Expansion Microscopy of trichomonads.

Light microscopy has significantly advanced in recent decades, especially concerning the increased resolution obtained in fluorescence images. Here we present the Expansion Microscopy (ExM) technique in two parasites, Trichomonas vaginalis and Tritrichomonas foetus, which significantly improved the localization of distinct proteins closely associated with cytoskeleton by immunofluorescence microscopy. The ExM techniques have been used in various cell types, tissues and other protist parasites. It requires the embedment of the samples in a swellable gel that is highly hydrophilic. As a result, cells are expanded 4.5 times in an isotropic manner, offering a spatial resolution of ∼70 nm. We used this new methodology not only to observe the structural organization of protozoa in more detail but also to increase the resolution by immunofluorescence microscopy of two major proteins such as tubulin, found in structures formed by microtubules, and costain 1, the only protein identified until now in the T. foetus's costa, a unique rod-shaped like structure. The individualized microtubules of the axostyle were seen for the first time in fluorescence microscopy and several other details are presented after this technique.

Costain 1 (ARM19800.1) - The first identified protein of the costa of the pathogenic protozoan Tritrichomonas foetus.

Protists members of the Trichomonadidae and Tritrichomonadidae families include agents of trichomoniasis that constitute important parasitic diseases in humans and in animals of veterinary interest. One of the characteristic features of these eukaryotic microorganisms is that they contain a fibrous structure known as the costa as an important cytoskeleton structure, that differs in several aspects from other cytoskeleton structures found in eukaryotic cells. Previous proteomic analysis of an enriched costa fraction revealed the presence of several hypothetical proteins. Here we describe the localization of one of the most prevalent protein found in this previously made proteomic assay to confirm its presence in the costa of Tritrichomonas foetus. A peptide sequence of the hypothetical protein ARM19800.1 was selected for the production of specific polyclonal antibodies and its specificity was confirmed by Western Blotting using an enriched costa fraction. Next, the specific localization of the selected protein was evaluated by immunofluorescence and electron microscopy immunocytochemistry. Our observations clearly showed that the ARM 19800.1 protein is indeed localized in the costa and displays an almost periodic labeling pattern. Since this is the first protein identified in the costa, it was designated as costain 1. A better understanding of a structure as peculiar as the costa is of great biological and evolutionary importance due to the fact that it contains unique proteins, it may represent a possible chemotherapy target and it may correspond to antigens of interest in immunodiagnosis and/or vaccine development.

Reductive evolution of virulence repertoire to drive the divergence between community- and hospital-associated methicillin-resistant Staphylococcus aureus of the ST1 lineage.

Methicillin-resistant Staphylococcus aureus (MRSA) of the ST1-SCCmecIV lineage has been associated with community-acquired (CA) infections in North America and Australia. In Brazil, multi-drug resistant ST1-SCCmecIV MRSA has emerged in hospital-associated (HA) diseases in Rio de Janeiro. To understand these epidemiological differences, genomic and phylogenetic analyses were performed. In addition, virulence assays were done for representative CA - and HA-MRSA strains. Despite the conservation of the virulence repertoire, some genes were missing in Brazilian ST1-SCCmecIV including lukSF-PV, fnbB, and several superantigen-encoded genes. Additionally, CA-MRSA lost the splDE while HA-MRSA strains conserved the complete operon. Most of these variable genes were located in mobile genetic elements (MGE). However, conservation and maintenance of MGEs were often observed despite the absence of their associated virulence markers. A Bayesian phylogenetic tree revealed the occurrence of more than one entrance of ST1 strains in Rio de Janeiro. The tree shape and chronology allowed us to infer that the hospital-associated ST1-SCCmecIV from Brazil and the community-acquired USA400 from North America are not closely related and that they might have originated from different MSSA strains that independently acquired SCCmecIV cassettes. As expected, representatives of ST1 strains from Brazil showed lower cytotoxicity and a greater ability to survive inside human host cells. We suggest that Brazilian ST1-SCCmecIV strains have adapted to the hospital setting by reducing virulence and gaining the ability to persist and survive inside host cells. Possibly, these evolutionary strategies may balance the biologic cost of retaining multiple antibiotic resistance genes.

A Type IIa crustin from the pink shrimp Farfantepenaeus paulensis (crusFpau) is constitutively synthesized and stored by specific granule-containing hemocyte subpopulations.

Crustins are cysteine-rich antimicrobial peptides (AMPs) widely distributed across crustaceans. From the four described crustin Types (I to IV), crustins from the subtype IIa are the most abundant and diverse members found in penaeid shrimp. Despite the critical role of Type IIa crustins in shrimp antimicrobial defenses, there is still limited information about their synthesis and antimicrobial properties. Here, we report the subcellular localization and the antibacterial spectrum of crusFpau, a Type IIa crustin from the pink shrimp Farfantepenaeus paulensis. The recombinantly expressed crusFpau showed antimicrobial activity against both Gram-positive and Gram-negative bacteria at low concentrations. Results from immunofluorescence using anti-rcrusFpau antiserum revealed that crusFpau is synthetized and stored by both granular and semigranular hemocytes, but not by hyaline cells. Interestingly, not all granular and semigranular hemocytes stained for crusFpau, revealing that this crustin is produced by specific granule-containing hemocyte subpopulations. Finally, we showed that the granule-stored peptides are not constitutively secreted into the plasma of healthy animals.

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination.

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations.

Molecular cloning of crustins from the hemocytes of Brazilian penaeid shrimps.

Crustins are antimicrobial peptides initially identified in the hemocytes of the crab Carcinus maenas (11.5-kDa peptide or carcinin) and recently also recognized in penaeid shrimps and other crustacean species. The aim of this study was to identify sequences encoding for crustins from the hemocytes of four Brazilian penaeid species: Farfantepenaeus paulensis, Farfantepenaeus subtilis, Farfantepenaeus brasiliensis and Litopenaeus schmitti. Using primers based on consensus nucleotide alignment of crustins from different crustaceans, cDNA sequences coding for crustins in all indigenous penaeid species were amplified. The obtained four crustin sequences encoded for peptides containing a hydrophobic N-terminal region rich in glycine repeats and a C-terminal part with 12 cysteine residues and a conserved whey acidic protein domain. All obtained crustin sequences showed high amino acidic similarity among each other and with crustins from litopenaeid shrimps (76-98%). This is the first report of crustins in native Brazilian penaeid shrimps.