strainswith distinct virulence traits or different mutant strains.

Conversion of alpha1,2-mannosidase E-I from Candida albicans to alpha1,2-mannosidase E-II by limited proteolysis.

Previous studies demonstrated the presence in Candida albicans ATCC 26555 of two soluble alpha1,2-mannosidases: E-I and E-II. In contrast, in the C. albicans CAI-4 mutant only E-I was detected and it could be processed by a membrane-bound proteolytic activity from the ATCC 26555 strain, generating an active 43 kDa polypeptide. Here, alpha1,2-mannosidase E-I from strain ATCC 26555 was purified by conventional methods of protein isolation and affinity chromatography in Concanavalin A-Sepharose 4B. Analytical electrophoresis of the purified enzyme revealed two polypeptides of 52 and 23 kDa, the former being responsible for enzyme activity as revealed by zymogram analysis. Time course proteolysis with an aspartyl protease from Aspergillus saitoi, converted alpha1,2-mannosidase E-I into an active polypeptide of 43 kDa which trimmed Man(9)GlcNAc(2), generating Man(8)GlcNAc(2) isomer B and mannose. Trimming was inhibited preferentially by 1-deoxymannojirimycin. Both, the molecular mass and the enzyme properties of the proteolytic product were identical to those described for alpha1,2-mannosidase E-II therefore supporting the notion that E-I is the precursor of E-II.